ICoFAB2019 Proceedings

Preface

The integrated ASEAN Economic Community (AEC) was launched in 2015. One of the main purposes of this integration is for the development of science and technology since it is a key factor in sustaining economic growth, enhancing community wellbeing and promoting integration in this region.

In order for ASEAN to become world class and be globally competitive, it requires the driving forces from the three main scientific areas of (1) Food science and technology (2) Agricultural technology and (3) Biotechnology. ASEAN is home to one of the world’s most precious natural resources, and the most diverse microbial community. Scientific strength in this region would be significantly enhanced provided that appropriate collaborative networks amongst member countries are promoted. In addition, education sectors should focus more on internationalizing. Their curricula and universities across this region should find more opportunities to collaborate in research and academic activities.

The Faculty of Technology (MSU) initiated the 1st International Postgraduate Symposium on Food, Agriculture and Biotechnology in 2014 (IPSFAB 2014). Our symposiums in the past four years (2014 – 2017) had gone successfully. We are honored by distinguished scientific committees and audiences from around the world. Since 2018, the name of symposium has been changed to “The International Conference on Food, Agriculture and Biotechnology (ICoFAB 2018)”. The revision has been made to broaden the conference scope, in which encourage more researchers, not limited to the postgraduates, in related research areas to take part and share their research experiences. Thus, the potential academic networks or collaboration amongst Thai and international researchers could be developed.

The 6th ICoFAB 2019 would be providing an atmosphere of collaborative networks amongst national and international researchers. All participants may find benefits from update research trends presented at the symposium.

(Assoc. Prof. Dr. Anuchita Moongngarm) Dean of the Faculty of Technology Mahasarakham University

ICoFAB2019 Proceedings

MSU Editorial Board

Assoc. Prof. Dr. Anuchita Moongngam Assoc. Prof. Dr. Maratree Plainsirichai Assoc. Prof. Dr. Thalisa Yuwa-amornpitak Assoc. Prof. Prasit Chutichudech Assoc. Prof. Dr. Anut Chantiratikul Asst. Prof. Dr. Sirirat Deeseenthum Asst. Prof. Dr. Wantana Sinsiri Asst. Prof. Dr. Pheeraya Chottanom Asst. Prof. Dr. Wasan Duangkhamchan Asst. Prof. Dr. Vijitra Luang-In Asst. Prof. Dr. Kedsirin Sakwiwatkul Asst. Prof. Dr. Luchai Butkhup Asst. Prof. Dr. Eakapol Wangkahart Asst. Prof. Dr. Panarat Phadee

Organizing Committee

Assoc. Prof. Dr. Anuchita Moongngam Asst. Prof. Dr. Sirirat Deeseenthum Asst. Prof. Dr. Eakapol Wangkahart Asst. Prof. Dr. Vijitra Luang-In Asst. Prof. Dr. Wasan Duangkhamchan Asst. Prof. Dr. Narendhirakannan RT Asst. Prof. Dr. Abdulhadi Albaser

Scientific Committee

Prof. He Chaoxing, Institute of Vegetable and Flowers Chinese Academy of Agricultural Sciences, Prof. Ping Zhang, Xishuangbanna Tropical Botanical Garden, China Prof. Yongqi Shao, Zhejiang University, China Prof. C. Hanny Wijaya, Bogor Agricultural University, Honorary Prof. Colin Wrigley, QAAFI, University of Queensland, Australia Prof. Emeritus Ian Warrington, Massey University, New Zealand Assoc. Prof. Dr. Ko-Tung Chang, National Pingtung University of Science and Technology, Asst. Prof. Dr. Abdulhadi Albaser, University of Sebha, Libya Assoc. Prof. Dr. Khamsah Suryati Mohd, Universiti Sultan Zainal Abidin, Terengganu, Prof. Jun Zou, Shanghai Ocean University, China Dr. Steve Bird, University of Waikato, New Zealand Assoc. Prof. Dr. Bei Wang, Ocean University, China Dr. Miguel Bao, Institute of Marine Research, Norway Asst. Prof. Dr. Narendhirakannan RT, Karunya Institute of Technology and Science, Assoc. Prof. Dr. Maratree Plainsirichai, Mahasarakham University, Assoc. Prof. Dr. Thalisa Yuwa-amornpitak, Mahasarakham University, Thailand ICoFAB2019 Proceedings

Assoc. Prof. Dr. Anut Chantiratikul, Mahasarakham University, Thailand Asst. Prof. Dr. Pheeraya Chottanom, Mahasarakham University, Thailand Asst. Prof. Dr. Karnnika Chokeatwatana, Mahasarakham University, Thailand Asst. Prof. Dr. Benjapon Kunlanit, Mahasarakham University, Thailand Asst. Prof. Dr. Srinual Jantathai, Mahasarakham University, Thailand Asst. Prof. Dr. Vijitra Luang-in, Mahasarakham University, Thailand Asst. Prof. Dr. Luchai Butkhup, Mahasarakham University, Thailand Asst. Prof. Dr. Kedsukon Maneewan, Mahasarakham University, Thailand Asst. Prof. Dr. Sirirat Deeseenthum, Mahasarakham University, Thailand Asst. Prof. Dr. Eakapol Wangkahart, Mahasarakham University, Thailand Asst. Prof. Dr. Kriengsak Bunleu, Mahasarakham University, Thailand Asst. Prof. Dr. Tatdao Phasipol, Mahasarakham University, Thailand Dr. Issaraporn Somboonwatthanakul, Mahasarakham University, Thailand Dr. Sunisa Roidoung, Mahasarakham University, Thailand

ICoFAB2019 Proceedings

Content Full papers: Page

Effect of Sugar Substitution by Stevia Extract on Sensory Acceptance, Color, and Texture 1 Profiles of Brownie Naruetit Sriharbutr

Phytochemical Screening, Total Phenolic Contents and Antioxidant Activity of the 6 Aqueous Extracts of Dendrocalamus membranaceus and Thyrsostachys siamensis Agarat Kamcharoen

Distribution of Melatonin and Serotonin in Germinated Rice 12 Jakkaphan Kaennok

Sensory Characteristics of No-Sugar Black-Rice Tea Drinks 18 Jutawan nuanchankong

Effect of Cinnamon Oil and Garlic Extract for Fresh Shrimp Preservation 22 Supraewpan Lohalaksnadech

Effects of Wood Vinegar and Cow Manure on Growth of Khao Dawk Pradoo Rice in 28 Experimental Field Wanida Sumranram

Kinetics Study on Hot-Air Drying Carrot Cubes 32 Wanwisa Suksamran

Effect of Acid-Alkaline Pretreatment on Reducing Sugar Yield and Lignocellulosic 39 Compositions of Rice (Oryza sativa L.) Residues Kaewkanlaya Sotthisawad

Mixture of Parawood Sawdust and Dried Napier Grass as a Substrate on Lentinus 44 squarrusulus Mont. Cultivation Niramai Fangkrathok

Variation of Inulin Content in Banana Peels at Different Maturity Stages 50 Ratchanee Puttha Phenolic and Antioxidant Properties of Male Bud Flowers and Fruit of Musa Genotypes 56 with Different Ploidy Somkit Jaitrong

Efficacy of Stephania pierrei Tuber Extract for Leaf Spot Disease Control in Greenhouse 61 and Field Condition Quanjai Rupitak

Phytochemical Contents and Antioxidant Activity of 64 Bagasse Sugarcane Extracts Panadda Sanarat

Locust Bean Gum Hydrolysis for Mannooligosaccharide (MOS) Production Using Bacillus 69 methylotrophicus KS1 Sunchai Phiwphech

ICoFAB2019 Proceedings

Content Full papers: Page

Screening of Yeasts from Thai Traditional Fermentation Starter (Loog-pang) for Alcoholic 74 Fermentation Products in Community Enterprise Pikulthong Paewlueng

Isolation and Identification of Lipase-Producing Bacteria from Soil in Nasinuan Forest, 79 Kantarawichai District, Mahasarakham Province Manatchanok Yotchaisarn

Screening and Identification of Bacteria that Produce Chitinase Enzymes from Soil in Na 88 Si Nuan Forest, Maha Sarakham, Thailand Ketsara Suwunnapukdee

Random Mutagenesis of Aspergillus sclerotiorum PSU-RSPG 178 for Improvement a 94 Lovastatin Production Supawan Meena

Genetic Variation among Thai Dugong (Dugong dugon) Populations from Cytochrome C 99 Oxidase Subunit 1 DNA Sequence Data Nattapong Srisamoot

Comparative Study of Proteome Pattern of Kluai Ta Nee and Kluai Nam Wa Leaf Proteins 106 Bung-on Prajanban

Characterisation of Microwave-assisted Pretreatment for Spent Coffee Grounds 112 Wipada Jamsai

Morphological Observation of Polylactide-b-Poly (Ethylene Glycol)-b-Polylactide Triblock 118 Copolymers Stereocomplex Films Pattarin Intaravichien

Plasma Technology and Abiotic Elicitor Effectively Increased Isothiocyanates, Bioactive 123 Compounds and Cytotoxicity against Caco2 Cells in Mustard Green Microgreen Extract Worachot Saengha

Phytochemical Screening, Antioxidant, and α-Glucosidase Inhibitory Activities of Different 132 Solvent Extracts from Leersia hexandra and Elephantopus scaber Saithong Sombutphoothorn

Evaluation on Phytochemical Constituents and Antioxidant Activities of Various Formula 139 from Ko-Klan Remedies by Aqueous Infusion Preparation Method Khwanchnok Maitnork

Plant Diversity in Burapha University, Sa Kaeo Campus 144 Chakkrapong Rattamanee

Parasitic Infection in Common Lowland Frog (Hoplobatrachus rugulosus Wiegmann) and 153 Disease Treatment Panarat Phadee

ICoFAB2019 Proceedings

doi:10.14457/MSU.res.2019.1 ICoFAB2019 Proceedings | 1

Effect of Sugar Substitution by Stevia Extract on Sensory Acceptance, Color, and Texture Profiles of Brownie

Naruetit Sriharbutr, Tanongsak Moontree and Anuchita Moongngarm*

Department of Food Technology and Nutrition, Nutrition for Health Research Unit, Faculty of Technology, Mahasarakham University, Mahasarakham 44150, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Currently, consumers pay more attention to health and consumption more healthy foods. Substitution of sugar with natural sweetening agents in sweet products is another interesting option. This research purposed to study the application of stevia extracts to replace sugar in brownies. Stevia leaves were extracted with water at 55ºC for 6 h prior to drying using a spray dryer to obtain stevia extract powder. The sugar was replaced by the stevia extract powder with the degree of substitution of 0.125, 0.25, 0.375, and 0.50%. The sensory evaluation of brownies was evaluated by 50 panelists using 9 points hedonic scale. The results indicated that the brownies contained 23.07% of sugar and 0.125% stevia extract powder received the highest liking score which is comparable to those of control brownie sample. For the study on the effect of the addition of stevia extract powder on the color of brownie, it was observed that the brightness value of brownie was affected by the stevia extract added, the more level of the stevia extract powder added, the lower the brightness obtained. For the texture characteristics of the brownie, it was found that the addition of stevia powder significant effect all texture profiles of brownies. Therefore, the application of stevia extract powder to replace sugar is possible. However, further studies on the effect of replacing sugar by stevia extract powder on texture characteristics and nutritional value are needed.

Keywords: Brownies, Stevia, Stevioside, Low sugar, Sugar replacement

Introduction

Excessive sugar intake may negatively affect health, including obesity, diabetes, and high blood sugar. Therefore, varieties of sweeteners have been produced to replace sugar including synthetic artificial and natural sweeteners which are low calories. However, some synthetic sweeteners such as saccharin and aspartame are restricted to be used in food because they may cause adverse effect to humans [1]. Stevioside is a natural sweetener with no calorie obtained from stevia leaves (Stevia rebaudiana Bertoni.). It is sweeter than sucrose approximately 300 times [2]. The sweetness of glycoside found in stevia relative to sucrose is indicated in Table 1. Since stevioside does not provide calories, makes it interesting in substituting for sugar. Purified stevioside is white, colorless, and odorless. Major active sweetener compounds extracted from stevia leaves are stevioside and rebaudioside A, which accounts for over 90% w/w of all sweeteners in the stevia [3]. The chemical structures of stevioside and rebaudioside A are presented in Fig. 1. Of all sweeteners in the stevia leaves, rebaudioside A gives a good taste without bitter taste [4]. Brownies are one of the most popular sweet bakery products, rich in sugar and chocolate provides a distinctive flavor. Brownies have a semi-cookie texture, consisting of flour, butter, chocolate, sugar [5]. As brownies contain very high sugar and chocolate content in one serving resulting in a sweet and bitter flavor which is associated with the taste of stevia extract that is very sweet and has a bitter after taste in the mouth. Therefore, this research was conducted to find out the suitable amount of stevia extract to replace sugar in brownies and to determine the effect of sugar substitution by stevia extract on texture profile and color of brownies.

Materials and methods

The extraction of stevia and stevia extract powder preparation Stevia (Stevia rebaudiana) (dried leaves) with moisture content approximately 10% was purchased from Chiangmai province, Thailand. The stevia leaves were cleaned and extracted with distilled water (ratio of 100 grams per 550 ml) at 80ºC for 6 hours. The extract was filtered using cloth cheese and 0.45 micron ICoFAB2019 Proceedings | 2

filter paper. After that, the filtrate was added with 2% maltodextrin prior to drying using a spray drier. The operation conditions were hot air temperature at 160°C, the flow rate of 12sec/ml. The stevia extract powder was obtained and kept in zip-lock plastic bags and storage at the temperature of 4ºC.

Table 1 Comparison of the sweet glycosides presented in S. rebaudiana

Glycoside name Content (%) Sweetness Molecular References relative to mass (g/mol) sucrose Stevioside 5.0-10.0 250-300 804.87 Bridel and Lavieille, 1931[7] RebaudiosideA 2.0-4.0 350-450 967.01 Wood et al. 1955[8] RebaudiosideB <<1 300-350 804.87 Bride land Lavieille,1931[7] RebaudiosideC 1.0-2.0 50-120 951.01 Sakamoto et al. 1977[9] RebaudiosideD <<1.0 200-300 1129.15 Sakamoto et al. 1977[9] RebaudiosideE <<1.0 250-300 967.01 Sakamoto et al. 1977[9] RebaudiosideF <<1.0 Nd 936.99 Sakamoto et al. 1977[9] Steviolbioside <<1.0 100-125 642.73 Kohda et al. 1976[10] Dulcoside A 0.4-0.7 50-120 788.87 Wood et al. 1955[8] Nd=not detected Source: Geuns 2003 [6]

A B

Figure 1 Chemical structure of Stevioside (A) and Rebaudioside A (B) [11]

Experimental design for substitution of stevia extract powder for sugar A mixture design was applied to investigate the suitable degree of sugar substitution with stevia extract powder. The program Design-Expert Version 7.0 was used to generate the formula of brownies. The amount of sugar used was varied from 0 to 30.76% and stevia extract powder was between 0.2 to 0.5% of the total amount of raw materials [12]. Five formulas of brownies were obtained, namely (1) control formula, (2) sugar 23.07% and stevia extract powder 0.125%, (3) sugar 15.38% and stevia extract powder 0.25%, (4) sugar 7.69% and stevia extract powder 0.375%, (5) sugar 0% and stevia extract powder 0.50%. Brownie preparation Brownies were prepared using the method of Selvakumaran (2017) [13] with some modifications. The ingredients included 75 g of wheat flour, 60 g of cocoa powder,150 g of unsalted butter, 150 g of chocolate chips, 50 g of eggs, 108 g of sugar, 200 g of baking powder 1.5 g of vanilla, 4 g of salt, and 1.5 g of salt. The chocolate chip and unsalted butter were melt and mixed well, then sugar was added and blended with a high-speed electric mixer until homogeneous with eggs. All-purpose wheat flour was sifted ICoFAB2019 Proceedings | 3

together with baking powder. Then all ingredients and liquid portion were mixed homogeneously. The mixture was baked in an oven at 180°C for 20 minutes. After taken out, the brownies were cooled down to room temperature for 1 hour and the kept in polyethylene bags stored at 4°C for sensory evaluation using the Hedonic 9 scale method. Sensory evaluation of brownies The tested samples of 1x1x1 inch brownie (room temperature) were served to 50 test panelists for sensory evaluation using the hedonic test 9 point scale. The evaluation attributes included appearance, color, smell, sweetness, bitter after-taste, overall preference. Color measurement of brownies Brownie samples were determined for color according to Guajardo Flores [14], using colorimeter (Minolta: Model CR-300). The measuring head was placed in the center of each brownie. Colour values were measured using CIE (Commission Internationale d’ Eclarirage) L*⁄a*⁄b*⁄ scale in triplicate and means were recorded as L* = lightness (0 = black, 100 = white), a* (-a = greenness, +a = redness) and b* (-b = blueness, +b = yellowness). Texture profile analysis Texture Profile Analysis was used as a method to evaluate the texture of brownies by following the method used by Guajardo Flores [14]. Brownies were cooled and analyzed after 24 h of baking. Measurements were made using a piece of brownie (3x3x3 cm) for two-cycle compression. The probe used was 50 mm compression plate (P/50) and the settings used for this analysis were strain 50% at a distance of 10 mm with a force of 5.0g. The texture parameters obtained included hardness, adhesiveness, springiness, cohesiveness, gumminess, and chewiness. The texture parameters of each brownie were averaged from 3 replicates.

Results and Discussion

Sensory evaluation of brownies Sensory evaluation results obtained from 50 panelists were shown in Table 2. The appearance scores ranged from 4.76. to 7.20 The significant highest score was observed in control sample, followed by brownies added with 0.125 and 0.25% of stevia powder and the lowest appearance scores were found with brownies added with 0.375 and 0.5% of stevia powder. The color scores of the sample varied between 5.02. and 7.13 The higher level of stevia powder added, the lower the color score obtained; however, the color score of brownies added with 0.125% of stevia powder was not significant difference from the control sample. The odor scores of the samples ranged from 5.20 to 6.90. The odor of the control sample was not significantly different from the sample added with 0.125% of stevia powder. The sweetness scores were between 5.20 and 7.22. The sweetness of control and brownie mixed with 0.125% of stevia powder had no significant difference in sweetness. The aftertaste scores varied from 3.07 to 6.20 which there was no significant difference between the control and the sample added with 0.125% of stevia powder. The overall liking score of the sample ranged between 3.310 and 7.60 which the liking score of each sample was significantly different from each other. These results were comparable with the study of Saniah's and Samsiah [15] whop reported that stevia extract could partially replace sucrose in carbonate drinks, with the highest acceptance of 33.13% sucrose with 0.43% stevia extract. This was also similar to the study of Lenc et al. [16] who found that consumers revealed the highest acceptance in yogurt samples with a mixture of sweeteners between sugar and stevia extract.

Color determination The color value of brownies obtained from the CIE (Commission Internationale d'Eclarirage) color measurement included brightness (L*), red (a*) and yellow. (b*). The results were presented in Table 3. The brownies had L* values between 19.420 and 27.110. The control sample revealed highest of L*, a*, and b* values. The more the stevia powder added, the lower the values of lightness, a*, and b* values. When the amount of stevia extract powder added increased, the brightness of the brownies decreased, this may due to the chlorophyll and porphyrin color of stevia leaves which in accordance with the research of Salazar et al. [17].

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Table 2 Sensory evaluation of brownies

Sweeteners (%) Bitter Appearance Color Odor Sweetness Overall Sucrose Stevia after-taste 30.67 - 7.20±1.10a 7.13±1.14a 6.90±1.40a 7.22±1.35a 6.20±1.55a 7.60±0.84a 23.07 0.125 6.42±1.51b 6.78±1.11a 6.27±1.62bc 6.80±1.42a 5.89±1.71a 7.02±1.06b 15.38 0.25 6.09±1.69b 5.98±1.60b 6.51±1.06ab 5.40±1.96b 4.93±1.72b 6.02±1.22c 7.69 0.375 5.47±1.46c 5.33±1.54c 5.76±1.49c 4.51±1.86c 4.53±1.71b 5.07±1.60d - 0.50 4.76±1.49d 5.02±1.62c 5.20±1.82d 5.20±1.82b 3.07±1.50c 3.31±1.50e The results are shown in mean ± sd (P <0.05)

Table 3 effect of stevia on the color of brownies

Sweeteners L* a* b* Sucrose (%) Stevia extract (%) 30.67 - 27.110±1.060a 3.240±0.180a 5.800±0.190a 23.07 0.125 24.430±0.730b 2.460±0.180b 4.960±0.290b 15.38 0.25 21.590±1.340c -0.250±0.140e 2.120±0.090d 7.69 0.375 21.110±0.710c 0.130±0.020d 2.210±0.030d - 0.50 19.420±0.470d 1.370±0.180c 3.060±0.880c The results are presented in mean ± sd (P <0.05)

Texture profile analysis Texture characteristics of brownies using a texture profile analysis (TPA) as shown in Table 4. Hardness values of brownies were between 22.54 and147.31N. Brownies added with stevia alone were significantly different in hardness from those added with some sugar, however, it was not significantly different from the control sample. Moreover, reducing sugar and increasing the proportion of sweetener from stevia leave extract increased the adhesiveness value of the brownie (-0.05 to - 0.46 N/s). When the proportion of sweetener from stevia leave extract was increased and the sugar added was reduced, the brownies adhered to the teeth was less. The sample's springiness values ranged between 4.35 and 7.80. The brownie with the addition of sugar only has the highest springiness value (7.80) whereas the brownies with stevia extract alone had the springiness value of 4.35. The brownies that less springiness, the more hardness. The cohesiveness value of the sample ranged between 0.22 and 0.42. The cohesiveness of the brownies varied from 0.22 to 0.42. Brownies with only sugar addition had the least cohesive value (0.22). When the sugar content was reduced and the addition of stevia leaf extract powder was increased, the cohesiveness of brownies increased. The gumminess of the brownies was between 0.22 and 60.70. The brownies that add sweeteners from the stevia extract powder alone had highest gumminess (60.70), while the brownies that add sugar alone was lowest gumminess. Lowest Chewiness values of brownies are between 38.17-263.47. Brownies added with stevia extract alone revealed the highest chewiness value (263.47) while the chewiness values of brownies with sugar of 23.07:0.125% stevia extract and sugar 30% were not significantly different (p <0.05).

Table 4 Texture analysis profiles of brownies

Sweeteners Texture profiles Sucrose Stevia Hardness Adhesiveness Springiness Cohesiveness Gumminess Chewiness % % (N) (N/s.) 30.67 0 22.54±1.72b -0.05±0.02a 7.80±0.39a 0.22±0.01c 0.22±0.01c 38.17±2.64b 23.07 0.125 35.36±17.81b -0.33±0.17bc 7.08±1.29ab 0.35±0.07ab 0.35±0.07ab 79.46±20.08b 15.38 0.25 23.38±5.24b -0.10±0.35a 6.40±0.46b 0.36±0.02ab 8.46±1.39bc 54.15±8.45b 7.69 0.375 79.95±16.16b -0.24±0.04b 6.55±0.17b 0.34±0.00b 27.74±5.90b 182.37±42.98a 0 0.50 147.31±70.48a -0.46±0.12c 4.35±0.23c 0.42±0.03a 60.70±25.00a 263.47±10.37a Values within a column represent mean ± standard deviation of replicate experiments (n = 3) with different letters indicating a significant difference (P<0.05)

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Conclusions

Based on the results of sensory evaluation, color values, and texture profiles, brownies which were replaced sugar by stevia extract powder of 0.125% was the suitable substitution degree. When increasing the amount of stevia extract powder, it significantly affected the sensory evaluation, color values, and texture profile of the brownies. Therefore, this research showed that the amount of sugar could be reduced from normal formulas by replacing by stevia extract powder.

Acknowledgements

This research has been funded by Mahasarakham University and the company Kidtueng bakery Co., Ltd.

References

[1] Fukushima S, Arai M, Nakanowatari J, Hibino T, Okuda M, Ito N. Differences in susceptibility to sodium saccharin among various strains of rats and other animal . GANN Japanese Journal of Cancer Research. 1983; 74(1), 8-20. [2] Gasmalla MA, Yang R, Amadou I, Hua X. Nutritional composition of Stevia rebaudiana Bertoni leaf. effect of drying method. Tropical Journal of Pharmaceutical Research. 2014; 13(1), 61-5. [3] Crammer, B. and R. Ikan. Sweet glycosides from the stevia . Chemistry in Britain, 1986; 22, p. 915. [4] Carakostas MC, Curry LL, Boileau AC, Brusick DJ. Overview: the history technical function and safety of rebaudioside A. a naturally occurring steviol glycoside for use in food and beverages. Food and Chemical Toxicology. 2008; 46(7), S1-S10. [5] Mary Gage. History of Brownies.(Chocolate), Available at:http://www.newenglandrecipes.org/html/brownies.html, accessed May 2019 [6] Geuns JM. Stevioside. Phytochemistry. 2003, 64(5), p.913-921. [7] Bridel M, Lavieille R. The principle of sweetness (Stevia rebaudiana Bertoni). Bull Soc Chem Biol. 1931; 13, 636–655. [8 Wood JRHB, Allerton R, Diehl HW, Fletcher HG Jr. Stevioside. I. The structure of the glucose moieties, J Org Chem, 1955, 20, 875–883. [9] Sakamoto I, Yamasaki K, Tanaka O. Application of 13C NMR spectroscopy to chemistry of natural glycosides: rebaudioside-C, a new sweet diterpene glycoside of Stevia rebaudiana. Chem Pharm Bull. 1977; 25, 844–846. [10] Kohda H, Kasai R, Yamasaki K, Murakami K, Tanaka O. New sweet diterpene glucosides from Stevia rebaudiana. Phytochem. 1976; 15, 981–983. [11] Mosettig, E. and W. R. Nes. Stevioside. II. The structure of the aglucon. The Journal of Organic Chemistry. 1955; 20(7), 884-899. [12] Saniah, K. and M. S. Samsiah. The application of Stevia as sugar substitute in carbonated drinks using Response Surface Methodology. J. Trop. Agric. and Fd. Sc. 2012; 40(1), 23-34. [13] Selvakumaran L, Shukri R, Ramli NS, Dek MS, Ibadullah WZ. Orange sweet potato (Ipomoea batatas) puree improved physicochemical properties and sensory acceptance of brownies. Journal of the Saudi Society of Agricultural Sciences. 2019; 18(3), 332-336. [14] Flores DG.2007, Effect of Antioxidants, Color and Sensory Attributes of Inclusion of Different Sorghum Brans in Model Baking Systems. Doctoral dissertation, Doctoral Dissertation, A&M University, Texas, USA. [15] Saniah, K. and M. S. Samsiah. The application of Stevia as sugar substitute in carbonated drinks using Response Surface Methodology. J. Trop. Agric. and Fd. Sc, 2012; 40(1), 23-34. [16] Lisak K, Lenc M, Jeličić I, Božanić R. Sensory evaluation of the strawberry flavored yoghurt with stevia and sucrose addition. Hrvatski časopis za prehrambenu tehnologiju, biotehnologiju i nutricionizam. 2012; 7(SPECIAL ISSUE-7th), 39-43. [17] Salazar VA, Encalada SV, Cruz AC, Campos MR. Stevia rebaudiana: A sweetener and potential bioactive ingredient in the development of functional cookies. Journal of functional foods. 2018; 44, 183-90. doi:10.14457/MSU.res.2019.2 ICoFAB2019 Proceedings | 6

Phytochemical Screening, Total Phenolic Contents and Antioxidant Activity of the Aqueous Extracts of Dendrocalamus membranaceus and Thyrsostachys siamensis

Sirilak Kamonwannasit, Chakkrapong Rattamanee and Agarat Kamcharoen*

Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo 27160, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

This study aims to determine the phytochemical screening, total phenolic contents, and antioxidant activity of leaf and shoot extracts of Dendrocalamus membranaceus and Thyrsostachys siamensis. Each part of plant was extracted by boiling with distilled water in the solid to liquid ratio of 1 to 5 for 30 min twice. Phytochemical screening involved the methods to detect the presence of alkaloids, flavonoids, saponins, tannins, cardiac glycosides and triterpenoids. Total phenolic contents were estimated with gallic acid as standard. Antioxidant activity was determined using 2, 2’-azino-bis (3-ethyl benzthiazoline-6- sulfonic acid) (ABTS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assays. The result showed that flavonoids and saponins were presented in the aqueous extracts of both leaf and shoot of D. membranaceus and T. siamensis. Total phenolic content was the highest in shoot extract of D. membranaceus and the lowest in leaf extract of D. membranaceus. The leaf extract of D. membranaceus exhibited the highest antioxidant activity in ABTS and DPPH assays, with 24.0 and 503.0 μg/ml, respectively, and was higher activity than its shoot extract.

Keywords: Antioxidant activity, , phytochemicals, total phenolic contents

Introduction

Free radicals cause human diseases such as cancer, heart disease and cerebrovascular disease through multiple mechanism [1]. Antioxidants are substances which inhibit the oxidation of other molecules in our body and prevent the formation of free radicals by scavenging them leading to prevention of cancer and degenerative diseases, slowing down the aging process and promotion of cardiovascular health [2]. Natural antioxidants present in plant origin protect against these radicals and are therefore important tools in obtaining and preserving good health. Moreover, the different parts of the plant have different bioactivity [3-5]. Therefore, search for new plant sources containing antioxidants is continued for their utilization in cosmetic, functional food and pharmaceutical industries. Tea from Camalia sinensis and herbal tea from the leaves, flowers, seeds, fruits and roots of other than C. sinensis are some of the most consumed beverages worldwide due to the attractive flavors and antioxidant activity [6]. Other plant infusions have become popular such as mulberry (Morus alba), lemon gress (Cymbopogon citratus), bamboo gress ( triandra), sappan (Caesalpinia sappan) and bamboo leaf (Sasa borealis) [7, 8]. Bamboo is a group of genera of evergreen plants belonging to Poaceae, or grass family and is a multipurpose plant known mostly from its industrial uses but is now being recognized as a source of bioactive compounds and natural antioxidants [4]. There are more than 1250 bamboo species distributed all around the world and all the parts of the bamboo have clinical applications [2]. Wroblewska et al. [4] studied phenolic contents and antioxidant activity of five native Brazilian bamboo species such as Aulonemia aristulata, Chusquea bambusoides, C. capituliflora, C. meyeriana and Merostachys pluriflora. They found that the antioxidant activity which expressed in IC50 varied between 137.55-260 μg/ml and phenolic contents ranged from 43.64-87.81 mg of gallic acid equivalents (GAE) per g of plant material. Moreover, bamboo in Sasa such as Sasa borealis and Sasa palmata were developed into herbal tea [7, 8]. Although many bamboo species have already been studied, little is known about the antioxidant activity of bamboo species in the East of Thailand, for example D. membranaceus and T. siamensis. In addition, water is a universal solvent and environmental friendly. Moreover, water soluble phenolic only important as antioxidant compound [9] and flavonoids and phenolic compounds [10]. For this reason, the aim of this study was to screen the phytochemicals and to determine the total phenolic contents and ICoFAB2019 Proceedings | 7

antioxidant activity of the water extraction of D. membranaceus and T. siamensis, which was further selected to develop into herbal tea.

Materials and methods

Plant materials Fresh leaf and shoot of (D. membranaceus and T. siamensis) (Figure 1) were collected from Burapha University, Sakaeo Campus. The leaves were washed with tap water, dried at 50˚C for 3 days in tray dryer and grinded to powder. The shoots were also washed with tap water, cut into small pieces, dried at 50˚C for 3 days and grinded to powder. The powder was contained in plastic bag and kept in the desiccator until used.

Figure 1 The figure of D. membranaceus (A) and Thyrsostachys siamemsis (B)

Plant extraction The powder sample of 60 g was extracted in boiling water of 300 ml for 30 min twice. The aqueous extract of the bamboo was filtered through filter paper (Whatman No. 1), evaporated at 80˚C using water bath for 12-24 h or until dried, and then kept in refrigerator until used.

Determination of percentage yield (%) The percentage yield of the extract was determined using the weight of extract after evaporation the solvent (a) and the weight of sample (b) using equation as followed:

푎 푃푒푟푐푒푛푡푎푔푒 푦푖푒푙푑 (%) = × 100 푏

Phytochemical screening The crude extracts of leaves and shoots were tested for the present of alkaloids, flavonoids, saponins, tannins, cardiac glycosides and triterpenoids according to the previous report [11-13].

Total phenolic contents The total phenolic contents were determined using Folin-Ciocalteau reagent as described by Prior et al. [14]. Briefly, 100 μl of 2.5 μg/ml extract were added to the test tube and combined with 2 ml of 2% Na2CO3. The tubes were vortexed and allowed to react at room temperature 2 min. After that, 100 μl of Folin-Ciocalteu reagent were added to the test tube and incubated in the dark at room temperature for 30 min. The absorbance of the mixture was measured at 750 nm. Standard calibration curve for gallic acid was prepared in the same manner and the results were expressed as miligrams gallic acid equivalents (GAE) per gram of extract (mg GAE/g extract).

Antioxidant activity determination

ABTS radical cation scavenging activity assay In this method, the radical scavenging capacity was measured by using ABTS radical cation (ABTS•+). The assay was carried out according to Re et al. [15]. For ABTS•+ generation from ABTS, 5 ml ICoFAB2019 Proceedings | 8

of 4.9 mM potassium persulfate (K2S2O8) was reacted with 5 ml of 14 mM ABTS in the dark at room temperature for 16 h so that it reached a stable oxidative state. The working solution was prepared by diluting the mixture with ethanol to achieve the absorbance of 0.700±0.020 at 734 nm. The extract of 100 µl at various concentrations (0.2-2.4 mg/ml) was added to 2 ml of ABTS•+ solution, mixed well and allowed to react at room temperature for 6 min. The absorbance was measured at 734 nm comparing to the butylated hydroxyl toluene (BHT) as standard.

DPPH radical scavenging activity assay The DPPH radical scavenging activity was measured using the methods by Bor et al. [16]. Briefly, 0.1 ml of different concentrations of the extract (0-50 µg/ml) and 0.9 ml of distilled water were added to 4 ml of 1 mM DPPH in methanol solution, mixed well followed by incubation in the dark at room temperature for 30 min. The absorbance of the mixture was measured at 517 nm. Antioxidant activity was expressed as IC50, which was defined as the concentration of the extract required to inhibit the formation of DPPH radicals by 50%. Ascorbic acid was used as reference standard.

Results and discussion

Percentage of yield The results showed that the percentage of yield of all extracts were found between 0.83-1.80% in terms of dry weight and the highest yield was associated with leaves of T. siamensis (1.80%), followed by shoots of T. siamensis (1.32%), shoots of D. membranaceus (0.93%) and leaves of D. membranaceus (0.83%) as shown in Figure 2.

Figure 2 Percentage of yield

Phytochemical screening of extracts of leaves and shoots of bamboo The leaf and shoot extracts of D. membranaceus and T. siamensis were subjected to qualitative phytochemical analysis. The result showed that alkaloids, cardiac glycosides and triterpenoids were absent in the leaf and shoot extracts and tannins were absent in the shoot extract of the both bamboos (Table 1). The conclusion is that the different parts of plant could have different phytochemical compound, which may contribute to different pharmacological effect of each part [3, 5]. The presence of flavonoids and saponins in the bamboo extract were also found in the Oxytenanthera abyssinica and Bambusa ventricosa in Ghana [17], Gigantochloa manggong [18] and Schizostachyum Lumampao in Philippine [19].

Total phenolic contents The total phenolic contents were estimated using gallic acid as standard. The result of total phenolic contents from aqueous extracts of different parts of D. membranaceus and T. siamensis are shown in Figure 3. Among the four crude extracts, shoot extract of D. membranaceus contained the highest (315.2±11.8 mg GAE/g) amount of total phenolic contents followed by shoot (174.0±16.4) and leaf (173.6±6.4) extract of T. siamensis, and then leaf (120.7±1.3) extract of D. membranaceus. Wroblewska et al. [4] reported that the total phenolic contents of five bamboo species were between 43.6-87.8 mg GAE/g, which was lower than total phenolic contents from D. membranaceus and T. siamensis (120.7-315.2 mg GAE/g).

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Table 1 Phytochemical screening of the aqueous extracts of D. membranaceus and T. siamensis

Aqueous extracts Phytochemicals D. membranaceus T. siamensis Leaf Shoot Leaf Shoot Alkaloids - - - - Flavonoids + + + + Saponins + + + + Tannins + - + - Cardiac glycosides - - - - Triterpenoids - - - - Present (+), Absent (-)

Figure 3 Total phenolic contents of the bamboo extracts

Antioxidant activity In this study, the antioxidant activity of D. membranaceus and T. siamemsis was measured using two different assays, namely ABTS and DPPH. The single assay to evaluate the antioxidant activity would not achieve the correct result since the bioactivity of plant extract is influenced by many factors [3]. The antioxidant activity of the extracts which reported in IC50 value are shown in Table 2. ABTS assay depends on the antioxidant ability to scavenge ABTS radical. The antioxidant capacity of lipophilic and hydrophilic compounds could measure in the same sample in this assay [3]. In this study, BHT was as positive control for ABTS radical scavenging activity assay. The leaf extract of D. membranaceus was found to be effective in scavenging the ABTS radical than the others. The IC50 of the leaf extract of D. membranaceus was 24.0 μg/ml while that of BHT was 27.4 μg/ml. This result indicated that the scavenging of the ABTS radical by the leaf extract of D. membranaceus was found to be higher capacity than standard. This result shows that the leaf extract of D. membranaceus presents a good ability to scavenge the ABTS radical. The effect of antioxidants on DPPH radical was thought to be due to their hydrogen donating ability to the free radicals and reducing it to nonreactive substances [20]. In this study, ascorbic acid was as positive control for DPPH radical scavenging activity assay. The DPPH radical scavenging activity of the aqueous leaf and shoot extracts of D. membranaceus was 503 and 581 μg/ml, respectively. The result revealed that the extracts of D. membranaceus were more activity than the extracts of T. siamensis which measured by the lower IC50 value, but it has lower antioxidant activity compared to ascorbic acid (1.85 μg/ml). When compared to DPPH scavenging ability of the five native Brazilian bamboo species (137-260 μg/ml) [4], the IC50 of the extract of D. membranaceus and T. siamensis exhibited a lower radical scavenging activity. Based on the phytochemical screening that has been done, the bamboo extracts contains a various bioactive compounds that have the potential as an antioxidant such as flavonoid and saponins, which might be synergistically work together to prevent oxidative stress and neutralize the negative impact of free radical [18].

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Table 2 Antioxidant activity of the bamboo extracts

IC50 Bamboo Parts Standard ABTS (μg/ml) DPPH (μg/ml) leaf 24.0±0.00 503.1±0.01 D. membranaceus shoot 27.6±0.01 581.1±0.00 leaf 61.2±0.00 1048.0±0.02 T. siamensis shoot 31.2±0.00 816.0±0.01 Control ascorbic acid ND 1.85±0.00 BHT 27.4±0.00 ND

ND = not determined, IC50: Inhibition concentration means the concentration needed to inhibit 50% of the radical formation.

Conclusions

Phytochemical screening of leaf and shoot extract of D. membranaceus and T. siamensis revealed that the presence of flavonoids and saponins. Among the extracts, the shoot extract of D. membranaceus shows the maximum total phenolic contents. The leaf extract has highest ABTS free radical scavenging activity. The next step of these research will be developing the bamboo leaf into herbal tea.

Acknowledgements

This work was financial supported by the Research Grant of Burapha University through National Research Council of Thailand (Grant no. 2/2562).

References

[1] Lee KW, Kim YJ, Lee HJ, Lee CY. Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine. J. Agri. Food Chem. 2003; 51, 7292-7295. [2] Nirmala C, Bisht MS, Bajwa HK, Santosh O. Bamboo: A rich source of natural antioxidants and its applications in the food and pharmaceutical industry. Trends Food Sci. Technol. 2018; 77, 91-99. [3] Iqbal E, Abu Salim K, Lim LBL. Phytochemical screening, total phenolics and antioxidant activities of bark and leaf extracts of velutinus (Airy Shaw) from Brunei Darussalam. J. King Saud Univ. Sci. 2015; 27, 224-232. [4] Wroblewska KB, Baby AR, Buaratini MTG, Moreno PRH. In vitro antioxidant and photoprotective activity of five native Brazilian bamboo species. Ind. Crops Prod. 2019; 130, 208-215. [5] Sembiring EN, Elya B, Sauriasari R. Phytochemical screening, total flavonoid and total phenolic content and antioxidant activity of different parts of Caesalpinia bonduc (L.) Roxb. Pharmacogn. J. 2018; 10, 123-127. [6] Deetae P, Parichanon P, Trakunleewatthana, P, Chanseetis C, Lertsiri S. Anantioxidant and anti- glycation properties of Thai herbal teas in comparison with conventional teas. Food Chem. 2012; 133, 953-959. [7] Oh J, Jo H, Cho AR, Kim SJ, Han J. Antioxidant and antimicrobial activities of various leafy herbal teas. Food Control. 2013; 31, 403-409. [8] Horn T, Haser A. Bamboo tea: reduction of taxonomic complexity and application of DNA dianostics based on rbcL and matK sequence data. PeerJ. 2016; 4. [9] Tiwari P, Kuma B, Kaur M, Kaur G, Kaur H. Phytochemical screening and extraction: A review. Internationals PHarmaceutica Sciencia. 2011; 1, 98-106. [10] Zhu D, Wang C, Zhang Y, Yang Y, Shang Y, Niu X, Sun L, Ma Y. Wei Z. Insight into solvent effects on phenolic content and antioxidant activity of bamboo leaves extracts by HPLC analysis. J. Food Meas. Charact. 2018; 12, 2240-2246. [11] Yadav R, Agarwala M. Phytochemical analysis of some medicinal plants. Journal of Phytology. 2011; 3, 10-14. [12] De S, Dey Y, Ghosh A. Phytochemical investigation and chromatographic evaluation of the different extracts of tuber of Amorphaphallus paeoniifolius (Araceae). Int. J. Pharm. Biol. Res. 2010; 1, 150- 157. [13] Gundidza M. Phytochemical screening of some Zimbabwean medicinal plants. Cent. Afr. J. Med. 1985; 31, 238-239. ICoFAB2019 Proceedings | 11

[14] Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in food and dietary suppliments. J. Agric. Food Chem. 2005; 53, 4290-302. [15] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Evans CR. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Fee Radical Biol Med. 1999; 26, 1231-1237. [16] Bor JY, Chen HY, Yen GC. Evaluation of antioxidant activity and inhibitory effect on nitric oxide production of some common vegetables. J Agri Food Chem. 2006; 54, 1680-1686. [17] Coffie GY, Antwi-Boasiako C, Darkwa NA. Phytochemical constituents of the leaves of three bamboo (Poaceae) species in Ghana. J. Pharmacogn. Phytochem. 2014; 2, 34-38. [18] Supriyatin RS, Sukmawati D. Phytochemical composition, antimicrobial and antioxidant activity of Manggong bamboo (Gigantochloa manggong) leaf extract. Asian J. Microbiol Biotech. Env. Sc. 2015; 17, 443-450. [19] Tongco JVV, Aguda RM, Razal RA. Proximate analysis, phytochemical screening, and total phenolic and flavonoid content of Philippine bamboo Schizostachyum lumampao. J. Chem. Pharm. Res. 2014; 6, 709-713. [20] Chandran P, Vysakhi MV, Manju S, Kannan M, Abdul Kader S, Sreekumaran Nair, A. In vitro free radical scavenging activity of aqueous and methanolic leaf extracts of Aegle tamilnadensis Abdul Kader (). Int. J. Pharm. Pharm. 2013; 5, 819-823.

doi:10.14457/MSU.res.2019.3 ICoFAB2019 Proceedings | 12

Distribution of Melatonin and Serotonin in Germinated Rice

Jakkaphan Kaennok, Siriporn Lawan and Anuchita Moongngarm*

Department of Food Technology and Nutrition, Research Unit of Nutrition for Life, Faculty of Technology, Mahasarakham University

*Corresponding author’s e-mail: [email protected]

Abstract:

Melatonin is a hormone synthesized in the pineal gland of the brain. It plays an important role in regulating the biological rhythm of the body, improve the sleeping quality, and exhibits strong antioxidant activity in animals and human. Melatonin can also be found in plants and plays a significant role in seed germination and regulates plant growth. This study was conducted to investigate the effect of germination on changes of melatonin and its derivative i.e. serotonin content distributed in different milling fractions of germinated rice including rice bran, polished rice, and brown rice. The paddy rice of non-waxy rice (Red Mali) and waxy-rice (RD 6) were soaked in water for 24 h and then germinated in room temperature for 1, 2, 3, and 4 days. The samples were dried and milled to obtain brown rice prior to polishing to obtain polished rice and rice bran. The results showed that germination improved melatonin content approximately 2 and 3 folds in both rice types after germinated from 2 to 4 days. In non-waxy rice, all milling fractions were a rich source of melatonin when germinated for 2 and 3 days whereas highest level of melatonin of waxy rice was found in brown rice and polished rice after germinated for 3 to 4 days. These results suggested that all fractions of germinated rice are a rich source of melatonin and rice bran fraction is a good source of serotonin revealing the potential use of each milling fraction of rice grain.

Keywords: Melatonin, Serotonin, Tryptophan, Germinated rice, Brown rice

Introduction

Germination has been widely used to enhance the concentration of bioactive compounds and various nutrients in seed plants. During germination, chemical compositions change dramatically, due to the chemical activity of the seeds for energy use in germination causing the degradation of large molecules to be smaller, such as starch molecules to be monosaccharide. In addition, germination can also improve the level of bioactive compounds such as ascorbic acid, tocopherols, tocotrienols, phenolic compounds, γ- aminobutyric acid (GABA), dietary fiber, ferulic acid, magnesium, potassium, zinc, gamma-oryzanol [1], [2], [3]. Moongngarm et al. (2010) [4] compared the bioactive compounds of un-germinated rice, germinated paddy rice, and germinated brown rice and found that the concentration of bioactive substances in germinated paddy rice was higher than in germinated brown rice and un-germinated rice. However, currently, there is little information about the effect of germination on melatonin and serotonin. Melatonin is a hormone in the nervous system that synthesized at the pineal gland in the brain [5]. Melatonin in humans plays an important role in controlling the biological rhythm and treating insomnia [6]. The structural formula melatonin, N-acetyl-5-methoxytryptamine or N-acetyl- 3- (2-aminoethyl) -5-methoxyindole is C13 H16 N2 O2, the molecular mass of 232.278 g/mol with a melting point at 116-118 °C, and the chemical structure is shown in Fig.1 [7]. It has amphipathic properties, which causes melatonin to dissolve with solutions containing polar and non-polar solutions. In addition, melatonin also has highly effective antioxidant [8], [9]. Therefore, melatonin can be used as a supplement. In plants, melatonin is a signaling hormone which influences development processes during germination, vegetative and reproductive growth. This study was carried out to study the effect of germination on melatonin and serotonin in rice. Including the study of the distribution of melatonin in various parts of rice obtained from rice milling.

Materials and methods

Materials Paddy rice (Oryza sativa L.) cultivar RD 6 (waxy rice) and Red Mali (non-waxy rice) were purchased from a local rice milling factory in Mahasarakham province, Thailand. Un-germinated rice was prepared by removing a husk to obtain brown rice using a rice milling machine. Some portions of brown ICoFAB2019 Proceedings | 13

rice were further polished to obtain polished or white rice and rice bran. Germinated rough rice was prepared by following the method reported by [4] with some modifications. Paddy rice seeds were soaked in tap water at room temperature for 1 day (40% moisture content) and water was changed every 7-8 h. The steeped kernels were placed in plastic baskets and covered by the cheesecloth. Germination took place in a germinating basket from 1 to 4 days at 28-30°C. The germinated seeds from each germination day were dried at 50°C to approximately 10% of moisture content. The hull, root, and shoot were separated. The rice grains were de-husked to obtain brown rice which divided into two portions, one for brown rice sample and the other for polishing to obtain rice bran and white rice using a rice milling machine. All samples were performed in triplicates. Germinated rice samples were stored at -20°C and finely ground (40 mesh) prior to analyses.

Figure 1 Chemical structure of melatonin [7]

Sample extraction Dried sample (10 g) was extracted with 50 mL of 80% methanol (MeOH) then shaking incubated for 16-22 h, at 150 rpm and room temperature. The extract was filtered and the supernatant was collected for melatonin and serotonin.

Determination of melatonin and serotonin Melatonin and serotonin content were determined according to the method of Kocadağlı, Yılmaz, & Gökmen, (2014) [10], and Pothinuch & Tongchitpakdee, (2011) [11]. The extracted sample was purified using Solid Phase Extraction (SPE). SPE was activated with 10 ml of methanol (MeOH) followed by 10 ml of deionized (DI) water. The extracted sample (5 ml) was loaded and the impurity in SPE was washed with 10 ml of 5% MeOH and then 5 ml of 80% MeOH was eluted. The eluted sample was filtered using a 0.2 µm filter before analysis using liquid chromatography-tandem mass spectrometry (LC/MS-MS). The mobile phase was prepared as (A) 0.45% formic acid in DI water and (B) acetonitrile in the ratio (50:50, v/v). InertSustain® C18 column (2.1×150 mm i.d., 3 µm) with the column oven set at 40oC was used. The isocratic elution was performed with 2 µl of injection volume, the flow rate at 0.2 ml/min and total running time for 10 min. Acquire of mass spectral data were positive mode and identification of melatonin using multiple reaction monitoring (MRM) with ESI settings. Nitrogen gas was used nebulizing and drying, flow rate 3L/min and 15L/min with 4.5kv of the interface voltage at 250oC and 400oC for the desolvation line (DL), and the heat block. The MS (Q3 scan) was selected for product ion scans using argon as the collision- induced dissociation (CID) gas at 230 kPa. The transition at 233.0174.0 (collision energy of -15ev) was used to detect the melatonin and dwell time at 100 ms.

Results and discussion

Comparison of melatonin content in various milling fractions of germinated waxy rice and non- waxy rice The study found that melatonin content in polished rice and brown rice of waxy rice (RD 6), germinated for 0, 1, and 2 days, had no significant increase whereas the melatonin in rice bran significantly increased after germinated for 1 day prior to decreased when germinated from 2 to 4 days, as shown in Figure 1. There was a significant increase in the amount of melatonin in polished rice and brown rice germinated for 3 and 4 days, with the content of 553.35ng/g and 507.69 ng/g, respectively. The highest melatonin content was observed in white rice and brown rice whereas there was no significant change in rice bran had, while the white rice obtained from the rice that grew 3 days had the highest amount and in the brown rice obtained from the germinated rice 4 The day has the highest volume. ICoFAB2019 Proceedings | 14

700 Rice bran Polished rice Brown rice 600

500

400

300

Melatonin (ng/g) Melatonin 200

100

0 0 1 2 3 4 Germination Tim (day)

Figure 2 Melatonin content in various milling fraction of waxy rice

From the analysis of melatonin content in various milling fractions of non-waxy rice (Red Mali rice), it showed that day 0 and 1 of germination did not affect the increase in melatonin content in all milling fractions. The germination day of 2, 3, and 4 days showed that melatonin in all parts of the rice kernel increased significantly (Fig. 3). The rice bran obtained from germinated rice for 2 days, germinated polished rice and brown rice obtained from the 4th day of germinated rice had the highest amount of melatonin with the amount of 685.95, 704.71 and 681.76 ng/g, respectively. The melatonin in germinated brown rice and polished rice trended to be increased on day 3 and 4 of germination while the amount of melatonin in rice bran revealed somewhat reduced but non-significant difference from the day 2 of germination.

900 Rice bran Polished rice Brown rice 800

700

600

500

400

Melatonin (ng/g) Melatonin 300

200

100

0 0 1 2 3 4 Germination Time (day) Figure 3 Melatonin content in various milling fraction of non-waxy rice (Red Mali rice)

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Comparison of serotonin content in various milling fractions of germinated waxy rice and non-waxy rice The effect of germination on the serotonin content and distribution in rice milling fraction in RD 6 cultivars, it was found that germination affected the serotonin content in all milling fractions of germinated rice as shown in Fig. 4. The highest content of serotonin was observed in rice bran fraction of day 1 and 2 germinated rice (76.10 and 71.40 ng/g, respectively), which increased as highs as 114.77% compared with rice bran fraction from un-germinated rice. The serotonin content also significantly increased in polished rice and brown rice of waxy rice but with a lower level than the rice bran fraction.

90 Rice bran Polished rice Brown rice 80

70

60

50

40

30 Serotonin (ng/g) Serotonin 20

10

0 0 24 48 72 96 Germination Time (h) Figure 4 Serotonin content in various milling fraction of waxy rice (RD 6)

The un-germinated red Mali had the serotonin content of 41.19 ng/g and the maximum serotonin content was found in the day 2 of germinated rice with the amount of 61.41 ng/g which increased approximately 49.09% of un-germinated rice. The serotonin in rice bran fraction of Red Mali significantly reduced when germinated for 4 days. The germination time did not affect the content of serotonin in polished rice and brown rice during germination for 1 to 2 days, however, the serotonin content slightly decreased after germination for 3 to 4 days.

Comparison of melatonin content of two germinated rice The comparison of melatonin and serotonin content of two germinated rice cultivars, namely, RD 6 (waxy) and Red Mali (Non-waxy), obtained from the most suitable day of germination are indicated in Table1. It was found that melatonin and serotonin content in Red Mali were higher than that of RD 6 and found that melatonin content distributed in all three fractions of the rice milling. For the waxy rice RD 6, it was found that melanin content is high in white polished rice and brown rice fraction while the serotonin was mainly distributed in rice bran fraction.

Discussion

Melatonin is a signaling hormone in plants which influence development processes during germination, vegetative and reproductive growth. It plays an important role on seed germination, therefore its level increase significantly during the germination process. The serotonin is a precursor of melatonin, even though it was found in a lower amount than melatonin, it also significantly increased during germination. These findings of this study was supported by several studies. Previous investigations have proved that exogenously applied melatonin had the potential to enhance seed germination and plant development. Many studies have suggested that melatonin can act as a plant growth regulator [12], [13] and biostimulator in stressful situations [14], [15], [16], [17]. Pre-treatment with melatonin has increased seed germination of Brassica oleracea rubrum [15], Cucumis sativus [18], and Phacelia tanacetifolia [19]. The use of melatonin in seed pre-treatment has also influenced the further growth of plants such as the study of Tan et al. (2007) in soybean seed priming, they found that melatonin increased leaf size, plant height, and seed number. ICoFAB2019 Proceedings | 16

80 Rice bran Polished rice Brown rice 70

60

50

40

30 Serotonin (ng/g) Serotonin

20

10

0 0 1 2 3 4 Germination Time (day) Figure 5 Serotonin content in various milling fraction of non-waxy rice (Red Mali rice)

Table 1 Comparison of melatonin and serotonin content (ng/g) of two germinated rice cultivars obtained from the suitable germination time of each cultivar

Melatonin Serotonin Milling fractions Waxy Non-waxy Waxy Non-waxy (RD 6) (Red Mali) (RD 6) (Red Mali) Rice bran 385.66±20.40Bb 685.95 ±9.49Aa 76.10 ±10.04Aa 61.41 ± 7.32Aa Polished rice 553.35 ± 8.86Ba 704.71 ± 76.41Aa 64.49 ± 13.02Ab 48.65 ± 4.01Ab

Brown rice 541.26 ± 32.83Ba 681.76 ± 10.64Aa 63.23 ± 1.96Ab 52.75 ± 7.69Aab Results are expressed as mean value ±SD Results in the same column with the same superscript (A, B) are not significantly (p<0.05) Results in the same row with the same superscript (a, b) are not significantly (p<0.05)

Conclusions

The melatonin content was the highest in polished rice and brown rice fraction in waxy rice germinated for 3-4 days whereas in non-waxy rice (Red Mali) the melatonin content was highest in the bran fraction after germinated for 2 to 4 days. The serotonin content was highest in rice bran fraction in both rice cultivars germinated for 1 to 2 days in waxy rice and germinated for 2 to 3 days in Red Mali rice. These results suggested that each rice milling fractions germinated rice is a rich source of melatonin and derivatives depending on cultivar revealing the potential use of each milling fraction of rice grain.

Acknowledgements

This research has been funded by the Faculty of Technology, Mahasarakham University, Thailand.

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References

[1] Fernandez-Orozco R, Frias J, Zielinski H, Piskula MK, Kozlowska H and Vidal-Valverde C. Kinetic study of the antioxidant compounds and antioxidant capacity during germination of Vigna radiate cv. emmerald, Glycine max cv. jutro and Glycine max cv. merit. Food Chemistry. 2008; 111(3), 622- 630. [2] Frias J, Miranda, ML Doblado R and Vidal-Valverde C. Effect of germination and fermentation on the antioxidant vitamin content and antioxidant capacity of Lupinus albus L. var. Multolupa. Food Chemistry. 2005; 92(2), 211–220. [3] Kayahara H (2001) Functional components of pre-germinated brown rice, and their health promotion and disease prevention and improvement (In Japanese). Weekly Agric Forest. 2001; 179, 4–6 [4] Moongngarm A, Saetang. Comparison of chemical compositions and bioactive compounds of germinated rough rice and brown rice. Food Chemistry. 2010; 122, 782–788 [5] Hardeland R and Pandi-Perumal SR, Melatonin, a potent agent in antioxidative defense: Actions as a natural food constituent, gastrointestinal factor, drug and prodrug. Nutrition & Metabolism. 2005; 2:22, DOI:10.1186/1743-7075-2-22. [6] Buscemi N, Vandermeer B, Pandya R, Hooton N, Tjosvold L, Hartling L, Baker G, Vohra S, Klassen T. Melatonin for Treatment of Sleep Disorders. Evid Rep Technol Assess (Summ). 2004; 108,1-7 [7] Gómez-Moreno G1, Guardia J, Ferrera MJ, Cutando A, Reiter RJ. Oral Dis. Melatonin in diseases of the oral cavity. 2010; 16, 242-7. DOI: 10.1111/j.1601-0825.2009.01610.x. [8] Reiter RJ, Manchester LC, Tan DX. Melatonin in walnuts: Influence on levels of melatonin and total antioxidant capacity of blood. Nutrition. 2005; 21: 920-924. [9] Wang X. The antiapoptotic activity of melatonin in neurodegenerative diseases. CNS Neurosci Ther. 2009; 15(4), 345-57. DOI: 10.1111/j.1755-5949.2009.00105.x. Epub 2009 Oct 10. [10] Kocadağlı, T, Yılmaz, C and Gökmen, V. Determination of melatonin and its isomer in foods by liquid chromatography tandem mass spectrometry. Food Chemistry. 2014; 153: 151-156. [11] Pothinuch P, and Tongchitpakdee S. Melatonin contents in mulberry (Morus spp.) leaves: effects of sample preparation, cultivar, leaf age and tea processing. Food Chemistry. 2011; 128(2), 415-419. [12] Murch SJ, Campbell SSB and Saxena PK. The role of serotonin and melatonin in plant morphogenesis: regulation of auxin-induced root organogenesis in in vitro-cultured explants of St. John’s wort (Hypericum perforatum L.). In Vitro Cellular & Developmental Biology Plant. 200; 37, 786–793. DOI: 10.1007/s11627-001-0130-y. [14] Arnao MB and Hernandez-Ruiz J. Growth conditions determine different melatonin levels in Lupinus albus L. Journal of Pineal Research. 2013; 55, 149–155. DOI: 10.1111/jpi.12055. [15] Posmyk M, Bałabusta M, Wieczorek M, Śliwińska E and Janas KM. Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. Journal of Pineal Research. 2009; 46, 214–223. DOI: 10.1111/j.1600-079X.2008.00652.x. [16] Tan DX, Manchester LC, Helton P and Reiter PJ. Phytoremediative capacity of plants enriched with melatonin. Plant Signaling & Behavior. 2007; 2:514–516. DOI: 10.4161/psb.2.6.4639. [17] Wei W, Li Q-T, Chu Y-N, Reiter RJ, Yu XM, Zhu DH, Zhang WK, Ma B, Lin Q, Zhang JS and Chen SY. Melatonin enhances plant growth and abiotic stress tolerance in soybean plants. Journal of Experimental Botany. 2015; 66, 695–707. DOI: 10.1093/jxb/eru392. [18] Posmyk M, Bałabusta M, Wieczorek M, Śliwińska E and Janas KM. Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. Journal of Pineal Research. 2009; 46, 214–223. DOI: 10.1111/j.1600-079X.2008.00652.x. [19] Tiryaki I and Keles H. Reversal of the inhibitory effect of light and high temperature on germination of Phacelia tanacetifolia seeds by melatonin. Journal of Pineal Research. 2012; 52, 332–339. DOI: 10.1111/j.1600-079X.2011.00947.x.

doi:10.14457/MSU.res.2019.4 ICoFAB2019 Proceedings | 18

Sensory Characteristics of No-Sugar Black-Rice Tea Drinks

1 1 1 Pattamaporn Jaroennon *, Sujarinee Sangwanna , Jutawan nuanchankong , Laddawan Kongplee2 and Sakunta manakla1

1Department of Nutrition and Dietetics, Faculty of Science and Technology, Valaya Alongkorn Rajabhat University under the Royal Patronage, Klongluang, Pathum thani, Thailand 2Department of Food and Beverage innovation for health, Faculty of Science and Technology, Valaya Alongkorn Rajabhat University under the Royal Patronage, Klongluang, Pathum thani, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

This research studied the sensory characteristics of no-sugar black rice tea drinks using various types of rice, including Luem-Pua glutinous rice, Mali-Nil rice and Sangyod rice with different rice bran:rice seed ratios. The sensory characteristics of no-sugar black rice tea drinks were tested on 50 people using the Hedonic 9-scale test. Statistical analysis of variance (ANOVA) followed by LSD multiple comparison post-hoc tests were employed to analyze the data obtained from the study (p<0.05) in appearance, color, odor, taste and overall acceptance. Rice-seed tea drink had a significantly lower score than rice-bran tea drink for all characteristics. Preferences of rice bran tea drink with various rice bran:rice seed ratios showed insignificant differences (p>0.05) for odor, taste and overall acceptance. The highest of appearance and color scores in Sangyod rice drinks were significant (p<0.05). Overall, the acceptance of Luem-Pua glutinous rice drink was the best. Therefore, Luem-Pua glutinous rice tea drink 1:2 formulation is the most suitable. This research demonstrates an application of rice bran to provide high nutritional ingredients for healthy drink products.

Keywords: Rice-tea drinks, Sensory characteristics, Black rice

Introduction

Recently, people have developed increased interest in health promotion. Consumption of grain products is popular in the form of tea drinks, especially in Thailand, where the hot weather results in the drinking of cold drink products. Nevertheless, grain tea drinks presently contain a large amount of sugar, providing energy to the body. When consumers have an unbalanced intake of sugar, the result may be health problems [1]. Thus the addition of sugar substitutes (sucralose) is necessary. Furthermore, the development of a no-sugar tea drink from rice (Oryza sativa Linn.) bran would provide a good choice for consumers. Rice bran is one of the valuable by-products of the rice milling process, with potential to supply beneficial health effects for the human body. In particular, rice bran is rich in dietary fiber and contains starch, protein, vitamins and dietary minerals [2]. Moreover, rice bran contains antioxidant phytochemicals such as α- tocopherol, tocotrienol and γ-oryzano [3]. Several studies have shown that α-tocopherol in rice bran can lower the risk of cancer formation, coronary heart diseases, Alzheimer’s disease and allergies [4]. Tocotrienols also reportedly inhibit cholesterol synthesis, lower serum-cholesterol levels in various animal models, and suppress tumor-cell proliferation [5]. It is also reported to γ-oryzanol can be used to reduce blood cholesterol levels, treat nerve imbalance, treat inflammatory processes, increase HDL cholesterol levels and inhibit platelet aggregation [6]. Many studies have found that black rice contains higher levels of γ-oryzanol than white rice [7]. The few well-known black varieties such as Luem-Pua glutinous, Mali- Nil and Sangyod are popular for consumption, but studies to develop cold drinks using these promising rice types are still lacking. Therefore, this study aimed to develop cold no-sugar black-rice tea drinks.

Materials and methods

Sample preparation Rice material of three varieties were collected from difference sources, Luem-Pua glutinous (Sisaket province), Mali-Nil (Surin province) and Sangyod (Phatthalung province). The paddy-rice samples were milled to obtain rice bran and milled grain. All rice brans were dried in a hot air oven at 60 ᵒC for 12 ICoFAB2019 Proceedings | 19

hours and the remaining grain samples were heated for 5 min. These rice bran and grain samples were used in the development of no-sugar black rice tea drinks.

Development of no-sugar black rice tea drinks In the first preliminary phase, six formulations were prepared in tea bags which were only rice bran or only grain (Table 1). All formulations were dipped in 200 ml of boiling water for 5 min. After cooling, to all drink products were added 0.0067 g of sucralose. Before the sensory test, the drink products were refrigerated at 5 ᵒC for 12 h.

Table 1 Formulation of cold drink products in preliminary phase

Formulation Rice bran(g) Grain)g( Luem-Pua glutinous 3 - Luem-Pua glutinous - 3 Mali-Nil 3 - Mali-Nil - 3 Sangyod 3 - Sangyod - 3

The secondary phase included nine formulations in tea bags (Table 2). All formulations were dipped in 200 ml of boiling water for 5 min. After cooling, to all drink products were added 0.0067 g of sucralose. Before the sensory test, the drink products were refrigerated at 5 ᵒC for 12 h.

Table 2 Formulation of cold drink products in secondary phase

Formulation Rice bran(g) Grain)g( Luem-Pua glutinous 1.0 2.0 Luem-Pua glutinous 1.5 1.5 Luem-Pua glutinous 2.0 1.0 Mali-Nil 1.0 2.0 Mali-Nil 1.5 1.5 Mali-Nil 2.0 1.0 Sangyod 1.0 2.0 Sangyod 1.5 1.5 Sangyod 2.0 1.0

Sensory characteristic of no-sugar black rice tea drinks Products of both the first preliminary phase and the second phase were labeled with a 3-digit random code number. Fifty subjects who were without rice allergy received the black rice tea drinks. Six sensory characteristics were evaluated by the sample’s appearance, color, odor, taste, flavor and overall preference. The study on sensory characteristics was tested by the 9-point hedonic test, 9 being most preferred to 1 being least preferred. The study was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Ministry of Public Health (Ref. no. 33/2561).

Results and discussion

Rice tea and rice bran tea drinks Results from the 9-scale hedonic analysis of rice bran tea and rice seed tea are presented in Table 3. The Luem-Pua glutinous rice, Mali-Nil rice and Sangyod only rice seed tea had overall acceptability scores of 5.62, 5.82 and 5.62, respectively. Overall acceptability score of Luem-Pua glutinous rice, Mali- Nil rice and Sangyod rice bran formulations were 3.92, 4.44 and 4.08, respectively. Mean score of rice bran drink was 3.92-4.64 while rice tea was 4.92-6.12. Rice tea drink had the significantly lower score than rice bran tea drink for all characteristic. Due to rice bran did possess unique odor influencing sensory evaluation. This odor consequent effect on the volatile compound in black rice bran [8]. However, rice bran contains active ingredient for health benefits. This the reason, this study incorporate between rice bran and rice seed to accept in consumer.

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Table 3 Sensory characteristics of rice bran and rice tea drinks

Rice tea drinks Characteristic Rice bran Rice 1. Luem-Pua glutinous rice Appearance 4.08 ± 1.24b 5.82 ± 1.48a Color 3.90 ± 1.23b 5.70 ± 1.68a Odor 4.26 ± 1.19b 4.92 ± 1.32a Taste 4.04 ± 1.29b 5.56 ± 1.57a Overall acceptance 3.92 ± 1.37b 5.62 ± 1.52a 2. Mali-Nil rice Appearance 4.58 ± 1.42b 6.12 ± 1.61a Color 4.22 ± 1.43b 6.06 ± 1.65a Odor 4.64 ± 1.44b 5.26 ± 1.66a Taste 4.44 ± 1.70b 5.82 ± 1.70a Overall acceptance 4.44 ± 1.69b 5.82 ± 1.59a 3. Sangyod rice Appearance 4.42 ± 1.43b 5.82 ± 1.73a Color 4.44 ± 1.53b 6.10 ± 1.74a Odor 4.38 ± 1.77b 5.40 ± 1.26a Taste 4.02 ± 1.78b 5.60 ± 1.59a Overall acceptance 4.08 ± 1.69b 5.62 ± 1.54a * Data are means of three replications ± standard deviation. Different letters within the columns indicate significant difference (p<0.05).

Ratio of rice bran and rice In the development of drinks, sensory properties such as appearance, color, odor, taste and overall acceptance are important factors determining consumer acceptability. The mean sensory scores of the parameters of the rice tea drink formulations, as affected by the rice bran : rice ratio 1 : 2, 1 : 1 and 2 : 1, are presented in Table 4. Preferences of rice bran tea drinks with various ratios of rice bran and rice seed showed insignificant differences (P>0.05) for odor, taste and overall acceptance (Table 4). The color of Sangyod rice drinks at 1 : 1 had preference scores significantly lower than Luem-Pua glutinous and Mali-nil rice tea drinks (P<0.05). Color preferences of Luem-Pua glutinous rice tea drink at 2:1 also were significantly lower than for other rice tea drinks. Sangyod rice tea drink had the higher scores for the appearance and color characteristics. Results demonstrated that the dark red purple of Sangyod rice tea drink was preferred. The color of Sangyod rice tea drink results from proanthocyanin which is a red purple pigment [9]. Nadjaree et al reported that the color of rice has high importance for product development [10]. Therefore, only color characteristic influenced the preferences of consumers. Rice tea drink ratios of 1 : 2 formulations had the highest means of sensory scores while the ratio 1 : 1 had the lowest score for overall acceptance (P>0.05). Mean scores of rice tea drinks with various rice types were significantly different (P<0.05) for appearance. Sangyod rice tea drink (4.92) had the highest preference followed by Luem-Pua glutinous rice (4.76) and Mali-nil rice tea drink (4.36). For the odor and taste evaluation, Luem-Pua glutinous rice drink had higher scores than Mali-nil rice and Sangyod rice formulations (P>0.05). Luem-Pua glutinous rice is highly pigmented with strongly flavored rice. For the 1 : 2 rice tea drinks, the Luem-Pua glutinous rice, Mali-nil rice and Sangyod rice tea formulations had significantly overall acceptability scores of 5.10, 4.44 and 4.58, respectively (P<0.05). Results showed that Luem-Pua glutinous rice with 1 : 2 formulation was the most acceptable one because of color and appearance. Luem-Pua glutinous rice 1 : 2 formulation tea drink had the highest score (5.10), which was evaluated as “average”. The consumers rated all the sensory attributes for all sample between 4 and 5, which were evaluated as ‘‘slightly unlike” to ‘‘average”, respectively. Although scores in preference of rice bran tea drinks were slightly low,this result demonstrated the possibility of rice bran tea drink development.

Conclusions

This research evaluated sensory characteristics of no-sugar black rice tea drinks with various combinations of rice bran and rice seed. The Luem-Pua glutinous rice exhibited the highest overall acceptability score for 1:2 ratio. Other rice tea drinks including rice bran and rice seed were evaluated as slightly unlike. Therefore, there are essential for pursuing further study of rice tea product development. ICoFAB2019 Proceedings | 21

However, our research demonstrated that rice bran tea possessing high nutritional ingredients could be employed for producing difference healthy drink products.

Table 4 Sensory characteristics of rice bran tea drink with difference ratio of rice bran and rice seed

Ratio of rice bran:rice seed 1:2 1:1 2:1 1. Luem-Pua glutinous rice Appearance 4.76 ± 1.48aAB 4.26 ± 1.77aB 4.24 ± 2.08aA Color 4.82 ± 1.60aA 4.16 ± 1.61abB 4.08 ± 2.09bB Odor 4.86 ± 1.74aA 4.78 ± 1.65aA 4.96 ± 1.98aA Taste 4.84 ± 1.65aA 4.92 ± 1.97aA 4.86 ± 2.04aA Overall acceptance 5.10 ± 1.59aA 4.72 ± 1.92aA 4.92 ± 1.93aA 2. Mali-Nil rice Appearance 4.36 ± 1.38aB 4.36 ± 1.38 aAB 4.22 ± 1.72aA Color 4.74 ± 1.29aA 4.12 ± 1.57bBC 4.22 ± 1.72abAB Odor 4.70 ± 1.13aA 4.40 ± 1.54aA 4.12 ± 1.72aA Taste 4.16 ± 1.53aA 4.24 ± 1.79aA 4.50 ± 1.74a Overall acceptance 4.44 ± 1.47aAB 4.24 ± 1.83aA 4.38 ± 1.64aA 3. Sangyod rice Appearance 4.92 ± 1.37aA 4.92 ± 1.37aA 4.84 ± 1.52 aA Color 4.98 ± 1.45aA 4.98 ± 1.45aA 4.80 ± 1.57aA Odor 4.64 ± 1.55aA 4.64 ± 1.55aA 4.58 ± 1.26aA Taste 4.38 ± 2.00aA 4.38 ± 2.00aA 4.16 ± 1.68aA Overall acceptance 4.58 ± 1.79aB 4.58 ± 1.79aA 4.46 ± 1.81aA * Data are mean of three replications ± standard deviation. Different letters within the columns indicate significant difference (P < 0.05).

References

[1] K Jorge, Brewtech Ltd, Jacarepagua and Rio de Janeiro. SOFT DRINKS.Chemical Composition.2003;5346-5352. [2] Cynthia Fabian, Aning Ayucitra, Suryadi Ismadji and Yi-Hsu Ju. Isolation and characterization of starch from defatted rice bran. Journal of the Taiwan Institute of Chemical Engineers. 2011; 42, 86– 91. [3] Renu Sharma, Tanuja Srivastava and D.C. Saxena. Studies on Rice Bran and its benefits- A Review. Journal of Engineering Research and Applications. 2015; 5, 107-112. [4] Anand Dutta BSc and Sudhir K. Dutta MD. Vitamin E and its Role in the Prevention of Atherosclerosis and Carcinogenesis: A Review. Journal of the American College of Nutrition. 2003; 22, 258–268. [5] M.-H. Chen and C.J. Bergman. A rapid procedure for analysing rice bran tocopherol, tocotrienol and g-oryzanol contents. Journal of Food Composition and Analysis. 2005; 18, 139-151. [6] Shanggong YU, Zachary T. Nehus, Thomas M. Badger, and Nianbai Fang. Quantification of Vitamin E and ç-Oryzanol Components in Rice Germ and Bran. Journal of agricultural and food chemistry. 2007; 55, 7308-7313 [7] Wipavadee Daiponmak, Chadapon Senakun and Sirithon Siriamornpun. Antiglycation capacity and antioxidant activities of different pigmented Thai rice. International Journal of Food Science and Technology. 2014; 49, 805–1810. [8] Sukhontha Sukhonthara, chockchai Theerakulkait and Mitsuo Miyazawa. Characterization of volatile aroma compounds from red and black bice bran. Journal of Oleo Science. 2009; 58, 155-161 [9] Yu-Ping Huang and Hsi-Mei Lai. Bioactive compounds and antioxidative activity of colored rice bran. Journal of food and drug analysis. 2016; 24, 564-574. [10] Nadjaree Poomipak, Rajnibhas Sukeaw Samakradhamrongthai and Niramon Utama-ang. Consumer Survey of Selected Thai Rice for Elderly Using Focus Group and Acceptance Test. Food and Applied Bioscience Journal. 2018; 6, 134-143.

doi:10.14457/MSU.res.2019.5 ICoFAB2019 Proceedings | 22

Effect of Cinnamon Oil and Garlic Extract for Fresh Shrimp Preservation

Supraewpan Lohalaksnadech1* and Dumrong Lohalaksnadech2

1 Department of Food Industry and Fishery Product, 2 Department of Fish Technology, Rajamangala University of Technology, Srivijaya, 179 Mu. 3, Maifad sub-district, Sikao district, Trang 92150, THAILAND

*Corresponding author’s e-mail: [email protected]

Abstract:

The effect of garlic extract, cinnamon oil on the quality changes of raw shrimp during refrigerated storage (4±1°C) of 15 days was investigated. Four different treatments were treated : control sample (T1), 8 % garlic extract solution (T2), 0.5 % cinnamon oil (T3) and 8% garlic extract incorporated with 0.5% cinnamon oil (T4). The results indicated that the cinnamon oil incorporated with garlic extract (T4) was efficient against the proliferation of microorganisms including total variable count. The results from chemical analysis indicated that the treated samples underwent a significant decrease (P<0.05) in the term of pH value and the total volatile base nitrogen content. However, the results of sensory attributes show that the high scores for the appearance, texture and odor demonstrated that 8%garlic extract incorporated with 0.5% cinnamon oil (T4) can help delay the proliferation of microorganisms spoilage, prevent the generation of undesirable chemicals, improve the levels of sensory attributes and extend the shelf life of raw shrimp during refrigerated storage. The samples treated with 8%garlic extract incorporated with 0.5% cinnamon oil (T4) had lowered change in chemical quality but similar total bacteria count, in comparison with T3, T2 and T1. Therefore, 8% garlic extract incorporated with 0.5% cinnamon oil (T4) could be a promising inhibition of raw shrimp quality loss with extended shelf life. While the use of cinnamon oil (T3) extended the shelf life of raw shrimp by 12 days, T3 for 9 days, compared to 6 days in T1 and T2 samples.

Keywords: Shrimp, Total volatile base nitrogen, Shelf life, Cinnamon oil, Garlic extract

Introduction

Shrimp is one of the popular seafoods in the world including East Asian countries such as Thailand, Korea, China, and Japan, because it has several applications in foods. However, seafood quality is easily lost due to microbiological contamination and/or chemical reactions because shrimp has high water content, large quantities of free amino acids, and autolytic enzymes among other factors [1]. After the capture of shrimp, a series of complex changes occurs in the seafood, resulting in a decrease of quality [2]. Therefore, shrimp should be frozen or kept at a cold temperature to limit or reduce enzymatic and microbial activities to keep its good quality [3]. Also, other treatments such as dipping or spraying food additives are being developed. To reduce the undesirable biochemical and physical changes, the kinds of food additives for dipping solutions in seafood are sodium acetate, sodium lactate, sodium citrate. [4] Although phosphate derivatives are commonly used among other additives, only a few researchers have studied the effects of phosphate derivatives in shrimp. Consumer perceive fresh seafood to be a superior product to its frozen equivalent. Being highly perishable, fresh seafood had a limited shelf life due to their biological composition. Spoilage of shrimp results from the oxidation of microorganism. Even if refrigeration can be applied to the products, these activity although slower, will over time lead to a shorter shelf life and a poorer safety and quality of shrimp products and consequently represent a high risk for consumer health and economic losses, therefore enhancing shelf life of shrimp with natural preservatives is an important issue to eliminate economic losses and provide safe and good quality seafood to consumer. Cinnamon oil is the natural preservative and flavoring agent that is not toxic when it is added in food products. It can inhibit the growth of Aspergillus flavus, Aspergillus parasiticus, Fusarium moniliforme [5], Lactobacillus sp., Bacillus thermoacidurans, Salmonella sp., Corynebacterium michiganense, Pseudomonas striafaciens, Clostridium botulinum and Penicillium roqueforti [6]. Cinnamon oil and thyme oils are active against Salmonella enterica, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes [7]. Therefore, cinnamon oil should be examined with respect to antimicrobial modifying activity. The most important cause of the raw shrimp spoilage are accumulation of undesirable compounds as a resulted of microbiological growth and biochemical reaction. Alginates are naturally occurring, indigestible ICoFAB2019 Proceedings | 23

polysaccharides commonly produced by and refined from various genera of brown algae .Alginate is widely used in various industries such as food, beverage, textile, printing, and pharmaceutical as a thickening agent, stabilizer, emulsifier, chelating agent, encapsulation, swelling, a suspending agent, or used to form gels, films, and membrane. Sodium alginate is the most common salt of alginate [8]. Application of edible film and coating with natural active compounds for enhancing storage stability of food product is a promising active packaging approach. Several biopolymers with the inclusion of antimicrobial and/or antioxidant active agent have been test in vitro. In this study, alginate coating with the inclusion of cinnamon oil or garlic extract were applied to raw shrimp as an alternative natural method to extend shelf life and prevent the quality change in during storage.

Materials and methods

Material Five kg middle size (80-95 shrimp/kg) of Vannamei shrimp (Litopenaeus vannamei) were purchased from a shrimp farm in Trang province, Thailand and transported to laboratory in ice-packed container immediately after being caught. Upon After arriving to the laboratory shrimp sample were immediately weighed and washing with tap water. The shrimp samples were de-headed, peeled, and deveined. The samples were divided into four equal groups and stored in refrigeration until further treatments. Cinnamon oil and garlic extract prepared by Specialty Natural Products Co.,Ltd.

Method Preparation of sample : Four factors were treats. Dipping treatments of shrimp batch was soaked for 3 mins in different four of cold solutions (4±1°C), The first one was dipped into distilled water (T1). The second one was dipped into alginate solution (20 µg ml-1 incorporated with 8% garlic extract (T2). The third was dipped into alginate solution (20 µg ml-1 incorporated with 0.5% cinnamon oil (T3). Then, the fourth one was dipped into alginate solution (20 µg ml-1) incorporated with 0.5% cinnamon oil and 8% garlic extract (T4). Then sample remove from the treatment solutions with a strainer (drained well for 1 min) prior to packed into sterile bags. Packaging and storage: Shrimp samples were packed in sterile bags with 250 g. in each bag, three bags for each groups) and stored in the refigerator (model Evermed, LR 625 WV.201) at 4±1°C. The sample were analysed to microbiological, physiological and sensory properties at day zero, then periodically every 3 days until decomposition or up to 15 days of refrigerated storage. Microbiological examination: The samples were taken aseptically in a vertical laminar-flow cabinet (Alpha Clean, Model 1300) and 25 g of shrimp were transferred to a stomacher bag, 225 ml of sterile Butterfield’s phosphate-buffered water (Difco) using a stomacher machine (Lab-blender 400, Seward Laboratory) for 60 sec. From the 10-1 dilution, other decimal dilutions were prepared. Total viable count (TVC) were determined by using pour plate method. [9] Plate count agar (Difco) were used and incubated at 35°C for 48 hours (Blender, model 28). The permissible limit of TVC in chilled fish was 6 log CFU g-1 recommended by ICMSF. [10] Chemical analyses: The total volatile basic nitrogen (TVB-N) expressed as mg TVB-N per 100 g muscle of shrimp meat was determined according to the method of Conway’s diffusion method [11]. For pH determination 10 g of shrimp samples were homogenized in 100 ml distilled water for 1 min by homogenizer and pH value were measured at room temperature using pH meter (SI Analytics, model lab 855) as described by Ozyurt et al [12]. The pH values above 7.10 are indicative of decomposition in fish [12]. Sensory assessment of raw shrimp: Scoring method with 10 trained panel was carried using 4- point scoring scale, according to National Bureau of Agricultural Commodity and Food Standard of Thailand, ACTF [13]. The samples served to the panellist to evaluate the appearance, odor and texture attributes of shrimps. Three parameters on a scale from 1 (extremely undesirable) to 4 (extremely desirable) were evaluated. They were asked to give a score for each of appearance, odor and texture while the samples were raw. Statistical analysis results were expressed means and standard deviation (Means±SD) from triplicate. Analysis of variance (ANOVA) was performed to compare the effect of dipping treatments. Differences among the mean values of the various treatments and storage periods were determined by Duncan new multiple rang test (DMRT) and Significant differences were defined as p<0.05, according to program package.

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Results and discussion

Microbiological examination Total viable count (TVC) of shrimp under investigation were analysed and log CFU g-1 were present in Table 1, which slight higher in TVC was showed in control samples when compared with other treatment at day zero, indicating that cinnamon oil and garlic extract caused sudden lethal effect for microorganism immediately after dipping process. TVC of raw shrimp samples steadily increased as the of cold storage progressed, TVC of T1 showed the highest number. According to the permissible limit of TVC in chilled fish was 6 log CFU g-1 recommended by ICMSF [10], from the Table 1 showed that sample of control (T1) exceeded such limit at 6 day, while the sample of T2, T3 and T4 exceeded such limit at 6, 9 and 12 day respectively. Various studies have shown that garlic is also effective against many gram- negative and gram-positive bacteria, such as Escherichia coli, Salmonella, Staphylococcus and Vibrio chlorelae. Many of these bacteria do not develop resistance to allicin, despite being resistant to antibiotics. [14] [15] One study demonstrated the potential for garlic to act as a meat preservative. Garlic extract was shown to kill about 75% of the E. coli and non-pathogenic Salmonella on chicken meat as well as a majority of the contaminating bacteria on the meat after 10 minutes of exposure. The study also showed that the garlic extract could limit the growth of all the bacteria tested. [16] Cinnamon showed as antibacterial properties against gram positive and gram negative bacteria. Also, agree with Irfiana et al. [17] who recorded that cinnamon oil showed decreasing count of total plat count microorganisms of vaccum packed ground beef during refrigerated storage at 4±1°C for 0, 3, 6, 9,12 and 15 day when compared with count of control samples, due to cinnamon contains active compounds such as polyphenols, cinnamaldehyde and antibacterial compounds.

Table 1 Changes in total viable count of shrimp stored at refrigerated temperature (4±1°C)

CFU/g 8%garlic extract+0.5% Days Control 8% garlic extract 0.5% cinnamon oil cinnamon oil (T1) (T2) (T3) (T4) 0 2.4 x107 2.5x106 6.8x105 8.3x105 3 6.7x107 3.4x106 2.9x105 5.4x105 6 2.1x106 9.6x106 8.6x105 2.6x104 9 4.5x106 3.3x106 4.1x104 4.8x104 12 6.0x106 1.4x105 3.4x104 3.5x103 15 3.5x105 2.8x104 4.4x104 8.8x103

Chemical examination

TVB changes The initial (day-0) TVB-N value of T1, T2, T3 and T4 was 5.93, 3.60, 4.10 and 2.80 mgN/100g, respectively (Table 2). It showed that TVB-N of all samples increased with different rates, depending on pre-treatment and time of storage. The lowest TVB-N was obtained from T4 followed by T3, T2 and T1 during storage period, respectively. The TVB-N, which is mainly composed of ammonia and primary, secondary and tertiary amines, resulted from degradation of proteins and non-protein nitrogenous compounds is well documented as a good index for the quality and shelf life of fish and fish products because its increase is related to spoilage by the activity of endogenous enzymes and bacterial growth. The TVB-N values of good quality fish are generally less 25 mg N/100 g muscle and above 25-30 mg TVB/100 g indicated that fish is decomposed and inedible [18]. Generally, when the TVB-N level exceeded the maximum value, samples were already refused by the panelists. Therefore, TVB-N values correlated well with the results of sensory analyses and microbiological examination, providing a good index for the assessment of freshness of raw shrimp during refrigerated storage. pH changes Table 3 shows pH values for raw sample fillet as a function of treatment and storage time at 4±1°C. Initial (day 0) pH value in fresh control fillet samples (T1) was 6.30. While T2 and T3 were 6.05 and 5.15 were observed. In contrast, cinnamon oil treated-sample (T4) exhibited the lowest pH values as compared with other sample. Our results (Table 3) further shows that during cold storage, pH of raw shrimp samples increased throughout the storage time, presumably due to the production of basic amines as a result of ICoFAB2019 Proceedings | 25

decomposition of nitrogenous compounds caused primarily by microbial activity. Such an increase in pH of fish could indicate bacterial growth, the reduction of quality and ultimately spoilage of fish [12][19]. The sharp increase was observed in pH of control samples to end day of storage. The pH of 8% garlic extract (T2), 0.5% cinnamon oil (T3) and 8%garlic extract+4% cinnamon oil (T4) treated samples was quite increased throughout the storage.

Table 2 Changes in total volatile base nitrogen (TVB-N) of shrimp stored at refrigerated temperature (4±1°C)

TVB-N (mg/100) 8%garlic extract+0.5% Days Control 8% garlic extract 0.5% cinnamon oil cinnamon oil (T1) (T2) (T3) (T4) 0 5.93±0.45a 3.60±1.98b 4.10±0.65b 2.80±1.05c 3 12.07±0.56a 5.93±0.76b 3.60±1.45c 2.36±0.54c 6 16.23±0.58a 11.52±0.56a 5.72±0.76b 4.23±0.78b 9 25.00±0.78a 15.08±1.45b 4.59±0.80c 4.54±0.61c 12 43.47±0.45a 23.19±0.87a 13.64±0.69b 11.58±0.98b 15 50.35±1.21a 37.01±0.65b 29.77±0.34b 29.05±0.32b Means followed by a different letter within each comparative category in a row are significantly different P≤0.05).

According to in Table 3, it is obvious that starting from the third day of cold storage control samples exhibit significantly higher (p<0.05) pH values than treated samples, conversely, T4 treatments had the lowest pH increment during the course of refrigerated storage. No significant differences (P>0.05) were noticed between pH values of T3 and T4 treated samples. Table3 also obvious that control raw shrimp were of good and acceptable quality with regard to pH value up to 6 days in comparison to 9 and 12 days noticed for T3 and T4 treated samples, respectively. In addition, T4 samples reached the critical pH value (7.19) at 15 day of refrigerated storage. These results are in accordance with that found by [12] who reported that pH values above 7.10 are indicative of decomposition in fish.

Table 3 Changes in pH of shrimp stored at refrigerated temperature (4±1°C)

8%garlic extract+0.5% Control 8% garlic extract 0.5% cinnamon oil Days cinnamon oil (T1) (T2) (T3) (T4) 0 6.30±0.00a 6.05±0.00a 5.15±0.01b 5.06±0.01b 3 6.52±0.00a 6.60±0.00a 6.40±0.01a 5.81±0.02b 6 6.99±0.00a 7.08±0.00a 6.70±0.02a 6.18±0.02b 9ns 7.48±0.00 7.42±0.00 6.98±0.00 6.95±0.01 12 8.20±0.00a 8.05±0.01a 7.15±0.00b 7.08±0.01b 15 8.50±0.00a 8.35±0.01a 7.28±0.01b 7.19±0.01b

Means followed by a different letter within each comparative category in a row are significantly different (P≤0.05)

Sensory evaluation Changes in the sensory attribute scores of raw control and treated raw shrimp samples during refrigerated storage at 4±1°C are depicted in Table 4. Fresh shrimp were generally considered to possess very high acceptability. All samples developed a fishy odor as the storage time increased with significantly differences (P<0.05) between treatments (Table 4). For the control samples (T1), the deterioration occurred after 6 days of storage as evidenced by strong fishy and putrid odor. Also, the deterioration in color occurred after 6 days during storage at 4±1°C. The treatment of T2, T3 and T4 exhibited higher scores (P<0.05) for odor and color and exhibited no negative effect on sensory characteristics during storage as compared with control samples. For each batches, extended shelf life of raw shrimp to 6, 9 and 12 days, respectively was observed. Results of Table 4 also indicate that immersing samples in cold solution containing 0.5% cinnamon incorporated with 8%garlic extract (T4) prior to air packaging and refrigeration process effectively retarded off-odor, maintained good color and extended the shelf life of raw shrimp samples to 12 days with the highest sensory scores (P<0.05) and pleasant herbal flavor. Similar trend of raw shrimp ICoFAB2019 Proceedings | 26

shelf life was achieved during refrigerated storage by other authors [20][21]. However, the protective effects of garlic extract and cinnamon oil reflect strong antimicrobial activities of such natural materials. While, undesired off-odors and flavors, color changes, slime formation and texture deterioration occurring during fillet spoilage are mainly caused by products of bacterial growth and metabolism. The present results indicate that sensory scores correlated well with the increase in TVB-N values and microbiological counts of the same samples.

Table 4 Changes in appearance odor and texture score of shrimp stored at refrigerated temperature (4±1°C)

8%garlic 8% garlic 0.5% cinnamon Control extract+0.5% Quality days extract oil (T1) cinnamon oil (T2) (T3) (T4) appearance 0 ns 4.0±0.05 3.8±0.03 3.7±0.09 4.2±0.09a 3 2.3±0.30c 3.3±0.05b 3.2±0.79 b 4.1±0.03a 6 2.3±0.67c 3.2±0.63b 3.3±0.67 b 4.0±0.63a 9 2.1±0.20c 3.0±0.67b 2.5±0.71 c 3.4±0.70a 12 1.4±0.57c 2.3±0.67b 2.0±0.00 c 2.8±0.23a 15 1.0±0.00b 1.0±0.00 b 1.0±0.00 b 1.6±0.52 a odor 0 3.5±0.05a 3.5±0.57a 3.2±0.52b 3.3±0.82b 3 2.5±0.09b 3.0±0.47a 3.0±0.82a 2.9±0.57b 6 1.8±0.08b 2.8±0.16a 2.7±0.94a 2.8±0.18a 9 1.4±0.52c 2.2±0.53b 2.3±0.95b 2.9±0.00a 12 1.4±0.52b 1.00±0.42b 2.0±0.52a 2.2±0.84a 15 1.0±0.00b 1.0±0.00 b 1.6±0.00a 1.6±0.52a texture 0ns 3.4±0.79 4.0±0.82 4.2±0.52 4.3±0.74 3 3.0±0.48b 3.7±0.79a 3.4±0.57a 3.8±0.79a 6 2.3±0.40a 3.0±0.32 a 3.1±0.57a 3.0±0.10 a 9 1.4±0.42c 2.3±0.67b 2.4±0.70b 3.0±0.50 a 12 1.2±0.00c 2.0±0.52b 2.4±0.00ab 2.5±0.52 a 15 1.0±0.00b 1.0±0.00b 1.0±0.00b 1.6±0.09 a Means followed by a different letter within each comparative category in a row are significantly different (P≤0.05).

Conclusions

This study demonstrated that dipping of raw shrimp in cinnamon oil and garlic solution could be prolong the shelf life and quality of samples. The resulted showed slower change in qualities of raw shrimp when compared with the control and the sample garlic extraction alone. Considering from microbiological and sensory quality, it was found that raw shrimp, dipped into cinnamon oil and garlic solution could be accepted at less than 12 days at refrigerated temperature storage whereas control samples were accepted in 6 days.

Acknowledgements

Financial support by Rajamangala University of Srivijaya, Trang Campus is greatly acknowledged.

References

[1] Fang X.B., Sun H.Y., Huang B.Y., and Yuan G.F. Effect of pomegranate peel extract on the melanosis of Pacific white shrimp (Litopenaeus vannamei) during iced storage. J. Food Agric.Environ. 2013;11:105–109. ICoFAB2019 Proceedings | 27

[2] Tsironi T., Dermesonlouoglou E., Giannakourou M., and Taoukis P. Shelf life modelling of frozen shrimp at variable temperature conditions. LWT-Food Sci Technol. 2009; 42 : 664-671. [3] Gonçalves A.A., Ribeiro J. and L.D. Do. Phosphates improve the seafood quality? Reality and legislation. Pan-Am J. Aquat. Sci. 2008;3:237–247 [4] Sallam K.I. Antimicrobial and antioxidant effects of sodium acetate, sodium lactate, and sodium citrate in refrigerated sliced salmon. Food Control.; 2007;18:566-575. [5] Soliman K.M., and R.I. Badeaa: Food Chem Toxicol. Vol.40, 2002; p.1669-1675 [6] Matan, N., H. Rimkeeree, and A.J. Mawson : Int. J. Food Microbiol. Vol. 107, 2006; p.180–185 [7] Smith-Palmer,A., J., Stewart and L., Fyfe: Lett. App.l Microbiol. Vol.26, 1998; p.118–122. [8] Kim Y.J., Yoon K.J., and Ko S.W. Preparation and properties of alginate superabsorbent filament fibers crosslinked with glutaraldehyde. J. Appl. Polym. Sci. 2000;78:1797–1804 [9] BAM. Bacteriological Analytical Manual Chapter 3 Aerobic Plate Count. Food and Drug Administation, US, 2001. [10] ICMSF. Microorganisms in Foods, Sampling for Microbiological Analysis : Principles and Specific Applications. 2nd Edn.,University of Toronto Press, Toronto, 1986. [11] Conway, E.J. Microdiffusion analysis and volumetric error. 5th ed., Crosby Lockwood and Son Ltd, London. 467 p. 1962. [12] Ozyurt, G., E.,Kuley, S. Ozkutuk and F. Ozogul. Sensory, microbiological and chemical assessment of the freshness of red mullet (Mullus barbatus) and goatfish (Upeneus moluccensis) during storage in ice. Food Chemistry, 2012; 114:505-510. [13] National Bureau of Agricultural Commodity and Food Standard of Thailand. Thai Agricultural .commodity and Food Standard TACFS 7020-2007 (Vannamei Shrimp). 2007. [14] Waag, T., Gelhaus, C., Rath, J., Stich, A., Leippe, M., and Schirmeister, T. Allicin and derivaates are cysteine protease inhibitors with antiparasitic activitiy. Bioorg Med Chem Lett., 2010; 20, 5541- 5543. [15] Strika, I.A, Basic, A.B, and Halilovic, N.B . Antimicrobial effects of garlic (Allium sativum L.). Bulletin of the Chemists and Technologists of Bosnia and Herzegovina. 2017; 70 (1) : 24-29. [16] Sarma N. Can Garlic (Allium sativum) Be Used as a Meat Preservative. Kansas Academy of Science Vol. 107 No. ¾ (Autumn, 2001) :2004; pp. 148-154. [17] Irfiana D, Utami R, Khasanah L.U. and Manuhara GJ. Preservation effect of two-stage cinnamon Bark ( burmanii) oleoresin microcapsules on vacuum-packed ground beef during refrigerated storage. International Conference On Food Science and Engineering 2016;1-7. [18] Connel, J.J. Control of fish quality. Fishing News Books. Farnham, Surrey. 179 p, 1975. [19] Gram, L. and Huss, H.H. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology, 1986; 33, 121-137. [20] Burt, S. Essential oils: their antibacterial properties and potential applications in foods-a review. International Journal of Food Microbiology, 2004; 94, 223–253. [21] Erkan, N., H. Dogruyol, A. Gunlu and I. Genc, Use of natural preservatives in seafood: Plant extracts, edible film and coating. Journal of Food and Health Science, 2015; 33-49.

doi:10.14457/MSU.res.2019.6 ICoFAB2019 Proceedings | 28

Effects of Wood Vinegar and Cow Manure on Growth of Khao Dawk Pradoo Rice in Experimental Field

Wanida Sumranram*, Thawat Suphason and Wittayakon Phliasanthia

Program of Agricultural Technology, Buriram Rajabhat University, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Effects of wood vinegar and cow manure on growth of Khao Dawk Pradoo (KDP) rice in experimental field. The objective of this study were to compared wood vinegar with manure cow manure on growth of KDP . The experiment was conducted 6 treatments: 1. No manure, cow manure and wood vinegar (control). 2. cow dung fertilizer rate 1,000 kg / rai and wood vinegar dilution rate 1:20. 3. cow dung fertilizer rate 1,000 kg / rai and wood vinegar diluted 1:40. 4. Wood vinegar diluted 1:20. 5. Wood vinegar diluted 1:40 and 6. cow dung fertilizer at 1,000 kg. / Rai. Completely Randomized Design (CRD) with 3 replications were experimented. The result was found that cow dung consists 1,000 kg / rai. make the height of plant, number of tillers, leaf area, leaf area index is highest on statistically significant. Therefore, wood vinegar at the rate 1:20 and 1:40 with no cow dung and cow dung fertilizer have make the number of tiller and the high of plant at 60 day after planting (DAP) are statistical significantly. In conclusion, the cow dung fertilizer at the rate 1000 kg/rai make highest the number of tillers as 10.66 shoot per tiller at significantly.

Keywords: Cow dung, Wood vinegar, Growth, Khao Dawk Pradoo, Native rice

Introduction

The current agricultural production uses a lot of agricultural chemicals and causes chemical residues on the production. Which directly affects the health of consumers. In addition, excessive use of chemical fertilizers causes nitrate contamination in underground water and surface water which is an important environmental problem. Farmers are more concerned about health and the environment; therefore, they want to consume safe food and are willing to pay higher prices than usual organic rice production is one way to meet the specific groups of consumers. However, organic rice production has quality criteria that farmers have to follow. That is not use all types of chemicals in production, including chemical fertilizers and pesticides (Wanida, 2015). Khao Dawk Pradoo rice :KDP rice is a native of Isan sticky rice, short, red, hard to eat, has high nutritional value, soft, sweet, delicious, and crunchy. It is attractive colors to eat because it is a black color and it is native rice. Farmers do not grow well because they are rice that is not good price, thus causing farmers to grow less or grow just enough to eat in the household. The factors that are important in rice cultivation are fertilizers, which in rice production, fertilizer is an important for produce yield which the main cost of rice production was from organic fertilizers contain nutrients that are essential to the growth of essential elements. In addition, the rate of nutrient release in cow manure occurs slowly because it has to go through the process of decomposition by the activity of microorganisms in the soil. Therefore, may cause plants grown by using cow manure to receive nutrients that may not be able to meet the needs of plants in each period of growth, which chemical fertilizers are fertilizers that contain the main food that is dissolved in the form that plants can use as soon. The high concentration of wood vinegar has a strong disinfectant effect due to the high acidity and contains compounds such as methanol and phenol which can be sterilized or pest repellent the beneficial and anti-bacterial microorganisms will increase more receiving nutrients from wood vinegar can be used in agriculture as well. Therefore, the researcher team has the objective to study wood vinegar and cow manure on growth, yield, yield components of Khao Dawk Pradoo rice in the field of Nong Khwang experimental plot.

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Materials and methods

Soil preparation Keep the soil in each the plot for analysis soil pH, available phosphorus, total nitrogen, potassium extraction and electrical conductivity before and after rice planting. To dry out the soil in each plot for about 1 week. Prepare the soil in the planting plot with an area of 6 × 2 meters per area, which has 6 treatments of experiment.

Rice planting Select the healthy seeds and take the seeds 5-10 seeds per hill in the bed plant. Measure the space length to 25 × 25 cm. Then cover the hill to protect animals or insects to bite them. The experimental was divided as 6 treatments as following as: 1; no fertilizer (control) 2; cow dung fertilizer 1,000 kg./rai + wood vinegar 1:20 3; cow dung fertilizer 1,000 kg./rai + wood vinegar 1:40 4; wood vinegar 1:20 5; wood vinegar 1:40 6; cow dung fertilizer 1,000 kg./rai.

Data Analysis Soil samples were collected before planting the rice and after harvest for soil analysis. Soil property were recorded for pH, electrical conductivity, organic matter by Walkley and Black method (Black, 1965), soil texture (%sand, silt, clay), available P (Bray II extraction (Drilon, 1980), total nitrogen by Kjeldahl (Black, 1965) and extractable K by 1 N Ammonium acetate (NH4OAC) method (Cottenie, 1980). For plants in each plot were randomly selected at tillering and maximum tillering stages, and the data were recorded for plant height and tiller number. At harvest, plants in each plot were harvested and the data were recorded for leaf area and leaf area index by direct methods can be easily applied on collecting leaves during leaf fall in traps of certain area distributed below the canopy. The area of the collected leaves can be measured using a leaf area meter.

Results and discussion

The purpose of this research was to study the effects of cow manure and wood vinegar on growth of Dawk Pradoo rice all 6 treatments in Completely Randomized Design (CRD). The results were analyzed in the following table.

Soil chemical properties before rice planting From the analysis, it is found that the total nitrogen in soil was very low as 0.0231 %, available phosphorus very high as 55.00 mm / kg., exchangeable potassium was low as 252.67 mm./kg. The soil organic matter was low as 0.411%. The acidity - alkalinity of the soil is slightly acidic, with a pH of 6.36. The soil does not have salinity at 0.028 EC. There is no effect on plant growth. The characteristics of soil series is Ban Phai Series (Bpi) are classified in the soil series 41, which classify sub-soil groups (loamy, siliceous, isohyperthermic Arenic Paleustalfs) with characteristics and soil properties. (Table 1)

Table 1 Chemical soil properties before rice planting

Chemical soil properties Total nitrogen (%) 0.0231 Available phosphorus (mg/kg) 55.00 Exchangeable potassium (mg/kg) 252.67 Organic matter (%) 0.411 pH 6.36 Electrical conductivity 0.028

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Effects of fertilizer application on the growth of Dawk Pradoo rice.

Height of rice. No statistical difference all of treatment at 30, 60, 90 and 120 days after planting (Table 2).

Table 2 Plant height of Dawk Pradoo rice in each treatment at 30, 60, 90 and 120 day after planting.

Height (cm.) Treatment 30 60 90 120 No fertilizer (control) 54.33 55.33 76.66 80.00 Cow dung 1,000 kg./rai+wood vinegar 1:20 47.66 58.00 90.33 86.73 Cow dung 1,000 kg./rai+wood vinegar 1:40 53.66 62.33 101.33 85.73 Wood vinegar 1:20 59.66 53.66 79.66 79.23 Wood vinegar 1:40 49.10 63.66 93.00 82.50 Cow dung 1,000 kg./rai 54.66 66.00 90.06 83.16 F-test ns ns ns ns CV (%) 2.76 1.65 2.14 4.03 Means in the same column with the same letter are not significantly different by DMRT at 0.05 probability level. Ns : not significant and significant at 0.05 probability level, respectively

Number of tiller per planting plot The application of cow dung fertilizer and without the fertilizer and the vinegar at the ratio of 1:20 and 1:40 resulted in the number of tiller per planting plot at 30, 90 and 120 days was no statistical difference. Cow dung fertilizer application at the rate of 1,000 kg / rai and vinegar at the rate of 1:20 and 1:40 resulted in a significant difference in the number of tiller per hill during the 60 DAP. The method of cow dung fertilizer at the ratio 1,000 kg./Rai have the highest number of tiller per hill at 10.66 tiller, followed by non- fertilizer application , cow dung fertilizer 1,000 kg ./Rai + wood vinegar at the rate of 1:40 , Wood vinegar rate 1:20 , cow dung fertilizer 1,000 kg / rai + wood vinegar rate 1:20 T2, wood vinegar rate 1:40 , with number of tiller per hill were 6.00 6.00 6.00 4.66 and 4.33 tiller per hill, respectively.(Table 3)

Table 3 Number of tiller per planting plot of Dawk Pradoo rice in each treatment at 30, 60,90 and 120 day after planting

Number of tiller per planting plot (tiller) Treatment 30 60 90 120 No fertilizer (control) 4.66 6.00b 5.33 5.66 Cow dung 1,000 kg./rai+wood vinegar 1:20 6.33 4.66c 6.33 5.66 Cow dung 1,000 kg./rai+wood vinegar 1:40 6.66 6.00b 6.33 5.66 Wood vinegar 1:20 6.33 6.00b 5.00 4.66 Wood vinegar 1:40 6.00 4.33c 5.33 5.00 Cow dung 1,000 kg./rai 5.33 10.66a 8.33 9.33 F-test ns * ns ns CV (%) 3.40 3.03 3.36 2.34 Means in the same column with the same letter are not significantly different by DMRT at 0.05 probability level. Ns : not significant and significant at 0.05 probability level, respectively

Leaf area and leaf area index Leaf area and leaf area index of rice after planting by cow dung fertilizer at the rate 1,000 kg./rail and no fertilizer and plus wood vinegar at the rate of 1:20 and 1:40 . No statistical difference with the highest of leaf area was cow dung fertilizer 1,000 kg / rai + wood vinegar at 1:40 with an area of 48.34 square centimeters, followed by cow dung fertilizer 1,000 kg / rai , without fertilizer , cow dung fertilizer 1,000 kg / rai + wood vinegar rate 1:20 , wood vinegar at 1:40 , and wood vinegar at the rate of 1:20 have average leaf area is 39.71 34.84 32.16 31. and 30.56 square centimeters, respectively. (Table 4)

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Table 4 Leaf area of Dawk Pradoo rice in each treatment at 30, 60, 90 and 120 day after planting

Leaf area Leaf area index Treatment (cm2) ( - ) No fertilizer (control) 34.84 0.87 Cow dung 1,000 kg./rai+wood vinegar 1:20 32.16 0.79 Cow dung 1,000 kg./rai+wood vinegar 1:40 48.34 1.20 Wood vinegar 1:20 30.56 0.76 Wood vinegar 1:40 31.67 0.79 Cow dung 1,000 kg./rai 39.71 0.98 F-test ns ns CV (%) 2.04 1.20 Means in the same column with the same letter are not significantly different by DMRT at 0.05 probability level. Ns: not significant and significant at 0.05 probability level, respectively

Conclusions

It was found that the nitrogen content in the soil is very low, available phosphorus and extractable potassium very high. The soil organic matter is low. The acidity - alkalinity of the soil is slightly acidic, with a pH of 6.36. No statistical difference all of treatment at 30, 60, 90 and 120 days after planting. The application of cow dung fertilizer and without the fertilizer and the wood vinegar at the rate of 1:20 and 1:40 resulted in the number of tiller per planting plot at 30, 90 and 120 DAP. was no statistical difference. In addition, the application of cow fertilizer and the vinegar at the rate of 1:20 and 1:40 resulted in the number of tiller per planting plot at 60 DAP was differently statistically significant because of it is high tillering to initial pregnant stage of rice growth stage for absorb fertilizer and wood vinegar for promote the tiller on this growth stage. Leaf area and leaf area index of rice after planting by cow dung fertilizer at the rate 1,000 kg./rail and no fertilizer and plus wood vinegar at the rate of 1:20 and 1:40 were no statistical difference.

Acknowledgements

The research project was financially supported by the Research and Development Institute of Buriram Rajabhat University.

References

[1] Black CA (1965) Methods of soil analysis: Part A. Agronomy 9, American Society of Agronomy, Madison, Wisconsin, USA. [2] Buarach K, Thongjoo C, Udomprasert N, Amkha S. (2014). Effects of tillage system and soil organic matter amendment on growth, yield of Pathumthani 80 rice and carbon sequestration in paddy soil. Mod Appl Sci. 8(4): 1-7. [3] Chang EH, Chung RS, Wang FN. (2008). Effect of different types of organic fertilizers on the chemical properties and enzymatic activities of an Oxisol under intensive cultivation of vegetables for 4 years. Soil Sci Plant Nutr. 54(4): 587-599. [4] Cottenie A. (1980). Soil and plant testing as a basis of fertilizer recommendations. Soil Bulletin 38/2. FAO, Rome.

doi:10.14457/MSU.res.2019.7 ICoFAB2019 Proceedings | 32

Kinetics Study on Hot-Air Drying Carrot Cubes

Wanwisa Suksamran1, Jakraphan Duangkhamchan2, Wasan Duangkhamchan3 and Kriangsak Banlue1*

1Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University, Khamriang, Kantarawichai, Maha Sarakham, 44150, Thailand 2Department of Agriculture Engineering, Faculty of Engineering, Khon Kaen University, Khon Kaen 40002, Thailand 3Electronics for Agriculture Research Unit, Faculty of Engineering, Mahasarakham University, Khamriang, Kantarawichai, Maha Sarakham, 44150, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Carrot is widely consumed as it contains bioactive compounds such as carotene. However these are very sensitive to heat. Therefore, degradation behaviors during thermal process should be considered. This study aimed to study kinetics of moisture content, shrinkage and -carotene degradation of carrot cubes subjected to hot-air drying. Several empirical models were fitted to experimental data obtained under different hot-air temperatures ranging from 60C to 80C. Based on the highest coefficient of determination (R2), lowest root mean square error (RMSE) and chi square (2), the suitable equations were selected and employed to describe the change behavior of moisture, shrinkage and -carotene. The results indicated that the Page and Modified Page models, the so-called Ratti model and the 2nd-order reaction kinetics equation suitably described the kinetics behaviors of moisture, shrinkage and degradation of -carotene, respectively. Consequently, all suitable kinetics model obtained in this work can be further used as a basis for design and optimization purposes.

Keywords: Mathematical modeling, Drying characteristics, -Carotene degradation, Shrinkage, Carrots

Introduction

Carrot (Daucus carota L.) is the one of vegetables widely consumed in the world due to its high nutritional values. Beta-carotene (-carotene), bioactive compound mostly found in carrot, assesses high antioxidant properties as it turns into vitamin A after intake into human body [1, 2]. In the food industry, carrots are mostly dried with purposes of reducing weight, extending shelf-life and broadening product availability [3]. With the use of heat in drying process, qualities including physical and chemical properties change, resulting in consumer unacceptability. Therefore, in this work kinetics of moisture, shrinkage and - carotene degradation were investigated in order to be served as a basis for process design and optimization.

Materials and methods

Experimental setup All experiments were conducted using a laboratory tray dryer with a size of 436939 cm. Air was supplied by centrifugal blowers connected to an inverter for adjusting air velocity which was kept constant as 0.5 m/s. The air was heated by 15-kW finned heater and its temperature was measured using K- type thermocouple. With a variation of air temperatures used in this work (60-80C), the air temperature was controlled by PID controller.

Drying procedure and Drying characteristics modeling Fresh carrots (Daucus carota L.) purchased from the local market in Nakhon Ratchasima province, Thailand were cleaned, peeled and subsequently cut into cubes with a size of 1 cm. Prior to experiments, initial moisture content of carrot cubes was randomly determined based on a standard method of AOAC [4]. Carrot cubes were placed into the drying chamber with the orientation in which hot air could pass through uniformly, as shown in Figure 1. The samples were subsequently subjected to hot-air drying with temperatures of 60, 70 and 80C. During a drying run, the sample weights were measured every 30 min ICoFAB2019 Proceedings | 33

until reaching equilibrium. With the weight recorded, the moisture content could be determined for each time interval. All experiments were triplicated and averaged data were presented.

Figure 1 Sample orientation

In order to describe the drying behavior of carrots subjected hot-air drying under temperatures of 60-80C, the experimental data of moisture ratio (MR) expressed in equation (1) were fitted to the proposed empirical drying model, as listed in Table 1. Statistical parameters including a coefficient of determination (R2), root mean sqaure (RMSE) and chi square (2) were used as criteria for model selection. M  M MR  t e (1) M 0  M e In equation (1), Mt, M0 and Me denotes the moisture content at time t, at initial time and at equilibrium, respectively. All values were expressed in wet basis.

Table 1 The model used with the drying kinetics of hot air drying of carrot cubes [5].

Model Model equation Lewis MR = exp(-kt) (2) Page MR = exp(-ktn) (3) Modified Page MR = exp(-kt)n (4) Henderson and Pabis MR = a exp(-kt) (5) Logarithmic MR = a exp(-kt)+c (6) Two term MR = a exp(-k0t)+b exp(-k1t) (7) Midilli MR = a exp(-ktn)+bt (8) a, b, c, n is constants in drying models and k is drying rate coefficient (1/minutes).

Kinetics modeling of Shrinkage Change in shrinkage during process was analyzed by means of fluid replacement (Archimedes’ principle) associated with n-heptane. Percentage of shrinkage was calculated using the volume before (V0) and after drying (Vt) at each time interval, as expressed in Eq.(9). V V %shrinkage  0 t 100 (9) Vt The shrinkage data were fitted to several empirical models summarized by [6] with little modification. The change in sample shrinkage was correlated with moisture content at specific drying time, as shown in Table 2. Again, the most suitable equation for describing the shrinkage kinetics was obtained using R2, RMSE and 2.

Table 2 Empirical equations describing the shrinkage.

Name of the equation Equation Eq.

Lozano [7] S  b1  MR  b2 (10)  b   6  Lozano [8] S  b3  b4  MR  b5  exp  (11)  b7  MR  Ratti [9] 2 3 (12) S  b8  b9  MR  b10  MR  b11  MR Vazquez [10] 3/2 (13) S  b12  b12  MR  b14  MR  b15 expb16  MR Mayor and Sereno [11] 2 (14) S  b17  b18  MR  b19  MR ICoFAB2019 Proceedings | 34

From Table 2, S is shrinkage coefficient, bi is numerical constants of empirical equations for shrinkage, MR is moisture content, g /g dry matter.

Determination of -carotene concentration and its kinetics modeling of degradation Concentration of -carotene was determined according to [12]. Three grams of dried carrot cubes was placed into a test tube containing 10 mL of acetone solution, and mixed using a vortex-mixed equipment for 30 second. The mixture was then subjected to a centrifuge at a speed of 2100g for 5 minutes. The supernatant was filtered through a paper Whatman No. 1, and subsequently evaporated under temperature of 50C. The evaporated supernatant was again extracted with 2-mL nitrile acetone, and finally filtered with a syringe filter with a pore size of 0.45 m. -carotene content was measured by means of high performance liquid chromatography (HPLC) method, following the protocol modified by [13]. Briefly, the mobile phase consisted of methanol and water (9:1 v/v) with a flow rate of 0.8 ml/min. UV detector was used at 472 nm under column temperature of 25C. As widely used to describe a change of reaction in biological materials, the kinetics equations with several orders (0th-2nd) were evaluated (see in equations (15-17)). The concentrations of -carotene determined at each drying time intervals were fitted to the 0th-, 0.5th-, 1st-, 1.5th-, and 2nd-order kinetics models. The best choice was chosen based on the highest R2, and lowest RMSE and 2. The equation of concentration ratio (CR) as a function of drying time for each temperature is expressed as following [14]:

d(CR)  kCR1n (15) dt

Equation 10 was converted to logarithm form for different reaction order [15] :

lnCR kt b ; (n = 1) (16) (CR)1n  kt b ; (n  1) (17) where CR is (C/C0, C, C0 denotes concentration of -carotene at a specific and initial time, respectively), t is drying time (min), k is the reaction rate constant, and b is an equation constant. All constants in equations (16) and (17) were determined by means of linear regression method.

Statistical analyses Besides the coefficient of determination, R2, root mean square (RMSE) and chi square (2) were evaluated using equations (18) and (19), respectively, at different temperatures.

1 n 2 RMSE  Vexp,i Vpre,i  (18) n i1

n 2 Vexp,i Vpre,i  2  i1 (19) n  z In equations (18) and (19), Vexp and Vpre is experimental value and predicted value. n is a number of experimental data recorded and z is a number of constants in kinetics equations.

Results and discussion

Drying characteristics modeling Commonly found in biological materials [16], moisture content (MC) of carrot cubes exponentially reduced with drying time for all temperatures, as shown in Figure 2. In addition, higher hot- air temperature resulted in increasing drying rate, as MC reduced to the desired level (10%wb) faster due to higher driving force. ICoFAB2019 Proceedings | 35

100 90 Moisture Content at 60 oC Moisture Content at 70 oC 80 Moisture Content at 80 oC 70 60 50 40 30

Moisture Content (%wb) Content Moisture 20 10 0 0 100 200 300 400 500 Drying Time (min)

Figure 2 Comparison of moisture contents as a function of drying time at various temperatures.

Among several mathematical models in Table 1, the drying characteristics of carrot cubes tested in this work could be suitably described using the Page and modified Page model as the result that these models gave the highest R2, and lowest RMSE and 2, as presented in Table 3.

Table 3 Statistical parameters of drying models at different temperatures.

Models Statistical parameters 60 °C 70 °C 80 °C Lewis R2 0.99543 0.99257 0.99543 χ2 0.00046 0.00065 0.00014 RMSE 0.02052 0.02440 0.01147 Page R2 0.99612 0.99900 0.99612 χ2 0.00044 9.82×10-5 3.76×10-5 RMSE 0.01892 0.00896 0.00555 Modified Page R2 0.99612 0.99900 0.99612 χ2 0.00044 9.82×10-5 3.77×10-5 RMSE 0.01892 0.00896 0.00555 Henderson and Pabis R2 0.99558 0.99269 0.99558 χ2 0.00050 0.00072 0.00016 RMSE 0.02019 0.02421 0.01145 Logarithmic R2 0.99558 0.99503 0.99558 χ2 0.00056 0.00055 5.15×10-5 RMSE 0.02015 0.01995 0.00612 Two term model R2 0.99558 0.99269 0.99558 χ2 0.00064 0.00092 0.00021 RMSE 0.02019 0.02421 0.01145 Midilli R2 0.99658 0.70797 0.99658 χ2 0.00049 0.03677 0.04701 RMSE 0.01775 0.15296 0.17297

With the use of non-linear regression method, all constants in each suitable equations were estimated, and therefore, the mathematical models that could best describe the drying characteristics of carrot cubes at different temperatures are summarized in Table 4. These findings were consistent with the results obtained by [17] of which the Page model suitably described the drying behavior of carrot shreds (4420 mm) subjected to hot-air convective drying at temperatures ranging from 50-70C.

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Table 4 Suitable drying models and their constants.

Models Conditions Equations Page 60 °C MR = exp(-0.53566t0.53566) 70 °C MR = exp(-1.14821t0.70908) 80 °C MR = exp(-1.34413t0.79351) Modified Page 60 °C MR = exp(-0.55986t)1.07616 70 °C MR = exp(-1.21520t)0.70908 80 °C MR = exp(-1.45166t)0.79351

Kinetics modeling of shrinkage Figure 3 shows volume ratio (V/V0) or shrinkage of carrot cubes as a function of drying time at different temperatures. It was found from Figure 3 that the sample volume decreased with drying time. This shrinkage was attributed to water evaporation inside the samples, resulting in a gap which caused the sample surface to collapse [18]. In addition, the samples shrank quicker when hot-air temperature increased, especially at the initial drying period (0-60 min), corresponding to higher drying rate, as seen in Figure 2. This results was also found in the work of [3]. 1 0.9 0.8

0.7 60C 0.6 70C

o 80C

0.5 V/V 0.4 0.3 0.2 0.1 0 0 60 120 180 240 300 360 420 Drying time (min) Figure 3 Comparison of shrinkage as a function of drying time at various temperatures.

Table 6 Statistical results for cubes mathematical models of shrinkage carrots.

Models Statistical parameters 60 °C 70 °C 80 °C Lozano [7] R2 0.97770 0.88920 0.98770 χ2 0.00260 0.01246 0.00118 RMSE 0.04498 0.09843 0.03024 Lozano [8] R2 0.97778 0.98686 0.99790 χ2 0.00453 0.02195 0.00035 RMSE 0.04486 0.03390 0.01252 Ratti [9] R2 0.99760 0.99670 0.99840 χ2 0.00111 0.10103 0.00073 RMSE 0.02485 0.23691 0.02009 Vazquez [10] R2 0.97778 0.88840 0.98674 χ2 0.00453 0.02195 0.00222 RMSE 0.04486 0.09876 0.03141 Mayor and Sereno [11] R2 0.99370 0.98530 0.99820 χ2 0.00086 0.00193 0.03418 RMSE 0.02392 0.03587 0.15094

Due to time independcy, experimental data of sample shrinkage were fitted to well-known empirical models (see Table 2). Table 6 summarizes the statistical parameters of various equations. Based on the statistical criteria, the model proposed by Ratti [9] gave the highest suitability for describing the change in volume of carrot cubes and their influence of temperature. Furthermore, its model parameters ICoFAB2019 Proceedings | 37

were estimated by means of non-linear regression. The appropriate shrinkage kinetic models for various temperature are therefore expressed as follows:

60C: S = 0.1192 + 0.0852·X + 0.0078·X2 – 0.0008·X3 (20a) 70 °C: S = 0.1283 + 0.0637·X + 0.0463·X2 – 0.0041·X3 (20b) 80 °C: S = 0.1111 + 0.1472·X – 0.0140·X2 + 0.0007·X3 (20c)

Degradation kinetics of beta-carotene Figure 4 shows -carotene content reduced from 58.03 mg/100g dry matter (fresh carrot) to approximately 2-4 mg/100g dry matter (at the end of drying process). With variation of hot-air temperature, -carotene degraded with different rate, the higher temperature, the higher degradation rate. Therefore, at the same drying time, higher retention of beta-carotene was found when using lower drying temperature. Again, kinetics was investigated for describing degradation of beta-carotene during drying process. Table 6 shows the statistical parameters of kinetic equations of reaction of carrot cubes dried under different temperatures. From this, the best parameters were found for the 2nd-order kinetic equation, R2 in a range of 0. 981-0. 995. The suitable models for predicting concentration of -carotene in carrot cubes during drying at different temperatures are summarized here,  1  60C:    0.0398t  0.1825 (21a)  CR   1  70 °C:    0.0573t  0.2053 (21b)  CR   1  80 °C:    0.0806t 0.7163 (21c)  CR  where CR denotes concentration ratio of -carotene and t stands for drying time (minutes). It could be seen from equations 21a-c that the inverse of concentration ratio of -carotene linearly correlated with process time. Therefore, the amount of -carotene could be predicted and controlled under a certain drying time.

Figure 4 Comparison of -carotene content as a function of drying time at different temperatures.

Conclusions

Carrot cubes were subjected to drying process with variation of temperature in order to investigate kinetic modeling for moisture content, shrinkage and -carotene. The suitable equations used for describing the change in these qualities were the Page and modified Page model, the Ratti model and the 2nd-order reaction model, respectively. Moreover, hot-air temperatures significantly affected all qualities, the higher temperature, the higher rate of decreasing. Consequently, these kinetic equations could be very useful for further applications and research in numerical study of drying sytems such as computational fluidynamics investigation which could be served as a basis for process design and optimization.

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Table 7 Statistical parameters of kinetic models of -carotene degradation.

Temperature Statistical parameters 0th 0.5th 1st 1.5th 2nd 60 °C R2 0.70370 0.8076 0.90490 0.97090 0.99010 χ2 0.00144 0.11060 0.01343 0.19635 0.17653 RMSE 0.20102 0.25760 0.08977 0.34324 0.32545 70 °C R2 0.57890 0.72300 0.87350 0.97120 0.99580 χ2 0.09198 0.13082 0.06006 0.22954 0.04177 RMSE 0.23492 0.28017 0.18983 0.37111 0.15831 80 °C R2 0.48380 0.66750 0.88080 0.99240 0.98190 χ2 0.10992 0.15278 0.08337 0.25417 0.07401 RMSE 0.25681 0.30277 0.22366 0.39052 0.21073

Acknowledgements

This study was supported by Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University.

References

[1] Zielinska M, Markowski M. Air drying characteristics and moisture diffusivity of carrots. Chem Eng Process. 2010; 49, 212–8. [2] Sumnu G, Turabi E, Oztop M. Drying of carrots in microwave and halogen lamp–microwave combination ovens. LWT-Food Sci Technol. 2005; 38, 549–533. [3] Jomlapelatikul A, Wiset L, Duangkhamchan W, Poomsa-ad N. Modelbased investigation of heat and mass transfer for selecting optimum intermediate moisture content in stepwise drying. Applied Thermal Engineering. 2016; 107, 987–993. [4] AOAC. 1990, Official method of analysis. Association of Official Analytical Chemists, Arlington [5] El-Sebaii, A.A., and S.M. Shalaby. Experimental investigation of an indirect-mode forced convection solar dryer for drying thymus and mint. Energy Conversion and Management. 2013; 74, 109-116. [6] Banu Koc, İsmail Eren, Figen Kaymak Ertekin, Modelling bulk density, porosity and shrinkage of quince during drying: The effect of drying method. J. Food Eng. 2008; 85, 340–349. [7] Lazano, J.E. Rotstein, E., and Urbicain, M.J. Total porosity and open porosity in the drying of fruits. Journal of Food Science. 1980; 45, 1403-1407. [8] Lazano, J.E. Rotstein, E., and Urbicain, M.J. Shrinkage, porosity and bulk density of food stuffs at changing moisture contents. Journal of Food Science.1983; 48, 1497-1502. [9] Ratt, C. Shrinkage during drying of foodstuffs. J. Food Eng. 1994; 23, 91-105. [10] Vazquez, G., Chenlo, F., Moreira, R., and Costoyas, A. The dehydration of garlic. 1. Desorption isotherms and modeling of drying kinetics. Drying Technology. 1990; 17, 1095-1108. [11] Mayor, L., and Sereno, A.M. Modeling shrinkage during convective drying of food materials: A review. Journal of Food Engineering. 2004; 61, 373-386. [12] Brinton, G. Structure and properties of carotenoids in relation to function. The Federation of American Societies for Experimental Biology. 1995; 9, 1551-1558. [13] Barba, A.I.O., Hurtado, M.C., Mata, M.C.S., Ruiz V.F., de Tejada, M.L.S. Application of a UV-vis detection-HPLC method for a rapid determination of lycopene and beta-carotene in vegetables. Food Chemistry. 2006; 95, 328-336. [14] L. Yang, L. Zhongxin, W. Ma, S. Yan, K. Cui, Thermal Death Kinetics of Fifth-Instar Corcyras cephalonica (Lepidoptera: Galleriidae). Journal of Insect Science. 2015; 15, 1-5. [15] Yan, R., Z. Huang, H. Zhu, J. A. Johnson, and S. Wang Thermal death kinetics of adult Sitophilus oryzae and effects of heating rate on thermotolerance. J. Stored Prod. Res. 2014; 59, 231–236. [16] Jamali, A., Kouhila, M., Mohamed, L. A., Idlimam, A., & Lamharrar, A. Moisture adsorption– desorption isotherms of Citrus reticulate leaves at three temperatures. J. Food Eng. 2006; 77, 71-78. [17] Raees-ul Haq, Pradyuman Kumar and Kamlesh Prasad, Hot air convective dehydration characteristics of Daucus carota var. Nantes. Cogent Food & Agriculture. 2015; 1, 1096184. [18] Yadollahinia, A. and Jahangiri, M. Shrinkage of potato slice during drying. J. Food Eng. 2009; 94, 52–58.

doi:10.14457/MSU.res.2019.8 ICoFAB2019 Proceedings | 39

Effect of Acid-Alkaline Pretreatment on Reducing Sugar Yield and Lignocellulosic Compositions of Rice (Oryza sativa L.) Residues

Kaewkanlaya Sotthisawad*, Suleepron Siripru and Kanyarat Poprom

Program of Biology, Faculty of Science and Technology, Sakon Nakhon Rajabhat University, Sakon Nakhon 47000, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Rice residues (RRs) are classified as an agricultural waste, which refer to rice bran, rice husk and rice straw. In the present study, the chemical compositions of each RRs were analyzed. The determination of lignocellulosic compositions of rice bran was revealed and represented as an outstanding biomass that consisted of high cellulose and low hemicellulose and lignin. While the compositions of rice husk and rice straw was obviously contrasted from rice bran. Furthermore, the acid-alkaline pretreatment of RRs under autoclaving at 121 ºC for 90 min was investigated for determination of the reducing sugar yield and lignocellulosic compositions change. The study consists of four pretreatment conditions for disruption of RRs structure with different solutions, including the condition (1): 1%(v/v) H2SO4, (2): 1%(w/v) NaOH, (3): 1%(w/v) NaOH following by 1%(v/v) H2SO4, and (4): distilled water (control). The result showed that the acid pretreatment with 1%(v/v) H2SO4 solution (condition 1) was the best condition that provided the highest reducing sugar yield from all RRs kinds. The dilute acid and alkaline pretreatment of RRs under 121 ºC for 90 min illustrated a clearly variation of its lignocellulosic compositions. The acid pretreated rice bran showed that cellulose reduction which correlated with concentration of reducing sugar yield released in the same experiment condition.

Keywords: Acid-alkaline pretreatment, Rice (Oryza sativa L.), Lignocellulosic biomass, Reducing sugar

Introduction

Lignocellulosic biomass such as agricultural and forest residues are considered as an alternative, inexpensive, renewable, and abundant source for fuel ethanol production [1]. This biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be produced from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials. In 2018, production of rice (Oriza sativa L.) in Thailand is approximately amounts to 24.22 million tons [2]. The residues of rice are one of alternative and potential crops that can use as lignocellulosic biomass to produce biofuel. The main by-products of rice called rice residues (RRs) includes rice bran, rice husk and rice straw, are an abundant, readily available agricultural waste, which shows as a potential feedstock for bioenergy production. The RRs cell walls consists of three main biopolymers, namely, cellulose, hemicellulose, and lignin. Cellulose is a homopolymer of cellobiose (two units of glucose), which are linked by 훽-1,4- glycosidic bonds. The complete hydrolysis of cellulose yields glucose, which is a preferable carbon source for commonly used fermenting microorganisms in industry [3]. Hemicelluloses are branched, heterogenous polymers of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose) and acetylated sugars [4]. Lignin is a large complex polymer of unrepeated phenolic monomers. It significantly contributes to the water conduction and defense systems in plants [3]. These residues can be recovered into a cheap and environmentally friendly renewable resource by converting them to sugars using pretreatment processes. The aim of pretreatment is to disrupt the crystalline structure of lignocellulosic materials and to remove or reduce hemicelluloses and/or lignin. Pretreatment is the most important and costly steps, which has a major influence on the cost of bioenergy production from lignocellulosic biomass [5]. Normally, pretreatment methods of lignocellulosic material include physical (milling and grinding), physico-chemical (steam pretreatment and hydrothermal lysis), chemical (alkaline and dilute acid) and biological (enzyme hydrolysis) or combination of these [6]. Among these pretreatment methods, dilute sulfuric acid pretreatment has been found to be cheap, and to produce high hemicellulose recoveries and cellulose digestibility [7]. The optimization of acid ICoFAB2019 Proceedings | 40

pretreatment of rice husks has been studied previously [8]. Whereas, alkaline pretreatment is well-known that improves cellulose digestibility by providing the effective delignification and chemical swelling of fibrous cellulose [9]. In the biological pretreatment, is provided a high biodegradability rate which realized at exorbitant cellulolytic enzyme costs and enzymes activity during biomass decomposition is very slow requiring long duration [9]. Therefore, the main purpose of this research was to compare the effectiveness of dilute acid-alkaline pretreatments on chemical composition and reducing sugar production of rice bran, rice husk and rice straw.

Materials and methods

Raw materials preparation Rice bran, rice husk and rice straw were collected in 2018 from agriculture field of Sakon Nakhon Province, Thailand. These RRs were mashed into small pieces and oven-dried overnight at 70 ºC. All RRs samples were stored in a sealed plastic bag at room temperature until further use.

Determination of chemical compositions The chemical compositions (such as cellulose, hemicellulose, lignin and ash) of dried RRs were analyzed both before and after pretreatment, and described the method by Serechodchawong and Sangkharak (2014) [11].

Acid-alkaline pretreatment The dilute acid-alkaline pretreatment of dried RRs was modified from Sotthisawad et al. (2017) [12]. 5 g of each RRs (rice bran, rice husk and rice straw) were treated and added as a substrate to solution at a ratio of 1:10, prior to autoclaving at 121 ºC for 90 min. The pretreatment was performed by four different pretreatment conditions including: (1)-Acid pretreatment with 1%(v/v) H2SO4 solution (2)-Alkaline pretreatment with 1%(w/v) NaOH solution (3)-Acid-alkaline pretreatment with 1%(w/v) NaOH and following by 1%(v/v) H2SO4 solution (4)- Pretreatment with distilled water (DW) and served as controls

Reducing sugar assay The amount of reducing sugar released after the pretreatment process was assayed by 3,5-di nitrosalicylic acid (DNS) analysis method of Miller (1959) [13] using spectrophotometer at 540 nm.

Results and discussion

Chemical compositions of RRs Chemical characteristics of rice bran, rice husk and rice straw are presented in Table 1. The carbohydrate composition refer as lignocellulosic compositions of rice husk and rice straw were quite similar, while rice bran was clearly differed from them. The composition analysis revealed that rice bran is wealthy of cellulose that can be hydrolyzed to produce glucose, was found to be 77.552.19%, (w/w). While, hemicellulose content was found that higher than other components in rice husk and rice straw to 42.671.15 and 53.331.15 %, (w/w), respectively. The results obtained in the present study are in agreement with Sridevi et al. (2015) [14] who also reported that rice bran contained highest cellulose content.

Table 1 Chemical compositions of raw rice residues

Contents (%, w/w) Components Rice bran Rice husk Rice straw Cellulose 77.552.19 12.671.15 10.002.00 Hemicellulose 3.001.41 42.671.15 53.331.15 Lignin 16.670.58 41.750.95 35.172.98 Ash 2.780.20 2.920.37 1.500.07

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Reducing sugar released from pretreatment Pretreatment processes are vital for efficient separation of the complex interlinked components and enhance the availability of every component, i.e., cellulose and hemicellulose [15]. The pretreatment of RRs with dilute acid and alkaline was carried out in this research. The yield of reducing sugar in the liquid fraction resulting from 5 g of each RRs after acid/alkaline pretreatments at 121 ºC for 90 min in four conditions of pretreatment was analyzed (Fig. 1). It was noted that reducing sugar yields from acid/alkaline pretreated RRs were higher than those from the control, indicating that pretreatment is critical for increasing the saccharification of RRs. Especially, the acid pretreatment with 1%(v/v) H2SO4 resulted in the highest reducing sugar yield from all RRs kinds. The maximum amount of reducing sugar liberated from acid pretreated rice bran was 20.040.49 g/L, followed by rice husk (18.010.91 g/L) and rice straw (9.760.16 g/L). Based on the findings in this study, pretreatment of RRs with 1%(v/v) H2SO4 was the best suited for enhancing the reducing sugar yield. Similarity, Deejing and Ketkorn (2009) [16] found the maximum reducing sugar concentration released from rice bran was obtained by hydrolysis with 1.0% (v/v) H2SO4 under 111 ºC for 30 min. Besides, the findings of the experiments carried were supported by recent reports about pretreatment of mushroom cultivation waste material with 1% (v/v) H2SO4 for 90 min resulted in the highest yield of reducing sugar of 14.09±0.38 g/L [12].

Rice bran Rice husk Rice straw 25

20.04 20 18.01

15 13.02

9.76 10 8.26

5.61 5 3.01 3.76 1.62 1.66

1.67 1.32 Reducing sugar Reducing yield (g/L) 0

1%H 2SO4 1%NaOH 1%NaOH + DW 1%H2SO4

Pretreatment conditions

Figure 1 Reducing sugar contents released after acid-alkaline pretreatment of RRs. Bars represent the means of three replicates, and error bars indicate the standard deviation.

Effect of pretreatments on lignocellulosic compositions of RRs Some chemicals such as acids, alkali, organic solvents, and ionic liquids have been reported to have significant effect on the native structure of lignocellulosic biomass [17]. Compositional analysis of lignocellulose, before and after pretreatment, is used in almost all second-generation biofuel production studies [18]. The effect of RRs pretreatment with dilute acid and alkaline under 121 ºC for 90 min showed a variation of lignocellulosic compositions of RRs (Table 2). All conditions of rice bran pretreatment found that caused of decreasing of cellulose content whereas hemicellulose and lignin increased. The rice bran pretreatment with 1%(v/v) H2SO4 (condition 1), revealed a lowest cellulose content which may be resulted by the breakdown of cellulose complex into glucose or other carbohydrates. Results of the present study on dilute sulfuric acid pretreatment has been studied for a wide range of lignocellulosic biomass [3]. It results in high recovery of the hemicellulosic sugars in the pretreatment liquid, and in a solid cellulose fraction [19].

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Table 2 The lignocellulosic composition changes of RRs after acid-alkaline pretreatment

Rice residues Pretreatment Contents (%, w/w) conditions* Cellulose Hemicellulose Lignin Ash untreated 77.552.19 3.001.41 16.670.58 2.780.20 1 17.0012.73 37.001.41 43.7510.82 2.250.49 Rice bran 2 46.008.49 29.009.90 23.371.23 1.630.18 3 36.002.83 11.001.41 49.150.89 3.852.31 4 50.002.83 12.005.66 35.662.63 2.340.20 untreated 12.671.15 42.671.15 41.750.95 2.920.37 1 22.008.49 32.002.83 43.655.64 2.350.01 Rice husk 2 36.002.83 2.000.00 41.702.83 2.300.00 3 30.002.83 16.000.00 52.114.23 1.891.40 4 31.001.41 34.002.83 31.711.60 3.290.18 untreated 10.002.00 53.331.15 35.172.98 1.500.07 1 46.0019.80 42.0011.31 21.6016.46 0.400.51 Rice straw 2 67.007.07 8.002.83 23.753.86 1.250.38 3 40.000.00 11.004.24 47.885.43 1.121.19 4 34.000.00 43.001.41 22.121.30 0.880.11

*Rice residues are both untreated and pretreated with i: 1%(v/v) H2SO4; ii: 1%(w/v) NaOH; iii: 1%(w/v) NaOH following by 1%(v/v) H2SO4; and iv: distilled water (served as control).

On the other hand, the dilute acid and alkaline pretreatment of rice husk and rice straw effected to cellulose content expanding while hemicellulose decrease. After pretreatment with 1%(w/v) NaOH following by 1%(v/v) H2SO4 (condition 3) found that lignin content of rice husk and rice straw were higher than other conditions (52.114.23 and 47.885.43%(w/w), respectively). Moreover, the pretreatment of rice straw and rice husk with 1%(w/v) NaOH (condition 2) illustrated the highest cellulose content of 67.007.07 and 36.002.83 %(w/w), respectively. This result correlated with Kobkam et al. (2018) [6] discussed that the alkaline pretreatment of rice straw affects the crystallinity decreasing of cellulose and increases hemicellulose disruption. The effect of alkali pretreatment (such as NaOH, KOH and anhydrous ammonia) cause swelling of biomass, which increases the internal surface area of the biomass, and decreases both the degree of polymerization, and cellulose crystallinity. Alkaline pretreatment disrupts the lignin structure and breaks the linkage between lignin and the other carbohydrate fractions in lignocellulosic biomass, thus making the carbohydrates in the heteromatrix more accessible [20].

Conclusions

The lignocellulosic compositional analysis of each RRs, both before and after pretreatment, is an important step for development of biofuel production studies. The biomass provided a high cellulose content will be considered and chosen to use as a raw material for biofuel generation. The acid-alkaline pretreatment of lignocellulosic biomasses is also essential process for alter the structure of biomass residues to enhance the reducing sugars yield. It is clear from this study that rice bran represents as a distinctive biomass provided high cellulose and low hemicellulose and lignin. The pretreatment of rice bran with 1%(v/v) H2SO4 at 121 ºC for 90 min was the optimal condition for liberation of reducing sugar yield from this substrate.

Acknowledgements

The authors are sincerely acknowledged to the Research and Development Institute (RDI) and the Program of Biology, Faculty of Science and Technology, Sakon Nakhon Rajabhat University (SNRU) for all supports and scientific aids.

ICoFAB2019 Proceedings | 43

References

[1] Banoth C., Sunkar B, Tondamanati PR and Bhukya B. Improved physicochemical pretreatment and enzymatic hydrolysis of rice straw for bioethanol production by yeast fermentation. 3Biotech. 2017; 7(5), 334. [2] Thai Rice Exporters Association. (2019). Rice products. Available at: http://www.thairiceexporters.or.th/ production.htm, accessed July 2019. [3] Hu F and Ragauskas A. Pretreatment and lignocellulosic chemistry. Bioenerg. Res. 2012; 5(4), 1043– 1066. [4] Saha BC. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 2003; 30, 279–291. [5] Yang B and Wyman CE. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Bioref. 2008; 2, 26–40. [6] Kobkam C, Tonoi J and Kittiwachana S. Alkaline pretreatment and enzyme hydrolysis to enhance the digestibility of rice straw cellulose for microbial oil production. KMUTNB Int. Appl. Sci. Technol. 2018; 11(4), 247-256. [7] Li Z, Jiang Z, Fei B, Cai Z and Pan X. Comparison of bamboo green, timber and yellow in sulfite, sulfuric acid and sodium hydroxide pretreatments for enzymatic saccharification. Bioresour. Technol. 2014; 151, 91–99. [8] Salimi MN, Lim SE, Yusoff AHM and Jamlos MF. Conversion of rice husk into fermentable sugar by two stage hydrolysis. J. Phys. 2017; Conf. Ser. 908, 012056. [9] Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. Mol. Sci. 2008; 9, 1621-1651. [10] Zhao X, Peng F, Cheng K and Liu D. Enhancement of the enzymatic digestibility of sugarcane bagasse by alkaline-peracetic acid pretreatment. Enzyme. Microb. Technol. 2009; 44, 17–23. [11] Serechodchawong P and Sangkharak K. The production of biodiesel and ethanol from pressed coconut. Thaksin. J. 2014; 17(3, special), 103-110. [12] Sotthisawad K, Mahakhan P, Vichitphan K, Vichitphan S and Sawaengkaew J. Bioconversion of mushroom caltivation waste materials into cellulolytic enzymes and bioethanol. Arab. J. Sci. Eng. 2017; 6(42), 2261-2271. [13] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry.1959; 31(3), 426–428. [14] Sridevi A, Narasimha G, Ramanjaneyulu G, Dileepkumar K, Rajasekhar RB and Suvarnalatha PD. Saccharification of pretreated sawdust by Aspergillus niger cellulase. 3 Biotech. 2015; 5, 883–892. [15] Tayyab M, Noman A, Islam W, Waheed S, Arafat Y, Ali F, Zaynab M, Lin S, Zhang H and LIN W. Bioethanol production from lignocellulosic biomass by environment-friendly pretreatment methods: a review. Appl. Ecol. Env. Res. 2017; 16(1), 225-249. [16] Deejing S and Ketkorn W. Comparison of hydrolysis conditions to recover reducing sugar from various lignocellulosic materials. Chiang Mai J. Sci. 2009; 36(3), 384-394. [17] Swatloski RP, Spear SK, Holbrey JD, Rogers RD. Ionic liquids: new solvents for nonderivitized cellulose dissolution. Abstr Pap Am Chem Soc, 2002; 224, U622. [18] Karimi K and Taherzadeh MJ. A critical review of analytical methods in pretreatment of lignocelluloses: Composition, imaging, and crystallinity. Bioresour. Technol. 2016; 200, 1008-1018. [19] Jönsson LJ and Martín C. Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects. Bioresour. Technol. 2016; 199, 103-112. [20] Chandra RP, Bura R, Mabee WE, Berlin A, Pan X, Saddler JN. Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics?. Adv Biochem Eng Biotechnol. 2007; 108, 67–93.

doi:10.14457/MSU.res.2019.9 ICoFAB2019 Proceedings | 44

Mixture of Parawood Sawdust and Dried Napier Grass as a Substrate on Lentinus squarrusulus Mont. Cultivation

Niramai Fangkrathok* and Songchai Wongwaitaweewong

Faculty of Agricultural Technology, Burapha University Sakaeo Campus, Wattana Nakorn, Sa Kaeo 27160, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Lentinus squarrosulus Mont. , or Hed Khon Khao, is a commercial mushroom in Thailand. Parawood sawdust is a main substrate for bag mushroom cultivation but it is quite expensive. Napier grass is a fast growing grass which can be used as an alternative source for mushroom cultivation. This study aims to investigate the effect of parawood sawdust and Napier grass mixture on mycelial growth and fruiting body production of L. squarrosulus. Parawood sawdust and Napier grass mixtures in the ratio of 100:0(T1), 90:10(T2), 80:20(T3), 70:30(T4) and 50:50(T5) were used as a main substrate in bag cultivation. C/N ratio, substrate pH, mycelial growth, fruiting body and biological efficiency (BE) were determined. The results showed that all substrates had pH value and C/N ratio in the range of 6.23-6.80 and 107.56- 160.57, respectively. Mycelial running on T1 to T4 substrates was faster than T5 substrate. T2 substrate gave higher fresh fruiting body weight (59.42±16.14 g/bag) than T1 (54.53 ± 10.67 g/bag), T3 (27.17 ± 11.98 g/bag), T4 (22.33 ± 13.70 g/bag) and T5 (4.14 ± 4.05 g/bag), respectively. In addition, there was no significant difference between BE of T1 (12.36±2.42%) and T2 (13.47±3.66%). These results suggested that 10% dried Napier grass mixture (T2) could be used to culture L. squarrosulus the same as parawood sawdust (T1) whereas higher amount of dried Napier grass resulted in significant decreasing in mushroom production. In conclusion, dried Napier grass can be used to mix with parawood sawdust for L. squarrosulus cultivation leading to the decrease of parawood sawdust usage.

Keywords: Lentinus squarrosulus, Parawood sawdust, Napier grass, C/N, Biological efficiency

Introduction

Edible mushroom is widely consumed in Thailand. Fast growing of mushroom farming business results in highly demand in raw materials for mushroom cultivation. Parawood sawdust is a main composition in bag mushroom cultivation. However, parawood sawdust is quite expensive because of delivery cost. The farmers have to buy this parawood sawdust in bulk to lower the cost and have to have a big area to keep the sawdust. In this study Napier grass was selected as an alternative source to mix with parawood sawdust in order to lower the parawood sawdust usage in the substrate bag. Pennisetum purpureum, also known as Napier grass or elephant grass is a rhizomatous perennial grass in Poaceae family. This grass is tall, fast growing and can be harvested several times a year [1]. Napier grass needs low water and nutrient inputs [2,3], therefore, this grass is widely grown in Thailand. Napier grass stem and leaf contain high carbon (44-48%), oxygen (44-49%), cellulose (29-38%), hemicellulose (15-20%), lignin (24-30%) and several elements especially potassium (49-64%) [3]. Carbon/Nitrogen (C/N) ratio of substrate is important for mushroom cultivation. High C/N ratio substrate is needed during mycelial growth period whereas low C/N ratio is needed during fruiting body formation [4,5]. Therefore, mycelial growth can be faster in high C/N ratio substrate than those of low C/N ratio substrate. Fruiting bodies need nitrogen for protein synthesis, thus, low C/N ratio substrate supports the formation of fruiting bodies better than high C/N ratio. Lentinus squarrosulus (Mont. ) or Hed Khon Khao is an edible mushroom commonly found in Thailand. This mushroom is in Polyporaceae family. Both fruiting bodies and mycelium of L. squarrosulus have high nutritional and pharmaceutical properties [6-8]. For commercial cultivation of L. squarrosulus, parawood sawdust is used as a main composition in a substrate bag. However, parawood sawdust price is expensive. In Thailand, parawood sawdust prices vary between 1,500-2,700 baht/ton depending on distance and purchase amount. And the farmers have to purchase at least 10 ton of sawdust in order to reduce the production cost. Finding an alternative source of substrate may lead to the reducing of mushroom production cost. Therefore, the objective of this study was to determine the effect of parawood sawdust and dried Napier grass mixture on mycelial growth and fruiting body production of L. squarrosulus. ICoFAB2019 Proceedings | 45

Materials and methods

Raw material preparation Napier grass was collected from Burapha University Sakaeo Campus farm. Fresh grass was cut and dried at 60ºC for 3 days by using hot air oven. The dried grass was mashed by using wood grinder and the approximate size of mashed grass was 1-5 mm. Dried parawood sawdust was bought from a farmer in Sa Kaeo Province. Rice bran was bought from a rice mill in Wattana Nakorn District, Sa Kaeo Province, Thailand.

Experimental design This study was performed at Burapha University Sakaeo Campus, Thailand, since September to December 2016. Completely Randomized Design (CRD) was used in this experiment. The treatments were divided into 5 treatments including parawood sawdust and Napier grass mixtures in the ratio of 100:0(T1), 90:10(T2), 80:20(T3), 70:30(T4) and 50:50(T5). The experiments were performed in triplicate and each treatment consisted of 12 mushroom bags. Substrate ingredients of each treatment were shown in Table 1. The ingredients were mixed and then packed into a mushroom plastic bag (1 kg/bag). The bags were streamed for 5 h. The bags were sampling for pH measuring and C/N ratio analysis. C/N ratio was analyzed by iLab, Chiang Mai Province, Thailand. Carbon was analyzed as described by Allison [9] and nitrogen was analyzed by using AOAC [10].

Table 1 Substrate ingredients of each treatment

Treatments (kg) Substrate ingredients T1 T2 T3 T4 T5 Parawood sawdust 12.00 10.80 9.60 8.40 6.00 Dried and mashed Napier grass 0.00 1.20 2.40 3.60 6.00 Sugar 0.12 0.12 0.12 0.12 0.12 Lime 0.12 0.12 0.12 0.12 0.12 Pumice powder 0.12 0.12 0.12 0.12 0.12 Gypsum powder 0.12 0.12 0.12 0.12 0.12 Magnesium sulfate 0.06 0.06 0.06 0.06 0.06 Water 7.00 7.00 7.00 7.00 7.00

Mushroom spawn, mycelial and fruiting body cultivation L. squarrosulus mycelia were isolated from fresh fruiting body and cultured in potato dextrose agar for 5 days. The mycelia were transferred to grow on sterile sorghum seeds. After incubation at room temperature for 5 days, the mushroom spawn completely grew on sorghum seeds. The mycelial spawn was inoculated into all substrate bags. The bags were incubated at room temperature until the mycelium completely grew (6 weeks). The bags were transferred into mushroom house and the upper part of bags was cut. During housing, water was sprayed twice a day to control the humidity (70-80%) and temperature (25-32ºC). Three or four days after primordium appearing, mature fruiting bodies were ready for picking up based on commercial size of this mushroom. In each day, fresh fruiting bodies were collected and weighed. After 48 days, accumulated weight of fresh fruiting body in each treatment was calculated for fresh fruiting body weight per bag and then calculated for biological efficiency (BE) as following equation. BE = (fresh fruiting body weight per bag ÷ substrate weight per bag)  100

Statistical analysis The results were expressed in mean ± S.D. One-Way ANOVA and Post-Hoc test (LSD) were analyzed by using SPSS version 16.0. Significant difference was p value < 0.05.

Results and discussion

The substrate pH and C/N ratio of each treatment were shown in Table 2. Substrate pH was in range of 6. 23 – 6. 80 and there was no significant difference between substrate pH of each treatment indicating that increased Napier grass mixing did not affect the whole substrate pH. The C/N ratio is important for mushroom production. During mycelial growth, the mycelia adsorb carbon to produce an energy for their growth, therefore, mycelia rapidly grow in high C/N ratio substrate [11, 12]. Whereas low C/N ratio is necessary for fruiting body formation because high nitrogen needs for enzyme and protein production in fruiting bodies [5]. The C/N ratio of substrate treatments in this study ICoFAB2019 Proceedings | 46

were between 107.56 to 160.57. In Figure 1, the mycelial growth of T1, T2, T3 and T4 was in the same rate and faster than those of T5. However, all bags were ready to be transferred into mushroom house at 6th week. These results indicated that variation of C/N ratio of substrate might not directly affect to mycelial growth. The mycelium of this mushroom may possibly grow in wide range of C/N ratio. Chang and Miles (2009) explained C/N ratio that the most appropriate for Pleurotus spp. production is vary between 32 to 150 [13]. After housing for 4 days, the primordium was formed and then subsequently developed to form mature fruiting bodies (Figure 2). T1 provided fruiting bodies faster and higher in yield than those of T2, T3, T4 and T5, respectively (Figure 3). However, at day 46 to 48 the cumulative weight of T2 became higher than those of T1. The results might indicate that T2 tended to give higher yield than T1 when housing longer. Interestingly, there was no significant difference between average weight of fresh fruiting bodies (59.42 ± 16.14 g/bag) and BE (13.47 ± 3.66 %) of T2 and those of T1 (54.53 ± 10.67 g/bag and 12.36 ± 2. 42 %, respectively) (Table 2). Whereas T3, T4 and T5 showed lower yield, average weight of fresh fruiting bodies per bag and BE than T1 and T2 (Figure 3 and Table 2). These results indicated that Napier grass mixing higher than 10% might decrease the yield of this mushroom.

Table 2 pH, C/N, fruiting body weight and BE of each treatment

Average weight of Parawood : Substrate Substrate Treatments fruiting bodies BE (%) Napier grass pH C/N ratio (g/bag) T1 100 : 0 6.28±0.47 125.75 54.53 ± 10.67a 12.36 ± 2.42A T2 90 : 10 6.23±0.42 107.56 59.42 ± 16.14a 13.47 ± 3.66A T3 80 : 20 6.80±0.53 160.57 27.17 ± 11.98b 6.16 ± 2.72B T4 70 : 30 6.27±0.15 119.71 22.33 ± 13.70b,c 5.06 ± 3.11B,C T5 50 : 50 6.27±0.12 122.42 4.14 ± 4.05c 0.94 ± 0.92C Note: a-c represents significant difference between average weight of fruiting bodies (p < 0.05) A-C represents significant difference between BE (p < 0.05)

Figure 1 Mycelial growth of each treatment during incubation period. ICoFAB2019 Proceedings | 47

Figure 2 Development of L. squarrosulus fruiting bodies. A white arrow indicates a primordium.

Figure 3 Cumulative weight of fruiting bodies from each treatment within 48 days.

Although low C/ N ratio is necessary for fruiting body formation, low C/N ratio may affect negatively fruiting body productivity. According to Donini et al. (2009), lowering C/N ratio by adding 10% (C/N ratio of 43) and 20% soybean bran (C/N ratio of 21) into Napier grass-based substrate did not increase Pleurotus ostreatus productivity and also lower in productivity than the grass without soybean bran supplement (C/N ratio of 162) [14]. Even though C/N ratio of 10% supplement was higher than 20% supplement, they found that there was no significant difference in productivity of P. ostreatus between using of 10% and 20% soybean, wheat, rice and corn brans. In addition, they also found the difference in productivity of different strains in the same species. They concluded that addition of 10% bran was superior and 20% supplement did not increase productivity nor BE [14]. According to Ruilova Cueva et al. (2017), they reported C/N ratio of the substrate mixture for P. ostreatus cultivation. The treatment M1 that contained corn stover (40%), rice husk (20%) and wheat straw (38%) had C/N of 104.63. Whereas M2 that contained stover lentil (30%), wheat straw (40%) and bagasse sugarcane (28%) had C/N of 72.40. Although C/N ratio of M1 and M2 were different, both treatments provided similar results in first harvest day, period of harvesting and also no significant difference between fresh fruiting body weight and BE [15]. According to de Leon and colleagues [16] reported productivity and BE of L. squarrosulus when supplementing rice straw and sawdust based substrate with 5% to 25% rice bran and rice hull for increasing nitrogen. They found that productivity and BE did not sequentially increase when increasing of nitrogen source enrichment [16]. Osibe and Chiejina (2015) reported C/N ratio of palm press fibre (33.54), mahogany sawdust (235.95), Gmelina saqdust (158.77) and wheat bran (17.76) that used in the substrate mixture for L. squarrosulus ICoFAB2019 Proceedings | 48

cultivation [17]. Although mahogany and Gmelina sawdust had very different in C/N ratio but both sawdust gave no difference in BE (8.96% and 8.83%, respectively) and yield (44.80 and 44.15 g/kg, respectively). And when supplementation with nitrogen source from wheat bran (3% to 18%) they found that addition of 3% and 8% wheat bran into mahogany sawdust gave higher BE (15.91% and 58.95%) than mahogany sawdust alone (8.96%). But addition of 13% and 18% wheat bran into this sawdust decreased BE to 45.47% and 13.39%, respectively [18]. These results suggest that nitrogen supplement may reduce the C/N ratio but not certainly increase the fruiting body production. There are several physical, chemical and biological factors that affect the growth of mycelium and fruiting body development including chemical composition, water activity, C/N ratio, minerals, surfactant, pH, moisture, source of nitrogen, particle size, light, CO2, temperature, salinity, inoculum and microbial contamination. These factors may affect individually or have interactive effects among the factors that may influence to mushroom growth and production [18,19]. In the production of each mushroom, therefore, the substrate and culture condition have to be optimize. By the way, T1 and T2 in our study provided higher BE than the previous reports that used the different substrate on L. squarrosulus cultivation. This mushroom was reported to grow on rice straw, parawood sawdust and their mixture with the BE of 2.78 – 7.83 % [16]. Substrate mixture of parawood sawdust and 20% oil palm fruit fiber could advance the time of primordial appearance, induce the fresh fruiting body weight with BE of 3.94 – 8.13 % and increase number of flushes of L. squarrosulus [20]. Therefore, agricultural residues or agro-industrial wastes that have high nitrogen content can be mixed with the main substrate for increasing of yield and/or decreasing the use of parawood sawdust.

Conclusions

Napier grass can be mixed with parawood sawdust for L. squarrosulus cultivation, especially ratio of 1:9, can produce fruiting bodies as well as parawood sawdust alone. Therefore, the amount of parawood sawdust usage in L. squarrosulus production can be reduced by adding of dried Napier grass. For further study, fermented Napier grass will be applied in the substrate development.

Acknowledgements

This study was a part of a special problem subject, Faculty of Agricultural Technology, Burapha University Sakaeo Campus. The research was performed by Mr. Songchai Wongwaitaweewong, an undergraduate student, and a research fund was supported by his advisor.

References

[1] Farrell G, Simons SA, Hillocks RJ. Pests, diseases, and weeds of Napier grass, Pennisetum purpureum: a review. Int. J. Pest Manage. 2002; 48(1), 39-48. [2] Strezov V, Evans TJ, Hayman C. Thermal conversion of elephant grass (Pennisetum purpureum Schum) to bio-gas, bio-oil and charcoal. Bioresour. Technol. 2008; 99, 8394-8399. [3] Mohammed IY, Abakr YA, Kazi FK, Yusup S, Alshareef I, Chin SA. Comprehensive characterization of Napier grass as a feedstock for thermochemical conversion. Energies. 2015; 8, 3403-3417. [4] Hoa HT, Wang CL, Wang CH. The effects of different substrates on the growth, yield, and nutritional composition of two oyster mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus). Mycobiology. 2015; 43(4), 423-434. [5] Alborés S, Pianzzola MJ, Soubes M, Cerdeiras MP. Biodegradation of agroindustrial wastes by Pleurotus spp. for its use as ruminant feed. Electron J. Biotechn. 2006; 9(3), 215-220. [6] Mhd Omar NA, Abdullah N, Kuppusamy UR, Abdulla MA, Sabaratnam V. Nutritional composition, antioxidant activities, and antiulcer potential of Lentinus squarrosulus (Mont.) mycelia extract. Evid. Based Complement Alternat. Med. 2011, DOI: 10.1155/2011/539356. [7] Mhd Omar NA, Abdullah S, Abdullah N, Kuppusamy UR, Abdulla MA, Sabaratnam V. Lentinus squarrosulus (Mont.) mycelium enhanced antioxidant status in rat model. Drug Des. Devel. Ther. 2015; 9, 5957-5964. [8] Attarat J, Phermthai T. Bioactive compounds in three edible Lentinus mushrooms. Walailak J. Sci. & Tech. 2015; 12(6), 491-504. [9] Allison LE. Organic carbon. In: Black CA (ed. ). Methods of soil analysis, Part 2, Chemical and microbiological properties. American Society of Agronomy, Madison. 1965, p. 1367-1378. [10] Latimer GW. AOAC official method 955.04. In: Official method of analysis of AOAC international. 19th ed. AOAC International, Gaithersburg. 2012. ICoFAB2019 Proceedings | 49

[11] Melo De Carvalho CS, Sales-Campos C, Nogueira de Andrade MC. Mushrooms of the Pleurotus genus: a review of cultivation techniques. Interciencia. 2010; 35(3), 177-182. [12] Anderson SJ, Merrill JK, Klopfenstein TJ. Soybean hulls as an energy supplement for the grazing ruminant. J Animal Sci. 1988; 66, 2959-2964. [13] Chang S, Miles P. 2nd ed. Mushrooms Cultivation, Nutritional Value, Medicinal Effect and Environmental Impact. Washington. 477 p. 2009. [14] Donini LP, Bernardi E, Minotto E, Nascimento JS. Cultivation of elephant grass substrate supplemented with different kinds of bran. Scientia Agraria. 2009; 10(1), 67-74. [15] Ruilova Cueva MB, Hernandez A, Nino-Ruiz Z. Influence of C/N ratio on productivity and the protein contents of Pleurotus osteatus grown in different residue mixtures. Rev. FCA UNCUYO. 2017; 49(2): 331-344. [16] de Leon AM, Reyes RG, dela Cruz TEE. Lentinus squarrosulus and Polyporus grammocephalus: Newly domesticated, wild edible macrofungi from the . Philipp. Agric. Scientist. 2013; 96(4), 411-418. [17] Osibe DA, Chiejima NV. Assessment of palm press fibre and sawdust-based substrate formulas for efficient carpophore production of Lentinus squarrosulus (Mont.) Singer. Mycobiology. 2015; 43(4): 467-474. [18] Bellettini MB, Fiorda FA, Maieves HA, Teixeira GL, Avila S, Hornung PS, Junior AM, Ribani RH. Factors affecting mushroom Pleurotus spp. Saudi J. Biol. Sci. 2019; 26: 633-646. [19] Khan F, Chandra R. Effect of physiochemical factors on fruiting body formation in mushroom. Int. J. Pharm. Pharm. Sci. 2017; 9(10): 33-36. [20] Ayodele SM, Akpaja EO. Yield evaluation of Lentinus squarosulus (Mont) Sing. on selected sawdust of economic tree species supplemented with 20% oil palm fruit fibers. Asian J Plant Sci. 2007; 6(7), 1098-1102.

doi:10.14457/MSU.res.2019.10 ICoFAB2019 Proceedings | 50

Variation of Inulin Content in Banana Peels at Different Maturity Stages

Ratchanee Puttha*, Mattana Khetpratum and Sureerat Nueksom

Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo 27160, Thailand

*Corresponding author’s e-mail: [email protected], [email protected]

Abstract:

Banana is an inexpensive source of vitamin and dietary fiber. The objective of the present study was to determine inulin content of Kluai Nam Thai (Musa acuminata) peel at different maturity stages. The experiment was conducted at the experimental farm of the Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo, Thailand. Banana peels at four maturity stages (light three-quarters, light full three-quarters, full three-quarters and full) were used and arranged in a completely randomized design with three replications. Data were recorded for number of fruits/finger, peel fresh weight/finger, peel dry weight/finger, total fruit (pulp and peel) fresh weight, peel fresh weight/fruit, percentage of peel fresh weight/fruit, total fruit dry weight, peel dry weight/fruit, percentage of peel dry weight/fruit and inulin content. Maturity stages were significantly different (P < 0.05) for total fruit fresh and dry weight, percentage of peel fresh and dry weight/fruit and inulin content. Banana peel harvested at light three-quarters stage had the highest inulin content of 0.57% dry weight, whereas banana peel at light full three-quarters stage, full three-quarters stage and full stage had inulin content of 0.39, 0.35 and 0.32% dry weight, respectively. Banana peels at light three-quarters had the highest of percentage of peel fresh and dry weight/fruit of 51.4 and 45.3%, respectively. The results indicated that banana peel at light three-quarters stage had a significant amount of inulin (0.57% dry weight) for use as an alternative source of a raw material for functional food producers and animal feed.

Keywords: Inulin, Prebiotic, Functional food, Banana by-products, Spectrophotometry

Introduction

Banana (Musa spp.) is an important fruit crop and cash crop for banana growers in the tropics. However, it is consumed in most parts of the world. Banana is a member of Musaceae family. Banana cultivars originated mainly from intra- and interspecific hybridizations between two diploid wild species, Musa acuminata Colla (‘A’ genome) and M. balbisiana Colla (‘B’ genome) [1]. Banana cultivars and hybrids in Thailand were classified based on morphological descriptors for grouping genomic (AA, AAA, AAB, ABB and BB) [2]. In this study, Kluai Nam Thai is a model of banana genome AA for determination of inulin content in peel because of its sweet taste, aromatic smell and thick peel. Peel is the major by-product of banana industry. Banana peels at different maturity stages were different in nutritional properties, functional properties and morphological traits [3] [4]. Commercially grown bananas are harvested at the green stage with varying degrees of maturity. According to FAO index of maturity based on the fullness of fruit fingers of the banana, four maturity stages of banana are defines as 1) light three-quarters (sharp angles are still present), 2) light full three quarters (the fingers are still angular), 3) full three quarters (the intermediate between “full” and “light three-quarters”) and 4) full (finger rounded) [5]. Inulin is a water soluble storage polysaccharide and belongs to a group of fructans (non-digestible carbohydrates) [6]. Inulin is a type of carbohydrate polymers that are neither digested nor absorbed in digastric system. Therefore, they are subjected to bacterial fermentation in the digestive tract and thus impact the ratio and activities of bacterial in stomach. Some dietary fibers can also be classified as prebiotic [7]. Inulin is classified as prebiotic dietary fiber that has beneficial effects on the host by selectively stimulating the growth of good bacteria and reducing number of bad bacteria in the stomach also improves immune system and health and adjusts gut microbiota dysbiosis [8] [9]. Because of its health promoting properties, inulin is defined as functional food and used in food and pharmaceutical industries [10]. In ICoFAB2019 Proceedings | 51

previous study, total dietary fiber values of banana peels at three different stages of ripeness from six varieties consisting of Grande Naine and Yankambi Km5 (plantain dessert banana, Musa AAA), French Clair and Big Ebanga (plantain, Musa AAB), Pelipita (cooking banana, Musa ABB) and CRBP039 (hybrid, Musa AAAB) varied from 32.9 to 51.9% [11]. Inulin is found at high concentrations in root crops and tuber crops such as in chicory and Jerusalem artichoke, which contain 36-48% and 16-20% dry weight, respectively, and it is also found at a traceable amount in raw banana (0.3-0.7%) and raw-dried banana (0.9-2.0%) [12]. Although inulin found in banana peels is low, the amount of peels from the banana industry is abundant. Numerous studies have been done so far to evaluate fructan, inulin content and dietary fiber in banana fruit, leaf, rhizome and banana blossom (banana male bud) [13] [14] [15]. The information on the variation in inulin content in banana peels is rather limited. A better understanding on the chances in the inulin content at different maturity stages is important for selection of the appropriate harvest times for high inulin content in banana peels. The aim of this study was to evaluate inulin content in peels of Kluai Nam Thai at different maturity stages. The information obtained in this study is important for appropriate utilization of banana peels, which are considered as agricultural waste from banana industry.

Materials and methods

Plant Materials and field experiment Banana variety Kluai Nam Thai used in this study was planted at the experimental farm of the Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo, Thailand (Lat 13°44´ N, Long 102°17´ W, 87 masl). The experiment was arranged in a completely randomized design with three replications.

Sample collection and preparation Data of 10 parameters were recorded for number of fruit/finger, peel fresh weight/finger, peel dry weight/finger, total fruit fresh weight, peel fresh weight/fruit, percentage of peel fresh weight/fruit, total fruit dry weight, peel dry weight/fruit, percentage of peel dry weight/fruit and inulin content. The bunches of banana were randomly collected and cut into fingers. Three banana fingers in the same bunches along the length of the bunches were identified as different maturity stages including light three-quarters (maturity 70%), light full three-quarters (maturity 80%), full three-quarters (maturity 90%) and full (maturity 100%) [16] (Table 1). The banana peels were sliced into thin pieces. The samples were oven-dried at 50 ºC for 10 h or until the weight was constant. The dried samples were milled and stored at -20 ºC until inulin extraction.

Extraction procedure Analysis of inulin content was performed by using the method described previously [17] [18]. The milled samples of each finger were divided into three replications each of which had two grams, and inulin was extracted by reverse osmosis water in a 25 mL flask at 80 ºC for 20 min on a water bath. The samples were shaken well, stored at room temperature to reduce heat and then sieved by filtering the solution through a Whatman no. 1 filter. The extract solution of 2 mL and 750 µL of hydrochloric acid (3% v/v HCl) were added to a 25 mL volumetric flask for hydrolysis. The volume was adjusted with reverse osmosis water to 25 mL and heated at 97 ± 2 ºC for 45 min on a hot plate. The aliquot of extracts kept to cool at room temperature and then stored at 4 ºC in plastic bottles until further analysis.

Analysis of inulin content Fructose was determined by spectrophotometry using periodate reaction. The reaction was conducted by mixing 4 mL of aliquot of extracts with 5 mL of 70 mmol L-1 citrate buffer pH 6.0, 550 µL of reverse osmosis water and 200 µL of 10 mmol L-1 sodium periodate reagent. After 5 min, 250 µL of 100 mmol L-1 potassium iodide was added, and the mixture was left for an additional 5 min. The solution absorbance was subsequently measured at 390 nm using a UV-Vis spectrophotometer. The concentration of free fructose was deduced from calibration curve of standard fructose.

Statistical Analysis Data for all parameters were analyzed statistically according to a completely randomized design using computer software STATISTIX8. Means were separated by least significant difference (LSD) at 0.05 probability level. ICoFAB2019 Proceedings | 52

Table 1 Cross sections of fruits, fruits and peels of Kluai Nam Thai at different maturity stages

Maturity stages Cross section of fruits Finger 1 Finger 2 Finger 3 Banana peels

Light three- quarters

Light full three-quarters

Full three-quarters

Full

ICoFAB2019 Proceedings

Results and discussion

Fruit maturity stages were not significantly different for number of fruits/ finger, peel fresh weight/finger, peel dry weight/finger, peel fresh weight/fruit and peel dry weight/fruit (Table 2). However, they were significantly different (P < 0.05) for total fruit fresh weight, percentage of peel fresh weight/fruit, total dry fruit weight, percentage of peel dry weight/fruit and inulin content (Table 2). The results indicated that fruit maturity stage is an important postharvest criterion for quantity and quality of pulps and peels of banana. Harvest at mature stage and over mature stage can result in low inulin content in peels.

Table 2 Mean squares for number of fruits/finger (NF/Fi), peel fresh weight/finger (PFW/Fi), peel dry weight/finger (PDW/Fi), total fruit fresh weight (TFFW), peel fresh weight/fruit (PFW/F), percentage of peel fresh weight/fruit (%PFW/F), total fruit dry weight/fruit (TFDW), peel dry weight/fruit (PDW/F), percentage of peel dry weight/fruit (%PDW/F) and inulin content (IC) of Kluai Nam Thai fruits at four maturity stages

Source df NF/Fi PFW/Fi PDW/Fi TFFW PFW/F %PFW/F TFDW PDW/F %PDW/F IC Maturity stages 3 3.6ns 551.4ns 7.8ns 133.4* 1.7ns 273.0** 26.4* 0.04ns 491.1** 0.039** Error 8 2.3 711.3 16.4 53.5 1.7 37.3 4.6 0.03 52.0 0.002 CV (%) 12.9 17.3 20.0 19.9 9.8 16.0 27.4 10.6 26.7 10.3 ns, *, ** non-significant and significant at P < 0.05 and P < 0.01 probability levels, respectively

Banana peels at full three-quarters stage had the highest total fruit fresh weight of 41.9 g, whereas banana peels at full stage, light full three-quarters stage and light three-quarters stage had total fruit fresh weights of 40.5, 37.1 and 27.3 g, respectively (Table3). Banana peels at full three-quarters and full stage had the highest total fruit dry weight of 10.2 and 10.0 g, respectively. Banana peels at light full three-quarters stage and light three-quarters stage had total fruit dry weight of 7.4 and 3.8 g, respectively (Table3). In previous report, yield and dry matter content of banana harvested at late fruit developmental stages were higher than banana harvested at early fruit maturity stages because dry matter content of banana slowly increased in opposition to the decrease in fruit moisture content with the development of physiological maturity of the fruits [19].

Table 3. Number of fruits/finger (NF/Fi), peel fresh weight/finger (PFW/Fi), peel dry weight/finger (PDW/Fi), total fruit fresh weight (TFFW), peel fresh weight/fruit (PFW/F), total fruit dry weight (TFDW) and peel dry weight/fruit (PDW/F) of Kluai Nam Thai fruits at four maturity stages

NF/Fi PFW/Fi PDW/Fi TFFW PFW/F TFDW PDW/F Maturity stages (fruit) (g) (g) (g) (g) (g) (g) Three-quarters 10 145 18.0 27.3b 14.0 3.8b 1.74 Light Full Three-quarters 13 173 20.7 37.1ab 13.3 7.4ab 1.59 Full Three-quarters 12 156 21.7 41.9a 13.4 10.2a 1.86 Full 12 144 21.0 40.5ab 12.3 10.0a 1.80 F-test ns ns ns * ns * ns Means with different letters in the same column are significantly different at P < 0.05 by LSD ns, * non-significant and significant at P < 0.05 probability levels, respectively

This study represented pulp to peel ratio by percentage of peel per one fruit. Banana peels at light three-quarters stage had the highest of percentage of peel fresh weight/fruit and percentage of peel dry weight/fruit (51.4 and 45.3%, respectively) (Figure 1). Banana peels at light full three-quarters, full three- quarters and full stages had percentage of peel fresh weight/fruit of 38.8, 31.9 and 30.4%, respectively. Banana peels at light full three-quarters, full three-quarters and full stages had percentage of peel dry ICoFAB2019 Proceedings | 54

weight/fruit of 26.6, 18.3 and 17.9 %, respectively (Figure 1). This finding support with Amin et al. [19] who reported the increasing trend of pulp to peel ratio with the increase of harvesting days of banana varieties BARI Kola 1 and Sabri Kola.

Figure 1 Peal fresh weight percentage and peel dry weight percentage in one fruit of Kluai Nam Thai at different maturity stages. Data are presented as means of three replications ± standard error.

Banana peels at light three-quarters stage had the highest inulin content of 0.57 % dry weight, whereas banana peels at light full three-quarters stage, full three-quarters stage and full stage had inulin contents of 0.39, 0.35 and 0.32 % dry weight, respectively (Fig 2). The results revealed that harvest of the banana at under maturity stage is optimum for height inulin yield because of height inulin content and thick peels.

Figure 2 Inulin content in the peels of Kluai Nam Thai harvested at different maturity stages. Data are presented as means of three replications ± standard error.

Conclusions

Kluai Nam Thai peels at light three quarter stage may be used as an alternative source of inulin for use as a raw material for health food products and animal feed, and the use of banana peels can increase the efficiency of banana production. However, this information is limited to one species of banana. Care must be taken to extrapolate the results to other genome groups of banana and further investigations in a wide range of banana genome groups and varieties within genome groups are required in order to obtain more conclusive results

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Acknowledgements

This work was financially supported by the Research Grant of Burapha University through National Research Council of Thailand (Grant no. 28/2562). The authors would like to acknowledge the Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo, Thailand.

References

[1] Kurtoğlu G, Yildiz S. Extraction of fructo-oligosaccaride components from banana peels. GU J Sci. 2011; 24(4), 877-882. [2] Shalini R, Antony U. Fructan distribution in banana cultivars and effect of ripening and processing on Nendran banana. J. Food Sci. Technol. 2015; 52, 8244-8251. [3] Emaga TH, Andrianaivo RH, Wathelet B, Tchango JT, Paquot M. Effects of the stage of maturation and varieties on the chemical composition of banana and plantain peels. Food Chem. 2007; 103, 590- 600. [4] Khawas P, Deka SC. Comparative nutritional, functional, morphological, and diffractogram study on culinary banana (Musa ABB) peel at various stages of development. Int J Food Prop. 2016; 19, 2832- 2853. [5] FAO. Postharvest management of banana for quality and safety assurance. Food and Agriculture Organization of the United Nations (FAO), Italy, 2018, p. 20. [6] Shoaiba M, Shehzada A, Omar M, Rakhaa A, Razaa H, Sharif HR, Shakeel A, Ansari A, Niazi S. Inulin: properties, health benefits and food applications. Carbohyd Polym. 2016; 147, 444-454. [7] Holscher HD. Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut microbes. 2017; 8(2),172-184. [8] Wanga H, Wei C-H, Min L, Zhu L-Y. Good or bad: gut bacteria in human health and diseases. Biotechnol Biotec Eq. 2018; 32(5), 1075-1080. [9] Vandeputte D, Falony G, Vieira-Silva S, Wang J, Sailer M, Theis S, Verbeke K, Raes J. Prebiotic inulin-type fructans induce specific changes in the human gut microbiota. Gut microbiota. 2017; 66, 1968-1974. [10] Roberfroid MB, Functional foods: concepts and application to inulin and oligofructose. Br J Nutr. 2002; 87(Suppl. 2), S139-S143. [11] Emaga TH, Andrianaivo RH, Wathelet B, Tchango JT, Paquot M. Effects of the stage of maturation and varieties on the chemical composition of banana and plantain peels. Food Chem. 2007; 103, 590- 600. [12] Moshfegh AJ, Friday JE, Goldman JP, Ahuja JKC. Presence of inulin and oligofructose in the diets of Americans. J Nutr. 1999; 129(7 Suppl):1407S-1411S. [13] Shalini R, Antony U. Fructan distribution in banana cultivars and effect of ripening and processing on Nendran banana. J Food Sci Technol. 2015; 52(12), 8244-8251. [14] Cruz-Cárdenas CI, Miranda-Ham ML, Castro-Concha LA, Ku-Cauich JR, Vergauwen R, Reijnders T, den Ende WV, Escobedo-GraciaMedrano RM. Fructans and other water soluble carbohydrates in vegetative organs and fruits of different Musa spp. accessions. Front. Plant Sci. 2015; 6, 1-10. [15] Florenta AW, Loha AMB, Thomas HE. Nutritive value of three varieties of banana and plantain blossoms from Cameroon. Greener J. Agric. Sci. 2017; 5(2), 52-61. [16] Silayoi B, Kluai, Kasetsart University Press, Bangkok, 2002, p. 130. [17] Saengkanuk A, Nuchadomrong S, Jogloy S, Patanothai A, Srijaranai S. A simplified spectrophotometric method for the determination of inulin in Jerusalem artichoke ( Helianthus tuberosus L.) tubers. Eur Food Res Technol. 2011; 233, 609-616. [18] Puttha R, Jogloy S, Wangsomnuk PP, Srijaranai S, Kesmala T, Patanothai A. Genotypic variability and genotype by environment interactions for inulin content of Jerusalem artichoke germplasm. Euphytica. 2012; 183, 119-131. [19] Amin MN, Hossain MM, Rahim MA, Uddin MB. Determination of optimum maturity stage of banana. Bangladesh J. Agril. Res. 2015; 40(2): 189-204.

doi:10.14457/MSU.res.2019.11 ICoFAB2019 Proceedings | 56

Phenolic and Antioxidant Properties of Male Bud Flowers and Fruit of Musa Genotypes with Different Ploidy

Somkit Jaitrong1* and John A. Manthey2

1Faculty of Agricultural Technology, Burapha University, Sakaeo campus, Sakaeo, Thailand 2United States Department of Agriculture, ARS, U.S. Horticultural Research Laboratory, Florida, USA

*Corresponding author’s e-mail: [email protected]

Abstract:

Banana (Musa X paradisiaca) fruits are important foods and considered to be especially rich sources of polyphenolics. The , abundant edible residues of banana production, were investigated as a potential source of natural colorants. The aim of this research was to investigate the total phenolic content and antioxidant activity of banana male bud flowers, unripe fruit peel and pulp at 80-90% mature of banana genotypes with different ploidy in Thailand. The total phenolics content and antioxidant activities were significantly different among banana cultivars. Total phenolic levels were higher in unripe peel than in male bud flowers and unripe pulp. The highest total phenolic contents in peel were 129.5 mg GAE/g DW in ‘Thep Panom’ (Musa ABB), followed by 128.9 mg GAE/g DW in ‘Nam Thai’ (Musa AA) and 72.9 mg GAE/g DW in ‘Thep Parod’ (Musa ABBB). This result is consistent with the antioxidant activity. The ABTS, DPPH and FRAP assays differed significantly (p<0.05) in different parts of banana with different ploidy (AA, ABB and ABBB). The highest DPPH antioxidant activity of unripe peel occurred in the ‘Nam Thai’ cultivar (970.2 mg TEAC/g DW), followed closely by ‘Thep Panom’ (963.9 mg TEAC/g DW) and ‘Thep Parod’ (887.9 mg TEAC/g DW). In conclusion, the amount of total phenolics and antioxidant activity were significantly different among banana cultivars with different ploidy. In addition, unripe peel extract showed stronger antioxidant activity than the male bud flowers and unripe pulp, respectively.

Keywords: Banana genotypes, Total phenolic content, Antioxidant activity, Fruit peel and pulp, Male bud flowers

Introduction

Early classification of banana (Musa) species was hindered by a poor understanding of the genetics of Musa, and a lack of knowledge of the complex hybridizations that readily occur within the genus. Yet, many diverse cultivars are now well studied and are known to occur as diploids (2n), triploids (3n), and tetraploids (4n). Simmonds and Shepherd [1] had previously suggested that edible bananas originated from two wild species, Musa acuminate Colla and Musa balbisiana Colla. The former species was designated as diploid genotype AA and Musa balbisiana Colla was designated diploid genotype BB. Because both species are native in common geographic areas, and because cross pollinations and hybridization readily occur among these two species and their hybrid progeny, numerous triploids (AAA, AAB, ABB, and BBB) as well as tetraploids (AAAA, AAAB, ABBB, and AABB) occur in nature and in commercial cultivation. Bananas contain bioactive compounds, such as phenolics, carotenoids, amines and phytosterols. Many of these compounds have antioxidant activities and potentially beneficial effects on human health [2]. Phenolic compounds are secondary metabolites produced in plants through the phenylpropanoid pathway and encompass a wide range of chemical classes, including phenolic acids, flavonoids, stilbenes and lignans [3]. Plant polyphenolics are generally considered essentials components of plant defense mechanisms, and exert health promoting effects in humans. They act as antioxidants and modulators of enzyme expression and thereby contribute to the allevation of wide range of chronic diseases [4]. The different parts of banana such as banana bracts [5], pseudostems [6], banana fruit peels and pulp have been found to be good source of antioxidants and food colorants [7]. Various phenolics present in banana have been identified as gallic acid, catechin, epicatechin, tannins, and anthocyanins [8]. All six of the common anthocyanidins (delphinidin, cyanidin, petunidin, pelargonidin, peonidin and malvidin) have been detected in bracts of different species of Musa [9]. In a recent study, the antioxidant activity of the banana peel extract, against lipid autoxidation, was stronger than that of the banana pulp extract, and the gallocatechins in their tissues may account for their high antioxidant effects [10]. In addition, banana fruits are important foods and ICoFAB2019 Proceedings | 57

considered to be especially rich sources of polyphenolics in banana peel and pulp. The bracts, abundant edible residues of banana production, were investigated as a potential source of natural colorants. The aim of this research was to investigate the total phenolic contents and antioxidant activities of banana male bud flowers of different banana genotypes in Thailand with different ploidy. The data will provide important information about the potentially beneficial effects of this plant tissue as a dietary component, and to suggest potential applications of new natural antioxidant-containing food ingredients in functional foods.

Materials and methods

Plant materials The banana specimens were collected from various locations in Thailand, characterized and identified based on Simmonds [11-12]. Banana fruit (Musa acuminate Juss.) cultivars Nam Thai (Musa AA group), Thep Panom (Musa ABB group) and Thep Parod (Musa ABBB group) were harvested at 90% maturity stage in the same plantation located in the Burapha University Farm, Sakaeo campus, Thailand. Analyses were made using three replications for each cultivars and results were reported as averages with calculated standard deviations. Banana male bud flowers were harvested at the flowering stage, fruit peel and pulp were harvested at 80-90% fruit maturity stage. The banana specimens harvested for chemical analyses were freeze dried (Labconco Corporation, USA) and ground to a dry powder. The dry powder was stored at -20 C for further analysis.

Sample extraction The dried power of banana specimens were prepared using the method of Pothavorn et al. [13], with some modifications and made in triplicate. Ten grams of banana male bud dry powder were mixed with 50 mL of 80% ethanol containing 100 mM NaCl, 0.2 mM ascorbic acid, 40 mM citric acid, 0.1 mM Na2S2O5, 0.25% Triton X-100 and 0.2 mM EDTA as antioxidants and inhibitors PPO. The sample was heated at 80C for 30 min, then collected the supernatant and re-extract twice, each time using 50 ml of extraction solutions. These three extracts were combined and centrifuged at 5000 rpm for 30 min at 4C using micro centrifuged (Beckman, J2-21, Beckman Instruments Inc., USA). The supernatants were collected and evaporated to dryness at 55C (rotary evaporator, Buchi Rotavapor R-205, Switzerland). The remaining residue was dissolved in 80% ethanol to 6 mL and stored at 4C under airtight and dark conditions for further analysis.

Total phenolic content (TPC) measurement Total phenolics contents were determined by the Folin-Ciocalteu method, which was adapted from Singleton and Rossi [14]. The 20 l of extract and 100 l of 1:10 diluted commercial Folin-Ciocalteu reagent (Sigma-Aldrich) were combined and mixed well using a vortex. The solution was allowed to react for 1 min in the dark and then 80 l of 7.5% (w/v) Na2CO3 was added and mixed well. The solution was incubated at room temperature (25C) in the dark for 30 min. The absorbance was measured at 765 nm using a microplate reader (EpochTM Microplate spectrometer, BioTek Instruments Inc., USA). The total amount of phenolics was expressed as milligrams of gallic acid equivalents (GAE) per gram dry weight (DW).

Antioxidant determinations The 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging (ABTS) assay followed the method of Thaipong et al. [15] with some modifications. The banana male bud extracts (10l) were allowed to react with 190l of ABTS cation radical reagent for 15 min in the dark. Then the absorbance was taken at 734 nm using a microplate reader. The results were expressed in mg Trolox equivalents (TEA) per gram DW. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was run according to the method of Prior et al. [16] with some modifications. The banana male bud extracts (10l) were allowed to react with 190l of DPPH solution for 30 min in the dark. Then the absorbance was taken at 515 nm using the Epoch microplate reader. The results were expressed in mg Trolox equivalents (TEA) per gram DW. The Ferric reducing antioxidant power (FRAP) assay was run according to Thaipong et al. [15] with some modifications. The banana male bud extracts (10l) were allowed to react with 190l of FRAP (Ferric reducing antioxidant power) reagent for 15 min in the dark. Readings of the colored product [Ferrous tripyridyltriazine complex] were then taken at 593 nm using the Epoch microplate reader. The results were expressed in mg Trolox equivalents (TEA) per gram DW. ICoFAB2019 Proceedings | 58

Statistical analysis The data of different parameters were subjected to one-way analysis of variance (ANOVA) and the significance of the difference between means was determined by Least Significant Difference (LSD) test (P<0.05) using STATISTIX8 [17]. Values expressed were means of three replications ±standard deviation (SD).

Results and discussion

Characteristic of banana male bud flowers and fruit Male bud shapes of banana show a diversity, including narrow ovate ‘Nam Thai’ (Musa AA), broadly ovate Thep Panom (Musa ABB), lanceolate Thep Parod (Musa ABBB) with acute and obtuse apex. Bract curling was bract reflex and roll back and wax on the bract. Colors were reddish-purple for the outside bract color, while reddish-orange to purple for the inside bract color. Bract color was reddish brown (‘Thep Panom’), reddish purple (‘Nam Thai’), and reddish brown with light purple (‘Thep Parod’). The pigmentations of male flowers were creamy yellow in ‘Nam Thai’, light pink in ‘Thep Panom’ and pinkish- purple in‘Thep Parod’ with yellow in the three cultivars (Figure 1). The variation in bract color is correlated with their anthocyanin content. Pazmino-Duran et al. [5] reported that banana bracts (Musa x Paradisiaca) contained anthocyanins such as cyaniding-3-rutinoside (32 mg anthocyanin/100 g bracts), and 3-rutinoside derivatives of delphinidin, pelargonidin, peonidin, and malvidin. They have suggested the use of anthocyanins from banana bracts (male bud flowers) as natural colorants. Many factors other than anthocyanin contents influence bract color, including pigment accumulation patterns, vacuolar pH, and copigments in each individual cell or tissue area [18]. Anthocyanins are considered to be a good food colorants due to their attractive colors, water solubility, and stability in processed foods [19] as well as their established health benefits in humans [20]. The fruit bunches were harvested at 75 days from removal of the blossom in ‘Nam Thai’, 111 days in ‘Thep Parod’ and 124 days in ‘Thep Panom’ bananas with mature green peel color at 80-90% mature was shown in figure 1. Degree of fruit maturity at each age of fruit bunch was assessed based on the standard maturity index for banana according to fullness of fingers stages; full three-quarters – angularity not prominent,>80-90% mature.

Nam Thai (Musa AA) Thep Panom (Musa ABB) Thep Parod (Musa ABBB)

Nam Thai (Musa AA) Thep Panom (Musa ABB) Thep Parod (Musa ABBB)

Figure 1 Male bud flowers (upper) and fruit bunch (lower) of Nam Thai, Thep Panom and Thep Parod bananas.

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Changes of TPC and antioxidant activities The amount of total phenolics and antioxidants were significantly different among the three banana cultivars. Total phenolics were more abundant in peel than in male bud flowers and pulp, respectively (Table 1). The highest total phenolic contents in peel was 129.5 mg GAE/g DW in ‘Thep Panom’ (Musa ABB), followed by 128.9 mg GAE/g DW in ‘Nam Thai’ (Musa AA) and 72.9 mg GAE/g DW in ‘Thep Parod’ (Musa ABBB). The ABTS, DPPH and FRAP assay values differed significantly (p<0.05) for different parts of banana with different ploidy (AA, ABB and ABBB). The unripe peel extract showed stronger antioxidant activity than the male bud flowers and unripe pulp, respectively. The highest antioxidant activity based on the DPPH assay for unripe peel occurred for the ‘Nam Thai’ cultivar (970.2 mg TEAC/g DW), followed by ‘Thep Panom’ (963.9 mg TEAC/g DW) and ‘Thep Parod’ (887.9 mg TEAC/g DW). The DPPH and FRAP assays showed no difference among determinations, while the ABTS differed. Banana peel and pulp of Musa Cavendish are reported to contain higher levels of total phenolics in peel (907 mg GAE/100g DW) than in pulp (232 mg GAE/100g DW). The peel extract showed 2.2 times stronger antioxidant activity than the pulp extract and may be attributed to their phenolic contents [10]. In addition, banana peel extracts have been shown to be rich in antioxidant dopamine, L-dopa, and catecholamines [21]. The comparison of dopamine with other natural antioxidants, such as ascorbic acid and phenolic acids (e.g. gallocatechin gallate), the dopamine showed higher antioxidant activity in vitro (DPPH assay) [22]. Recently, Vu et al. [23] have also reviewed the phenolic compounds and their potential health benefits coming from banana peel. They have suggested the use of this valuable by-product from banana fruit processing industry in food and pharmaceutical industry. Banana peel and male bud flowers, which is usually discarded, should also be considered to be a good source of natural antioxidants and to be a functional food.

Table 1 Antioxidant activity of male bud flowers, unripe banana fruit peel and pulp by the ABTS, DPPH, and FRAP assays from three banana genotypes.

Banana cultivar Parts of banana TPCa Antioxidant activityb (mgTEAC/gDW) (mgGAE/gDW) ABTS DPPH FRAP Nam Thai Male bud flowers 22.6±0.9c 600.1±9.6d 203.3±9.9d 33.6±1.3h (Musa AA) Peel 128.9±5.2a 1246.2±49.1b 970.2±7.6a 681.6±48.4a Pulp 15.9±1.1d 196.9±2.4e 72.6±9.3g 68.3±5.6e

Thep Panom Male bud flowers 17.01±1.3d 83.1±3.7gh 70.4±2.5h 49.7±2.5g (Musa ABB) Peel 129.5±2.8a 1737.5±17.5a 963.9±8.0b 670.2±28.2b Pulp 8.7±0.5e 117.7±6.8f 65.8±5.8i 34.6±2.7h

Thep Parod Male bud flowers 21.9±2.2c 95.1±2.4g 106.6±7.1e 78.9±9.5d (Musa ABBB) Peel 72.9±1.9b 1096.4±28.4c 887.9±26.7c 458.3±23.2c Pulp 3.2±0.4f 74.0±4.6h 93.6±1.4f 52.5±5.3f Value in each column marked by the same letter are not significantly different at P<0.05. Results showed mean ±SD a Total phenolic content milligrams of gallic acid equivalent (GAE) per gram of dry weight (DW) b Milligrams of Trolox equivalent antioxidant capacity (TEAC) per gram of dry weight (DW)

Conclusions

The amount of total phenolics and antioxidant activity were significantly different among banana cultivars with different ploidy. Total phenolics were more abundant in peel than in male bud flowers and pulp. The highest antioxidant activity was shown in ‘Nam Thai’ (Musa AA), followed by ‘Thep Panom’ (Musa ABB) and ‘Thep Parod’ (Musa ABBB). The unripe peel extract showed stronger antioxidant activity than the male bud flowers and unripe pulp, respectively.

Acknowledgements

This work as financially supported by the Research Grant of Burapha University through National Research Council of Thailand (Grant no. 27/2562).

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References

[1] Simmonds NW, and Shepherd K. The and origins of the cultivated banana. J. Linn. Soc. (Botany). 1955; 55, 302-312. [2] Singh B, Singh JP, Kaur A, and Singh N. Bioactive compounds in banana and their associated health benefits-A review. Food Chem. 2016; 206, 1-11. [3] Manach C, Scalbert A, Morand C, Remesy C, and Jimenez L. Polyphenols: food source and bioavaibility. Am. J. Clin. Nutr. 2004; 79(5), 727-747. [4] Liu RH. Potential synergy of phytochemicals in cancer prevention: mechanism of action. J. Nutr. 2004; 134 (12 Suppl), 3479S-3485S. Doi: 10.1093/jn/134.12.3479S. [5] Pazmino-Duran EA, Giusti M M, and Wrolstad RE, Gloria M B A. Anthocyanins from banana bracts (Musa x paradisiaca) as potential food colorants. Food Chem. 2001; 73, 327-332. [6] Aziz, N A A, Ho LH, Azahari B, Bhat R, Cheng LH, and Ibrahim M N M. Chemical and functional properties of the native banana (Musa acuminata x balbisiana Colla. Cv. Awak) pseuso-stem and pseudo-stem tender core flours. 2011; 128, 748-753. [7] Sidhu JS and Zarfar TA. Review: Bioactive compounds in banana fruits and their health benefits. J. Food Saf. Food Qual. 2018; 2, 183-188. [8] Bennett RN, Shiga TM, Hassimotto NMA, Rosa EAS, Lajolo FM, and Cordenunsi BR. Phenolics and antioxidant properties of fruit pulp and cell wall fractions of postharvest banana (Musa acuminate Juss.) cultivars. J. Agric. Food Chem. 2010; 58, 7991-8003. [9] Kitdamrongsont K, Pothavorn P, Swangpol S, Wongniam S, Atawongsa K, Svasti J, and Somana J. Anthocyanin composition of wild bananas in Thailand. J. Agric. Food Chem. 2008; 56(22), 10853- 10857. Doi: 10.1021/jf8018529. [10] Someya S, Yoshiki Y, and Okubo K. Antioxidant compounds from banana (Musa Cavendish). Food Chem. 2002; 79. 351-354. [11] Simmonds NW, and Shepherd K. The taxonomy and origins of the cultivated bananas. Bot. J. Linn Soc. 1955; 55(359), 302-312. Doi: 10.1111/j.1095-8339.1955.tb00015.x. [12] Silayoi B. Banana (in Thai). 4th Edition. Kasetsart University Press, Bangkok (Thailand), 2015, 512p. [13] Pothavorn P, Ditdamrongsont K, Swangpol S, Wongniam S, Atawongsa K, Svasti J, and Somana J. Sap physochemical compositions of some bananas in Thailand. J. Agric. Food Chem. 2010; 58, 8782- 8787. [14] Singleton VL, and Rossi JA. Colorimetry of total phenolics with phosphomolypdic-phosphotugstic acid reagents. Am. J. Enol. Vitic. 1965; 16, 144-158. [15] Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L and Byrne DH. Comparison of ABTS, DPPH, FRAP and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Compos. Anal. 2006; 19, 669-675. [16] Prior RL, Wu X, and Schaich K. Standardization methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005; 53, 4290-4302. [17] Statistix8. 2003. Statistix8: Analytical Software User’s Manual. Tallahassee, Florida. [18] Grotewold, E. The genetics and biochemistry of floral pigments. Annu Rev Plant Biol. 2006; 57, 761- 780. doi:10.1146/annurev.arplant.57.032905.105248. [19] Torskangerpoll, K., and Andersen, Ø. M. Colour stability of anthocyanins in aqueous solutions at various pH values. Food Chemistry. 2005; 89(3), 427-440. Doi:10.1016/j.foodchem.2004.03.002. [20] Konczak, I., and Zhang, W. Anthocyanins—More Than Nature's Colours. J. Biomed Biotechnol. 2004; 5, 239-240. Doi: 10.1155/s1110724304407013. [21] Gonzalez-Montelongo R, Lobo MG, and Gonzalez M. Antioxidant activity in banana peel extracts: testing extraction conditions and related bioactive compounds. Food Chem. 2010; 119, 1030-1039. [22] Kanazawa K, and Sakakibara H. High content of dopamine, a strong antioxidant, in Cavendish banana. J. Agric. Food Chem. 2000; 48, 844-848. [23] Vu HT, Scarlett CJ, and Vuong QV. Phenolic compounds within banana peel and their potential users: a review. J. Funct. Foods. 2018; 40, 238-248. doi:10.14457/MSU.res.2019.12 ICoFAB2019 Proceedings | 61

Efficacy of Stephania pierrei Tuber Extract for Leaf Spot Disease Control in Greenhouse and Field Condition

Sirilak Kamonwannasit, Agarat Kamcharoen and Quanjai Rupitak*

Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Wattana-nakhon, Sakaeo 27160, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

This study evaluated the efficacy of Stephania pierrei tuber extract for control of leaf spot disease in two cultivars of tomatoes and chili under greenhouse and field condition. The experiment was conducted Both experiments were conducted in Completely Randomized Design with 3 replications. Treatments included 1) control, 2) fungal, 3) Stephania pierrei tuber extract, 4) fungicide (mancozeb), 5) fungal with Stephania pierrei tuber extract and 6) fungal with fungicide (mancozeb). The results showed that tomatoes plants treated with the fungal with Stephania pierrei tuber extract decreased the disease severity to 8.0% and 3. 8% in Puang- chom-poo and Sida cultivar, respectively after prevention but not differed from chemical fungicide under greenhouse and field condition. The efficacy of extracts depends on plant genotype, plant species and environmental factors. Further studies are needed to find out the way to use under conventional condition.

Keywords: Tomato, Leaf spot disease, Stephania pierrei

Introduction

The plants of the genus Stephania (Family: ) are slender climbers with peltate and membranous leaves. The inflorescences are axillary and arising from old leafless stem. The plants in this family are mostly herbs or shrubs but rarely trees. They are widely distributed and are traditionally used for the treatment of various ailments such as asthma, tuberculosis, fever, dysentery, hyperglycemia, cancer, and malaria. The biological activities of their extraction were found such as antimicrobial activity (fungi, bacteria), anti-malarial activity, anthelmintic activity, anti-viral activity and anti-inflammatory and analgesic activity. The extracts were used from tubers, stem or flowers [1]. Fungi are the main pathogen in plant causing developmental stage, fruit quality and yield. Alternaria alternata causes black spot in many fruits and vegetables around the world [2]. Fungi pathogen is usually controlled by fungicides which harmful effects on human health and environment. Therefore, the plant extracts/products are interested as the alternative control. Many plant extracts are used for against plant pathogen (fungi, bacteria or virus) or insects. The extracts of tuba root and clove can inhibit growth of Alternaria sp. causal agent of leaf spot disease of lettuce under hydroponics systems [3]. Many medicinal plant extracts were investigated the antifungal activity for control Alternaria alternata in laboratory. Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined ranging between 1.25-25 µg/mL and 1.25-10 µg/mL, respectively. The extracts of Salvia sclarea, Salvia officinalis and Rosmarinus officinalis had potential for treating diseases in plant [4]. Previously, we found that the minimum fungicidal concentration (MFC) was 4 mg/ml of Stephania pierrei tuber extract, while Mancozeb® (chemical fungicide) was 0.125 mg/ml in laboratory scale. The present study tested the antifungal activity of Stephania pierrei tuber extract under greenhouse and field condition

Materials and methods

Plant material culture and experiment design The experiments were carried out in the greenhouse and field at Burapha University, Sakaeo campus, Sakaeo. Both experiments were conducted in Completely Randomized Design with 3 replications (10 plants/replication). Treatments included control (T1), fungal (T2), Stephania pierrei tuber extract (T3), fungicide (mancozeb) (T4), fungal with Stephania pierrei tuber extract (T5) and fungal with fungicide (0.125 mg/ml of mancozeb) (T6). Seeding of Tomato (Puang-chom-poo cultivar; PCP and Sida cultivar; ICoFAB2019 Proceedings | 62

SiD) and chili (Prik Num cultivar; PN) in tray for 3 weeks and then transplanted to plastic bag for greenhouse experiment and to soil for field experiment. At 1 month after transplanting, plants were treated according to the experimental design in both experiments.

Alternaria inoculation Alternaria alternata TISTR 3435 strain was obtained from the Thailand Institute of Scientific and Technological Research (TISTR). Fungal suspension was prepared according to Kamonwannasit et al. [5]. Alternaria alternata was grown in potato dextrose agar (PDA) plates for 7 days at room temperature. After that, the cultured plates were washed with sterile distilled water. The spore suspension was transferred into sterilized 250 ml flask for use for inoculum after its concentration of 105 spores/ml. The tomato leaves were inoculated with Alternaria alternata suspension according to the experimental design in both experiments.

Stephania pierrei tuber extraction Stephania pierrei tuber extract was prepared according to Kamonwannasit et al. [5] to 2 mg/ml in 1% of dimethyl sulfoxide (DMSO). The tubers of Stephania pierrei were extracted by soxhlet apparatus using chloroform as solvent. The tuber extract was evaporated using rotary evaporator and kept at 4˚C. The extract was dissolved in 1% of dimethyl sulfoxide. After ten days of fungal inoculation the disease symptom appeared. Then the Stephania pierrei tuber extract and fungicide were used according to experimental design.

Disease incidence Disease incidence was recorded on a scale of 0 – 5 in accordance to the degree of leaf spot. Scale zero refers to not suffer any leaf spot symptoms and scale 5 indicates the disease severity with the >50% of spot leaves.

Disease severity = [Sum of all disease rating/Total number of rating x Maximum disease grade] x 100

Disease severity of leaf spot was rating at two times; first time at 10 days after fungal inoculation and second time at 7 days after plant extract or fungicide prevention. Data between before and after prevention were compared by using Pair t-test and were analyzed by one-way ANOVA (analysis of variance) test. Pairwise comparison was carried out with Duncan’s Multiple Range Test.

Results and discussion

Table 1 Mean of leaf spot disease severity (%) on plant leaves under greenhouse and field condition before and after prevention.

Type Times T1 T2 T3 T4 T5 T6 P-value Greenhouse condition PCP After 4.89c 10.44ab 8.00bc 6.67bc 13.78a 7.33bc * Before 5.56c 10.22abc 6.89bc 8.44abc 11.78a 11.33ab * Pair t-test ns ns ns ns ns ns SiD After 7.33 7.78 6.00 6.89 7.56 5.33 ns Before 5.56 14.89 10.67 13.11 12.00 13.33 ns Pair t-test ** * ns ns ns * PN After 1.11 2.44 2.89 2.22 3.56 3.56 ns Before 1.78 4.67 2.00 2.22 5.11 4.89 ns Pair t-test ns ns ns ns ns ns Field condition PCP After 7.56 11.78 6.00 9.78 8.00 7.78 ns Before 2.44b 9.78ab 4.67b 8.89ab 14.67a 6.22ab *** Pair t-test * ns ns ns * ns SiD After 7.11 7.11 5.56 7.33 3.78 4.22 ns Before 6.44 9.33 11.33 8.22 11.33 11.78 ns Pair t-test ns ns ns ns * * PN After 3.56 5.11 4.89 2.44 5.33 4.00 ns Before 2.89 4.44 2.67 2.67 5.78 4.89 ns Pair t-test ns ns ns ns ns ns The same letter in a row are not significantly different according to Duncan’s Multiple Range Test ICoFAB2019 Proceedings | 63

Table 1 showed the mean of leaf spot disease severity on plant leaves under greenhouse and field condition before and after prevention. Under greenhouse condition, the leaf spot disease severity of Puang- chom-poo cultivar showed a significant difference between treatments both at before and after prevention but there was no difference in another tomato cultivar and chili. And under field condition, there was only a significant difference of leaf spot disease severity between treatments in Puang-chom-poo cultivar at before prevention. This susceptibility to environment and fungal inoculation might depended on genotype of plants. The treatment of fungal with Stephania pierrei tuber extract (T5) showed a significant difference of disease severity between before and after prevention in both genotypes under field condition. It determined that Stephania pierrei tuber extract could reduce disease symptom comparison to before prevention. The efficacy of plant extracts appeared only under field condition due to weather condition (moisture and temperature) of greenhouse might be suitable for fungi growth [6]. While the Sida cultivar showed a significant difference between before and after prevention on fungal with fungicide treatment (T6) in greenhouse and field condition. It determined that chemical fungicide effected in some species and some cultivars. Considering Sida cultivar under field condition, the plant extract reduced disease severity 7.55% while chemical fungicide reduced 7.56% which efficacy of plant extract was similarly to chemical fungicide. However, the potential of plant extracts was not widely controlled the leaf spot disease in all species and all genotypes under greenhouse and field condition. Further studies are needed to find out the way to use under conventional condition.

Conclusions

The results of this work showed that efficiency of Stephania pierrei tubers extracts depends on plant species and environmental factors. Nevertheless, the natural plant- derived fungicide is still an alternative instead of chemical fungicide for organic agriculture.

Acknowledgements

This work was financially supported by the Research Grant of Burapha University through National Research Council of Thailand (Grant no. 3/2562).

References

[1] Semwal D K, Badoni R, Semwal R, Kothiyal S K, Singh G J P, Rawat U. The genus Stephania (Menispermaceae): chemical and pharmacological perspectives. J Ethnopharmacology 2010; 132, 369-383. [2] Peever T L, Ibañez A, Akimitsu K, Timmer L W. Worldwide phylogeography of the citrus brown spot pathogen, Alternaria alternata. Phytopathology. 2002; 92, 794-802. [3] Nuangmek W, Nakhonthaisong R. Studies of herbal extracts for the lettuce leaf spot control in hydroponic system. Naresuan Phayao Journal. 2014; 7, 131-136. [4] Dellavalle P D, Cabrera A, Alem D, Larrañaga P, Ferreira F, Rizza M D. Antifungal activity of medicinal plant extracts against phytopathogenic fungus Alternaria spp. Chilean J. Agri Res. 2011; 71(2), 231-239. [5] Kamonwannasit S, Rupitak Q, Kamcharoen A. Antifungal and antioxidant activities of the extract of Stephania pierrei tubers. In Proceeding of RSU International Research Conference 2019. Available at https://rsucon.rsu.ac.th/proceedings. [6] Talley S M, Coley P D, Kursar T A. The effects of weather on fungal abundance and richness among 25 communities in the Intermountain West. BMC Ecology. 2002; 2:7. Available at: http://www.biomedcentral.com/1472-6785/2/7.

doi:10.14457/MSU.res.2019.22 ICoFAB2019 Proceedings | 64

Phytochemical Contents and Antioxidant Activity of Bagasse Sugarcane Extracts

Panadda Sanarat and Prasong Srihanam*

The Center of Excellence in Chemistry (PERCH-CIC) and Creative and Innovation Chemistry Research Unit, Department of Chemistry Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

The objective of this work is to determine phytochemical contents including total phenolic, total flavonoids and total triterpenoid in ethanolic extracts of 2 varieties of sugarcane bagasse. The results showed that the phytochemicals were variable in contents by the sugarcane variety. The highest contents of phytochemical were found in Khon Kaen II extracts which resulted to highest antioxidant activity as well. All phytochemicals were positively corelated to antioxidant activity, especially the free radical scavenging activity when tested by DPPH and ABTS assays and also corelated to reducing power activity by FRAP with modulate value. The obtained results indicated that bagasse is a natural good source of phytochemicals composed of antioxidant activity which would be developed these extracts for further health benefit products.

Keywords: Sugarcane bagasse, Phytochemical Crude extract, Antioxidant activity

Introduction

In recent, study on phytochemicals has been gradually increased. Among the phytochemicals, phenolic compounds are the largest group in plant metabolites [1]. Different types of phenolic compounds including phenols, flavonoid, tannin [2] and terpenoids such as carotenoids [3] have been reported. It has been proved that phytochemicals have various biological activities. Flavonoid is an important substance for antioxidation and could be protected degenerative diseases causing from free radicals [4]. Phytosterols helped to decrease lipid in human blood. Carotenoids and alkaloids have anti-inflammatory which used as active ingredient for various diseases protection [3, 5]. Sugarcane (Saccharum officinarum L.) is a main economic crop of many countries includes Thailand. It is planted in all parts of Thailand, especially in the northeastern area. However, the main application of sugarcane is sugar production since the sugarcane composes of high sucrose content (17- 35%). Sugarcane was also used for ethanol production as fuel instead of petroleum. Moreover, some reports about phytochemicals in sugarcane have been discovered [6]. The phytochemicals were varied following strain and geographic area planted [7-9]. In the sugar production process, the residuals after juice extraction are bagasse. This bagasse was limited to apply for value added productions and still remaining as waste which gradually increased every year. Therefore, the authors are interesting for studying the phytochemicals in bagasse extract as well as their biological activities. The obtained results would be used as basic information for further studying and value added of this waste.

Materials and methods

Materials The bagasse of 2 strains of sugarcane for this work are Khon Kaen I and Khon Kaen II which planted in Burirum Province. The bagasse was obtained from Burirum Sugarcane Factory. The sugarcane tree was placed into manual crusher for juice extraction at room temperature to obtain the bagasse and then dried in an oven at 60 °C for 18 h. The bagasse was grinded into small pieces and kept in a seal bag at room temperature.

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Preparation of crude extract The crude extract of bagasse was extracted by ethanol. The 1g of bagasse was weighed and 25 mL of ethanol was then added into the bagasse. The mixture contained in volumetric flask was shaken for 48 h. All samples were extracted in triplicate. The extracts were pooled and evaporated the solvent by rotary evaporator. The powder of extract was separated from the round bottom bottle and weighed using balance. The exactly dried weight of crude extracts was weighed before adding ethanol for dissolving the prepared crude extract.

Total phenolic content The total phenolic content (TPC) was determined using a modified colorimetric method [10]. A 1 mL of crude solution was mixed with 5 mL of 10% Folin-Ciocalteu reagent, before incubating at room temperature for 5 min. After that, 4 mL of 7.5% of Na2CO3 solution was added into the mixture solution before standing at room temperature for 1 h. Then, the mixture was measured at 765 nm using UV-Vis spectrophotometer. Gallic acid was used as standard for a calibration curve. The TPC was indicated as mg gallic acid equivalent (mgGAE)/100g of dried weight.

Total flavonoid content The total flavonoid content (TFC) of the bagasse extract was measured using a modified previous method [11]. Briefly, 2 mL of crude solution was mixed with 0.4 mL of distilled water and 0.4 mL of 5% (w/v) NaNO2 was subsequently added. The mixture was then incubated at room temperature for 6 min before adding 0.6 mL of 10% AlCl3, then standing for 6 min. The mixture solution was then mixed with 4 mL of 0.1 M NaOH and left for 15 min at room temperature. The absorbance at 510 nm was measured using UV-Vis spectrophotometer. Quercetin was used as standard for a calibration curve. The TFC was indicated as mg quercetin equivalent (mgQE)/100g of dried weight.

Total triterpenoid content The total triterpenoid content (TTC) was determined using a modified method [12]. A 0.3 mL of crude solution was evaporated at water bath at 100 °C, then 0.5 mL vanillin-acetic acid (5:95 w/v) and 0.8 mL perchloric acid (HClO4) was added before incubating at 60 °C for 15 min. After that, 5 mL of acetic acid (CH3COOH) was added into the mixture solution before standing at room temperature for 15 min. The mixture solution was then measured at 548 nm using UV-Vis spectrophotometer. Ursolic acid was used as a standard for a calibration curve. The triterpenoid content was indicated as mg ursolic acid equivalent (mgUA)/100 g dried weight.

Antioxidant activity by DPPH assay DPPH assay was carried out to measure the free radical scavenging activity following previous method [13]. A 0.5 mL of crude solution was concentrated in methanol followed by mixing with of 0.1 mM DPPH solution in methanol. After incubation at room temperature in the dark for 30 min, the absorbance was read at 517 nm. Trolox (6-hydroxy-2,5,7,8-tetramethychlorman-2-carboxylic acid) was used as positive control for comparison and solvent mixed with 0. 1 mM DPPH solution was taken as negative control. The percent scavenging was calculated by following formula

DPPH radical scavenging (%) = [(A0–As)/A0]  100 where A0 of control is the absorbance of the solvent mixed with DPPH solution and As is the absorbance of the extract solution. DPPH radical scavenging was indicated as mg Trolox equivalent (mgTE)/g dried weight.

Antioxidant activity by ABTS assay The ABTS assay was performed following the method of Trolox equivalent antioxidant capacity (TEAC)[14]. The stock solution included a 7 mM ABTS and 2. 45 mM potassium persulfate (K2S2O8) solutions were mixed. The working solution was then prepared by adding 10 mL K2S2O8 to 10 mL ABTS solution. The two solutions were mixed well and allowed to react for 16 h at room temperature in the dark. The absorbance of solution at 0.7 ± 0.02 was used as working solution. Trolox was used as positive control for comparison. Crude solution (0.5 mL) was allowed to react with 1 mL ABTS working solution in the dark at room temperature for 5 min, and then the absorbance was measured at 734 nm using UV-Vis spectrophotometer. The results were expressed as mg Trolox equivalent (mgTE)/g dried weight.

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Ferric reducing antioxidant potential (FRAP) assay The FRAP assay was conducted according to previous method [14]. The working solution was prepared by mixing 25 mL of acetate buffer pH 3.6 (3.1 g of CH3COONa.3H2O and 16 mL of CH3COOH), 2. 5 mL TPTZ solution (10 mM TPTZ in 40 mM HCl) and 2. 5 mL of 20 mM FeCl3. 6H2O solution and incubating at 37 °C before use. Crude extracts as samples or distilled water as blank (200 μL) were allowed to react with 2.8 mL of the working solution for 30 min in dark at 37°C. Absorbance was measured at 593 nm using UV-Vis spectrophotometer. Ferus sulfate (FeSO4) was used as standard to establish a standard curve. The FRAP antioxidant activity was expressed as mmol of Fe2+ equivalents per g of dried weight (mM FeSO4/ g DW).

Statistical analysis Statistical analyses are performed using Excel software (Microsorft Office 2013) for calculating the means and the standard error of the mean. Results are expressed as the mean ± standard deviation (SD). Determination of f- test ( one- way ANOVA) and correlation ( Pearson correlation coefficient, r) on phytochemical and antioxidant activity is analyzed by using SPSS software for Windows (version 19).

Results and discussion

Phytochemical contents Table 1 showed phytochemical contents found in bagasse extracts. The results indicated that the crude extract of Khon Kaen II had higher phytochemical contents than Khon Kaen I. The TPC, TFC and TTC were 35.19 mg GAE/100 g DW, 101.47 mg QE/ 100 g DW and 7.73 mg QE/ 100 g DW, respectively. These contents higher than Khon Kaen I about 13, 28 and 44%, respectively even the yield of crude extracts from Khon Kaen I was higher than Knon Kaen II about 26.5%. In general, the obtained phytochemicals from the bagasse have lower than phytochemicals content found in the sugarcane [7-9] about 33%. However, the phytochemicals content in bagasse have the same content as found in partially purified fraction of sugarcane extract [8]. The types and contents of phytochemicals were varied by some factors including planted regions, climates, strain, parts of plants, harvest times, instrument analysis, solvents, method and procedures analysis [9,16,17].

Table 1 Phytochemical contents of sugarcane bagasse crude extracts.

Extraction TPC TFC TTC Extracts Yield (%) (mg GAE/100 g DW) (mg QE/ 100 g DW) (mg UA/ 100 g DW) Khon Kaen I 2.256±0.08 30.75±0.38 73.07±2.05 4.34±0.34 Khon Kaen II 1.783±0.02 35.19±1.04 101.47±3.89 7.73±0.57

Antioxidant activity The antioxidant activity of bagasse crude extracts was shown in Table 2. The results found that Khon Kaen II had higher antioxidant activities than Khon Kaen I. The radicals scavenging of Khon Kaen II extract for DPPH and ABTS were 17.32 and 150.85 mg TE/ g DW, respectively which were higher than Khon Kaen I extract about 25%. Moreover, the bagasse extract of Khon Kaen II showed higher reducing 2+ power of Fe (26.59 mM FeSO4/ g DW) than Khon Kaen I extract about 15%. Comparison with previous reports, the reducing power of bagasse extract has closed to sugarcane tree extract fractionated by silica gel column chromatography [8], but the bagasse extract had higher scavenging free radical activity [7]. This might be according to the bagasse extract composed high content of ortho-dihydroxyl polyphenols such as flavonoids (quercetin, catechin myricetin) which could be interacted well with Fe2+ via coordinate linkages [17,18]. Moreover, phenolic compounds which composed high hydroxyl groups are the main antioxidant substances in plants [20,21].

Table 2 Antioxidant activity of sugarcane bagasse crude extracts.

DPPH assay ABTS assay FRAP assay Extracts (mg TE/ g DW) (mg TE/ g DW) (mM FeSO4/ g DW) Khon Kaen I 13.87±0.99 110.07±2.97 22.69±2.22 Khon Kaen II 17.32±0.92 150.85±5.50 26.59±1.56 ICoFAB2019 Proceedings | 67

Correlation analysis The correlation between phytochemicals and antioxidant activity of the bagasse extract as shown in Table 3. The results indicated that all phytochemicals; TPC, TFC and TTC positively correlated to antioxidant activity. This indicated that all obtained substances synergistic effect on free radicals. Considering each phytochemical about its antioxidation mechanism, the TPC and TFC preferred scavenging activity than reducing power. However, the TFC showed higher ABTS scavenging than DPPH and reducing Fe2+. The TTC has lower antioxidant activity than others. This indicated that the main mechanism on free radicals of the bagasse extract was scavenging activity. The obtained results were in agree with previously reported [22-24].

Table 3 Correlation (r) of phytochemical contents and antioxidant activity of sugarcane bagasse crude extracts.

Factors TPC TTC TFC DPPH. ABTS+ FRAP TPC 1 .947** .947** .957** .916* .622 TTC - 1 .939** .934** .822* .480 TFC - - 1 .950** .967** .721 DPPH. - - - 1 .876* .533 ABTS+ - - - - 1 .860* FRAP - - - - - 1 ** Correlation is significant at the 0.01 level. * Correlation is significant at the 0.05 level.

Conclusions

Bagasse composed of different phytochemicals containing antioxidant activity. The types and contents of phytochemicals varied by the strain of sugarcane. The crude extract of Khon Kaen II has higher phytochemicals contents and antioxidant activity than Khon Kaen I. The phytochemicals found in the bagasse extracts showed positively related to their antioxidant activity with statistical significance. The mechanism of phytochemicals was clearly acted of free radical scavenging more than reducing power. This work suggested that the bagasse is a good source of some phytochemicals with high antioxidant potential, especially flavonoids and phenolic acids. Therefore, it might be possible to apply this bagasse extract as human health supplement.

Acknowledgements

The authors would like to thank Burirum Sugarcane Faculty, Burirum Province for supplying bagasse. Thank you also extend to the PERCH-CIC, Department of Chemistry, Faculty of Science, for chemical and instrument supports. We also thank you Mahasarakham University for financial support of this work.

References

[1] Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev. 1998; 56, 317-333. [2] Decker EA. The role of phenolics, conjugated linoleic acid, carnosine and pyrrolquinolinequinone as nonessential dietary antioxidants. Nutr Rev. 1995; 5, 49-53. [3] González-Castejón M, Rodriguez-Casado A. Dietary phytochemicals and their potential effects on obesity: a review. Pharmacol Res. 2011; 64, 438-455. [4] Pham-Huy, LA., He, H., and Pham-Huyc C. Free radicals, antioxidants in disease and health. Int J Biol Sci. 2008; 4(2), 89-96. [5] Beghyn T, Deprez-Poulain R, Willand N, Folleas B. Natural compounds: leads or ideas? Bioinspired molecules for drug discovery. Chem Biol Drug Des. 2008; 72, 3-15. [6] Duarte-Almeida JM, Negri G, Salatino A, de Carvalho JE, Lajolo FM. Antiproliferative and antioxidant activities of a tricin acylated glycoside from sugarcane (Saccharum officinarum) juice. Phytochemistry 2007; 68(8), 1165-1171. [7] Krapankiow W, Srihanam P. Investigation of phytochemical and antioxidant activity of different parts of sugarcane planted in Buriram province. J Sci Tech MSU 2016; The 12thMahasarakham University Research Conference, 706-713. ICoFAB2019 Proceedings | 68

[8] Naowaset D, Srihanam P. Phytochemical contents and antioxidant activity of partially purified sugarcane extract by silica gel column. J Sci Tech MSU 2017; The 13thMahasarakham University Research Conference, 444-453. [9] Feng S, Luo Z, Zhang Y, Zhong Z, Lu B. Phytochemical contents and antioxidant capacities of different parts of two sugarcane (Saccharum officinarum L.) cultivars. J Food Chem. 2014; 151, 425- 458. [10] Škerget M, Kotnik P, Hadolin M, Hraš AR, Simonic M, Knez Ž. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem. 2005; 89, 191-198. [11] Jia Z, Tang MC, Wu JM. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999; 64(4), 555-559. [12] Ni QX, Xu GZ, Wang ZQ, Gao QX, Wang S, Zhang YZ. Seasonal variations of the antioxidant composition in ground bamboo sasa argenteastriatus leaves. Int J Mol Sci. 2012; 13(2), 2249-2262. [13] Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Byrne DH, Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J Food Compos Anal. 2006; 19, 669-675. [14] Berg R, Haenen G, Berg H, Bast A. Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evalua- tion of antioxidant capacity measurements of mixtures. Food Chem. 1999; 66, 511-517. [15] Benzie IFF, Szeto YT. Total antioxidant capacity of teas by the ferric reducing/antioxidant power assay. J. Agric. Food Chem. 1999; 47, 633-636. [16] Antoniolli A, Fontana AR, Piccoli P, Bottini R. Characterization of polyphenols and evaluation of antioxidant capacity in grape pomace of the cv. Malbec. Food Chem. 2015; 178, 172-178. [17] Berli FJ, Alonso R, Bressan-Smith R, Bottini R. UV-B impairs growth and gas exchange in grapevies grown in high attitude. Physiol Plantarum 2012; 149(1), 127-140. [18] Andjelkovic M, Camp JV, Meulenaer BD, Depaemelaere G, Socaciu C, Verloo M Verhe R. Iron- chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem. 2006; 98, 23- 31. [19] Moran FJ, Klucas RV, Grayer RJ, Abian J, Becana M. Complexes of iron with phenolic compounds from soybean nodules and other legume tissue: prooxidant and antioxidant properties. Free Radical Bio Med. 1997; 22, 861-870 [20] Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010; 2(12), 1231-1246. [21] Visioli F, Lastra CA, Andres-Lacueva C, Aviram M, Calhau C, Cassano A. Polyphenols and human health: a prospectus. Crit Rev Food Sci. 2011; 51, 524-546. [22] Abu Bakar MF, Mohamed M, Rahmat A, Fry J. Phytochemicals and antioxidant activity of different parts of bambangan (Mangiferapajang) andtarap (Artocarpusodoratissimus). Food Chem. 2009; 113, 479-483. [23] Butsat S, Weerapreeyakul N, Siriamornpun S. Changes in phenolic acids and antioxidant activity in Thai rice husk at five growth stages during grain development. J Agic Food Chem. 2009; 57, 4566- 4571. [24] Rice-Evans CA, Miller NT and Paganga G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997; 4, 304-330.

doi:10.14457/MSU.res.2019.14 ICoFAB2019 Proceedings | 69

Locust Bean Gum Hydrolysis for Mannooligosaccharide (MOS) Production Using Bacillus methylotrophicus KS1

Sunchai Phiwphech1*, Vijitra Luang-In1, Sirirat Deeseenthum1 and Surachai Rattanasuk2

1Natural Antioxidant Innovation Research Unit, Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham 44150 Thailand 2 Major of General Science, Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, 45120 Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Locust bean gum is a source of galactomannan that can be used as substrate for mannooligosaccharide production with prebiotic potential through mannanase-catalyzed hydrolysis. The aim of this research was to hydrolyze the locust bean gum using Bacillus methylotrophicus KS1 isolated from soil collected from Roi Et, Thailand. Locust bean gum hydrolysis conditions were performed using 1% Bacillus methylotrophicus KS1 inoculum (108 CFU/ml) in nutrient broth containing 1% locust bean gum in 500 ml Erlenmeyer flask and incubated at 37 oC and 150 rpm for 24 h. Five milliliters of cultures were collected at the specific time intervals for mannanase activity determination. The result indicated that the highest mannanase activity was found at 18 h with 14.10 U/ml. Mannooligosaccharide composition of locust bean gum hydrolysis was identified using High Performance Liquid Chromatography method. The result showed that the produced mannooligosaccharide consisted of mannotriose, mannotetraose and mannohexose. These may have prebiotic attributes that will be investigated in the future.

Keywords: Bacillus methylotrophicus KS1, Locust bean gum, Mannanase, Mannooligosaccharide, HPLC

Introduction

Locust bean gum (LBG) is a source of galactomannan consists obtained from seed endosperm of fruit pod of Ceratonia siliqua L. Chemical structure of LBG of a β-1,4-linkage mannose backbone with galactose monomers linked to it randomly by α-1,6 bonds [1, 2]. It has been used for food, pharmaceuticals, paper, textile, oil well drilling, cosmetics and also beneficial for human health [3]. LBG can be used as substrate for mannooligosaccharides (MOS) production as prebiotics by mannanase-catalyzed hydrolysis. MOS are an oligosaccharides comprised of mannose residues obtained from mannan hydrolysis including mannan, glucomannan, galactomannan and galactoglucomannan by β-mannanase [4]. MOS are prebiotics that can be used as a feed additive to reduce pathogenic bacteria such as Vibrio, Coliforms, Clostridia and Salmonella and modulate the immune system of host animals and also stimulate the growth of probiotics such as Bifidobacterium sp. and Lactobacillus sp. [5-7]. Mannanase is an enzyme that randomly hydrolyzes of mannan and heteromannan at the 1,4-β-D- mannosidic linkage [1, 8]. Many microorganisms are sources of mannanase such as Chryseobacterium indologenes, Geobacillus stearothermophilus, Neosartorya fischeri P1, Bacillus pumilus GBSW19, Bacillus sp. and Kitasatospora sp. [1, 9-12]. The aim of this research was to produce MOS from LBG by isolated bacteria, Bacillus methylotrophicus KS1.

Materials and methods

Chemical and reagents LBG was purchased from Sigma (USA). Nutrient broth (NB) was purchased from Himedia Laboratories (India). 3,5-Dinitrosalicylic acid and sodium potassium tartrate were purchased from Sigma (USA). Standard sugars including mannose, mannobiose, mannotriose, mannotetraose, mannopentose and mannohexose were purchased from Megazyme (Wicklow, Ireland).

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Bacterial strain Bacillus methylotrophicus KS1 was isolated from soil in Roi-Et and stored at microbiology laboratory, Department of Science and Technology, Roi Et Rajabhat University, Roi Et, Thailand.

Bacterial growth and staining Single colony of B. methylotrophicus KS1was cultured using nutrient broth (NB) medium and incubated at 37 oC with 150 rpm for 18 h. One percentage (v/v) of B. methylotrophicus KS1 was mixed with 200 ml NB containing 1% LBG and incubated at 37 oC with 150 rpm for 24 h. Five milliliters of cultured medium broth were collected at 0 - 72 h and centrifuged. Supernatant was discarded and cell pellet was resuspended using sterile normal saline. Bacterial cell count was determined by ten-fold dilution method. One hundred microliters of each dilution were spread on to nutrient agar (NA) incubated at 37 oC for 18 h. Bacterial population was calculated from plate containing 30-300 colonies. B. methylotrophicus was stained by Gram-staining and endospore staining method.

Mannanase activity assay B. methylotrophicus KS1was cultured using nutrient broth (NB) medium at 37 oC with 150 rpm for 18 h. The turbidity of the bacterial cell suspension was adjusted at OD600 to the required concentration of 108 CFU/ml. One percentage (v/v) of B. methylotrophicus KS1 was mixed with 200 ml NB containing 1% LBG and incubated at 37 oC with 150 rpm for 24 h. Five milliliters of cultured medium broth were collected at 0, 3, 6, 12, 18 and 24 h and centrifuged. Mannanase activities assay was described previously [9]. Briefly, 500 µl of supernatant was mixed with 500 µl of 1% LBG, pH 7 (phosphate buffer) and incubated at 60 oC for 5 min. The reaction was inhibited by adding DNS solution. The mixture was boiled for 5 minutes and cooled on ice. Two thousand and five hundred microliters of water were added. The amount of reducing sugar was determined at OD540 nm. One unit of mannanase activity is defined as the amount of mannanase that hydrolyzes LBG and liberates 1 µmol D-mannose within 1 min of reaction at 60 oC.

Mannooligosaccharide composition analysis MOS composition was analyzed by high pressure liquid chromatography (HPLC) condition described as previously [13]. Mannose, mannobiose, mannotriose, mannotetraose, mannopentose and mannohexose were used as standard sugars.

Results and discussion

Bacterial growth and staining B. methylotrophicus KS1 was cultured using NB and cultured broth samples were collected for bacterial count. The result indicated that at lag phase was at 0 - 12 h, exponential phase was at 12 - 18 h, stationary phase was at 18 - 48 h and decline phase was after 48 h (Fig. 1). Result of Gram staining and endospore staining showed that B. methylotrophicus KS1 was Gram-positive bacilli, rod shape and produced oval subterminal spores after 18 h. (Fig. 2). This growth curve presented that late exponential phase that had the highest growth rate. This incubation time of 18 h will be used for future experiment and it was similar to the reported value in ref [14].

10 8.62 8.87 8.76 9 8.2 7.9 7.35 8 6.87 6.51 6.56 7 5.82 5.92

(log CFU/ml) (log 6 5.2 stationary 4.63 5 phase log phase 24 - 48 h death phase 4 12 - 18 h 48 - 72 h 3 lag phase 2 0 - 12 h 1

Bacterial population Bacterial 0 0 10 20 30 40 50 60 70 80 Time (h)

Figure 1 Growth curve of Bacillus methylotrophicus KS1 over 72 h. ICoFAB2019 Proceedings | 71

Endospore

Vegetative cell

A B

Figure 2 Bacillus methylotrophicus KS1 Gram staining and endospore staining under light microscope 1000x. A. Gram positive bacilli; B. Oval subterminal endospore

Mannanase activity assay Mannanase activity of B. methylotrophicus KS1 was gradually increased until presented the high activity at 18 h (14.10 U/ml) (Fig. 3). Form the above result that showed at 18 h after incubation was late log phase that indicated that high cell growth.

16 14.1 14 13.24 12.97

12 10.92

10 8.82

8

6

4 Mannanase activity (U/ml) activity Mannanase 2 0 0 0 5 10 15 20 25 30 Incubation time (h)

Figure 3 Mannanase activity of Bacillus methylotrophicus KS1

Mannooligosaccharide composition analysis The result from this research found the MOS composition from LBG hydrolysis composed of mannotrios M3 (2.42±0.04 mg/ml), mannotetraose M4 (36.74±0.45 mg/ml) and mannohexose M6 (1.77±0.08 mg/ml) (Table. 1.) obtained from locust bean gum hydrolysis. This result was similar to previous reports [15-17]. However, the composition of MOS is also found from the copra meal by mannase enzyme from Bacillus circulans NT6.7 composed of mannotrios M3, mannotetraose M4 and mannohexose M6 [18]. MOS composition from copra meal hydrolysis by mannanase enzyme from Streptomyces sp. BF3.1 composed of mannotrios M3, mannotetraose M4, mannopentose M5 and mannohexose M6 [19].

Acknowledgements

This research was supported by King Bhumibol scholarship and research grants for graduate students (master's degree) for the year 2019 from Faculty of Technology, Mahasarakham University.

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Table 1 Mannooligosaccharide composition from LBG hydrolysis by Bacillus methylotrophicus KS1

Mannooligosaccharide (mg/ml)

Replicate Total M6 M4 M3 M1 (M6-M3) 1 1.71 36.42 2.44 0.61 40.57

2 1.83 37.05 2.39 0.64 41.27

X̅ ± SD 1.77±0.08 36.74±0.45 2.42±0.04 0.63±0.02 40.92±0.49

References

[1] Zang H, Xie S, Wu H, Wang W, Shao X, Wu L, et al. A novel thermostable GH5_7 beta-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production. Enzyme Microb Technol. 2015;78:1-9. [2] Kim S, Lee M-H, Lee E-S, Nam Y-D, Seo D-H. Characterization of mannanase from Bacillus sp., a novel Codium fragile cell wall-degrading bacterium. Food Sci Biotechnol. 2017;27(1):115-22. [3] Barak S, Mudgil D. Locust bean gum: processing, properties and food applications a review. International journal of biological macromolecules. 2014;66:74-80. [4] McCleary BV. β-D-Mannanase. Methods in Enzymology. 1988;160:596-610. [5] Phothichitto K, Nitisinprasert S, Keawsompong S. Isolation, screening and identification of mannanase producing microorganisms. Kasetsart J(Nat Sci). 2006;40:26-38. [6] Rungrassamee W, Kingcha Y, Srimarut Y, Maibunkaew S, Karoonuthaisiri N, Visessanguan W. Mannooligosaccharides from copra meal improves survival of the Pacific white shrimp (Litopenaeus vannamei) after exposure to Vibrio harveyi. Aquaculture. 2014;434:403-10. [7] Cuong D, Dung V, Hien N, Thu D. Prebiotic evaluation of copra-derived mannooligosaccharides in white-leg shrimps. Journal of Aquaculture Research and Development. 2013;4(5). [8] Rattanasuk S, Prasertsang K, Phiwphech S, editors. Isolation of thermophilic mannanase-producing bacteria useful for mannooligosaccharide (MOS) production. International Conference on Science and Technology (TICST), 2015; 2015: IEEE. [9] Rattanasuk S, Ketudat-Cairns M. Chryseobacterium indologenes, novel mannanase-producing bacteria. Sonklanakarin Journal of Science and Technology. 2009;31(4):395. [10] Suwanto A, T Henawidjaja M, T Resnawati PU. Isolation and characterization of mannanolytic thermophilic bacteria from palm oil shell and their mannanase enzyme production properties. BIOTROPIA-The Southeast Asian Journal of Tropical Biology. 2005(25). [11] Yang H, Shi P, Lu H, Wang H, Luo H, Huang H, et al. A thermophilic beta-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers. Food Chem. 2015;173:283-9. [12] Rahmani N, Kashiwagi N, Lee J, Niimi-Nakamura S, Matsumoto H, Kahar P, et al. Mannan endo- 1,4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers. AMB Express. 2017;7(1):100. [13] Tanimoto T, Ikuta A, Sugiyama M, Koizumi K. HPLC analysis of manno-oligosaccharides derived from Saccharomyces cerevisiae mannan using an amino column or a graphitized carbon column. Chemical and pharmaceutical bulletin. 2002;50(2):280-3. [14] Ge B, Liu B, Nwet TT, Zhao W, Shi L, Zhang K. Bacillus methylotrophicus Strain NKG-1, Isolated from Changbai Mountain, China, Has Potential Applications as a Biofertilizer or Biocontrol Agent. PloS one. 2016;11(11):e0166079-e. [15] Nguyen H-M, Pham M-L, Stelzer EM, Plattner E, Grabherr R, Mathiesen G, et al. Constitutive expression and cell-surface display of a bacterial β-mannanase in Lactobacillus plantarum. Microbial cell factories. 2019;18(1):76. [16] Xie S, Zhu B, Yang X, Gu C, Hu B, Gao T, et al. Mannan oligosaccharides trigger multiple defence responses in rice and tobacco as a novel danger-associated molecular pattern. Molecular plant pathology. 2019;20(8) [17] Pangestu R, Rahmani N, Palar R, Lisdiyanti P, Yopi. The effect of biomass particle size and chemical structure on the enzymatic hydrolysis reaction of galactomannan from sugar palm fruit by β- mannanase from Kitasatospora sp. KY576672. Earth and Environmental Science. 2019(251). ICoFAB2019 Proceedings | 73

[18] Rungruangsaphakun J, Keawsompong S. Optimization of hydrolysis conditions for the manno- oligosaccharides copra meal hydrolysate production. Journal for Biotechnology. 2018;8:169. [19] Ariandi, Yopi, Meryadini A. Enzymatic hydrolysis of copra meal by mannanase from Streptomyces sp. BF3.1 for the production of manno-oligosaccharide. Journal of Biosciences. 2015;22(2):79-86.

doi:10.14457/MSU.res.2019.15 ICoFAB2019 Proceedings | 74

Screening of Yeasts from Thai Traditional Fermentation Starter (Loog-pang) for Alcoholic Fermentation Products in Community Enterprise

Pikulthong Paewlueng1*, Sirirat Deeseenthum1 and Surachai Rattanasuk2

1 Master degree student, Natural Antioxidant Innovation Research Unit, Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham 44150 Thailand 2 Major of General Science, Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, 45120 Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Thai traditional fermentation starter or Loog-pang is a starter culture in dry including bacteria, yeast and fungi used for traditional Sato processing in Thailand. Alcoholic fermentation products in community enterprise are not good quality enough because of alcohol content are not stable. Thus, this study aimed to isolation of yeasts from Loog-pang. Microbes in Loog-pang Sato from 3 sources including Roi-Et Province, Maha Sarakham Province and Phangnga Province were isolated. Yeasts were isolated using yeast extract-peptone-dextrose (YPD) agar and incubated at 30 oC for 3 days. Yeast culture in YPD broth was incubated in a temperature controlled agitator at 37 oC at 200 rpm for 48 hours. The yeast concentration was adjusted to 108 cells /ml to be used as a starter. The isolates were determined for total dissolved solid (TDS), alcohol production and alcohol tolerance. The result found that isolate LPR5 showed the highest total dissolved solid at 19.07 oBrix, the highest alcohol production capacity at 1.54% and gave the highest alcohol tolerance. Consequently, the yeast isolate LPR5 was identified by DNA sequencing and morphology. The results indicated that LPR5 as Wickerhamomyces anomalus.

Keywords: Yeast, Thai traditional fermentation starter, Loog-pang, Alcoholic fermentation, Sato

Introduction

Loog-pang is a Thai traditional fermentation starter culture crafted in a form of ball shape with an important role in providing quality and flavors of Sato fermentation. Many herbs such as Allium sativa, Zingiber officinale and Alpinia siamensis were mixed with bacteria, yeasts and fungi for dried starter preparation. Loog-pang is the source of beneficial microorganisms for ethanol fermentation process [1]. Microorganisms that frequently found in Loog-pang are yeasts such as Saccharomyces, Pichia, Issatchenkia, Candida and Saccharomycopsis [2-4]. Sato or Thai rice wine is made using glutinous rice as raw material and produced under non-sterile conditions at home scale using Loog-pang [5]. The fungi are mainly responsible for the hydrolysis of starch in the glutinous rice into sugar and yeasts play basic roles in converting sugars into ethanol, carbon dioxide and hundreds of other secondary products [1, 6]. If Loog-pang was kept for too long, it may result in less effective fermentation. The problem of home scale production of Sato is inconsistency in Sato quality produced from each batch. This problem can be related to the variability of the microbial communities between different Loog- pangs. The use of good pure culture of desirable microorganisms should lead to better consistency in Sato quality from each bacth of fermentation [1]. The aim of this research was to isolate yeasts in Loog-pang with a potential to produce high alcohol in Sato.

Materials and methods

Yeast isolation Loog-pang balls were collected from 3 provinces including Roi Et, Mahasarakham and Phangnga, Thailand. Each Loog-pang was milled using sterile mortar before used. Fifty grams of steamed sticky rice were mixed with each milled Loog-pang (250 mg) in a sterile-glass bottle and incubated at room temperature for 3 days. The produced water from fermentation was used as source of yeasts. Yeast was ICoFAB2019 Proceedings | 75

isolated by spreading the produced water on yeast extract peptone dextrose agar (YPD agar) and incubated at 30 oC for 3 days. Single colony was picked and cross streaked on YPD agar for further experiment.

Determination the efficiency of sugar fermentation and ethanol production One milliliter of each isolated yeast was cultured in 10 ml of sterile 20 oBrix sugar solution mixed with 0.05% (W/V) diammonium phosphate (DAP) and contained glass Durham tube. The tubes were static incubated at 37 oC for 5 days. The remaining sugar was determined using hand refractometer. Alcohol content was measured by Gas chromatography (GC). Yeast that can produce the highest amount of alcohol was selected.

Determination of alcohol tolerance One milliliter of all isolate were tested for alcohol tolerance so how did the represent active isolate was select according to this parameter was cultured in 10 ml of sterile 20 oBrix sugar solution mixed with 0.05% (W/V) diammonium phosphate (DAP), 10% and 15% ethanol and contained glass Durham tube. The tubes were static incubated at 37 oC for 5 days. The alcohol tolerance was indirectly observed by gas production in Durham tube.

Yeast identification Isolated yeast with high efficiency in ethanol production was streaked onto YPD agar and cultured using YBD broth for 2 days. Yeast colony and cell morphology were observed under the microscope. For yeast identification, isolated yeast was sent to Thailand institute of scientific and technology research (TISTR) for automated DNA sequencing. The resulting sequences were compared with nucleotide database from PubMed using the BLAST program.

Results and discussion

Determination the efficiency of sugar fermentation and ethanol production The results found that 9 yeast isolates were isolated from Loog-pang collected from Roi Et (2 isolates), Mahasarakham (2 isolates) and Phangnga (5 isolates). All isolated yeasts were determined for the efficiency of sugar consumption and ethanol production. The results found that LPR5 isolates showed the lowest remaining sugar and the highest ethanol production at 19.07 oBrix and 1.54%, respectively (Table 1.)

Table 1 The efficiency of sugar fermentation and ethanol production from isolated yeasts.

Gas level at Gas level at Isolate number Remaining sugar (oBrix) Ethanol content (%) Day 1 Day 5 LPR4 - ++ 19.47±0.06 1.15±0.04

LPR5 ++ ++++ 19.07±0.06 1.54±0.05

LPS5 - + 19.37±0.12 0.67±0.03

LPS6 - +++ 19.20±0.10 1.20±0.03

LPP5 - - 19.80±0.10 0.07±0.03

LPP6 - - 19.77±0.06 0.05±0.04

LPP7 - - 20.03±0.06 0.01±0.01

LPP8 - - 19.87±0.06 0.03±0.02

LPP9 - - 19.87±0.06 0.03±0.01

+; gas level at ¼ Durham tube, ++; gas level at ½ Durham tube, +++; gas level at ¾ Durham tube, ++++; gas level at full Durham tube

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Determination of alcohol tolerance The result found that LPR5 isolate was the most tolerant against culture broth containing 10% and 15% ethanol followed by LPR6 and LPR4 at 5 day incubation (Table 2).

Table 2 Alcohol tolerance of isolated yeasts

10% Ethanol 15% Ethanol Isolate Gas level at Gas level at Gas level at Gas level at Day 1 Day 5 Day 1 Day 5

LPR4 - ++ - +

LPR5 + ++++ - +++

LPS5 - + - -

LPS6 - +++ - +

LPP5 - - - -

LPP6 - - - -

LPP7 - - - -

LPP8 - - - -

LPP9 - - - -

+; gas level at ¼ Durham tube, ++; gas level at ½ Durham tube, +++; gas level at ¾ Durham tube, ++++; gas level at full Durham tube

Yeast characterization and identification Colony of LPR5 has a morphology of white to cream-colored, smooth and pherical to ellipsoidal budding blastoconidia form. DNA sequences were compared with nucleotide database from PubMed using the BLAST program. The result indicated that LPR5 was identified as Wickerhamomyces anomalus at 100% identity (1-589 bases). The LPR5 sequences were subjected to phylogenetic tree using MAGA X program. Many yeast strains were isolated from Loog-pang including Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Pichia anomala, Issatchenkia orientalis, Tolulaspora delbrueckii and Candida glabrata were isolated from 114 Loog-pangs collected from small-scale factories and villages in central, northern and northeastern Thailand [1]. In addition, another study [6] presented that Hanseniaspora uvarum, Hanseniaspora occidentalis, Metschnikowia pulcherrima, Candida zemplinina, Hanseniaspera vineae, Issatchenkia orientalis, Zygosaccharomyces bailii, Pichia kluyveri and Saccharomyces cerevisiae were isolated from Cabernet Sauvignon musts in three vineyardsat the beginning, middle and final stages of spontaneous fermentations. One finding [7] reported that S. cerevisiae, Pichia anomala, Trichosporon sp, Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana were found in 54 ‘Hamei’ samples collected from household rice wine preparations in tribal villages of Manipur. It was also reported [8] that 3 isolates, Saccharomyces cerevisiae, Pichia kudriavzevii and Candida glabrata were isolated from 35 from rice wine starters (Loogpang) in Chiang Mai province, Thailand. They found 3 isolated yeast, Candida tropicalis, Saccharomyces cerevisiae and Saccharomycopsis spp. from Medombae [9].

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A B

Figure 1 Cell morphology of LPR5 isolates A: Colony of LPR5, B: Cell structure of LPR5

Conclusions

LPR 5 was isolated from Loog-pang collected from Roi Et province, Thailand. This isolate showed the highest sugar consumption, alcohol production (1.54%) and alcohol tolerance and was identified as Wickerhamomyces anomalus. This isolated yeast will combine with potential fungi and herbs for Loogpang preparation. The produced Loogpang will be sent to the community enterprise for improving sato production.

Acknowledgements

This research was supported by STEM (Science, Technology Engineering, and Mathematics) grant from NSTDA (National Science and Technology Development Agency), Thailand and Faculty of Technology, Mahasarakham University.

References

[1] Taechavasonyoo A, Thaniyavarn J, Yompakdee C. Identification of the molds and yeasts characteristic of a superior Loogpang, starter of Thai rice based alcoholic beverage Sato. Asian J Food Agro Ind. 2013;6:24-38. [2] Limtong S, Sintara S, Suwanarit P, Lotong N. Species diversity of molds in Thai traditional fermentation starters (Loog-Pang). Kasetsart J(Nat Sci). 2005;39:511-8. [3] Limtong S, Sintara S, Suwanarit P, Lotong N. Yeast diversity in Thai traditional fermentation starter (Loog-pang). Kasetsart J (Nat Sci). 2002;36:149-58. [4] Aidoo KE, Rob Nout M, Sarkar PK. Occurrence and function of yeasts in Asian indigenous fermented foods. FEMS yeast research. 2006;6(1):30-9. [5] Amatayakul T, Somsap N, Rotsatchakul P. Study of volatile compounds in Thai rice wine (Sato) produced from wheat. Asia-Pacific Journal of Science and Technology. 2012;17(6):939-49. [6] Li E, Liu A, Xue B, Liu Y. Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia, China. World Journal of Microbiology and Biotechnology. 2011;27(10):2475-82. [7] Jeyaram K, Singh WM, Capece A, Romano P. Molecular identification of yeast species associated with ‘Hamei’—a traditional starter used for rice wine production in Manipur, India. International journal of food microbiology. 2008;124(2):115-25. [8] Cheenacharoen S, Juntachai w. Diversity and Genetic Relationship of Ethanol Tolerant Yeasts Isolated from Rice Wine Starters (Loog-Pang) in Chiang Mai Province, Thailand. Thai Science and Technology Journal. 2018:478-89. ICoFAB2019 Proceedings | 78

[9] Chay C, Dizon EI, Elegado FB, Norng C, Hurtada WA, Raymundo LC. Isolation and identification of mold and yeast in medombae, a rice wine starter culture from Kompong Cham Province, . Food Research. 2017;(6):213-220.

doi:10.14457/MSU.res.2019.16 ICoFAB2019 Proceedings | 79

Isolation and Identification of Lipase-Producing Bacteria from Soil in Nasinuan Forest, Kantarawichai District, Mahasarakham Province

Manatchanok Yotchaisarn*, Sirirat Deeseenthum, Kedsukon Maneewan Vijitra Luang-In

Natural Antioxidant Innovation Research Unit, Department of Biotechnology, Faculty of Technology, Mahasarakham University, Khamriang, Kantarawichai, Mahasarakham 44150 Thailand.

*Corresponding author’s e-mail: [email protected]

Abstract:

Nasinuan Forest was located at Kantarawichai District, Mahasarakham Province Thailand. This area has been considered to be quite saline; however contains a variety of bacteria that may be able to produce industrial enzymes. Lipase is one of the industrial enzymes which play an important role in industry in Thailand such as detergent industry, food, fat and oil industries and cosmetics. This study aimed to identify lipase-producing bacteria from soil in Nasinuan Forest and determine the optimum conditions for lipase production and lipase activity. The results showed that 35 isolates were positive for lipase after being tested on tributyrin agar. The top five isolates with the highest halo : colony ratios included Bacillus subtilis 1.3LP2 > Brevibacillus brevis 2.1LP4 > Micrococcus sp. 3.2LP3 > Bacillus thuringiensis 1.1LP3 > Staphylococcus sp. 1.4LP4, respectively. The results of gram staining found that four isolates were gram- positive with rod-shaped and only gram-positive with cocci-shaped was found as Staphylococcus sp. 1.4LP4. These lipase-producing bacteria were closest to Bacillus spp., Staphylococcus sp., Brevibacillus sp. and Micrococcus sp. using 16S rRNA gene analysis whose from Italy, India, China, Egypt and Brazil. The optimum conditions for the highest lipase activity by Bacillus subtilis 1.3LP2 are pH 7.0 at 55 °C with the lipase activity of 160.92 ± 0.91 U/mL. This is the first report of lipase-producing bacteria isolated from soil in Nasinuan forest, Mahasarakham, Thailand. These bacterial enzymes can be applied in various industries in Thailand such as detergent industry, food, fat and oil industries and cosmetics.

Keywords: Bacteria, Soil, Lipase enzyme, Industry, 16S rRNA gene

Introduction

Lipase (EC 3.1.1.3) is an enzyme that can be found in nature, this enzyme acted by catalyzing hydrolysis of molecular ester bonds with triglycerol and long chain fatty acids which containing monoglycerides, dioxins and free fatty acids as the final product [1]. Lipase producing bacteria often have a variety of habitats such as waste, processing plants, vegetable oil, soil contaminated from oil mill area, etc. There have many factors of inducing lipase production such as temperature, pH, nitrogen and carbon source, inorganic salts, agitation and dissolved oxygen concentration [2]. The reliable way to detect microorganisms that can produce lipase is to use tributyrin as substrate and degrading zone around the colony was visualized [3]. Most of lipase- producing bacteria are gram-negative bacteria especially Pseudomonas, which have at least seven species that can produce lipase, namely Pseudomonos aeruginosa, Pseudomonos alcaligenes, Pseudomonos fragi, Pseudomonos glumae, Pseudomonos cepacia, Pseudomonos fluorescens and Pseudomonos putida [4]. Bacillus spp. can produce lipase as reported by Chaturvedi et al. [5] mentioned to lipase-producing bacteria isolated from contaminated soil areas, India. Solid State Fermentation (SSF) technique occurred led to discovery of Bacillus subtilis with lipase production. In addition, several reports mentioned that bacteria species Staphylococcus sp. [6] and Brevibacillus sp. [7] separated from soil and hot spring source can produce lipases as well as those were found in India. In general, soils are often the source of a variety of microorganisms due to richness in various nutrients and minerals. The advantages of using microorganisms for enzyme production are the ease to cultivate and low production costs. Therefore, this work studied optimum condition for bacterial lipase production. There are many factors that are very important for enzyme production such as substrate concentration, the starter cultures, acidity and temperature. This research aimed to isolated lipase-producing bacteria obtained from the soil in the Nasinuan Forest, Kantarawichai District, Mahasarakham Province and optimization conditions for lipase activity were evaluated. ICoFAB2019 Proceedings | 80

Materials and methods

Collection of soil sample The soil samples were collected from Nasinuan Forest, Kantarawichai District, Mahasarakham Province, Thailand. They were collected from 60 rai randomly in zones 1-3 (Figure 1).

Figure 1 Studied area. Zone 1, 2 and 3 of 9.6 hectare where soil samples were collected

Bacterial isolation and screening for lipase activity Soil sample (10 g.) was suspended in 90 mL of sterile 0.85% NaCl solution and serial dilution was performed. The suspension of 100 µL was spread on tributyrin agar (g/ 100 mL) consists of 0.5% peptone, 0.3% yeast extract, 1% (v/v) tributyrin and 1.5% agar (pH 7.0) and incubated at 37 °C for 3 days. The degrading zone was visualized and halo : colony ratios were calculated following the method of Haq et al [8]. The highest halo : colony ratios from 5 isolates were collected and subculture to obtain pure isolates using streaking technique on general bacterial medium (g/L) consists of 1.0% yeast extract, 0.5% tryptone and 0.5% NaCl (pH 7.0) then incubated at 37 °C overnight. The morphological properties of each bacterial isolate were identified including color, shape, surface and edge of colony and Gram staining results were recorded.

Screening of lipase production Five of pure isolates of lipase-producing bacteria were cultured and adjusted at 108 CFU/mL equivalent to 0.5 McFarland [9]. These isolates were inoculated into 30 mL lipase liquid medium (g/100 mL) consists of 0.2% peptone, 0.1% NH4H2PO4, 0.25% NaCl, 0.04% MgSO4.7H2O, 0.04% CaCl2.2H2O, 1.0% olive oil and 2% (v/v) tween 80 in flask (50 mL) incubated at 37 °C and shaking at 150 rpm for 24, 48 and 72 h. The extracellular enzyme (crude enzyme extract) in supernatants were obtained from centrifugation at 10,000g and kept at 4 °C for lipase activity analysis following a method of Winkler and Stuckman [10]. The crude enzyme extract with highest lipase activity was determined.

Optimization of lipase activity pH The crude extract of the highest lipase activity from above analysis was used to determine its optimum pH for activity using various pHs in suitable buffers in a range of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 incubated at 37 °C for 30 min and lipase activity was recorded.

Temperature The crude extract of the highest lipase activity with optimum pH was used to determine its optimum temperature for activity using various temperatures at 4, 25, 35, 45, 55, 65, 75, 85 and 95 °C for 30 min and lipase activity was recorded.

Protein quantitative analysis Protein content was determined following a method of Lowry [11] using BSA as standard agent.

Statistical analysis Measurements were obtained in triplicates as means ± standard deviation (SD). Statistical analysis ICoFAB2019 Proceedings | 81

was performed using One-way analysis of variance and Duncan Multiple’s Range Test by the software SPSS package version 17.0 at P < 0.05.

16S rRNA gene sequencing and phylogenetic analysis The pure isolates were inoculated from 20% glycerol stock to general bacterial medium for 18 h and identified using genomic DNAs obtained from the above method and using universal primers : 27F 5’- GAGAGTTTGATYCTGGCTCAG-3’ and reverse primer 1492R 5’-AAGGAGGTGATCCARCCGCA- 3’. In 25 µL PCR mixture, it was composed of genomic DNA 0.5 ng, 2X Master Mix (One PCR) of 100 mM Tris-HCl (pH 9.1), 0.1% TritonTMX-100, 200 mM dNTP, 1.5 mM MgCl2, 0.005 U Taq DNA Polymerase and 0.2 µM forward and reverse primers with volume adjustment with nuclease-free water sterile. PCR thermocycler (Thermo Scientific Hybaid Px2) was programed as follows : (1) initial denaturation for 2 min at 94 °C for 1 cycle ; (2) denaturation at 94 °C for 45 s ; annealing at 54 °C for 45 s, and extension at 72 °C for 1 min for 32 cycles ; (3) final extension at 72 °C for 7 min. Samples were held at 4 °C till further analysis. The PCR products of 16S rRNAs (1,500 bp) were detected on 0.8% agarose gel, purified using the PCR product purification kit (Vivantis, Malaysia), sent to First Base Co. Ltd. (Malaysia) for DNA sequencing. The 16S rRNA gene sequences were then compared with others available in GenBank using BLASTN program (Basic Local Alignment Search Tools) [12]. The phylogenetic tree was constructed using Muscle method for sequence alignment and neighbor-joining method using MEGA7 (www.megasoftware.net) with 1,000 replicates of bootstrap values [13].

Results and discussion

Soil sample information Lipase can be found in several sources such as plants, animals and microorganisms. At present, microorganisms are popular to use in enzymes production. Especially, bacteria have advantages over the production of enzymes derived from plants and animals as bacteria can increase the number of cells quickly within a short period of time in a cheap culture medium. In general, the selection of bacteria with the ability to produce lipase is commonly used to observe degrading zone around colonies indicate the ability to synthesize lipase [14]. There are several reports mentioned the use of olive oil and palm oil as substrates for lipase production [15].

Table 1 Information on soil samples from Nasinuan Forest

Electroconductivity Location of soil collection Surrounding area ( µS/cm ) 1.1 Azadirachta indica and Senna siamea 3.62 (N 16 20 17.945, E 103 12 14.731) were grown around in this area Low saline (0.15% NaCl) 1.3 Unknown trees 2.23 (N 16 20 6.619, E 103 11 0.985) Low saline (0.1% NaCl) 1.4 Found termite in this area 2.09 (N 16 20 23.547, E 103 12 35.244) Low saline (0.1% NaCl) 2.1 Found Ageratum conyzoides in this area 2.88 (N 16 20 36.882, E 103 12 32.315) Low saline (0.15% NaCl) 3.2 Catunaregam tomentosa 2.24 (N 16 20 35.406, E 103 12 35.157) was grown in this area Low saline (0.1% NaCl)

In this study, the pH and electrical conductivity of soils were recorded. These factors are related to salinity of soil samples collected from Nasinuan Forest, Kantarawichai District, Mahasarakham Province. The top 5 bacterial isolates were collected and isolated with highest lipase activity based on highest halo : colony ratios from the soil at locations 1.1, 1.3, 1.4, 2.1 and 3.2 with a pH range between 6.0 and 8.0 (Table 1). Normally, electrical conductivity values at 2-3 indicates that salinity or NaCl exists approximately about 0.1-0.15% in soil [16]. The soil showed slight salinity when the electrical conductivity ICoFAB2019 Proceedings | 82

of the soil is more than 2 µS/cm, it is considered as having some effect on the plants that grow around. Thus, in this case, low salinity in soil has little effect on plant growth in Nasinuan Forest.

Bacterial lipase isolation In this study, 35 lipase-positive isolates were isolated and identified. The top 5 lipase-producing bacterial isolates (1.1LP3, 1.3LP2, 2.1LP4 and 3.2LP3) showed the highest degrading zones on 1% tributyrin agar with different halo : colony ratios. Four bacterial representative strains showed similar colony morphologies and appeared to be gram-positive and rod-shaped; however another strain appeared to be gram-positive and cocci-shaped (Table 2). The results showed halo : colony ratios ranged from 1.2 to 2.4 (Table 2). Our finding was similar to Ertugrul et al. [3] and these 35 isolates were submitted to 16S rRNA gene analysis in order to identify bacterial isolates.

Table 2 Characteristics of 5 lipase-producing bacterial representative strains

Isolate Colony morphology Halo : colony ratios Gram staining

Irregular shape 1.1LP3 Flat structure Curled margin Cloudy white color 1.35 G+, rod

Irregular shape 1.3LP2 Flat structure Curled margin Cloudy white color 2.40 G+, rod

Circular shape Flat structure 1.4LP4 Undulate margin Cloudy white color 1.25 G+, cocci

Punctiform shape Flat structure 2.1LP4 Entire margin Cloudy white color 2.35 G+, rod

Filamentous shape Flat structure 3.2LP3 Filamentous margin

White color

1.60 G+, rod

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Identification of lipase-producing bacteria In this study, five lipase-positive bacteria (1.1LP3, 1.3LP2, 2.1LP4 and 3.2LP3) were identified using 16S rRNA gene product analysis (1,500 bp) on agarose gel electrophoresis (Figure 2). A 16S rRNA gene analysis is the most popular technique on bacterial identification [18]. The 16S rRNA gene, approximately 1,500 bp can determine the difference between bacterial species because it has certain functions. There is a large size and there is very little change in each generation of evolution, so it is easy to classify bacteria species [19]. It is also correlate with the analysis of the phylogenetic tree [20] and more accurate than API kit as previously reported [21].This study found that 5 strains 1.1LP3, 1.3LP2, 1.4LP4, 2.1LP4 and 3.2LP3 were closest to Bacillus thuringiensis, Bacillus subtilis, Staphylococcus sp., Brevibacillus brevis and Micrococcus sp., respectively. These bacteria were isolated from the Nasinuan forest and showed 88-99 % sequence identity to those bacteria found in Italy, India, China, Egypt and Brazil (Table 3). Our results were similar to previous reports, Bacillus subtilis I-4 isolated from oil contaminated effluents of various industries from Pakistan, this strain was produced lipase with an optimum conditions pH 7.0 and temperature 50 °C [22] . Furthermore, our results were similar to previous reports were included Staphylococcus sp. Lp12 isolated from an oil contaminated soil in India [23], Bacillus thuringiensis TS11BP isolated from soil in Bhimavaram , India [24] and Brevibacillus spp. isolated from hot springs located in Gazan area in Saudi Arabia [25]. However, this current work discovered for the first time that Micrococcus sp. isolated from Nasinuan forest, Kantarawichai District, Mahasarakham Province can be a lipase producer.

Figure 2 DNA bands of 16S rRNA gene from PCR-based 16S rRNA analysis on agarose gel (Approximately 1,500 bp)

Table 3 The 5 lipase-producing bacterial strains identified by 16S rRNA analysis.

Isolate Closest relative* Accession No.* %Identity* Origin* 1.1LP3 Bacillus thuringiensis HF584771.1 99 Grapevine root system, BD17-E12 Italy 1.3LP2 Bacillus subtilis MG733629.1 99 Bio aerosols, India APBSWPTB156 1.4LP4 Staphylococcus sp. MH518206.1 98 China CLC-F24 2.1LP4 Brevibacillus brevis KU973528.1 88 Soil, Egypt ABS9 3.2LP3 Micrococcus sp. JQ658425.1 98 Todos os Santos Bay oil HEXBA06 contaminated Mangrove, Brazil *Based on results from BLAST search (www.blast.ncbi.nlm.nih.gov/Blast.cgi) ICoFAB2019 Proceedings | 84

Phylogenetic tree analysis Phylogenetic tree of five lipase-producing bacteria strains showed a relationship between the 16S rRNA genes of five strains Bacillus spp., Staphylococcus sp., Micrococcus sp. and Brevibacillus sp. The evolution of five lipase bacterial strains was constructed by MEGA 7.0 (www.megasoftware.net). In our study finding, B. thuringiensis 1.1LP3 was evolutionarily similar to B. thuringiensis IA RI-IIWP-38 (NCBI accession no. KF054891.1) isolated from the soil of the rhizosphere grown in wheat, India. Staphylococcus sp. 1.4LP4 was similar to Staphylococcus cohnii RCB1038 (NCBI accession no. KT261250.1) isolated from soil sources in the cave ceiling, India. B. subtilis 1.3LP2 was evolved similarly to B. subtilis JM1C6 (NCBI accession no. EU221334.1) isolated from rhizosphere grown in wheat, India. Micrococcus sp. 3.2LP3 was similar to Micrococcus sp. SK13 (NCBI accession no. LC068961.1) isolated from commercial gasoline sources, Korea and Brevibacillus brevis 2.1LP4 was evolved similarly to Brevibacillus brevis YQH20 (NCBI accession no. HQ202569.1) isolated from soil sources, China (Figure 3).

Bacillus thuringiensis IARI-IIWP-38 100

Bacillus thuringiensis 1.1LP3 81

Staphylococcus cohnii RCB1038

81 100 Staphylococcus sp. 1.4LP4

Bacillus subtilis JM1C6

100 Bacillus subtilis 1.3LP2

Micrococcus sp. SK13

100 Micrococcus sp. 3.2LP3

Brevibacillus brevis YQH20

100 Brevibacillus brevis 2.1LP4

0.020

Figure 3 phylogenetic tree of 5 lipase bacterial strains from this study and other 5 reference strains from previous reports

Optimum conditions for lipase production Mazhar et al. [26], isolated lipase-producing bacteria from soil sources in Pakistan and found that the optimum conditions for lipase production and lipase activity of B. subtilis PCSIRNL-39 were temperature at 45 °C and pH 7.0. In addition, Femi-Ola et al. [27] mentioned that optimum conditions for lipase activity of B. subtilis was incubated at 60 °C (pH 8.0). In general, production and function of lipase is found in the pH range of 6.0-10.0 [28]. In this study, the optimum conditions for lipase production were evaluated. Our study found that B. subtilis 1.3LP2 gave the highest lipase activity at 97.78 ± 2.41 U/mL at 37 °C for 72 h compared to the other 2 bacterial species including B. thuringiensis 1.1LP3 and Micrococcus sp. 3.2LP3 (Figure 4A). Therefore, crude extract of B. subtilis 1.3LP2 at 72 h induction time was used to determine optimum conditions of lipase activity. The results showed that crude extract of B. subtilis 1.3LP2 gave the highest enzyme activity of 105.12 ± 12.04 U/mL at pH 7.0 and 37 °C after 30 min (Figure 4B) and highest enzyme activity with 160.92 ± 0.91 U/mL after incubation with 1% olive oil induction at 55 °C, pH 7.0 after curing ICoFAB2019 Proceedings | 85

for 30 min (Figure 4C), respectively. Olive oil used as substrate for screening of lipase producing bacterial has been referred as one of the best inductors and substrate for lipase production [29]. Our results show that B. subtilis 1.3LP2 has optimum temperature at 55 °C (pH 7.0). This results were similar with previous report that investigated the optimum conditions of the Bacillus sp. PD-12 on lipase production, incubation at 55 °C (pH 7.0) using olive oil as substrate and incubated for 1 h [30]. This is very interesting result because they are in agreement with our finding, but crude extract in this study was incubated only for 30 min and gave lipase activity. This is first report of lipase from B. subtilis 1.3LP2 isolated from Nasinuan Forest that offers possible advantage for food enhancement, biofuel production, removal of fat-containing stain and cosmetics ingredients.

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Figure 4 Lipase activity from lipase-producing bacteria (A) Lipase activity from B. thuringiensis 1.1LP3, B. subtilis 1.3LP2 and Micrococcus sp. 3.2LP3 (B) Optimal pH for lipase from B. subtilis 1.3LP2 and (C) Optimal temperature for lipase from B. subtilis 1.3LP2.

ICoFAB2019 Proceedings | 86

Conclusion

This is the first report of identification of lipase-producing bacteria isolated from soil samples in Nasinuan Forest, Nasinuan Sub-district, Kantarawichai District, Mahasarakham Province, Thailand. The 5 strains of lipase- positive bacteria were identified as Bacillus spp., Staphylococcus sp., Brevibacillus sp. and Micrococcus sp., respectively. The Bacillus subtilis 1.3.LP2 has the highest lipase activity after screening through liquid medium containing 1% olive oil as substrate. Optimum conditions for lipase activity were pH 7.0 and 55 °C. This lipase might be applied in a various industry in Thailand such as detergent industry, food (shelf life enhancement), fat and oil industries and cosmetics.

Acknowledgment

This research was financially supported by the National Research Council of Thailand (Year 2018) and awarded to Manatchanok Yotchaisarn.

References

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[17] Spratt DA. Significance of bacterial identification by molecular biology methods. Endodontic Topics. 2004; 9 (1) : 5–14. [18] Janda JM and Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. Journal of Clinical Microbiology. 2007; 45 (9) : 2761–4. [19] Clarridge Jill E. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews. 2004; 17 (4) : 840–62. [20] Bosshard PP, Zbinden R, Abels S, Bo B, Altwegg M and Bo EC. 16S rRNA Gene Sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of non-fermenting gram-negative Bacteria in the Clinical Laboratory. Journal of Clinical Microbiology. 2006; 44 (4) : 1359–66. [21] Iqbal SA and Rehman A. Characterization of lipase from Bacillus subtilis I-4 and its potential use in oil contaminated wastewater. Brazilian Archives of Biology and Technology. 2015; 58 (5) : 789–97. [22] Pogaku P, Suresh A SP and RS. Optimization of lipase production by Staphylococcus sp. Lp12. African Journal Biotechnology. 2010; 9 (6) : 882–6. [23] Badrud Duza M and Mastan DS. Optimization of lipase production from Bacillus thuringiensis (TS11BP), achromobacter xylosoxidans J2 (TS2MCN) isolated from soil sediments near oilseed farm. IOSR journal of pharmacy and biological sciences. 2014; 9 (2) : 66–76. [24] Amjad K. Isolation and characterization of three thermophilic bacterial strains (lipase, cellulose and amylase producers) from hot springs in Saudi Arabia. African Journal of Biotechnology. 2016; 10 (44) : 8834–9. [25] Mazhar H, Abbas N, Hussain Z and Ali S. Optimized production of lipase from Bacillus subtilis PCSIRNL-39. African Journal of Biotechnology. 2017; 16 (19) : 1106-15. [26] Femi-Ola TO, Odeyemi AT, Olaiya BS and Ojo OO. Characterization of lipase from Bacillus subtilis isolated from oil contaminated soil. Journal Applied and Environmental Microbiology. 2018; 6 (1) : 10–17. [27] Kambourova M, Kirilova N, Mandeva R and Derekova A. Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC. Journal of Molecular Catalyst and Biological Enzymes. 2003; 7 (22) : 307-13. [28] Javed S, Azeem F, Hussain S, Rasul I, Siddique MH and Riaz M. Bacterial lipases: A review on purification and characterization. Progress in Biophysics and Molecular Biology. 2018; 132 : 23–34. [29] Praveen, D and Sharmishtha P. Isolation and screening of cellulolytic bacteria from soil and optimization of cellulase production. Journal of Biological Sciences. 2017; 5 (2) : 277–82.

doi:10.14457/MSU.res.2019.17 ICoFAB2019 Proceedings | 88

Screening and Identification of Bacteria that Produce Chitinase Enzymes from Soil in Na Si Nuan Forest, Maha Sarakham, Thailand

Ketsara Suwunnapukdee*, Sirirat Deeseenthum, Vijitra Luang-In, Kedsukon Maneewan

Natural Antioxidant Innovation Research Unit,Department of Biotechnology, Faculty of Technology, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand.

*Corresponding author: E-mail: [email protected]

Abstract:

This research aimed to screen the bacteria that produce chitinase enzymes from soil in Na Si Nuan forest located at Kantarawichai District, Maha Sarakham Province, Thailand. The conservation project in response to Plant Genetic Conservation Project under the Royal initiative of Her Royal Highness Princess Maha Chakri Sirindhorn has occupied the community forest of 19.2 ha in this study. Isolation of bacteria was performed from soil on chitin agar. Our results were found two isolates of chitinase-positive bacterial strains. The isolate of 3.2 CT1 was showed the highest of halo: colony ratios with 4.5. The morphology isolate of 3.2 CT1 is gram negative, and rod shaped. The optimum temperature for chitinase activity was 40 ˚C with enzyme activity of 0.40 ± 0.01 U/mL and given a specific enzyme activity of 0.49 ± 0.01 U/mg. protein. The optimum pH was 6.0 with activity of 0.70 ± 0.01 U /mL. This chitinase may be used for agricultural purposes, such as plant pathogen inhibition and as insecticide for insect pest control.

Keywords: Chitinase enzymes, Bacteria, Soil, Na Si Nuan Forest, Agriculture.

Introduction

The conservation forest area, which is a community forest in Na Si Nuan, is located at Maha Sarakham, Thailand, has occupied 19.2 ha. The nature of the soil in this forest area is moderately saline and strongly saline, which was found in the basin area [9]. In addition, many species of edible and inedible mushrooms were also found in this forest. Local people [4] in the nearby area often come to use the bioresources for foods. Chitinase enzyme is derived enzyme from many types of microorganisms. Qualified to decompose chitin which is a component of fungal cell wall. Chitinase is possible that it can be used in combination with anti-fungal drugs to increase the effectiveness of the drug in inhibiting and destroying fungi. Chitinase enzymes in bacteria were found Aeromonas sp. 10s-24 [2][13] and Bacillus stearothermophilus CH-34 [10] fungus also found chitinase enzymes is Trichoderma harzianum [14] Aspergillus cameus [1][12]etc. The mechanism of chitinase found that living organisms produce chitinase enzymes to protect themselves against the invasion of pathogens. Chitinases hydrolysis of chitin, linear homopolymer of p-1,4-linked N-acetyl-D- glucosamine residues. This polysaccharide is attendant in the cell wall of fungal and in exoskeleton of insects. In addition to control of phytopathogens fungal other different applications of chitinase such as mosquito control, Estimation of fungal biomass, target for biopesticides and morphogenesis have been discovered. Biocontrol of plant pathogens furnish an interesting choice for handle of plant disease without the negative impact of chemical fungicides that can cause environmental pollution and usually expensive, and may cause disease resistance [16]. This research purpose this to study of the chitinase enzyme bacteria screening and to identify from soil in Na Si Nuan forest and determine the activity of chitinase enzyme that can inhibit the fungi that destroy plants.

Materials and methods

Soil sampling Soil samples were collected from Na Si Nuan forest, Maha Sarakham province, Thailand (Fig. 1) at a depth of 10-15 cm from the surface or Rhizosphere area [5][11]. Then the soil samples were placed in ICoFAB2019 Proceedings | 89

sterile polythene bags and kept at room temperature until analysis. The physical properties of the soil were analyzed by measuring electrical conductivity and pH.

Figure 1 Soil sampling area in Na Si Nuan forest in Maha Sarakham province, Thailand.

Isolation of bacteria from soil samples Ten grams of soil were weighed, and dissolved in 90 mL of sterile 0.85% NaCl. Soil samples were diluted to obtain serial dilutions at 10-2- 10-6. After that, 0.1 mL of solution was spread on chitin agar [(g/100 mL); 3% (w/v) colloidal chitin; 0.1% KH2PO4; 0.05% MgSO4.7H2O; 50 mM sodium phosphate buffer, (pH 6.0)] [15] and incubated at 37 °C for 3 days. Iodine solution was poured over the agar plates and cleared zones. It was observed around colony in the presence of bacterial chitinase that can hydrolyze chitin agar. These colonies of bacteria that produce chitinase were purified on LB agar using streak plate method and cell morphology, which was expressed under microscope (1,000 X).

Halo: colony ratio Bacterial isolates were cultured in LB media for 18 h. A toothpick was used to touch the culture and the point inoculation on a solid agar was performed, incubated at 37 °C for 2 days. The ratio of halo: colony was measured as the radius of clear area over the radius of the colony.

Bacterial chitinase activity Select of pure isolates of chitinase-producing bacteria [6] were cultured and adjusted at 108 CFU/mL equivalent to 0.5 McFarland were inoculated into 10 mL chitin liquid medium (100 ml): 3% w/v colloidal chitin [0.1% KH2PO4; 0.05% MgSO4.7H2O; 50 mM sodium phosphate buffer (pH 6.0)]. They were incubated at 37 ˚C with shaking at 150 rpm for 24, 48 and 72 h, and centrifuged at 12,000 g for 20 min at 4 ˚C as well as kept the supernatant (extracellular enzyme) reducing volume, for chitinase enzyme assay following a method of Mandana Zarei et al. [7][8]. The crude enzyme extracted with highest chitinase activity was determined [3].

Enzyme stability for chitinase enzyme activity pH The crude extract of the highest chitinase activity from above analysis was used to determine its optimum pH for activity using various pHs in suitable buffers (0.1 M sodium phosphate buffer: pH 6.0, 7.0 and 0.1 M Tris-HCl buffer: pH 8.0, 9.0, 10.0 and 11.0) in a range of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 as well as incubated at 37 °C for 30 min. Then, chitinase activity was recorded (3 repeated).

Temperatures The crude extract of the highest chitinase activity with optimum pH was used to determine its optimum temperature for activity using various temperatures at 45, 55, 65, 75, 85, 95 ˚C for 0, 5, 10, 15 and 30 °C for 30 min and lipase activity was recorded (3 repeated).

Statistical analysis Measurements were obtained in triplicates as means ± standard deviation (SD). Statistical analysis was performed using One-way analysis (One-way ANOVA) of variance and Duncan Multiple’s Range Test by the software SPSS package version 17.0 at P < 0.05.

Results and discussion

Chitinase can also be detected in human blood and possibly cartilage. As in plant chitinases, this may be related to pathogen resistance, which is derived enzyme from many types of microorganisms. Qualified to ICoFAB2019 Proceedings | 90

decompose chitin which is a component of fungal cell wall. In addition control of phytopathogens fungal other different applications of chitinase such as mosquito control, estimation of fungal biomass, target for biopesticides and morphogenesis have been discovered. In this study, the pH and electrical conductivity of soils were recorded. These factors are related to salinity of soil samples collected from Na Si Nuan forest, Kantarawichai district, Maha Sarakham province. (Table 1).

Table 1 Sources of soil samples and physical property analysis of soil.

Surrounding EC Temperature Area Longitude/Latitude pH (µS/cm) (°C) 1.1 N 16 20 17.945/ E 103 12 Under tree Azadirachta indica 36.2 31.6 8.69 14.731 1.2 N 16 20 17.945/ E 103 12 Under tree Terminalia chebula 19.9 31.0 7.72 14.731 1.3 N 16 20 6.619/ E 103 11 Unknown 22.3 31.0 7.06 0.985 1.4 N 16 20 23.547/E 103 12 Tectona grandis 20.9 31.0 6.40 35.244 2.1 N 16 20 36.882/ E 103 12 The open space was found around 28.8 31.0 6.27 32.315 tree Ageratum conyzoides 2.2 N 16 20 36.863/ E 103 12 Dipterocarpus intricatus forest 27.7 30.9 5.77 32.416 2.3 N 16 19 39.152/E 103 12 Eucalyptus nearby Pomegranate 184 31.0 4.49 33.655 2.4 N 16 20 37.157/E 103 12 hahinii sincl 24.1 31.0 5.39 33.579 3.1 N 16 20 36.44/E 103 12 Under the Mimosa pudica 23.4 31.0 5.66 34.072 3.2 N 16 20 35.406/E 103 12 Catunaregam spathulifolia 22.4 31.4 5.46 35.157 3.3 N 16 20 35.405/ E 103 12 Near tree anamensis Hance 26.3 32.0 5.40 35.154 3.4 N 16 20 35.425/E 103 12 Under tree Polyalthia debilis Finet & 24.2 33.8 5.26 35.178 Gagnep 3.5 N 16 20 36/E 103 12 36 Russula virescens area 23.5 32.5 5.39 3.6 N 16 20 36/E 103 12 36 Soil surface found near Russula 30.2 31.3 5.25 virescens

Separate bacteria that chitinase enzyme In this study, the pH and electrical conductivity of soils were recorded. These factors are related to salinity of soil samples collected from Na Si Nuan forest, Kantarawichai district, Maha sarakham province. The top 2 bacterial isolates were collected and isolated with highest lipase activity based on highest halo : colony ratios from the soil at locations 2.2 and 3.2 with a pH range 6.0 In this study, two chitinase-positive isolates, selected of pure isolates of chitinase-producing bacterial isolates (3.2CT1), showed the highest degrading zones on 3% chitin agar with different halo : colony ratios. Four bacterial representative strains showed similar colony morphologies and appeared to be gram-negative and rod-shaped. The results showed that halo: colony ratios ranged from 4.5. (3.2 is the soil, the point 3.2, PS is chitinase, 1 is the first bacteria)

2.2 CT1 (G-, Cocci shaped) 3.2 CT1 (G-, rod shaped) Halo : Colony ratio = 2.74 Halo : Colony ratio = 4.50

Figure 2 chitinase enzyme bacteria isolates

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Chitinase production The results of the bacterial dissolution of chitinase enzyme bacteria with the highest halo value ratio 3.2 CT1 had the highest bacteria chitinase enzyme activity. After induction bacteria in chitin medium containing colloidal chitin is substrate incubate at 37 ° C for 24, 48 and 72 h. Isolates 3.2 CT1 have enzyme specific activity at 0.32 ± 0.01 U/mg protein after incubation at 37 °C for 72 h (Fig. 3).

0.35 0.3 0.25 0.2 0.15

0.1 protein) 0.05 0 4 hr. 48 hr. 72 hr.

Specific Specific enzymeactivity (U/mg Time 3.2 CT1

Figure 3 Specific bacteria activity of chitinase enzyme producing by 3.2 CT1

Chitinase optimization In this study, the optimum conditions for chitinase enzyme were evaluated. Our studied found 3.2 CT1 gave the highest lipase activity at 0.32 ± 0.01 U /mL at 37 °C for 72 h after incubation with 3% colloidal chitin induction at 40 °C, pH 6.0 after curing for 30 min (Fig. 4), and highest enzyme activity with 0.70 ± 0.01 U/mL. It has been mentioned that colloidal chitin was used as substrate for screening of chitinase enzyme bacterial and has been referred as one of the best inductors

Optimum temperature Substrate for chitinase enzyme our results show that isolate 3.2 CT1 its optimum at 40°C cultured in chitin medium, incubated for 72 h. pH 7, various temperatures from 30, 40, 50, 60, 70 and 80 °C for 30 min. (Fig. 4)

0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2

protein) 0.1 0.1 0 0 protein)

20 30 40 50 60 70 80 Enzyme activity (µmol/mlactivity Enzyme

Temperature (˚C) Specific enzyme activity (U/mg activity enzyme Specific Enzyme activity (µmol/ml protein) Specific enzyme activity (U/mg protein)

Figure 4 Optimum temperatures

pH pH determination in various pH buffers were studied from pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively. Phosphate buffer at various pH incubated at 40 °C for 30 min. It was found that pH 6 isolates 3.2 CT1 had ICoFAB2019 Proceedings | 92

the highest enzyme activity value of 0.70 ± 0.01 U/mL, while the enzyme specific activity value was 0.70 ± 0.01 U/mg protein (Fig. 5)

0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0

Enzyme activity (µmol/ml protein) (µmol/mlactivity Enzyme 3 4 5 6 7 8 9 10

pH protein) (U/mg activity enzyme Specific

Enzyme activity (µmol/ml protein) Specific enzyme activity (U/mg protein)

Figure 5 Optimum pH

Conclusions

The conservation forest area, which is a community forest in Na Si Nuan, Maha Sarakham, Thailand, has occupied 19.2 ha. The nature of the soil in this forest area is moderately saline and strongly saline was found in the basin area. In addition, many species of edible and inedible mushrooms were also found in this forest. Local people in the nearby area often come to use the bioresources as foods from this forest. Isolation of bacteria was performed from soil on chitin agar. The results found 2 isolates of bacteria that produce chitinase enzymes. The results showed that 2 isolates select with the highest halo: colony ration is 3.2 CT1 at 4.5 for test the morphology of bacteria is G-, and rod shaped. The optimum temperature for chitinase activity was 40 ˚C with enzyme activity of 0.40 ± 0.01 U/ml and specific enzyme activity was 0.49 ± 0.01 U/mg protein. The optimum pH was 6.0 with activity of 0.70 ± 0.01 U /ml.

Acknowledgement

This research was financially supported by the Plant Genetic Conservation Project under His Majesty the King's initiative Princess Maha Chakri Sirindhorn (Year 2018) awarded to Ketsara Suwunnapukdee.

References

[1] Abdel Naby MA, Mohamed A, Kwon DY. (1992). The production of xylanase and Beta-xylosidase by Aspergillus niger NRC 107. App Microbiol Biotechnol. 20:543–550. [2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410. [3] APHA-AWWA-WEF (1995) Standard Methods for the Examination of Water and Wastewater. 19th Edition, Washington DC. [4] Arbhabhirama, A., D, Phantunvanit, J.Elklngton, Phaitoon Jngkasuwan. (1988). Thailand Natural Resources Profite. Oxford University Press. Oxford, New York, 431 pp. [5] Arnoldus, H.M.J. (1977).Predicting Soil Loss Due to Sheet and Rill Erosion. Food and Agriculture Organization (FAO) Conservation Guide Volume I, p.88-98 [6] Chang, W. T., Chen, C. S., and Wang, S. L. (2003) An antifungal chitinase produced by Bacillus cereus with shrimp and crab shell powder as a carbon source, Curr Microbiol 47, 102-108. [7] Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J. (1998), “Protein measurement with theFolin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951. ICoFAB2019 Proceedings | 93

[8] Mendpara J., Parekh V., Vaghela S., Makasana A., Kunjadia P.D., Sanghvi G., Vaishnav D. and ‘Dave G.S. (2013), Isolation and characterization of high salt tolerant bacteria from agricultural soil. European Journal of Experimental Biology, 3(6):351-358 [9] Norman, B.W. (1984), Report on the Comparison of New and Old Development Areas.Thai- Australia-World Bank Land Development Project, Chiang Mai, Thailand 339. [10] Sakai Y, et al. (1994) High-level ATP production by a genetically-engineered Candida yeast. Biotechnology (N Y) 12(3):291-3 [11] Smith, R.F and R. van den Bosch. (1967), Integrated ontrol. Pest Control. Edited by W. Kilgoreand R. doutte. Academic Prees, 298-299 p. [12] Tamura, K., Nei, M. (1993). Estimation of the numbr of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Bioloy and Evolution, 10, 512-526. [13] Ueda, M. and Arai, M (1992) Purification and some properties of chitinase from Aeromonas sp. No. 10S-24. Biosci Biotechnol Biochem 56, 460–464. [14] Ulhoa, C. J. and Peberdy, J. F. (1992) Purification and some proper ties of the extracellular chitinase produced by Trichoderma harzianum. Enzyme Microb. Technol., 14, 236-240. [15] Vincy, V., Vinu, S.M., Viveka, S., Mary, V.T., Jasmin, B.R. (2014), Isolation and characterization of chitinase from bacteria of Shrimp pond. Euro. J. Exp.Bio, 4(3): 7882. [16] Wang, R.F., Cao, W.W., and Cerniglia, C.E. (1996), PCR detection and quantitation of predominantanaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol, 62: 1242-124

doi:10.14457/MSU.res.2019.18 ICoFAB2019 Proceedings | 94

Random Mutagenesis of Aspergillus sclerotiorum PSU-RSPG 178 for Improvement a Lovastatin Production

Supawan Meena1, Wilaiwan Chotigeat1,2, Yaowapa Sukpondma3, Souwalak Phongpaichit4, Vatcharin Rukachaisirikul3 and Monwadee Wonglapsuwan1,2*

1Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand 2Center for Genomics and Bioinformatics Research Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand 3Department of Chemistry, Faculty of Science, Prince of Songkla University, Songkhla, 90112, Thailand 4Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Aspergillus sclerotiorum PSU-RSPG 178 is a natural fungus producing lovastatin which is commonly used as cholesterol reduction medication. Random mutagenesis method was used to increase the production of lovastatin via UV radiation (255 nm) with a different time of exposure at 30, 45 and 60 min, fifty colonies appeared and screened for lovastatin production by agar plug and Neurospora crassa bioassay. The results showed seven colonies had greater diameter of clear zone than that of wild type. After analysis of the quantity of lovastatin by HPLC, the mutant colonies from UV mutation at 30 min provided the maximum amount of lovastatin at 354.69 mg/L compared with that from the control at 113.4 mg/L. However, after sub-culture until 3rd generation, the potential in lovastatin production declined. This evidence indicated that UV mutagenesis could be used for improving the lovastatin production.

Keywords: Lovastatin, Aspergillus, Aspergillus sclerotiorum PSU-RSPG 178, random mutagenesis

Introduction

A. sclerotiorum PSU- RSPG 178 is a fungus that produced lovastatin. It was isolated from soil sample collected from the Plant Genetic Conservation project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand [1] Lovastatin (C24 H36 O5) is a secondary metabolite produced from fungi such as Monascus purpureus, Monascus ruber, Monascus pilosus, Monascus pubigerus, Monascus vitreus and Aspergillus terreus [2]. The important function of lovastatin known as a reducer of plasma cholesterol level by competitive inhibition of 3-hydroxy 3-methylglutaryl Coenzyme A (HMG-CoA) reductase which is the rate-limiting enzyme in cholesterol biosynthesis [2] and also found to be effective in treatments of hypercholesterolemia, Parkinson’s disease, bone fracture, atherosclerosis, Alzheimer’s disease, cerebrovascular disease and cancer [3]. Lovastatin consisted of two forms including lactone and acid form which is soluble in water. Ninety percent of lovastatin fermentation exists as β-hydroxy acid form which can be function as an anti-fungal exhibitor against N. crassa [4]. Therefore, agar plug and N. crassa bioassay was performed to screen for lovastatin production. Due to the low amount of lovastatin production from natural A. sclerotiorum PSU-RSPG 178, in this study, random mutagenesis was performed by using UV radiations method with a different time intervals (30, 45 and 60 min) to enhance the production of lovastatin.

Materials and methods

Microorganism A. sclerotiorum PSU-RSPG 178 was cultured on potato dextrose agar (PDA) and incubated at 28º C for 7 days.

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Strain improvement by random mutagenesis The spore was collected in 0.85% NaCl containing 2% Tween 20 and shaken for 10 min, then it was counted using hemocytometer and further diluted with PBS (pH 7.4) to 1x106 spores/mL followed by spread on PDA. After that, plates were exposed to UV radiation at 255 nm with a different time interval (30, 45 and 60 min) at the distance of 5 cm from the UV source and incubated at 28ºC for 5 days.

Screening of lovastatin by using Neurospora crassa bioassay The colonies were grown on PDA to screening the lovastatin producing fungi by using N. crassa bioassay [5]. The colonies with the biggest in diameter of clear zone were selected to culture on potato dextrose broth (PDB) at 28ºC for 14 days, and then extraction by ethyl acetate was performed.

Cultivation and extraction A. sclerotiorum PSU-RSPG 178 and mutant colonyies were culture on PDA at 28 ºC for 7 days. Five pieces (0.5×0.5 cm2) of mycelial agar plugs were incubated in PDB (potato dextrose broth) 150 ml in 250 ml Erlenmeyer flask at 28º C for 14 days. After incubation, the mycelia and medium were separated by filtration. The medium was extracted with an equal volume of ethyl acetate and the cells were cut into small pieces and incubated with an equal volume of methanol at room temperature for 3 days. The medium was extracted twice with ethyl acetate (2x300 ml). The organic layer was evaporated by an evaporator to dryness under a reduced pressure (BE). The mycelia were separated from methanol by filtration. The methanol was removed by evaporator and hexane: H2O (1:1) 200 ml, was added. Then, upper layer was evaporated by an evaporator to dryness under a reduced pressure (CH) and the lower layer was extracted twice with ethyl acetate (2x300 ml) and also evaporated to dryness (CE). All of crude extract were determined by Nuclear Magnetic Resonance (1HNMR).

Analysis qualitative of lovastatin by High-performance liquid chromatography (HPLC) Estimation of lovastatin by HPLC system (Agilent 1200 series DAD using an ACE ® Generix 5 C-18 column) was performed by using acetonitrile and 0.1 % phosphoric acid in a ratio of 60:40 (V/V) as a mobile phase. Sample was injected by flow rate adjustment at 1.0 ml/min and further detected at 238 nm. The identity of sample was confirmed by lovastatin standard.

Results and discussion

Genetic improvement Genetic improvement is one of the rising approaches for increasing the production of secondary metabolites by UV radiation method. Culture plates with the mutant colonies survival rate at less than 90% compared with control plate were selected (Fig.1).

A

Figure 1 Comparison of mutant strains of A. sclerotiorum PSU-RSPG 178 grown on PDA between normal strain with irradiated (A) and radiated to UV radiation (B).

Screening and isolation of lovastatin production through bioassay method The results of bioassay revealed seven colonies (Fig. 2) out of fifty showed the bigger size in diameter of zone of inhibition (cm) than control. Then, those seven colonies were cultured on PDB for 14 days and further extraction for lovastatin by ethyl acetate.

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Figure 2 Bioassay of samples from different fungal mutants. Clear zone of selected seven colonies were showed by red arrow (E = negative control, C = A. sclerotiorum PSU-RSPG 178) mutant colony UV07.30_13 (A), UV07.30_14 and UV07.30_15 (B), UV07.30_18 (C), UV07.30_22 (D), UV07.45_33 and UV07.60_35 (E)

Analysis qualitative of lovastatin by HPLC To ensure whether the maximum zone of inhibition by mutant colony UV07.30_22 on bioassay plate was appeared due to lovastatin activity, crude extract was injected to HPLC for quantitatively confirming the amount of lovastatin in the fungal extraction. The resemblance in retention time (RT) of peak shown in chromatogram of control (A. sclerotiorum PSU-RSPG 178) and mutant colony UV07.30_22 was similar at 16.6 min (Fig. 2A and B). The results found the maximum amount of lovastatin by N. crassa bioassay was 354.69 mg/L (UV07.30_22) followed by 297.68 mg/L (UV07.30_18), 296.02 mg/L (UV07.45_33), 221.84 mg/L (UV07.60_35), 186.33 mg/L (UV07.30_13), 175.6 mg/L (UV07.30_15), 144.93 (UV07.30_14) and 113.4 mg/L (control) (Fig. 4). This result indicated that UV mutagenesis could improve the lovastatin yield.

Figure 3 High-performance liquid chromatography chromatogram of A. sclerotiorum PSU-RSPG 178 (A) and mutant colony UV07.30_22 (B) ICoFAB2019 Proceedings | 97

Figure 4 Concentration of lovastatin produced by the fungal A. sclerotiorum PSU- RSPG 178 and mutants

1 Analysis qualitative of lovastatin by Nuclear Magnetic Resonance ( HNMR) Qualitative of lovastatin was observed by 1HNMR spectrum. Both the 1HNMR spectrum of A. sclerotiorum PSU- RSPG 178 and mutant colony UV07.30_22 showed the similar signal of 1HNMR peak. This evidence indicated that the mutant colony UV07.30_22 could produced lovastatin (Fig.5).

Figure 5 1H NMR spctrum of A. sclerotiorum PSU- RSPG 178 (A) and mutant colony UV07.30_22 (B).

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Conclusions

Considering these references as discussed above, it could be concluded that A. sclerotiorum PSU- RSPG 178 UV07.30_22 exposed to UV radiation could produce an increasing of lovastatin yield at 354.69 mg/L suggesting the physical mutagenesis (UV random mutation) can probably be used for lovastatin yield improvement.

Acknowledgements

The authors thank acknowledge the NSTDA Chair Professor grant of the Crown Property Bureau and the National Science and Technology Development Agency to Professor Dr. Vatcharin Rukachaisirikul and Center for Genomics and Bioinformatics research, Department of Molecular Biotechnology and Bioinformatic, Department of Microbiology and Department of Chemistry, Faculty of science, Prince of Songkla University, Hatyai, Songkhla, Thailand. And thank you scholarship from Research Grant for Thesis for support this research.

References

[1] P. Phainuphong, V. Rukachaisirikul, S. Saithong, S. Phongpaichit, K. Bowornwiriyapan, C. Muanprasat, C. Srimaroeng, A. Duangjai, J. Sakayaroj, J. Nat. Prod. 79 (2016) 1500–1507. [2] B. Janani, G. Saibaba, G. Archunan, K. Vidhya, J. Karunyadevi, J. Angayarkanni, Asian J. Pharm. Clin. Res. 10 (2017) 258–262. [3] R.S. Upendra, P. Khandelwal, Int. J. Pharm. Pharm. Sci. 8 (2016) 163–167. [4] V.K. Nigam, Asian J. Biomed. Pharm. Sci. 05 (2015) 24–29. [5] M.S. Kumar, P.M. Kumar, H.M. Sarnaik, A.K. Sadhukhan, J. Microbiol. Methods 40 (2000) 99–104.

doi:10.14457/MSU.res.2019.19 ICoFAB2019 Proceedings | 99

Genetic Variation among Thai Dugong (Dugong dugon) Populations from Cytochrome C Oxidase Subunit 1 DNA Sequence Data

Kongkiat Kittiwatanawong1 and Nattapong Srisamoot 2*

1Phuket Marine Biological Center, Phuket 83000 2Department of Biotechnology, Faculty of Agricultural Technology, Kalasin University, Kalasin 46000

*Corresponding author’s e-mail: [email protected]

Abstract:

Dugong (Dugong dugon) was classified as vulnerable and currently considered rare over most of this range. The population of dugong in Thailand was found in fragmented habitats along the Thai coast. Genetic studies were used in this study to determine the variation of cytochrome c oxidase subunit 1 (COI) DNA sequence of ten dugong samples from Krabi, Phuket, Trang and Satun Province. The DNA extraction result showed that the samples stored in ethanol can maintain the integrity of the genetic material for a long time. The length of COI sequence was 794 to 859 bp and the average length was 823.4 bp. All COI sequences in this study were identical to GenBank accession numbers AY075116.1 which have an average of 91.34%. The lowest genetic distance (0.005) was between dugong 3 and dugong 4, while the highest (0.163) was between dugong 6 and dugong 10 with an average of 0.070. The low genetic distance demonstrated that the gene flow between the dugong population in each area still occurred. The dendrogram constructed from COI sequence comparisons using the Maximum Likelihood method based on the Tamura- Nei model divided ten dugong samples into three subgroups. The major group comprised of seven dugong samples which were collected around Koh Libong and the coastal areas of Trang Province. The clustering corresponds to the area with the largest and abundant seagrass.

Keywords: Dugong, Dugong dugon, Genetic variation, Cytochrome C oxidase, COI

Introduction

The dugong (Dugong dugon) is unique among mammals in that it is the only fully marine herbivore. It is a member of the genus Dugong, which is the only extant member of the family Dugongidae [1]. The Dugongidae is one of two families in the order Sirenia, the other one is the Trichechidae. All members of the Trichechidae, Trichechus manatus, T. inunguis and T. senegalensis, require fresh water to survive while the dugong is exclusively marine [2]. Dugongs were classified as the most endangered among CITES-listed according to Appendix I of CITES (Convention of International Trade in Endangered Species of Wild Flora and Fauna) [3]. The main causes of the decreasing in dugong populations are inadvertent capture and intentional collision with ships, habitat degradation, hunting for meat or medicinal purposes and slow rate of reproduction. Thailand has long coastlines of some 2,600 km facing the South China Sea to the east and the Andaman Sea on the west side [4] that is rich in biodiversity. Dugongs were classified as wildlife preservation and were protected in accordance with the Thai Fisheries Act since 1947 [5]. Although the dugong hunting is illegal, the population of both the Andaman Sea and the Gulf of Thailand had declined drastically. In 2017, it is believed that there are no more than 200 dugongs in the Thai waters. Most of them, 130-150, are found in Hat Chao Mai National Marine Park and Mu Ko Libong Non-Hunting Area in Trang province where seagrass is still fertile. Moreover, about 15 are found around Ko Samet islands of Rayong and some areas in Chon Buri, Chanthaburi and Trat provinces, and about 10 others in the Chaiya Bay of Surat Thani [6]. Therefore, data supporting and developing conservation strategies are extremely important to the dugong population in Thailand. Currently, bioinformatics is essential for the manipulation of biological data involving genetic resources conservation, remodeling phylogenetic, assessment of gene dispersal and search of genomic markers. These genetic techniques can infer parameters like the population structure and movement of their genes. An understanding of the levels of genetic diversity within a population will allow for an understanding of the genetic stability of the population. Higher levels of genetic diversity may allow populations to adapt to environmental changes more efficiently than populations with little genetic diversity [7]. The resulting non-adaptive variation will lead to the greater susceptibility of a population to further environmental change [8]. ICoFAB2019 Proceedings | 100

The analysis of sequence data is one of the bioinformatics used for population studies and measuring genetic diversity is. The most commonly gene used for these approaches is the cytochrome c oxidase gene. The cytochrome c oxidase plays a central role in the metabolism of eukaryotic aerobic organisms. It is a key enzyme in the electron transport chain which located in the inner mitochondrial membrane. It consists of several subunits, and the catalytic cytochrome c oxidase subunit 1 (COI) is encoded in the mitochondrial genome [9]. Due to its mutation rate is fast enough to distinguish closely related species, the COI gene is suitable for the comparative studies of genetic variation among dugong populations. In this study, we amplified and sequenced the COI gene of Thai dugong populations for its genetic variation and phylogenetic analysis. The information obtained may be used to consider that populations of dugong found in Thailand should be managed as connected or as separate stocks and additionally elucidate population structure of dugongs in the region.

Materials and methods

Dugong tissue samples (N = 10) were collected at four localities in the Andaman Sea (Krabi, Phuket, Trang and Satun) by responding to reports of dead specimens (Table 1). Tissue samples were stored in ethanol and then maintained at the Phuket Marine Biological Center from 2010-2017. Genomic DNA was extracted from dehydrated tissue by phenol-chloroform method with minor modification. This protocol starts with 10 mg of tissue being soaked and washed three times with distilled water. Then the tissue was ground in the isolation buffer (0.075 M NaCl, 0.025 M EDTA, 0.5% SDS) and incubated with protease K at 65oC overnight. Chloroform was then used to extract the proteins from the digested tissue. DNA precipitate was washed two times with 70% ethanol. The remaining DNA pellet was air-dried and resuspended in TE buffer (10 mM Tris, 1 mM EDTA).

Table 1 Dugong samples in this study, their observation area and date.

Sample Collection Area Latitude Longitude Collection date No. 1 Ban Phra Muang, Naklua, Kantang, Trang 7.2977300 99.439820 Jan 22, 2010 2 Ao Prao, Koh Libong, Kantang, Trang 7.2634300 99.409940 Jan 24, 2010 3 Koh Nok, Koh Libong, Kantang, Trang 7.2660500 99.464090 Nov 14, 2010 4 Toh Chai Cape, Koh Libong, Kantang, Trang 7.2827500 99.384910 Mar 26, 2011 5 Koh Luk Mai, Koh Libong, Kantang, Trang 7.2514600 99.455900 Apr 11, 2011 6 Thungbulang, Thungbulang, Thunwa, Satun 7.0249900 99.672680 Sep 11, 2011 7 Ao Prao, Koh Libong, Kantang, Trang 7.2634300 99.409940 Dec 23, 2011 8 Yamu Cape, Pa Klok, Maung, Phuket 7.9922600 98.422170 Nov 6, 2012 9 Had Yao Beach, Koh Libong, Kantang, Trang 7.3122310 99.387595 Jul 7, 2013 10 Loh Dalum Beach, Ao Nang, Maung, Krabi 7.7405610 98.770677 Jun 18, 2017

A partial region of COI was amplified using a set of specific primers with sequences as follows: COI-F3, 5’-CCT GCA GGA GGA GGA GGA GAG CC-3’ and COI-R3, 5’-AGT ATA AGC GTC TGG GTA GTC-3’ [10]. The total volume of 25 uL contains 1X PCR buffer, 0.4 mM dNTP, 2.0 mM MgCl2, 0.25 uM forward primer, 0.25 uM reverse primer, 0.5 unit Taq polymerase (Vivantis) and 20 ng DNA template. PCR amplification was performed with the following program: pre-PCR incubation at 95oC for 15 min, 35 cycles of 95oC for 20 s, annealing at 60oC for 45 s, and extension at 72oC for 30 s, with a final extension at 72oC for 5 min. PCR products were electrophoresed in 1.5% agarose gels in 1 × TAE buffer. The gel was stained with ethidium bromide for 30 minutes and then visualized under UV light. For sequencing, PCR products were excised from a 1.5% agarose gel and purified using a PureLink Quick Gel Extraction Kit (Invitrogen) following the manufacturer's instructions. The purified DNA fragments were sequenced on an ABI Prism 3730 automatic sequencer (Gibthai Co., Ltd) using both forward (COI-F3) and reverse primers (COI-R3). Genetic variations were estimated from COI sequences by using the Maximum Likelihood method based on the Tamura-Nei model [11]. The dendrogram was drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There was a total of 891 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 software [12].

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Results and discussion

The extracted DNA fragment by the modified phenol-chloroform method has a large genomic band and locate at the same size as the genomic DNA of the Lambda / HinDIII marker (Figure 1A). Spectrophotometer results showed that the quality of the extracted DNA was good and can be used to amplify the COI gene by polymerase chain reaction (data not showed). This indicates that the samples stored in ethanol can maintain the integrity of the genetic material for a long time, even if collected from dead samples. Amplification of the COI gene located at approximately size 800 bp on 1.5% agarose gel (Figure 1B) in all the dugong accessions used in this study. The sequencing was carried out 3 times for each sample. The results showed that the nucleotide sequence of most samples was not different. Some examples are slightly different (data not showed). This difference occurs only at the end of the nucleotide sequence, which may be caused by the limitations of the nucleotide sequence analysis system. The sequencing errors can occur from a large number of nucleotides being sequenced and higher error rate at the ends of sequenced [13]. The similarity of these COI sequences compared to the GenBank database by Blastn found that all of them were identical to accession number AY075116.1 with 90.56 -91.59 % Identical (Table 2). These accession number was the Dugong dugon mitochondrion, complete genome.

A L 1 2 3 4 5 6 7 8 9 10 B M 1 2 3 4 5 6 7 8 9 10 5,148 bp 2,000 bp

2,027 bp 600 bp

Figure 1 Extracted DNA (A) and amplification products of the COI gene (B) from ten samples of Thai dugong. A 1-10 number of dugong accessions were shown in Table 1. The dash arrow indicates genomic DNA band. The arrow indicates COI amplified fragment. L was Lambda / HinDIII marker and M was a 1kb DNA ladder marker.

The COI sequences of the ten dugong samples were aligned and resulted in 891 positions with 466 variable sites (52.30%) as show in Figure 2. The alignment result indicated that each sample has a different length of the COI sequence. The length was 794 to 859 bp and the average length was 823.4 bp. The COI gene has an average CG content of 39.05%. The highest was sample dugong No. 2 and the lowest was dugong No. 1 with CG content of 40.13% and 38.07%, respectively. The AT content of the COI sequence is high across the dugong sample, it does not have a simple explanation but often use the transcription hypothesis of codon usage as an explanation instead [14]. The cell has a high availability of ATP and relatively low availability of the other three rNTPs, so the transcription efficiency can be increased by using A in the third codon position of the protein encoding gene [15]. The third codon position, the usage tends toward A or U because not only do they pair well with optimal codons, they also cloud pair with other synonymous codons. On the other hand, if the third codon base is G or C, they cannot completely pair with optimal codons [14]. The genetic distance matrix is showed in Table 3. The average genetic distance between the ten dugong samples from the Kimura 2-parameter model was 0.070. The minimum value was between dugong 3 and dugong 4, while the maximum value was between dugong 6 and dugong 10 with genetic distance value 0.005 and 0.163, respectively. The minimum and maximum genetic distance between the specimens of the dugong corresponds to the location of the collecting area. Distance from the location of dugong 6 to dugong 10 is approximately 125 kilometers apart, while dugong 3 and dugong 4 are only about 9 kilometers away. Close distance makes it easy to communicate with each other. The gene flow between populations is always occurring, resulting in minor variations in population genetics. Gene flow within a population can increase the genetic variation of the population, whereas gene flow between genetically distant populations can reduce the genetic difference between the populations [16]. For dugong 6 and dugong 10, geographical isolation could increase opportunities for genetic divergence. A similar result was reported by Bushell (2013) [17], which found a strong genetic differentiation between the dugong populations form the Gulf of Thailand and the North Andaman Sea and between the Gulf of Thailand and Trang Province through ICoFAB2019 Proceedings | 102

pairwise comparison of microsatellite alleles. However, there is no significant difference between Trang Province and the North Andaman Sea populations.

Figure 2 Sequence comparisons of COI sequence from ten dugong samples. Dots (.) indicate the nucleotides and dashes (-) are introduced to gap.

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Table 2 The length and nucleotide composition of the COI gene of the dugong specimens in this study and their identical GenBank accession number.

Base Content Identical Length Sample No. GenBank % Identical (bp) A C G T %GC Acc. No. Dugongs 1 838 236 191 128 283 38.07 AY075116.1 91.59% Dugongs 2 795 207 187 132 269 40.13 AY075116.1 91.59% Dugongs 3 830 228 185 133 284 38.31 AY075116.1 91.59% Dugongs 4 821 223 192 131 275 39.34 AY075116.1 91.59% Dugongs 5 830 224 187 140 279 39.40 AY075116.1 91.59% Dugongs 6 833 222 193 132 286 39.02 AY075116.1 90.56% Dugongs 7 804 214 185 132 273 39.43 AY075116.1 91.59% Dugongs 8 859 242 194 135 288 38.30 AY075116.1 90.85% Dugongs 9 830 218 198 132 282 39.76 AY075116.1 91.59% Dugongs 10 794 218 183 125 268 38.79 AY075116.1 90.85% Average 823.4 223.2 189.5 132 278.7 39.05 91.34%

Table 3 Estimates of genetic distance matrix between dugong COI sequences.

Sample No. 1 2 3 4 5 6 7 8 9 10 Dugongs 1 0.000 Dugongs 2 0.010 0.000 Dugongs 3 0.011 0.008 0.000 Dugongs 4 0.011 0.007 0.005 0.000 Dugongs 5 0.015 0.016 0.019 0.016 0.000 Dugongs 6 0.151 0.153 0.151 0.151 0.151 0.000 Dugongs 7 0.008 0.010 0.011 0.008 0.011 0.150 0.000 Dugongs 8 0.089 0.092 0.092 0.092 0.097 0.165 0.091 0.000 Dugongs 9 0.008 0.007 0.010 0.008 0.015 0.155 0.010 0.092 0.000 Dugongs 10 0.111 0.116 0.119 0.119 0.112 0.163 0.109 0.101 0.116 0.000 Average 0.070

The dendrogram constructed from COI sequence comparisons using the Maximum Likelihood method based on the Tamura-Nei model demonstrated that the 10 taxa were divided into three subgroups (Figure 3). The first major group (I) comprised of seven dugong samples; 1, 2, 3, 4, 5, 7 and 9. The second group (II) comprised of two dugong samples; 8 and 10. The last group (III) has only one member; dugong 6. The dugong 6 was separated from the other members, which was not consistent with the location of the sampling. Because the distance from Koh Libong, the collection area of a major group, was only 40 kilometers away from the location of dugong 6 sampling. While the location of dugong 8 sampling was 140 kilometers away and along with many islands that are blocked such as Koh Yoa Yai, Koh Yao Noi, Phi Phi Island and Koh Lanta. However, the clustering of dugongs 8 and dugongs 10 (II) corresponds to the sampling location. The distance of about 50 kilometers between Yamu Cape, Pa Klok, Maung, Phuket and Loh Dalum Beach, Phi Phi Island, Ao Nang, Maung, Krabi resulting in both populations being able mating, thus forming a gene flow. However, the average genetic distance in this study was 0.07, which is considered very low. This demonstrated that the gene flow between the dugong population in each area still occurred. The genetic differences are likely caused by the variation of each individual. Furthermore, genetic grouping was not differentiated by region indicating maternal dispersal over long distances [17].

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Figure 3 A dendrogram of the 10 dugong samples constructed from sequence comparisons of the COI gene using the Maximum Likelihood method based on the Tamura-Nei model.

The sampling area of the major group was around Koh Libong and the coastal areas of Trang Province. This is because Trang Province has the largest existing population of dugong and largest and healthiest seagrass meadows in the country with at least 11 species present [18]. The seagrass beds and islands near Trang possibly house the largest group of dugongs remaining in [19]. Therefore, the conservation of dugongs must be conserved along the sea-grass.

Conclusions

The average length of the COI sequence of ten dugong samples was 823.4 bp. All COI sequence in this study were identical to GenBank accession number AY075116.1 with an average of 91.34%. The average genetic distance from the Kimura 2-parameter model was 0.070. The dendrogram divided ten dugong samples into three subgroups. The major group contains seven dugong samples from around Koh Libong and the coastal areas of Trang Province. The low genetic distance demonstrated a gene flow between populations, especially in areas with the largest and abundant seagrass.

Acknowledgements

The authors would like to thank you to the Phuket Marine Biological Center, Phuket for providing examples in this study. Thank you to the Department of Biotechnology, Faculty of Agricultural Technology, Kalasin University, Kalasin for the premises and the tools to conduct this research.

References

[1] H Marsh. Dugong: Status Reports and Action Plans for Countries and Territories. UNEP, 2002, p. 5-18. [2] AR Martin and RR Reeves. Diversity and Zoogeography. In: AR Hoelzel (eds) Marine Mammal Biology: An Evolutionary Approach. Blackwell Science ltd Oxford, 2002, p. 1-37. [3] CITES. Convention on International Trade in Endangered Species of Wild Fauna and Flora, 2019, Available at: http://www.cites.org, accessed April 2019. [4] Baimai V. Biodiversity in Thailand. The Journal of the Royal Institute of Thailand. 2010, 2, 107-114. [5] Hines E, Adulyanukosol K, Duffus DA. Dugong (Dugong dugon) abundance along the Andaman Coast of Thailand. Marine Mammal Science. 2005, 21, 536-549. ICoFAB2019 Proceedings | 105

[6] Thai PBS. New Survey on Dugong Population to be Launched. Thai Public Broadcasting Service. Breaking News, October 18, 2017. [7] R Frankham, JD Ballou and DA Briscoe. Introduction to Conservation Genetics. Cambridge University Press Cambridge, 2002, p. 1-617. [8] Schierenbeck KA. Population-level genetic variation and climate change in a biodiversity hotspot. Annals of Botany. 2017, 119, 215–228. [9] Strüder-Kypke MC, Lynn DH. Comparative analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in ciliates (Alveolata, Ciliophora) and evaluation of its suitability as a biodiversity marker. Systematics and Biodiversity. 2010, 8(1), 131-148. [10] Sun P, Shi ZH, Yin F, Peng SM. Genetic variation analysis of Mugil cephalus in China sea based on mitochondrial COI gene sequences. Biochemical Genetics. 2012; 50, 180-191. [11] Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution. 1993, 10, 512- 526. [12] Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. 2016, 33, 1870-1874. [13] Goldfeder RL, Wall DP, Khoury MJ, Ioannidis JPA, Ashley EA. Human genome sequencing at the population scale: a primer on high-throughput DNA sequencing and analysis. American Journal of Epidemiology, 2017, 186, 1000–1009. [14] Sun Z, Wan DG, Murphy RW, Ma L, Sheng XS, Huang DW. Comparison of base composition and codon usage in mitochondrial genome. Gene & Genomics, 2009, 31, 65-71. [15] Xia X. Mutation and selection on the anticodon of tRNA genes in vertebrate mitochondrial genome. Gene, 2005, 345, 13-20. [16] S Choudhuri. Bioinformatics for Beginners, Academic Press, Massachusetts, 2014, p. 27-53. [17] JB Bushell. 2013. The Genetic Diversity and Population Structure of the Dugongs (Dugong dugon) of Thailand. Master's Theses. San Jose State University, California, USA. [18] K Adulyanukosol and S Thongsukdee. The Results of the Survey on Dugong, Dolphin, Sea Turtle, and Seagrass in Trang Province. Report of Phuket Marine Biological Center and Marine and Coastal Resources Research Center (Bangkok). 2005. [19] Dugongs in Trang Province, Thailand: Recommendations for Conservation Strategy. Available at: https://bit.ly/32AEIP0, accessed July 2019.

doi:10.14457/MSU.res.2019.20 ICoFAB2019 Proceedings | 106

Comparative Study of Proteome Pattern of Kluai Ta Nee and Kluai Nam Wa Leaf Proteins

Bung-on Prajanban1*, Wichuda Jankangram2 and Nisachon Jangpromma3,4

1Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo 27160, Thailand 2Faculty of Science and Social Sciences, Burapha University, Sakaeo Campus, Sakaeo 27160, Thailand 3Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 4Department of Integrated Science, Forensic Science Program, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.

*Corresponding author’s e-mail: [email protected]

Abstract:

This study compared the protein profiles in leaves of diploid and triploid banana to identify the differentially accumulated proteins unique to each type of banana. Protein samples were analyzed by 2D- PAGE using IPG strip pH3-10. Fifty and 44 protein spots were detected in leaves of Musa acumimata (BB group) “Kluai Ta Nee” and Musa Xparadisiaca (ABB group) “Kluai Nam Wa”, respectively. Many spots of leaf proteins were corresponding to leaf proteins of Musa acuminate Colla (banana) reported previously such as ribulose-1,5-bisphosphate carboxylase, oxygen-evolving enhancer protein and superoxide dismutase. Ribulose-1,5-bisphosphate carboxylase, oxygen-evolving enhancer protein and superoxide dismutase in Kluai Ta Nee leaves had pIs and number of isoforms different from those in Kluai Nam Wa leaves. Three isoforms of endochitinase were found in Kluai Ta Nee leaves only. The major protein found in Kluai Ta Nee leaves, which had higher intensity than that in Kluai Nam Wa leaves, was identified as ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit. A low quality protein: oxygen-evolving enhancer protein 1, chloroplastic-like, found in Kluai Nam Wa leaves had higher intensity than that in Kluai Ta Nee leaves. The polymorphism of these proteins observed between leaves of Kluai Ta Nee and Kluai Nam Wa would be possibly due to different gene expressions in each banana type and may result in different functionalities of the proteins.

Keywords: 2D-PAGE, Kluai Ta Nee, Kluai Nam Wa, banana leaves

Introduction

Banana is one of the most important food crop of Musaceae family distributes in South East Asia, Africa and Australia [1]. Banana varieties grown in most parts of the world are hybrids derived from natural inter- and intraspecific crosses between two diploid wide species, Musa acuminate (genome A) and Musa balbisiana (genome B) [2]. Protein patterns can be used to study genetic diversity in banana. Proteins play a central role in biological processes, and proteomics differential assay can be used to determine the proteins that are affected by genetic variation during plant growth and development [3]. The majority of the identified leaf proteins from Musa acuminate Colla (AA) were found to be involved in energy metabolism. Moreover, the minority of leaf proteins identified by 2D-PAGE and MALDI-TOF MS were found to be involved in immunity and defense mechanisms [4]. In the case of leaves from normal banana and giant banana cultivar Prata Ana (AAB) with different genome from Musa acuminate Colla (AA) that were analyzed by 2D-PAGE and MALDI-TOF/TOF, the abundant proteins from the leaves of both normal banana and giant banana cultivar Prata Ana (AAB) were identified as being related to root metabolism, photosynthesis, protein translation carbon assimilation and nitrogen fixation [3]. In contrast, M. acumimata (BB group) “Kluai-Tanee” and M. Xparadisiaca (ABB group) “Kluai Nam Wa” with different genomes from Musa acuminate Colla (AA) and banana cultivar Prata Ana (AAB) have not been reported in the protein reference map. In Thailand, M. acumimata BB (Kluai-Tanee) and M. Xparadisiaca ABB group (Kluai Nam Wa) are important types for leaf and fruit production. The analysis of protein compositions in banana leaves can increase the potential applications of these types of banana for both production efficiency and further improvement of banana through breeding. The aims of this study were to analyze and compare leaf proteins from different genomes of BB (Kluai-Tanee) and ABB (Kluai Nam Wa) by 2D-PAGE using known Musa acuminate Colla (banana) leaf proteins that were previously ICoFAB2019 Proceedings | 107

reported for comparison. The proteins from leaves of both banana types were compared, and two protein spots with different intensity between two banana types were selected for identification of each type by using LC-ESI-MS/MS method [5].

Materials and methods

Plant materials and field experiment The banana varieties of both types used in this study were planted at the experimental farm of the Faculty of Agricultural Technology, Burapha University, Sakaeo Campus, Sakaeo, Thailand.

Protein extraction from leaves Leaf samples were extracted by the method described previously [6] with some modifications. The leaf samples were ground into powder in a mortar with liquid nitrogen. The ground samples were suspended in lysis buffer (7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 2 % (w/v) DTT). After centrifugation at 12, 0000 rpm for 30 min at 4๐C, the supernatant for each sample was collected and cleaned with the 2D- clean up kit to remove contaminants such as carbohydrate, lipids and others. (Amersham Bioscience, Sweden). The protein pellets were suspended in rehydration buffer (7 M urea, 2 M thiourea, 2 % (w/v) CHAPS, 2 mM DTT, 0.8 % (w/v) IPG buffer and 0.2 % bromophenol blue). The protein concentration was determined using the Bradford assay with bovine serum albumin (Amersham Biosciences, USA) as the standard.

2D-PAGE electrophoresis Proteins were analyzed by 2D-PAGE as described by O'Farrel [7] and some modified method on linear (3-10) pH gradient. Protein samples of 100 μg each were solubilized in a focusing solution containing 7 M urea, 2 M thiourea, 0.3% DTT, 2% CHAPS and 2% IPG buffer corresponding to the pH gradient used. Isoelectric focusing (IEF) was conducted with a Ettan IPG phor 3 (Amersham Bioscience, USA) on 7 cm IPG strips using a gradient mode yielding 9,750 Vhr. After focalization, the gel strips were equilibrated for 15 min in buffer containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 1% SDS and 25 mM DTT. A second 15 min equilibration step in the same solution containing 250 mM iodoacetamide instead of DTT was then performed. Proteins were then subjected to SDS-PAGE. IPG strip was placed on second dimension 15% SDS polyacrylamide gels. The gel was then stained with colloidal coomassie brilliant blue G250 solution. These gels were then washed with double distilled water until the background was clear. The gel images were captured using ImageQuant LAS 500 (GE Healthcare Bio-Sciences AB, Sweden) and protein spots were then analyzed by Image Master 2D Platinum v. 5.0 software (Amersham Biosciences, USA).

Protein Identification by LC-ESI-MS/MS The protein spots with differential expressions were excised with trypsin using sequencing grade reagent (Promega, USA) according to the manufacturer’s specifications. Each tryptic peptides were analyzed with a nano-liquid chromatography system (EASY-nLC II, Bruker) coupled to an ion trap mass spectrometer (Amazon Speed ETD, Bruker) equipped with an ESI nano-sprayer. The ESI-TRAP instrument was calibrated in the m/z range of 50-3000 using an internal calibration standard (Tune mix solution), which was supplied from Agilent. Bruker Daltonics software package, HyStar v.3.2, was used to control the ion trap device. LC–MS/MS spectra were analyzed using Compass Data Analysis v.4.0. Compound lists were exported as Mascot generic files (mgf) for further identification of proteins, which was performed by searching against the protein database from NCBIprot (Other green plants) using MASCOT MS/MS Ion Search program (www.matrixscience.com).

Results and discussion

In ours preliminary study, we separated Kluai Ta Nee and Kluai Nam Wa leaves proteins by 2D-PAGE using linear IPG strip (pH3-10) size 7 cm. Figure 1 showed the protein patterns of Kluai Ta Nee (Figure 1A) and Kluai Nam Wa (Figure 1B) leaves. The results demonstrated that approximately 50 and 44 protein spots were reproducibly detected for Kluai Ta Nee and Kluai Nam Wa leaves, respectively. The overall protein patterns of Kluai Ta Nee leaves were different from those of Kluai Nam Wa leaves. However, the molecular weights of major leaf proteins common to both Kluai Ta Nee and Kluai Nam Wa ranged from approximately 25 kDa to 70.0 kDa and pI (isoelectric point) range was between 4 and 7. The ranges of protein molecular weights and pI in previous study were wider than in our study. According to Lu et al.[4], the protein molecular weights of M. acuminate Colla (banana) leaves ranged ICoFAB2019 Proceedings | 108

from approximately 10 kDa to 100 kDa and the range of pI was from 3 to 10. However, the major leaf proteins from both normal and giant plants of Musa spp. cultivar Prata Ana (AAB) had molecular weights ranging from 14.0 kDa to 50.0 kDa and pI range was between 3 and 10 [3]. The protein patterns of two banana genomes were compared and analyzed. Some protein spots from each genome were separated and identified by Image Master 2D Trial. Table 1 summarized leaf proteins identified by pI and molecular weight on two referent gels compared with M. acuminate Colla (banana) leaf proteins reported previously [4]. The well-separated protein spots are known banana leaf proteins such as ribulose-1,5-bisphosphate carboxylase, oxygen-evolving enhancer protein and superoxide dismutase. Table 2 summarized proteins identified by LC-ESI-MS/MS. Ribulose-1,5-bisphosphate carboxylase is an important primary enzyme in photosynthesis with a carboxylase that catalyzes the assimilation of carbon dioxide into organic compounds. Therefore, it is not surprising that we found ribulose-1,5-bisphosphate carboxylase in both Kluai Ta Nee and Kluai Nam Wa leaves, and these enzymes were also found in M. acuminate Colla leaf and Musa spp. cultivar Prata Ana (AAB) leaves [3, 4]. However, Kluai Nam Wa leaves had higher number of ribulose-1,5-bisphosphate carboxylase spots than Kluai Ta Nee leaves. In addition, ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit found in Kluai Ta Nee leaves had higher intensity than in Kluai Nam Wa leaves. Moreover, oxygen- evolving enhancer protein is one of protein complex of photosystem II found in thylakoid membranes [8]. We found that oxygen evolving enhancer protein from Kluai Nam Wa leaves had higher number of multiple spots than M. acuminate Colla leaves. Moreover, the authors found that a low quality protein: oxygen-evolving enhancer protein 1, chloroplastic-like, in Kluai Nam Wa leaves had higher intensity of protein bands than in Kluai Ta Nee leaves. Interestingly, we found that superoxide dismutase from Kluai Ta Nee and Kluai Nam Wa leaves had multiple spots similar to that found in M. acuminate Colla leaves [4]. Superoxide dismutase acts as the first line of defense against ROS. It is induced by cold stress in plantain (Musa paradisiaca L.; ABB group) [2]. As the leaves of Kluai Ta Nee and Kluai Nam Wa were collected on 1st December, 2018, superoxide dismutase found in this study may respond to low temperature in the winter season. In addition, endochitinase was found in Kluai Ta Nee leaves, which was in agreement with that found in M. acuminate Colla (banana) leaves [4]. The general function of endochitinase is believed to be the hydrolysis of chitin in the cell walls of fungi and bacteria. Most of plant endochitinases are inducible with wounding, cold, pathogen infections, or hormones, like ethylene, methyl jasmonate and gibberellins [9]. In this study, four proteins (ribulose-1,5-bisphosphate carboxylase, oxygen-evolving enhancer protein superoxide dismutase and endochitinase) in Kluai Ta Nee and Kluai Nam Wa leaves had multiple spots. The differentiations of pI and/or molecular weight of these proteins are the results of post–translational modification [4].

. Figure 1 2D-PAGE of Kluai Ta Nee (A) and Kluai Nam Wa (B) leaf proteins. ICoFAB2019 Proceedings | 109

Table 1 Summary of proteins identified by pI and molecular weight. pI Molecular weight (kDa) Banana leaves protein Experimental Theory Experimental Theory Kluai Ta Nee - Ribulose-1,5- 5.09 6.40 [4] 43 44.778 [4] bisphosphate 6.71 44 carboxylase 5.40 40 5.90 40 6.54 40 6.90 40 - Oxygen-evolving 5.26 8.26 [4] 28 27.775 [4] enhance protein 6.63 28 - Superoxide dismutase 5.12 7.1 [4] 25 25.823 [4] 5.41 25 5.85 25 - Endochitinase 6.94 6.07 [4] 15 14.295 [4] 7.60 14 7.94 14 Kluai Nam Wa - Ribulose-1,5- 5.82 6.40 [4] 44 44.778 [4] bisphosphate 5.12 43 carboxylase 5.48 40 5.67 39 5.85 40 6.13 40 6.47 40 6.72 40 - Oxygen-evolving 5.14 8.26 [4] 28 27.775 [4] enhance protein 5.45 28 5.70 28 6.06 27 6.38 27 - Superoxide dismutase 5.48 7.1 [4] 25 25.823 [4] 5.71 25 5.95 25

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Table 2 Identification of the selected proteins from leaves of Kluai Ta Nee and Kluai Nam Wa identified by LC-ESI-MS/MS.

Theory Calculated Spot Sequence Match to Protein name Score No. Mw. Mw. coverage Peptide sequences pI pI (kDa) (kDa) (%)

Kluai Ta Nee

13 YP_00885 Ribulose 1,5- 522 6.23 54.68 5.91 57 17% KDTDILAAFRV, 4433.1 bisphosphate RFLFCA EAIFKA, carboxylase/oxygen REMTLGFVDLLD ase large subunit ,REMTLGFVDLLR [Musa textilis] D, KDDE NVNSQ PFMRW, KTFQGP PHGIQVERD, KD DENVNSQPFMR W, KWSPELAAA CEVWKE, KGHY LNATAGTCEEM MKR

Kluai Nam Wa

18 XP_00941 LOW QUALITY 508 6.77 36.23 5.43 31 22% KRLTYDEIQSKT, 2420.1 PROTEIN: oxygen- KRLTY DEIQSKT, evolving enhancer KRLTYDEIQ SKT, protein 1, KRLTYDEIQSKT, chloroplastic-like RLTYDEIQSKT, [Musa acuminata KDGIDYAAVTV subsp. malaccensis] QLPGGERV, KD GIDYAAVTVQLP GGERV, KDGIDY AAVTVQLPG GE RV, RVPFLFTIKQ, RGGSTGYDNAV ALPAGGRG, RGD EEELSKENI KN, KIQGVWYAQLEQ

Conclusions

The 2D-PAGE patterns of Kluai Ta Nee leaves revealed ribulose-1,5-bisphosphate carboxylase, oxygen-evolving enhancer protein and superoxide dismutase with different pI and number of isoforms compared with the 2D-PAGE patterns of Kluai Nam Wa leaves. Moreover, 3 isoforms of endochitinase were found in Kluai Ta Nee leaves only. The major protein, ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit found in Kluai Ta Nee leaves had higher intensity than in Kluai Nam Wa leaves. However, a low quality protein: oxygen-evolving enhancer protein 1, chloroplastic-like found in Kluai Nam Wa leaves had higher intensity than in Kluai Ta Nee leaves. The polymorphism of these proteins found in Kluai Ta Nee leaves and Kluai Nam Wa leaves might be responsible for different biological functions in each type. The further investigations are required to use other techniques for identification, characterization and better understanding on functional properties of the proteins.

Acknowledgements

This work was financially supported by the Research Grant of Burapha University through National Research Council of Thailand (Grant no. 29/2562).

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References

[1] Padam BS, Tin HS, Chye FY, Abdullah MI. Banana by-products: an under-utilized renewable food biomass with great potential. J. Food Sci. Technol. 2014; 51(12), 3527-3545. [2] Yang QS, Wu JH, Li CY, Wei YR, Sheng O, Hu CH, et al. Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) seedlings. Mol. Cell Proteomics. 2012; 11(12), 1853-1869. [3] Livramento K, Fialho L, Santos A, Livramento D, Cardoso T, Paiva L. Proteomic analysis reveals differentially accumulated proteins in banana somaclonal variants. J. Exp. Agric. Int. 2018; 21(6), 1- 13. [4] Lu Y, Qi YX, Zhang H, Zhang HQ, Pu JJ, Xie YX. Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry. Genet. Mol. Res. 2013; 12(4), 6871-6881. [5] Janwan P, Intapan PM, Laummaunwai P, Rodpai R, Wongkham C, Insawang T, et al. Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae. Exp. Parasitol. 2015; 159: 53-58. [6] Khueychai S, Jangpromma N, Daduang S, Jaisil P, Lomthaisong K, Dhiravisit A, et al. Comparative proteomic analysis of leaves, leaf sheaths, and roots of drought-contrasting sugarcane cultivars in response to drought stress. Acta Physiol. Plant. 2015; 37 (4), 88. [7] O' Farrel, P.H. High-Resolution Two-dimensional Electrophoresis of Proteins, J.Biol. Chem. 1975; 250, 4007-4021. [8] Sasi S, Venkatesh J, Daneshi RF, Gururani MA. Photosystem II extrinsic proteins and their putative role in abiotic stress tolerance in higher plants. Plants (Basel.) 2018; 7(4), 100. [9] Liu, JH, Zhang, J, Xu, BY, Zhang, JB, Jia, CH, Wang, JS, et al. Expression analysis of bananaMaECHI1 during fruit ripening with different treatments. Afr. J. Biotechnol. 2012; 11, 12951- 12957.

doi:10.14457/MSU.res.2019.21 ICoFAB2019 Proceedings | 112

Characterisation of Microwave-assisted Pretreatment for Spent Coffee Grounds

Wipada Jamsai 1*, Jirapa Phetsom1, Teerachai Kuntothom2 and Anuwat Wanthong1

1 Department of Biology, Faculty of Science, Mahasarakham University, Khamriang, Kantarawichai, Mahasarakham 44150 Thailand 2 Department of Chemistry, Faculty of Science, Mahasarakham University, Khamriang, Kantarawichai, Mahasarakham 44150 Thailand

*Corresponding author’s e-mail : [email protected]

Abstract:

Spent coffee grounds (SCGs) is the natural waste from industry and cafe. It mainly composes of cellulose, hemicellulose, and lignin. The pretreatment process could alter the structure and composition of pretreated materials. This study was to characterise the pretreated SCGs based on microwave irradiation assisted NaCl. The process was carried on the various concentration of NaCl (1, 2 and 3 % w/v) and time of irradiation (1, 2 and 3 min). The power consumption of microwave was 300 W for all treatments. The morphological changes of SCGs residue from pretreatment process was characterised by Scanning Electron Microscopy (SEM), the functional groups of main components by Fourier transform infrared spectroscopy (FTIR), and crystallinity index by X-ray diffraction (XRD) methods. The pretreated SCGs with microwave and NaCl showed clear changes in structural surface with rougher and pores when increasing the NaCl concentration and irradiation time. The hemicellulose and lignin in SCGs residues were reduced after the pretreatment process with observation by the different FTIR spectra profiles between before and after SCGs treatment. After pretreatment with microwave power at 300 W for 3 min, the crystalline index was reduced to 31.20, 24.89, 23.39 and 21.82% with increased the NaCl concentration of 1, 2, and 3 % (w/v), respectively. However, the crystalline region was decreased with an increase in the amorphous region. These results could enhance the hydrolysis step and apply in the cellulose conversion process.

Keywords: Pretreatment, Spent coffee grounds, Microwave

Introduction

The production of energy from waste materials is an attractive alternative to replace the conventional process. Coffee is one of the most popular drinks around the world. Spent coffee grounds (SCGs) are products through the process of instant and fresh coffee productions. These processes are related to pretreatment methods with steam, hot water and high pressure that conduct coffee powder to be treated [13, 14]. The chemical composition of SCGs mainly of cellulose, hemicelluloses and lignin [15]. The cellulose composes of crystalline domains (highly ordered microfibrils) and amorphous domains (randomly ordered microfibrils) [2]. The pretreatment steps involved in a reduction in biomass size, depolymerization, and separation of the major components of biomass [16]. There are various pretreatment methods, such as physical, chemical, biological methods, and the combination of physicochemical method. The aim of pretreatment is to produce high sugar yield with the mild condition [18]. The use of inorganic salts as a catalyst to pretreat the lignocellulosic biomass has non-toxicity and low-cost [20]. Moreover, the catalytic activity of inorganic salts has been shown higher than that of acids [19]. Moreover, salts have unique characteristic activities when exposed to an electromagnetic field, owing to their Lewis acid nature [8]. The combination of Microwave-inorganic pretreatment is a promising green methodology in the cellulose production due to its unique characteristics of rapid volumetric heating, increased reaction rates, and energy saving, compared with other conventional heating methods [17]. Effect of microwave irradiation has been reported to change the chemical structure in the lignocellulosic materials [25]. Microwave irradiation has benefits in creating the thermal hot spots, accelerating the collision of ions with neighbouring molecules, and higher heat power than conventional heating method [8]. Therefore, this work aims to alter the structural cellulose of spent coffee grounds by applying microwave and sodium chloride in a pretreatment step. The pretreatment process could reduce the crystalline region and enhance chemical accessibility during the hydrolysis.

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Table 1 Validation of the optimized pretreatment on different lignocellulosic residues and crystallinity index.

Sample Pretreatment method % Crystallinity Reference Rubber wood Laccase enzyme 70 U/g, at 40-55°C for 8 h + sodium 65 [1] fibers chlorite at 75°C for 45 min + potassium hydroxide (3% w/w) at 80°C for 2 h + potassium hydroxide (6% w/w) at 90°C for 2 h Dissolving pulp Sulfuric acid (65% w/w) at 45°C for 1 h + 67 [2] ultrasonicated for 45 min Paper Ball milling + sulfuric acid (47 wt%) at 45°C for 1.5 h 89 [3]

Para rubber wood Sodium hydroxide (1% w/v) at 80°C for 2 h + sodium 70.2 [4] sawdust sulfide (1% w/v) at 80°C for 1.5 h + bleached by sodium chlorite (0.7% v/v) at 80°C for 1.5 h + hydrolyzed sulfuric acid (64% w/w) at 45°C for 3 h

Lime residues Autoclaving at 120°C for 2 h + homogenization 50.35 [5]

Sugarcane Sodium hydroxyl (1% w/v) at 90°C for 1.5 h + ionic 36% [6] bagasse liquid 130°C for 2 h at 400 W of microwave + homogenization

Materials and methods

Microwave-NaCl pretreatment The SCGs was obtained from the Amazon Cafe in Maha-sarakham province, Thailand. The SCGs was dried at 60°C until the weight was constant and stored in a desiccator. The SCGs pretreatment was carried out in a household type microwave (Samsung, ME81KS-1/ST, Korea). The output power range of the equipment was 100–800 W (Microwave test procedure IEC-705) with an operating frequency of 2450 MHz. The power consumption was subjected to 300 W for 1, 2 and 3 min. The sodium chloride (NaCl) concentration of 1, 2 and 3 % w/v was applied on SCGs with solid loading 1:10 (g/mL). After pretreatment, SCGs was thoroughly washed with distilled water and air-dried. The dried SCGs residue was used for further studies.

Characterization of microwave-NaCl pretreated

SEM analysis The morphological changes of SCGs before and after microwave-NaCl pretreatment was analysed by scanning electron microscopy (SEM, JEOL JSM-5600, Japan). The surface of SCGs residue was observed at magnification 1000X. The specimens were mounted on conductive tape and coated with gold palladium.

FTIR analysis Fourier Transform Infrared spectroscopy (Bruker, TENSOR27, USA) was used to analyse the changes in functional groups that occurred by pretreatment. FTIR spectrum was recorded between 4000 and 600 cm-1 using a Shimadzu Spectrometer with a detector at 4 cm-1 resolution and 25 scans per sample. Samples were prepared by mixing 1 mg of the dried sample with 100 mg of KBr (Spectroscopic grade) in the hydraulic pump with 10 Mpa pressure.

XRD analysis The crystallinity of SCGs was analysed by XRD (Bruker, D8, USA) Advance data collection in two dimensions, 2Theta (2θ) and Gamma (γ) angular range 360o. The instrument was set at 40 kV, 40 mA; ICoFAB2019 Proceedings | 114

radiation was Cu K� (�=1.54 Å), and grade range between 5 and 40o with a step size of 0.03o. Crystallinity of cellulose was calculated according to the empirical method proposed by Segal et al. [21]

o CrI (%) = [(I002 - I18.0 )/I002] x 100

o CrI is the crystalline index, I002 is the maximum intensity of the (002) lattice diffraction, and I18.0 is the intensity diffraction at 18.0o, 2�.

Results and discussion

SEM analysis Figure 1(a-d) illustrates the surface features of untreated SCGs and microwave-NaCl treated SCGs by using the scanning electron microscopy (SEM). The untreated and treated SCGs were comparatively observed which pretreated SCGs surface was clearly ruined and exposed the inner surface. The untreated SCGs surface presented a contiguous and smooth surface since the fibres were all intact. All of pretreat SCGs were loosening of fibres and cellular distortion that due to the microwave irradiation combined with NaCl. Moreover, increasing the incubation time from 1 to 3 minutes could enhance pore size and number. This occurrence revealed that the SCGs had the structural changes after microwave-NaCl pretreatment. This result is consistent with those reports in microwave-inorganic salt pretreatment by P. Moodley and E.B.G. Kana (2017) [8]. The highly fractionated-surface appearance was observed when pretreated SCGs with microwave-NaCl (300 W, 3%, for 3 min).

FTIR analysis FTIR spectroscopy was used to investigate the changes of cellulose structures after microwave-NaCl pretreatment. Figure. 2 shows the FTIR spectra of untreated SCGs, microwave-NaCl treated SCGs with 1%, 2% and 3% at microwave power 300 W. The effects of pretreatments can be analysed by changes in functional groups which relevant to the lignocellulosic biomass [9]. The FTIR spectra of 3200-3400 cm-1 were associated with the O-H stretching of hydrogen bonds [20]. The results showed FTIR spectra of microwave-NaCl treated SCGs that revealed the higher altered cellulose than untreated SCGs by increasing the NaCl concentration. These results were according to the spectrum at 1730 cm-1 and 1246 cm-1 these peaks indicated the changes of hemicellulose in all microwave- NaCl treated SCGs. The previous studies, the spectrum at 1730 cm-1 and 1246 cm-1 were attributed to the C=O stretching and C-O stretching vibration of hemicellulose, respectively [4, 9]. The spectrum at 1590 cm-1 and 1505 cm-1 were assigned to the aromatic parts of lignin [10]. The profiles of the FTIR spectra were different for untreated SCGs and microwave-NaCl treated SCGs. The results revealed that the lignin was decreased by pretreatment process with increasing NaCl concentration. It had been reported that the microwave irradiation enhances the saponification of intermolecular ester bonds cross-linking xylan and other components such as lignin [21]. Moreover, salts have unique characteristic activities when exposed to an electromagnetic field, owing to their Lewis acid nature [8]. The FTIR spectra for microwave-NaCl treated SCGs indicate the structural changes after microwave-NaCl treatment. These changes were according to the SEM results, which obtained from the pretreated SCGs with microwave-NaCl (300 W, 3%, for 3 min).

XRD analysis Figure 3 showed the XRD patterns of untreated and pretreated SCGs. The major diffraction peaks of the cellulose crystallographic plane were identified at 2휃 = 22°, signifying a highly organised crystalline region whereas the peaks at 2휃 = 18° indicated the less organised amorphous region [2]. The results showed that the XRD patterns of the untreated SCGs had changed after the microwave-NaCl treatment. The crystallinity index of the untreated SCGs and microwave-NaCl treated SCGs with different NaCl concentrations (1, 2 and 3 % w/v) at 300 W for 3 min were 31.20, 24.89, 23.39 and 21.82%, respectively. The crystallinity index significantly decreased because the crystalline cellulose became amorphous form after pretreatment. The amorphous structure is more easily degradable and susceptible to chemical attacks. The lignocellulosic materials, the crystalline region could be altered to the amorphous region after the pretreatment process [7]. The decreased crystalllininty in microwave-NaCl treated SCGs might be the result of removal of crystalline cellulose and hemicellulose from the biomass. A similar result was observed by J. Li et al. (2012) [6] that reported in breaking intermolecular hydrogen bonds of cellulose by using ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). Then, the loss of hydrogen bond caused the collapse of crystal structure after pretreatment [6]. The crystallinity index of untreated SCGs was higher ICoFAB2019 Proceedings | 115

than microwave-NaCl treated SCGs. Moreover, the crystalline structure is less accessible to chemical attacks due to hydrogen strong interactions between the microfibers.

(a) (b)

(c) (d)

Figure 1 SEM of (a) untreated (b) 300 W, 1% for 3 min (c) 300 W, 2% for 3 min and (d) 300 W, 3% for 3 min.

untreated mw-NaCl 1% mw-NaCl 2% mw-NaCl 3% 1.0

0.9

0.8

transmittance (%) transmittance 0.7

0.6

4000 3500 3000 2500 2000 1500 1000 500

-1 wavenumber cm

Figure 2 FTIR spectra of SCGs by using microwave-NaCl pretreatment

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MW-NaCl 3%

MW-NaCl 2%

Intensity MW-NaCl 1%

Untreated

0 10 20 30 40 50 2-theta

Figure 3 X-Ray diffraction of untreated and treated SCGs.

Conclusions

In this study, the pretreated microwave-NaCl SCGs was observed the changes in structural surface and crystallinity. The experiments indicated that the pretreatment of microwave-assisted 3% NaCl concentration, 300 W, 3 min obtained the crystallinity index 21.82% with more amorphous region. The morphology changes in pretreated SCGs had “sponge-puff” like appearance. This changes showed the rough surface with more porosity. Moreover, this pretreatment process can also remove lignin and hemicellulose that can be used to develop the pretreated SCGs for value-added products such as nano- carbon, nano cellulose or super capacity. These findings open up the possibility to develop SCGs into other applications.

Acknowledgements

This study was supported by Mahasarakham University and the Department of Biology. The authors would like to thank cafe Amazon in Mahasarakham for providing the spent coffee grounds.

References

[1] Podder PK, et al. Isolation of nano cellulose from rubber wood fibre and fibrillation effects on nano cellulose reinforced poly (ethylene oxide). The National Conference for Postgraduate Research. 2016; 704-711. [2] Gibril M, et al. Optimisation and enhancement of crystalline nanocellulose production by ultrasonic preatment of dissolving wood pulp fibres. Cellulose chemistry and technology. 2018; 10, 711-727. [3] Phanthong P, et al. Effect of ball milling on the production of nanocellulose using mild acid hydrolysis method. Journal of the Taiwan Institute of Chemical Engineers. 2016; 60, 617-622. [4] Laksika O, and Wunpen C. Nanofibrillar cellulose from para rubber wood sawdust as reinforcement in polylactic acid composites. SWU Sci. J. 2018; 34, 263-275 [5] Jongaroontaprangsee S, Chiewchan N, and Devahastin S. Production of nanocellulose from lime residues using chemical-free technology. Materials Today: Proceedings. 2018; 5, 11095-11100. [6] Li J, et al. Homogeneous isolation of nanocellulose from sugarcane bagasse by high pressure homogenization. Carbohydrate polymers. 2012; 4, 1609-1613. [7] Zhao D, et al. Dissolution of cellulose in phosphate-based ionic liquids. Carbohydrate Polymers. 2012; 87.2, 1490-1494. ICoFAB2019 Proceedings | 117

[8] Moodley P, and Kana EG. Microwave-assisted inorganic salt pretreatment of sugarcane leaf waste: effect on physiochemical structure and enzymatic saccharification. Bioresource technology. 2017; 235. 35-42. [9] Ravindran R, et al. Two-step sequential pretreatment for the enhanced enzymatic hydrolysis of coffee spent waste. Bioresource technology. 2017; 239, 276-284. [10] Ma Y, et al. Production of nanocellulose using hydrated deep eutectic solvent combined with ultrasonic treatment. ACS Omega. 2019; 4.5, 8539-8547. Aspects. Institute of Nutrition. 2017; 32, 67- 79. [11] Itoh T, and Brown RM. The assembly of cellulose microfibrils in Valonia macrophysa Kütz. Planta. 1984; 160.4, 372-381. [12] Oun AA, and Rhim, J. W. Preparation and characterization of sodium carboxymethyl cellulose/cotton linter cellulose nanofibril composite films. Carbohydrate Polymers. 2015; 127, 101-109. [13] Thunnalin W, Yuraporn S, and Numphung R. Nanocellulose: Food Application and Food Safety Aspects. Institute of Nutrition. 2017; 32, 67-79 [14] New York State Energy Research and Development Authority, Available at: https://www.nyserda.ny.gov/-/media/Files/Publications/Research/Biomass-Solar-Wind/evaluation- of-microwave-pretreatment, accessed July 2019. [15] Fang Z, Smith RL and Qi X (Eds.). Production of Biofuels and Chemicals with microwave. 2015. [16] Lai, LW, and Idris A. Comparison of steam-alkali-chemical and microwave-alkali pretreatment for enhancing the enzymatic saccharification of oil palm trunk. Renewable energy. 2016; 99, 738-746. [17] YU Qiang, et al. The effect of metal salts on the decomposition of sweet sorghum bagasse in flow- through liquid hot water. Bioresource technology. 2011; 102.3, 3445-3450. [18] Kang KE, Park DH, and Jeong GT. Effects of inorganic salts on pretreatment of Miscanthus straw. Bioresource technology. 2013; 132, 160-165. [19] Segal LGJMA, et al. An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Textile research journal. 1959; 29.10, 786-794. [20] Hsu TC, et al. Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis. Bioresource technology. 2010; 101.13, 4907-4913. [21] Jin QH, Dai SS, and Huang KM. Microwave chemistry. China Science Press. 1999. [22] Zhu Z, et al. Efficient sugar production from sugarcane bagasse by microwave assisted acid and alkali pretreatment. Biomass and bioenergy. 2016; 93, 269-278. doi:10.14457/MSU.res.2019.13 ICoFAB2019 Proceedings | 118

Morphological Observation of Polylactide-b-Poly (Ethylene Glycol)-b- Polylactide Triblock Copolymers Stereocomplex Films

Pattarin Intaravichien and Prasong Srihanam*

The Center of Excellence in Chemistry (PERCH-CIC) and Creative and Innovation Chemistry Research Unit, Department of Chemistry Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

The objectives of this work were to prepare PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films with and without tetracycline by solvent casting method and to observe their morphology drug under the scanning electron microscope (SEM). The results found that film prepared from pure PLLA-PEG-PLLA had smooth surfaces but composed of some small pores with tetracycline. The PLLA-PEG-PLLA/PDLA- PEG-PDLA blend films showed varied in surfaces and textures depending on the PDLA-PEG-PDLA ratios used. All blend films had small pores on their surfaces but still had homogeneous texture. The number of pores smooth and dense texture of the blend films were gradually increased when the ratio of PDLA-PEG- PDLA increased. This confirmed that stereocomplexation formation between both copolymers. The obtained results indicated the potential of blend films for drug delivery system development.

Keywords: Biodegradable polymer, film, morphology, PLA, stereocomplex

Introduction Controlled drug release systems are well established as an efficient approach for the treatment of several diseases [1]. This was due to their offer of several advantages, such as appropriate control of release kinetics, reduction of drug concentration variability in the blood which may cause adverse effects [2], decrease of dosage times and improvement of patient compliance [3]. Various types of controlled-release drug delivery systems have been prepared including particles, fibers, powders, gels and films [4]. The drug delivery systems prepared from biodegradable polymers have the advantage over nonbiodegradable polymers in that the removal of these devices at the completion of therapy is not required [5]. Polylactic acid (PLA) is a biodegradable polymer synthesized by ring-opening polymerization of lactic acids. These substances are derived by lactic fermentation of carbohydrates such as beetroot, sweet potato, corn, potato and cassava [6]. Generally, lactic acid ( LA) , a PLA monomer, exists into 2 stereoisomers, D- and L-lactic acids which further synthesized into poly (D-lactic acid) (PDLA) and poly (L-lactic acid) (PLLA), respectively [7]. The PLA has been used in various applications, especially in biomedical applications such as artificial bones, sutures, scaffold-based tissue engineering and drug delivery system [8]. Many reports have been proved that the PLA is safety in use, sustainability, high biocompatibility, bio-absorbability, good mechanical properties (especially in strength and modulus) and easy processability [9]. However, PLA has some draw back properties which restricted for applications such as inherent brittleness, low toughness and low crystallization ability [10]. Stereocomplexation of PLA is one of an approach to improve its physical properties. The method could be performed by combining poly (L-lactide) (PLLA) and poly (D- lactide) (PDLA). With stereocomplex crystallites, the melting temperature of PLA is enhanced more than 50°C as well as enhancement of barrier, mechanical and thermal properties [11]. The stereocomplex films of PLLA-PEG- PLLA/ PDLA-PEG-PDLA have been prepared by compression method [12]. Therefore, in this work polylactide-b poly (ethylene glycol) -b-polylactide triblock copolymers stereocomplex films by solvent casting method were prepared and the effects of PDLA-PEG-PDLA ratios and tetracycline on the morphology of the prepared films were also observed.

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Material and methods

Materials PLLA-PEG-PLLA and PDLA-PEG-PDLA copolymers were synthesized by ring-opening polymerization in bulk at 165 ∘C under a nitrogen atmosphere for 6 h using 0.075 mol% stannous octoate (Sn(Oct)2) as a catalyst. Polyethylene glycol (PEG) was used as an initiator [12]. Number-average molecular weight (Mn) of the PLLA-PEGPLLA and PDLA-PEG-PDLA obtained from Gel Permeation Chromatography (GPC) were 90,000 and 85,400 g-/mol, respectively, as well as dispersity indices were 2.8 and 2.1, respectively. As measure by Differential Scanning (DSC), glass transition temperatures (Tg) of the PLLA-PEG-PLLA and PDLA-PEG-PDLA were 31 and 29 ∘C, respectively. Their melting temperatures ∘ (Tm) were 171 and 170 C, respectively. The obtained copolymers were granulated before vacuum drying ∘ at 110 C for 3 h to remove any unreacted lactide. Tetracycline (C22H24N2O8) was used as a water-insoluble model drug. Dichloromethane (CH2Cl2) was used as a dissolving solvent.

Preparation of blend film PLLA-PEG-PLLA and PDLA-PEG-PDLA copolymers were firstly mixed at the ratio of 100/0, 90/10, 80/20, 70/30, 60/40 and 50/50 in a total weight of 0.2 g and then dissolved in 10 mL dichloromethane. The mixture of PLLA-PEG-PLLA and PDLA-PEG-PDLA copolymer solutions were vigorously stirred for 3 h, and then casted onto a 4.5 cm diameter petri dish followed by solvent evaporation at room temperature for 24 h. The films were peeled off and dried in a vacuum oven at 50 °C for 72 h to evaporate any residue solvent. For drug-loaded blend films, the tetracycline model drug was added into the polylactide-b-poly (ethylene glycol)-b-polylactide triblock copolymer films at 2% wt of each blend film before dissolving the mixture using 100 mL dichloromethane. The drug-loaded blend films were prepared by film casting as described above. The obtained films were kept in a desiccator until investigation.

Morphological observation All of the films were dehydrated and cut into ~1cm length before observing their morphology using a scanning electron microscope (SEM) (JEOL, JSM-6460LV, Tokyo, Japan). The film fractures were coated with gold (Au) to enhance conductivity before scanning.

Results and discussion

The morphology of all films was observed under scanning electron microscope. Figure 1 shows PLLA-PEG-PLLA/ PDLA-PEG-PDLA blend films at six different ratios without tetracycline at 1000X magnification. The pure PLLA-PEG-PLLA film had smooth both in surfaces and cross-section without phase separation (Fig. 1a). The PLLA-PEG-PLLA/ PDLA-PEG-PDLA blend films (Fig. 1b-f) had irregular patterns of pores in their surfaces with inconsistent shapes and sizes. At 20% (Fig. 1c) and 40% (Fig. 1e) of PDLA-PEG-PDLA ratios, the surfaces of films had the highest number of small pores in range of 1-10 µm. However, the smoothness of the blend films gradually increased when PDLA-PEG-PDLA increased as shown by cross-sections. The details of pores and textures were clearly by increasing higher magnification as shown in Figure 2. The results indicated that the small pores appeared only on the surfaces of films. This was due to the evaporation of the dissolving solvent in the dried process. In addition, the dense and smooth textures of the PLLA-PEG-PLLA/ PDLA-PEG-PDLA blend films were caused by stereocomplexation.

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Figure 1 SEM micrographs of the PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films at different ratios; (a) 100/0, (b) 90/10, (c) 80/20, (d) 70/30, (e) 60/40 and (f) 50/50 without tetracycline under 1000X magnifications. I and II present the surface and cross section of films, respectively. (All scale bars = 10 µm).

aI bI cI dI eI fI

aII bII cII dII eII fII

Figure 2 SEM micrographs of the PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films at different ratios; (a) 100/0, (b) 90/10, (c) 80/20, (d) 70/30, (e) 60/40 and (f) 50/50 without tetracycline under 3000X magnifications. I and II present the surface and cross section of films, respectively. (All scale bars = 5 µm).

Figure 3 shows the SEM micrographs of films containing tetracycline prepared from different ratios of PLLA-PEG-PLLA and PDLA-PEG-PDLA both surface and cross-section. The PLLA-PEG-PLLA film (Fig.3a) showed a smooth surface without phase separation as like as a cross-section. The texture of films found some particles (lower 2 µm in size) distributed covered the surfaces which suspected that were tetracycline model drug. Considering the obtained PLLA-PEG-PLLA/ PDLA-PEG-PDLA blend films as shown in Fig. 3b-f, all films had pores with about 2-3 µm on their surfaces. The pores in the surfaces gradually increased in sizes and the number. The results indicated that increasing of PDLA-PEG-PDLA ratio resulted to obtain a high number of pores and consistency of pore sizes. However, cross-sections of all blend films still homogeneous texture and gradually increased when the PDLA-PEG-PDLA increased. At high magnification (Fig.4), the pure PLLA-PEG-PLLA film showed some small pores in its surfaces but still homogeneous texture as shown in cross-section (Fig.4a). In the PLLA-PEG-PLLA/ PDLA-PEG- PDLA blend films (Fig. 4b-f), the surfaces had smoother than pure PLLA-PEG-PLLA film and gradually increased of smooth surfaces by increasing of the PDLA-PEG-PDLA ratios. This might be caused by stereocomplexation between the copolymers. Moreover, pores at the surfaces of films were also increased by increasing PDLA-PEG-PDLA contents but not found as shown in cross-section textures. This was confirmed the stereocomplex formation occurred inside the center of films and resulting in dense and smooth textures. The results indicated that the tetracycline did not interfere the formation of stereocomplex of PLLA-PEG-PLLA/ PDLA-PEG-PDLA blends. Comparison between non-drug-loaded and drug-loaded blend films, differences in their morphology were observed, especially texture and pore distribution. The drug-loaded blend films slightly had denser in texture than the non-drug-loaded blend films and also had consistent pore on the surfaces. This might be caused by the model drug supported the stereocomlexation of the copolymers. The result was in agree with the previous report about the blend films of this copolymer which prepared by compression method [12]. The obtained results advantaged for using the blended films for encapsulation water-insoluble drugs for drug delivery applications.

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aI bI cI dI eI fI

aII bII cII dII eII fII

Figure 3 SEM micrographs of the PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films at different ratios; (a) 100/ 0, (b) 90/ 10, (c) 80/ 20, (d) 70/ 30, (e) 60/ 40 and (f) 50/ 50 with tetracycline under 1000X magnifications. I and II present the surface and cross section of films, respectively. (All scale bars = 10 µm).

aI bI cI dI eI fI

aII bII cII dII eII fII

Figure 4 SEM micrographs of the PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films at different ratios; (a) 100/ 0, (b) 90/ 10, (c) 80/ 20, (d) 70/ 30, (e) 60/ 40 and (f) 50/ 50 with tetracycline under 3000X magnifications. I and II present the surface and cross section of films, respectively. (All scale bars = 5 µm).

Conclusions

PLLA-PEG-PLLA/ PDLA-PEG-PDLA blend films were successfully prepared by the solvent casting method. The morphology of the PLLA-PEG-PLLA/PDLA-PEG-PDLA blend films varied by the ratios of PDLA-PEG-PDLA used. The surfaces of the blend films appeared small pores but still had homogeneous texture. The dense and smooth texture of the blend films was caused by stereocomplexation between PLLA-PEG-PLLA and PDLA-PEG-PDLA copolymers which gradually increased by increasing of PDLA-PEG-PDLA ratios. The obtained results could be useful information for the development of controlled-release delivery material, especially water-insoluble drugs.

Acknowledgements

The authors would like to thank the Central Instrument and Department of Chemistry, Faculty of Science, and Mahasarakham University for chemicals support and staff for SEM guidance. Thank you is extended to the project for the Promotion of Science and Mathematics Talented Teachers (PSMT) for scholarship.

References

[1] Alves PE, Soares BG, Lins LC, Livi S, Santos P. Controlled delivery of dexamethasone and betamethasone from PLA electrospun fibers: a comparative study. Eur Polym J. 2019; 117, 1-9. [2] Mehuys E, Vervact C. Oral controlled release dosage forms. J Pharm Belg. 2010; 2, 34-38. ICoFAB2019 Proceedings | 122

[3] Busatto C, Pesoa J, Helbling I, Luna J, Estenoz D. Effect of particle size, polydispersity and polymer degradation on progesterone release from PLGA microparticles: Experimental and mathematical modeling. Int J Pharm. 2018; 536(1), 360-369. [4] Tran PHL, Duan W, Lee B, Tran TTD. The use of zein in the controlled release of poorly water- soluble drugs. Int J Pharm. 2019; 566, 557-564. [5] Srisuwan Y, Baimark Y. Preparation of biodegradable silk fibroin/alginate blend films for controlled release of antimicrobial drugs. Adv Mater Sci Eng. 2013; 2013, 1-6. [6] Gupta A, Mulchandani N, Shah M, Kumar S, Katiyar V. Functionalized chitosan mediated stereocomplexation of poly (lactic acid): Influence on crystallization, oxygen permeability, wettability and biocompatibility behavior. Polymer. 2018; 142, 196-208. [7] Jin F, Hu R, Park S. Improvement of thermal behaviors of biodegradable poly (lactic acid) polymer: A review. Compos Part B-Eng. 2019; 164, 287-296. [8] Pastorino L, Dellacasa E, Petrini P, Monticelli O. Stereocomplex poly (lactic acid) nanocoated chitosan microparticles for the sustained release of hydrophilic drugs. Mater Sci Eng C. 2017; 76, 1129-1135. [9] Li, L., Cao, Z., Bao, R., Xie, B., Yang, M., & Yang, W. Poly(l-lactic acid)-polyethylene glycol- poly(l-lactic acid) triblock copolymer: A novel macromolecular plasticizer to enhance the crystallization of poly(l-lactic acid). Eur Polym J. 2017; 97, 272-281. [10] Srithep Y, Pholharn D, Turng L, Veang-in O. Injection molding and characterization of polylactide stereocomplex. Polym Degrad Stab. 2015; 120, 290-299. [11] Xie Y, Lan XR, Bao RY, Lei Y, Cao ZQ, Yang MB, Yang W, Wang, YB. High-performance porous polylactide stereocomplex crystallite scaffolds prepared by solution blending and salt leaching. Mater Sci Eng. C. 2018; 90, 602-609. [12] Pasee S, Baimark Y. Improvement in mechanical properties and heat resistance of PLLA- b -PEG- b -PLLA by melt blending with PDLA- b -PEG- b -PDLA for potential use as high-performance bioplastics. Adv Polym Technol. 2019; 2019, 1-9.

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Plasma Technology and Abiotic Elicitor Effectively Increased Isothiocyanates, Bioactive Compounds and Cytotoxicity against Caco2 Cells in Mustard Green Microgreen Extract

Worachot Saengha1*, Thipphiya Karirat1, Benjaporn Buranrat2, Khanit Matra3, Sirirat Deeseenthum1 and Vijitra Luang-In1

1Natural Antioxidant Innovation Research Unit, Department of Biotechnology, Faculty of Technology, Mahasarakham University 44150, Thailand. 2 Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand. 3 Department of Electrical Engineering, Faculty of Engineering, Srinakharinwirot University, Nakhon Nayok 26120, Thailand

*Corresponding author’s e-mail:[email protected]

Abstract:

The objective of this study was to study the effect of plasma technology and abiotic elicitor on percent germination, length of stem, fresh weight, total isothiocyanate, bioactive compounds and cytotoxicity against Caco2 cells of mustard green (Brassica juncea L. Czern. et Coss) microgreens germinated on vermiculite at 25 ºC in 12 h light/12 dark cycle for 7 days. The results showed that plasma treatment and abiotic elicitor including sucrose, NaCl and CaCl2 did not have any effect on the % germination, but it affected the length of mustard green stems. Using only plasma treatment was able to increase the total isothiocyanate content (2.65 mmol/100g DW), total phenolic content (5.08 mg GAE/g DW), total flavonoid content (0.17 mg RE/g DW), antioxidant activity by DPPH assay (7.93 mg Trolox /g DW) and FRAP assay (15.42 mg Fe2+/g DW). The lowest IC50 of mustard green extract against CaCo2 cells was found when incubated for 72 h at 30.61 µg/mL. Treated Caco2 cells showed signs of shrinkage and membrane blebbing i.e. apoptosis. This study indicated that using plasma technology can improve the physiology of mustard microgreens and enhance the isothiocyanate content, bioactive compounds and cytotoxicity. Therefore, this technology can be used for improving the nutritive value and phytochemicals of microgreens as functional foods.

Keywords: Isothiocyanate, Cytotoxicity, Mustard green, Plasma, Elicitor

Introduction

Microgreens have recently become popular forms of vegetables and widely consumed around the world because they contain higher content of nutritive compounds such vitamins and minerals when compared to the same type of fully grown vegetables [1]. Microgreen vegetables have high nutritional value and also contain phytochemicals which exhibit antioxidant property, anti-inflammatory, reducing an ischemic heart disease, stroke, rate of illness and death rate from cancers [2][3][4]. It has been well-known that consuming vegetables in Cruciferae family has a positive effect on the metabolism and has remedial effects on various illnesses and preventing chronic diseases. Cruciferae family contains high glucosinolates (GSLs) whichare precursors of isothiocyanates (ITCs). ITCs are degradation products occurred during the hydrolysis process of GSLs by myrosinase enzyme (MYR) of plant tissues or many bacteria that live in the intestine [5][6]. One of the ITC products that have been extensively studied is sulforaphane (SFN) which is a product from the hydrolysis of glucoraphanin in broccoli. Previous research reported that using plasma technology makes the radish sprouts grow faster approximately 9-12% [7]. Plasma technology causes the surface of the seeds attract more oxygen-containing molecules and makes the seeds absorb more water molecules, therefore, seeds are easier to germinate. Plasma is also a temporary source of Reactive Oxygen Species (ROS) during the treatment of seeds. It induces the metabolism of secondary metabolites by stress such as flavonoids synthesis without destroying plant tissue [8][9]. In addition, it was found that using organic elicitors such as salt or sugar can lead to increased amount of GSLs in plants. High concentrations of salt caused physical stress on broccoli sprouts and resulted in increased activation of MYR[10]. Using high concentration of sucrose influences aliphatic GSL synthesis and anthocyanins in broccoli sprouts [11], but there is no report on using plasma technology or organic stimulants in the cultivation of local microgreens in Thailand. Therefore, this research aims to study the influence of cold plasma technology ICoFAB2019 Proceedings | 124

and abiotic elicitors on percentage germination, length of stem, fresh weight, ITC content and bioactive compounds of Thai mustard green microgreen and also its cytotoxicity against Caco2 cells.

Materials and Methods

Chemicals and materials Chemicals including 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), Folin-Ciocalteu phenol reagent (FCR) and gallic acid, Benzyl isothiocyanate (BITC) were obtained from Sigma-Aldrich Chemical Co., (St. Louis, MO, USA). Trolox standard (TE) (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and 2,4,6-Tripyridyl-5-Triazine (TPTZ) were purchased from Fluka Chemicals (Buchs, Switzerland). Methanol for HPLC grade was obtained from BDH (Poole, UK). Dulbecco's modifed Eagle's medium (DMEM), Fetal bovine serum (FBS) and all other reagents used in cell culture were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). MTT (Amresco, OH, USA). Mustard green seeds were purchased from online shop (https://www.pwcmallonline.com). Human Colorectal Adenocarcinoma (CaCo2 ATCC® HTB-37™) was obtained from American Type Culture Collections (ATCC, Manassas, VA, USA).

Plant material and cultivation conditions Seed treatment with plasma technology was performed at Faculty of Engineering Srinakharinwirot University, Nakhon Nayok Province. The procedure was done according to the previous method [3]. Mustard green seeds (100 seeds/treatment) were treated with plasma at the power level of 19 kV for 5 min. After that, seeds were washed and soaked with distilled water overnight. The seeds were sowed in a layer of vermiculite in a tray at 25ºC (42 µmol/sec/m2 of light concentration, 12 h light/12 h dark cycle) in plant tissue culture room. Twenty mL of the tested elicitors; mL, 176 mM sucrose, 160 mM NaCl,10 mM CaCl2 were individually sprayed on the leaves of tested microgreens; however the control was spraying with deionized water (DI) for 7 days. Microgreens of 7-d olds were gently cut and collected. The germination percentage, length of stem and fresh weight were measured. Microgreen samples were packed in polyethylene bags and stored at -80 ºC for next analysis.

Extraction and determination of ITC Extraction of ITC followed the previous method [12]. The freeze dried microgreens (250 mg) were homogenize with 4 mL of 0.1 M citrate-phosphate buffer (pH 7.0) and incubated in shaking incubator (LSI- 1005R, Lab Tech, Korea) at 37 ºC for 1 h at 250 rpm. Extracted ITC with dichloromethane (DCM) in the ratio of 1:1 and shaken for 30 min at 250 rpm. The mixtures were centrifuged at 10,000 g for 5 min using centrifuge (Universal 320R, Germany). The supernatants were added with 0.5 g magnesium sulfate (MgSO4) for removing the moisture before mixing and centrifuged at the same condition. The clear supernatants were mixed with methanol in the ratio of 1:4 for ITC determination. Determination of total ITC was performed as previously [13]. Ten microliters of sample were added in the 96-well plate and mixed with 90 µL of methanol and 90 µL of 0.1 M phosphate buffer (pH 8.0). After 2 min, 10 µl of 0.08 M benzene 1,2 dithiol solution were added to the well plate and mixed using the pipette and incubated at 60 ºC for 2 h. The absorbance was measured at 360 nm using microplate reader (Synergy 4HT Microplate Reader). Results were expressed as mmol equivalents in 100 g of dried sample (mmol/100 g DW). Benzene-isothiocyanate (BITC) was used as a standard.

Microgreen extraction for bioactive compounds and antioxidant activity Dried microgreens (0.1 g) were extracted with 5 mL of 80% methanol and incubated at 37 ºC on an orbital shaker at 200 rpm. After 24 h, the mixture was centrifuged at 10,000 g for 20 min and repeated extracting twice. The supernatant was filtered through Whatman (No.1). The extracted sample was stored at -80 ºC.

Determination of total phenolic content (TPC) TPC was determined using Folin–Ciocalteu reagent according to the previous reported [14][15]. Twenty microliters of sample and 100 µL of 10% Folin-Ciocalteu reagent were pipetted into 96-well plate. After 5 min, 80 µL of 7.5% NaHCO3 were added, mixed, and incubated at room temperature for 30 min. The reaction was measured at 750 nm using microplate reader. Gallic acid was used as a standard and the results were reported as mg gallic acid equivalent (GAE)/g DW.

Determination of total flavonoid content (TFC) The TFC was evaluated according to the previous reported [16] with a minor modification. Microgreen extracts (20 µL) were mixed with 60 µL of DI water in well plate. After that, 10 µL of 5% ICoFAB2019 Proceedings | 125

NaNO3 and 10 µL of 10% AlCl3.6H2O were added, mixed and left for 5 min at room temperature. The mixture was reacted by adding with 100 µLof 1 M NaOH, mixed and kept for 30 min before measuring at 510 nm. Rutin was used as standard and expressed as mg rutin equivalents (mg RE/g DW).

Free-radical scavenging activity on DPPH· The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of microgreen extract was determined according to the previous reported [17] with some modifications. Three hundred microliter of microgreen samples or control (80% methanol) were mixed with 180 µl of 0.2 mM of DPPH solution in methanol. After 30 min,The mixture was measured measured at 595 nm. A standard curve of trolox was used and expressed as mg trolox equivalent (mg TE/g DW).

Ferric reducing antioxidant power (FRAP) The total reducing capacity of micreogreem extracted was evaluated using FRAP assay according to the previous reported [18] with some modifications. Working FRAP reagent was initially prepared consisting of 300 mM acetate buffer pH 3.6 by, 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) solution in 40 mM HCl and 20 mM iron (III) chloride solution (FeCl3) in the ratio of 1:1:10 (v/v/v), respectively, and incubated at 37 ºC for 30 min before using. Microgreen extract s(20 µL) were mixed with 180 µL of FRAP reagent and were then incubated for 90 min at room temperature before measured at 593 nm. FRAP values were reported as mg Fe(II)/g DW.

Cell line and cell culture The Human Colorectal Adenocarcinoma CaCo2 cancer cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained according to the recommendations of the ATCC at 37 ºC and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with penicillin, 100 µL/mL of streptomycin and 10% Fetal bovine serum. DMEM media was renewed every 3 days and trypsinized with 0.05% trypsin-EDTA, and 106 cells were seeded into fresh DMEM for next analysis.

Isothiocyanate extraction for cell line Microgreen extraction for cell lines with some modifications was done according to the previous reported [19]. Fresh microgreens (50 g) were homogenized in 50 mL of 0.1 M citrate phosphate buffer, pH 7.0 at 37 ºC in shaker incubator at 250 rpm for 2 h. GSLs were hydrolyzed to isothiocyanates (ITCs) form by MYR. Then, 50 mL of homogenates and 50 mL of DCM in the ratio of 1:1 were mixed together and were then dehydrated using MgSO4. The DCM layer was filtered through Whatman No. 1 and evaporated using rotary evaporator (Buchi R-114, Switzerland) before drying using freeze dryer. The stock solution of the extract was dissolved in 1% of dimethyl sulfoxide (DMSO) for further analysis.

Cytotoxicity assay The effect of crude extract on human CaCo2 colon cancer cell cytotoxicity was measured using the 3-(4, 5-dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide (MTT) assay according to the previous reported [20]. The density of CaCo2 cells at 5×103 cells/well was placed into the 96-well plate and incubated at 37ºC in 5% CO2 for 24 h. Cells were treated with crude microgreen extract with serum free medium concentration 0-250 µg/mL by serial dilutions. After the incubated at 37ºC in 5% CO2 for 24, 48 and 72 h, 20 µL of MTT reagent (5 mg/mL) were then added to each well plate and incubated at 37ºC under 5% CO2 for 4 h. The solution in the plate was replaced with 200 µL of DMSO to dissolve the formazan crystal and absorbance at 600 nm was measuredusing microplate reader. The IC50 value was calculated based on graphs ofusing % cytotoxicity and concentrations of extracts.

Cell morphology The human CaCo2 colon cancer cells were seeded into a well plate at a density 5×104 cells/mL. Cells were prepared in same the conditions as above. After treatment for 24 h at 37ºC under 5% CO2, cell morphology of cancer cells was monitored using a microscope at 400X.

Statistical analysis of data Data were reported as means ± standard deviation (SD). Statistical analysis was performed using One-way analysis of variance (ANOVA) using software SPSS (demo version). Statistically significant differences were considered at p<0.05.

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Results and discussion

Effect of plasma technology and abiotic elicitor on mustard green microgreens growth The effects of cold plasma technology and abiotic elicitor on % seed germination (Fig. 1A) and length of stem (Fig. 1B) of mustard green microgreens were not significantly different when compared with the control except the treatment with plasma plus NaCl and only NaCl led to the lowest % seed germination. Plasma plus NaCl and sucrose, and NaCl treantments led to the lowest length of stem in mustard green microgreens. A significant increase in the level of fresh weight was observed in plasma treatment (38.33 mg/microgreen) (Fig. 2A) in mustrad green compared with the control. However, after treated using plama with NaCl, plama with CaCl2, NaCl, and CaCl2 led to fresh weight reduction. Ling et al. [21] found that seeds treated with plasma at low temperature can differently absorbed the moisture depending on the power of plasma used. Low temperature plasma can react with the chemical structure and shell surface of the seeds and it allows the seeds to absorb the water from the outside better. Yuan et al. [22] reported that radish sprout was inhibited after receiving 50 and 100 mM of NaCl. The salinity affects the metabolic processes of the radish and causes changes in anatomy and physiology and influence on seed germination. Esfandiari et al. [10] found that the concentration of 160 mM NaCl did not effect on the weight of broccoli sprout which was similar to the study of Lee et al. [23] who mentioned that NaCl inhibited the germination of seeds. NaCl produces hydrogen peroxide [24][25][26] and calcium helps the cell wall and cell membrane to become stronger and increased membrane permeability.

Figure 1 Effect of plasma treatment and abiotic elicitor on % germination and length of stem of mustard green microgreen. (A) % gemination (B) Length of stem. Different letters above columns indicate significant differences (p<0.05)

Figure 2 Effect of plasma treatment and abiotic elicitor on fresh weight and total ITC of mustard green microgreen. (A) Fresh weight. (B) Total ITC. Different letters above columns indicate significant differences (p<0.05)

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Total ITC content The results showed significantly different ITC contents among Thai mustard green microgreens treated with plasma (2.65 mmol/100g DW) at the highest level of total ITC followed by plasma with CaCl2, sucrose and CaCl2, respectively (Fig. 2B). Plasma technology and abiotic elicitor increased the total ITC compared with the control, except for plasma with sucrose, plasma with NaCl and only NaCl. Yang et al. [27] reported that using CaCl2improved the biochemical properties in GSL synthesis by enhancing the BrST5b gene (sulfotransferase 5b) and BrAOP2 gene (2-oxoglutarate-dependent dioxygenase 2) expression and 10 mM CaCl2 increased the amount of GSLs in broccoli. Increasing amount of GSLs and increased activity of the MYR resulted in increased ITC content. Verkerk et al. [28] found many genes including BrST and BrFMOGSOX related to the GSL synthesis.

Bioactive compounds and antioxidant capacity The results showed The TPC and TFC of mustard green microgreen were significantly increased after treatment with plasma technology and abiotic elicitor (p<0.05) when compared with the control. After treated with plasma, the highest level of TPC (5.37 mg GAE/g DW) (Fig. 3A) and TFC (1.42 mg RE/g DW) (Fig. 3B) were detected. Nevertheless, NaCl treatment led to TPC decrease. Antioxidant activity by DPPH and FRAP assays were also significantly different in plasma treatments (p<0.05). Results found that only plasma treatment showed the highest antioxidant activity by DPPH (7.44 mg TE/ g DW) (Fig. 4A) and FRAP assay (15.42 mg FeII/g DW) (Fig. 4B). Tsai et al. [29] found that sucrose was an essential energy source for plant cells and can also add sugar to the aglycone in the process of glycosylation, which is the final reaction of the anthocyanin synthesis. Mano et al. [30] reported that flavonoids synthesis and enzymes were related to the synthesis of anthocyanins in Arabidopsis such as flavanone 3β-hydroxylase chalcone synthase chalcone isomerase and anthocyanidin synthase. Sucrose, glucose and fructose are promoting the synthesis of GSLs and anthocyanin in cruciferous plants. Sucrose is the most effective substance to stimulate accumulation of secondary substances [31]. Researchers [32] reported that plasma stimulated gene growth regulating factors, antioxidant enzymes, and energy metabolic genes which influenced on plant growth. Reports of Bußler et al. [9] stated that plasma caused ROS and UV which induced stress in plants and thus induced plant synthesis of the secondary metabolites, antioxidant, molecules/enzymes that helped to resist those stresses.

Figure 3 TPC and TFC of mustard green microgreens. (A) TPC and (B) TFC. Different letters above columns indicate significant differences (p<0.05)

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Figure 4 Antioxidant capacity of mustard green microgreens. (A) DPPH assay. (B) FRAP assay. Different letters above columns indicate significant differences (p<0.05)

Figure 5 Effects of 21 kV plasma treated mustard green microgreen on CaCo2 cell death. (A); % Cytotoxicity. (B) ; The IC50 value of 21 kV plasma treated mustard green microgreen on Caco2 cells. Data represent means±SEM of three independent experiments. Different letters above columns indicate significant differences (p<0.05)

Cytotoxicity and cell morphology of CaCo2 cell The principle of MTT is that the alive cancer cells have intact succinate dehydrogenase enzyme in mitochondria which turns tetrazolium salt into formazan by reduction process. The quantity of formazan is directly varied according to the number of the living cells. Cytotoxicity against CaCo2 cancer cells of crude mustard green microgreen extract treated with plasma and incubated for 24, 28 and 72 h showed the IC50 value of 55.89, 43.42 and 30.61 µg/mL, respectively (Fig. 5A-5B). Cells incubation for 72 h had the lowest IC50 indicating most effective effect. CaCo2 cancer cells treated with crude mustard green microgreen extract at 24, 48 and 72 h (Fig. 6) showed some signs of shrinkage, membrane blebbing, organelle condensation, and fragmented cells. The severity of cells morphological changes varied with increasing concentrations of the extract. The contraction of cells and broken into small and the cell membrane aneurysm are indicators ofdead cells i.e. apoptosis. Oberhamme et al. [33] and Webb et al. [34] pointed out that the characteristic of cells death caused by apoptosis consisted of cell shrinkage, chromatin condensation, nuclear fragmentation, membrane blebbing and apoptotic bodies. Apoptosis cell death can occur via two routes, either extrinsic (activation of death receptors) or intrinsic (mitochondrial-mediated) pathways.

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Figure 6 Effect of mustard green microgreens treated with plasma on cell morphology of CaCo2 cancer cells at 24, 48 and 72 h of cell incubation.

Conclusion

The plasma technology and abiotic elicitor or only abiotic elicitor have no effect on the percentage of germination in mustard green seeds. Plasma treatment affected the length of stem and the fresh weight of mustard green microgreens. It can induced ITC production and increase the bioactive compounds, antioxidant activity and cytotoxicity against CaCo2 cancer cells. This is the first report of using cold plasma technology and abiotic elicitor for Thai local vegetable microgreen cultivation. Plasma technology and abiotic elicitor can be used to improve nutrients and bioactive compounds in other plants.

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Acknowledgements

This research was funded by National Research Council of Thailand (Year 2019) through Master students funding awarded to WS . Authors would like to thank Department of Biotechnology, Mahasarakham University and Central Laboratory for laboratory facilities.

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Phytochemical Screening, Antioxidant, and α-Glucosidase Inhibitory Activities of Different Solvent Extracts from Leersia hexandra and Elephantopus scaber

Saithong Sombutphoothorn 1 and Ampa Konsue2*

1 Graduate student of Health Science program, Faculty of Medicine, Mahasarakham University, Maha Sarakham, 44000, THAILAND. 2 Applied Thai Traditional Medicine, Thai Traditional Medicine Research Unit, Faculty of Medicine, Mahasarakham University, Maha Sarakham, 44000, THAILAND.

*Corresponding author’s e-mail: e-mail: [email protected]

Abstract:

The aim of this research was to determine on phytochemical screening, antioxidant and α- glucosidase inhibitory activities of Leersia hexandra and Elephantopus scaber by using different solvent extractions. Both plants (1:1;w:w) of recipe were extracted using by different solvents including aqueous (ALE) and 80% ethanol (ELE). Phytochemical screening was determined on total phenolic (TPC) and flavonoid (TFC) contents in plants. The antioxidant activities were determined by, 2,2-diphenyl-1- picrylhydrazy (DPPH) radical scavenging and 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) assay. The α-glucosidase inhibitory assay was evaluated on glucose transferase mechanism. This experimental study found that the recipe showed high among of total phenolic and flavonoid contents especially ELE (40.024±0.952 mgGE/gExt and 0.072±0.001 mgQE/gExt,). The ELE (IC50 = 0.082±0.0025 mg/mL) was significantly exerted on free radical scavenging activity higher than ALE (IC50 = 0.122±0.0033 + mg/mL). ABTS radical scavenging activity, the ELE (IC50 = 0.0048±0.00018 mg/mL) was significantly stronger than Trolox (IC50 = 0.0086±0.00063 mg/mL), known as standard substances. α-glucosidase inhibitory activity, ALE (IC50 = 0.022±0.001 mg/mL) and ELE (IC50 = 0.098±0.002 mg/mL) were significantly more effect in inhibited α-glucosidase enzyme than acarbose (IC50 =1.054±0.113 mg/mL), known anti-diabetic drug. The recipe has been having the phenolic and flavonoid contents which chemical substances were known as anti-oxidation and anti-diabetic property, Pharmaceutical activities were showed on antioxidant and α-glucosidase inhibitory activities. Further, also chemical compositions, major active compound(s) and in vivo were clarified in next study.

Keywords: Phytochemical screening, Phenol, Flavonoids, Antioxidant, α-glucosidase

Introduction

Herbal medicines have been developed not only to improve ancient traditional therapeutics, but also as an alternative solution for health problems [1]. Thai traditional medicine is folklore medicine inherited from Thai ancestor [2]. The drugs have been used for healing since the past until now. In each recipe might be consist with also approximately of some plant, some dosage, some herbal part and some indication to disease treatment. This traditional medicine recipe is once recipe from Thai traditional medicine which composed with two kinds of medicinal plants. First plant, Leersia hexandra, Swamp Rice grass, name in Thai is Yaa-Sai [3] and second plant, Elephantopus scaber, Prickly-leaved elephant's foot, name in Thai is Do-Mai-Ru-Lom in weight ratio 1:1 (w:w) [4]. Thai ancestor still believed that this recipe has been claimed usage to treatment of diabetes. The review literatures of each plant in the recipe was revealed in many scientific reports. L. hexandra (family: Poaceae) is grass species used in traditional medicine to treat many diseases including hypertension and diuretic. The phytochemical studies have demonstrated the presence of plants are polyphenol, flavonoids and terpenoids. The pharmacological activities were reported such as hypertension, antioxidant and anticancer [5-6]. E. scaber (family: Asteraceae) has been used as traditional medicine in many countries. It has been popular as a medicinal herb in many countries of Southeast Asia [7]. In Thailand, it has been used as traditional medicine including diuretic, tonic, antihelminthic and aphrodisiac [7]. The most important of these biologically active constituents of plants are elephantopin, triterpenes, stigmasterol, epifriedelinol and lupeol. Other compounds are copaene, isopropyl, dimethyl, hexahydronaphthalene, cyclosativene and ICoFAB2019 Proceedings | 133

Zingiberene from the essential oils [8]. This plant has been extensively screened and proved for anticancer activity, which is mainly for its deoxyelephantopin containing. Many other biological activities such as antimicrobial [9-12], hepatoprotective [13], antioxidant [14-16], antidiabetic [17-19], anti-inflammatory [14], analgesic [17], anti-asthamatic[20] and anticancer [21] have been reported in various research articles[7-8]. Free radical stress is a common theme which underlines etiology of several degenerative disorders. The production of free radicals is linked to the inflammatory process. Free radicals prime the immune response, recruit inflammatory cells and are innately bactericidal [22-23]. The most common reactive • oxygen species (ROS) in particular free radicals include superoxide (O2 ) anion, hydrogen peroxide (H202), peroxyl (ROO-) radicals, and reactive hydroxyl (OH•) radicals. The nitrogen derived free radicals are nitric oxide (NO•) and peroxynitrite anion (ONOO•). In treatment of these diseases, antioxidant therapy has gained an immense importance. Synthetic antioxidants like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) commonly used in processed foods have side effects and are carcinogenic [24-25]. Diabetes mellitusis a well-known metabolic disorder, which is characterized by an abnormal postprandial increase of blood glucose level. The control of postprandial hyperglycemia is believed to be important in the treatment of diabetes mellitus. α-Glucosidase secreted from intestinal chorionic epithelium is responsible for the degradation of carbohydrates. α-Glucosidase inhibitors slow down the process of digestion and absorption of carbohydrates by competitively blocking the activity of glucosidase. Consequently, the peak concentration of postprandial blood glucose is reduced and the blood sugar level comes under control. α-Glucosidase inhibitors can offer several advantages and has been recommend by the Third Asia-Pacific Region Diabetes Treatment Guidelines as the first-line of treatment for lowering postprandial hyperglycemia [26]. Several α-glucosidase inhibitors, acarbose was obtained from natural sources, can effectively control blood glucose levels after food intake and have been used clinically in the treatment of diabetes mellitus [27]. In clinically, they have been associated with serious gastrointestinal side effects. It is necessary to search for alternatives that can display α-glucosidase inhibitory activity but without side reactions [28]. However, this traditional medicine recipe was widely used to treat many diseases, but there is no any scientific report. Therefore, this study aimed to determine on phytochemical screening (TPC and TFC), anti-oxidant activity (DPPH and ABTS) and α-glucosidase inhibitory for confirmed pharmaceutical preliminary.

Materials and methods

Sample Collection Leersia hexandra and Elephantopus scaber in recipe were collected from Maha Sarakham and Amnat Charoen province, northeastern of Thailand. The specimens were identified and deposited at the Faculty of Medicine, Mahasarakham University, Thailand (code; L. hexandra: MSU.MED‑ LH0001/SS and E. scaber: MED‑ ES0001/SS). All of the fresh materials were cleaned and dried at 40°C for 18 hr in a hot air oven and then powdered.

Preparation of Extracts The aqueous extract (ALE) was prepared by distilled water for 15 min at 100°C in Electric boiler (1:10 w/v). The boiling process was repeated twice. The ethanolic extract (ELE) was macerated with 80% ethanol for 3 hr at 60°C in sonication bath (1:10 w/v). The sonicating process was repeated three times. The residue powder was excluded by using filter papers. The filtrate was evaporated using by a rotary evaporator (Heidolph Laborota 4000, Germany) and freeze-dried to obtain brown extract. The extracts were kept in the fridge at -20°C until be used.

Total phenolic content assay Total phenolic content was determined according to a modified procedure [29]. Sample (100 µL) will be oxidized with 500 µL of 0.2 N Folin-Ciocalteu’s reagent and neutralized by adding 400 µL of 7.5% Na2CO3. The absorbance measured at 765 nm by UV-Vis Spectrophotometer after mixed and incubated in room temperature for 30 min. The results were expressed as gallic acid equivalents (mgGE/gExt).

Total Flavonoid Content Assay Flavonoid content was estimated using the aluminum chloride colorimetric method [30]. The extracts from recipe will be mixed with 100 µL of 5% aluminum chloride (w/v), 400 µL of 2.5% NaNO2 After 5 min, 500 µL of 5%AlCl3. The mixture was allowed to stand at room temperature for 10 min. The solution was mixed 2,000 µL distilled water. The samples were measured at 415 nm. The TFC was calculated from a standard quercetin equivalent (mgQE/gExt). ICoFAB2019 Proceedings | 134

DPPH radical scavenging assay 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging capacities of wheat extracts were estimated by the reduction of the reaction color between DPPH solution and sample extracts as previously described by prior method [31]. DPPH was dissolved in ethanol to a 0.039 mg/mL. The plant extract at various concentrations was diluted with distilled water to get sample solution. The sample solution with 100 µL following which 900 µL DPPH (0.1 mM) working solution. After a 30 min reaction kept in the dark at ambient temperature then absorbance of the solution was measured at 515 nm. In this study, Trolox and ascorbic acid were used as standard substances. Blank was run in each assay. DPPH radical ability was expressed as IC50 (mg/mL) and the inhibition percentage calculated using the following formula: DPPH scavenging activity (%) = (A0-A1)/ A0 x 100 where A0 is the absorbance of the control and A1 is the absorbance of the sample.

ABTS+ Radical Scavenging assay In ABTS assay, the plants extract was allowed to react with ABTS+, a model stable free radical derived from 2,2-azinobis (3-ethylvenzothiazolin-6-sulphonic acid) (ABTS+) [32]. The ABTS+ (900 µL) was added to the extracts (100 µL) and thoroughly mixed. The mixture was held at room temperature for 6 min and absorbance was immediately measured at 734 nm. Trolox and ascorbic acid solution in distilled water was prepared and assayed under the same conditions. ABTS scavenging ability was expressed as IC50 (mg/mL) and the inhibition percentage calculated using the following formula: ABTS scavenging activity (%) = (A0-A1)/ A0 x 100 where A0 is the absorbance of the control and A1 is the absorbance of the sample.

α-Glucosidase inhibitory assay All extracts were tested for their ability in inhibiting α‑ glucosidase using in vitro assay. The assay method was assessed using Taepongsorat, et al. (2019) with slight modifications [33]. Briefly, a volume of 120 µL of the sample solution and 100 µL of 0.1 M phosphate buffer (pH 6.8) containing α‑ glucosidase solution (0.2 U/mL) was incubated at 37ºC for 20 min. After preincubation, 100 µL of 5 mM p‑ nitrophenyl‑ α‑ D‑ glucopyranoside solution in 0.1‑ M phosphate buffer (pH 6.8) was added to each well and incubated at 37°C for another 20 min. Then, the reaction was stopped by adding 320 µL of 0.2‑ M Na2CO3 into each well, and absorbance were read (A) and recorded at 405 nm by UV-Vis Spectrophotometer and compared to a control which had 120 µL of buffer solution. The system without α‑ glucosidase was used as blank, and acarbose was used as positive control. The α‑ glucosidase inhibitory activity was expressed as inhibition (%) and was calculated as follows: %inhibition = (A0–A1)/A0 × 100 where A0 is the absorbance of the control and A1 is the absorbance of the sample. IC50 values were calculated by the graphic method.

Statistical analysis All assays were expressed as means ± standard deviation (SD) from five separate experiments (n = 5). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. Differences at P < 0.05 were considered to be significant.

Results and discussion

The ELE and ALE from the recipe has been have both TPC and TFC which ELE (40.024±0.952 mgGE/gExt; 0.072±0.0007 mgQE/gExt) showed significantly higher contents than ALE (11.424±0.158 mgGE/gExt; (0.017±0.0002 mgQE/gExt). (Table 1). Phenolic compounds have an aromatic ring with one or more hydroxyl groups and act as antioxidants [47]. In the study, extraction methods, solvent polarity is frequently used for recovering phenolic compounds from plant. Ethanol is an organic solvent which has been known as a good solvent for phenolic substance extraction and lowly hazard to human consumption [34]. Some organic molecules are more polar and therefore more soluble in water. Thus, the chemical composition on aqueous extraction method were composed with polysaccharide, proteins and glycoside substances. The aqueous extraction may either contain non-phenolic or possess phenolic compounds that contain a smaller number of active groups than the other solvents [35]. Total phenolic compounds in methanol extract of E. scaber showed high TPC and significant antioxidant activity. The antioxidant activity increased with increasing concentration of extract [39]. E. scaber ingredients composed with phenolic, flavonoid, terpenoid and another compound might play a role in the antioxidant activity. These phytochemicals showed possess wound healing, anti-venom, anti-microbial, anti-inflammatory, anti- diabetic, cytotoxic and anti-tumour activities. In this study DPPH radical scavenging activity, standard substances, ascorbic acid (IC50 = 0.004±0.0002 mg/mL) and trolox (0.016±0.0012 mg/mL) were show significantly more potent than all of ICoFAB2019 Proceedings | 135

the extracts from this recipe. While, The ELE (IC50 = 0.082±0.0025 mg/mL) was significantly exerted on free radical scavenging activity higher than ALE (IC50 = 0.122±0.0033 mg/mL). (Table 1). Surprisingly, + ABTS radical scavenging assay, the ELE (IC50 = 0.0048±0.00018 mg/mL) was significantly stronger on this method than trolox (IC50 = 0.0086±0.00063 mg/mL), known as standard substances but not ascorbic acid (IC50 = 0.0019±0.00004 mg/mL). (Table 1). The antioxidant activity in the experiment found that the extraction by using by ethanol provided high significantly free radical scavenging both DPPH and ABTS+ methods which is related to the quantity of TPC and TFC. The ethanol extract gave the strong antioxidant capacity in the study which showed low values of IC50 [36]. The antioxidant activity of extracts varied depending on the polarity of solvent and the method used to extract bioactive compounds. Changing in solvent polarity alters its ability to dissolve a selected group of antioxidant compounds and influences the antioxidant activity estimation [37]. The antioxidant activity could be therapeutic importance in preventing oxidative stress involving in the development of several diseases [38]. There were some reports regarding the ethanol and aqueous extracts of L. hexandra showed that it has an antioxidant effect that protects the tissues from the deleterious effects of free radicals resulting in hypertension rat [5]. Methanol extract of E. scaber showed high TPC and the antioxidant activity increased with increasing concentration of extract [39]. In this experiment, α-glucosidase inhibitory activity, the result found that ALE (IC50 = 0.022±0.001 mg/mL) and ELE (IC50 = 0.098±0.002 mg/mL) which can inhibit activity of α-glucosidase enzyme more than acarbose (IC50 = 1.054±0.113 mg/mL). (Table 1). In this study, the α-glucosidase inhibitory activity was obtained stronger than the positive control, acarbose. Phytochemicals have also been isolated from plants includes elephantopin, triterpenes, stigmasterol epifriedelinol and lupeol [8]. They showed strong inhibitory activity against α-glucosidase. These studies showed α-glucosidase inhibitors slow down the process of digestion and absorption of carbohydrates by competitively blocking the activity of glucosidase [26]. On the basis of literatures published worldwide, summarized in a list of 411 natural products isolated from medicinal plants that showed α-glucosidase inhibitory activity [38]. Structurally these natural product inhibitors compose of terpene, alkaloid, quinine, flavonoid, phenol, phenylpropanoid, and steroid frameworks. These inhibitors are rich in organic acid, ester, alcohol, and allyl functional groups. A majority of the compounds reported contain flavonoid, terpene, and phenylpropanoid ring structures [38]. Recently, several studies have determined that flavonoid compounds can be very effective in inhibiting α- glucosidase activity [45]. A total of 103 flavonoids showed glycosidase inhibitory activity including xanthones, flavanones, flavans, anthocyamins, chalcones, and other structural motifs [38]. Thirty-seven polyphenols from plants have shown promising α-glucosidase inhibitory activity. Gallic acid, an important constituent of many plants species showed strong inhibitory activity against glucosidase both in vitro and in vivo [46]. The effect of phytomedicines can be evaluated by studying synergistic effects through multitarget effects or effects on pharmacokinetic or physicochemical properties. There has been some reports regarding 28Nor-22(R) with a 2, 6, 23-trienolide, a major steroid isolated from the acetone extract of the E. scaber decreased blood level glucose in STZ diabetic rats [41]. This may be due to a stimulating effect on insulin release from regenerated β-cells of the pancreas or increased cellularity of the islet tissues [41]. Bioactive compounds of E. scaber had also successfully identified in some compounds such as deoxyelephantopin, isodeoxyelephantopin, scabertopin, isoscabertopin, elescaberin, 17, 19- dihydrodeoxyelephantopin and a terpenoid which showed a broad range of biological functions [8]. The ethyl acetate root extract and methanol leaf extract of E. scaber showed antihyperglycemic effect by reducing the blood glucose level, glycosylated hemoglobin, a change in the lipid profile and kidney functions, liver and muscle glycogen, serum insulin levels and histopathological studies [42]. Aqueous extract of leaves and roots into alloxan induced diabetic rats significantly reduced serum glucose, glycosylated hemoglobin and the activity of gluconeogenic enzyme glucose-6-phosphatase [43]. However, could be linked to more than one mechanism including insulin sensitizing, insulin releasing, gluconeogenesis inhibition and α-glucosidase inhibition [44]. Thus, it is worthwhile to evaluate further the effective components of isolated compounds in vivo rather than make a conclusion based on enzyme inhibition assay only [45].

Conclusions

The recipe are combines from L. hexandra and E. scaber as a Thai traditional remedy, present experiments provides a preliminary data that the recipe ingredients with phenolic compounds and flavonoid contents. The pharmaceutical activities were show more potent on antioxidant and α-glucosidase inhibitory activities. In this study, the pharmaceutical preliminary was confirmed to use of this recipe from Thai traditional medicine. However, chemical compositions, major active compound(s) and in vivo need to be clarified in next study.

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Table 1 Total phenolic (TPC), and flavonoid contents (TFC), DPPH, and ABTS radical scavenging activities, and α-glucosidase inhibitory activities of different solvent extracts from combination of Leersia hexandra and Elephantopus scaber.

TPC TFC DPPH ABTS+ α-Glucosidase Samples mgGE/gExt mgQE/gExt IC50(mg/mL) IC50(mg/mL) IC50 (mg/mL) ALE 11.424±0.158b 0.017±0.0002b 0.122±0.0033d 0.0095±0.00054d 0.022±0.001a ELE 40.024±0.952a 0.072±0.0007a 0.082±0.0025c 0.0048±0.00018b 0.098±0.002a ascorbic - - 0.004±0.0002a 0.0019±0.00004a - acid - - 0.016±0.0012b 0.0086±0.00063c - trolox - - - - 1.054±0.113b acarbose ALE was extracted with aqueous. ELE was extracted with 80% ethanol. TPC was measured with gallic acid equivalents (mgGE/gExt). TFC was measured with quercetin equivalent (mgQE/gExt). Antioxidant activities and α-glucosidase + inhibitory activities showed IC50 of different extracts from recipe. DPPH and ABTS radical scavenging activity were used trolox and ascorbic acid as standard substances. α-glucosidase inhibitory activities was used Acarbose as a positive control. Different letters indicated significantly different at p < 0.05.

Acknowledgements

The study was supported by Faculty of Medicine, Mahasarakham University, Maha Sarakham, Thailand.

References

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doi:10.14457/MSU.res.2019.27 ICoFAB2019 Proceedings | 139

Evaluation on Phytochemical Constituents and Antioxidant Activities of Various Formula from Ko-Klan Remedies by Aqueous Infusion Preparation Method

Khwanchnok Maitnork1 and Ampa Konsue 2*

1Graduate student in Health Sciences program, Faculty of Medicine, Mahasarakham University, Maha Sarakham, 44000 Thailand 2 Thai Traditional Medicine Research Unit, Applied Thai Traditional Medicine program, Faculty of Medicine, Mahasarakham University, Maha Sarakham, 44000 Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Ko-Klan remedy is Thai folklore medicine appear on list of Thai herbal medicinal products which have been widely used to relief of muscle pain. The remedy were separated to 3 recipes; first, Mallotus repandus: Rhinacanthus nasutus: Elephantopus scaber: Aegle marmelos ; 1: 1:1:1 (w:w:w:w), second, Mallotus repandus: Rhinacanthus nasutus: Elephantopus scaber: Aegle marmelos; 5:2.5:1.5:1.5 (w:w:w:w), third, Mallotus repandus: Rhinacanthus nasutus: Elephantopus scabe: Ceasalpinia sappan: buchanani: Piper interruptum; 2:1:1:2:2:2 (w:w:w:w). Aims of this study were evaluated to total phenolic (TPC) and flavonoids (TFC) content, and antioxidant activities of various formulas from Ko- Klan remedy extracts by water infusion preparation. The TPC and TFC were determined to chemical composition. The antioxidations were tested using by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging, and 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) assay. The results showed that, the 1st remedy were higher contents of TPC and TFC (12.7927±0.0762 mgQE/gExt and 40.7925± nd 0.5372 mgGE/gExt), the 2 remedy was highest antioxidant activity by ABTS assay (IC50 = 0.0022± rd 0.00002 mg/mL), the 3 remedy were highest free radical scavenging by DPPH assay (IC50 = 0.0122± 0.00005 mg/mL). The finding studies suggest that all of the remedy were antioxidation higher than Ascorbic acid and Trolox as standard substances. Further study, some chemical compound(s) and pharmaceutical activity were clarified to medicinal plant use on health promotion.

Keywords: Ko-Klan, Phenol, Flavonoids, Antioxidantion, Infusion, DPPH, (ABTS+).

Introduction

Ko-Klan remedies are Thai folklore medicine on list Thai herbal medicinal products which have been widely used for relief of muscle pain. There are according in National List of Essential Medicines, 2012 [1]. Recent studies, the authers were selected to 3 formulas from Ko-Klan remedies hich but not have some sciencetific report in each recipe. The reviews of some literature revealed to the plants in the each formulas of the recipe. M. repandus the main medicinal plant of remedy, is scandent and hard wood plant. It composed of phytochemicals such as polyphenols, terpenoid, benzopyran, coumarin and steroid [2]. M. repandus stem had the highest bergenin (polyphenol) content of 12.67±0.26% and 19.38±0.63% of dry% weight, respectively [3]. Chemical structure of phenolic compound contents of flavonoid, tannins, coumarin, lignans, quinone and curcuminoids [4]. Moreover, by DPPH assay and FRAP assay, the methanol extract of M. repandus had IC50 = 24.45 µg/mL and 99.01±4.56 GEAC/g, respectively [5]. R. nasutus is s shrub plant, composing of phytochemicals such as naphthoquinone, lignans, flavonoid, tritepene and steroid. By DPPH assay and FRAP assay, the methanol extract of R. nasutus had IC50 = 55.56±7.71 µg/mL and IC50= 215.19±20.69 µg/mL, respectively [6]. E. scaber is herbaceous plant [7,8] whole plant composing of phytochemicals such as alkaloid, flavonoid, tannins [9], terpinoid, steroid [10], tritepene and flavone [11]. By DPPH assay, the 70% ethanol extract of leaf and the separate extraction by ethyl acetate (ESEAF) revealed that ESEAF 1,000 µg/mL had IC50 69.70±0.01 µg/mL. By superoxide anion radical scavenging activity (SOD), there is antioxidant property of IC50 3.79±0.16 µg/ [12]. The methanol extract of root had superoxide anion radical scavenging activity of IC50= 48±5 µg mL-1, hydroxyl radical scavenging activity of IC50=72±12 µg mL-1 and lipid peroxidation inhibition of IC50=103±18 µg mL-1 [13]. By The 3 hours 80 °C refluxed ethanol extract of whole plant had SC50= 12.4 µg/ml-1. By ICoFAB2019 Proceedings | 140

DPPH assay, ethanol extract of whole plant had xanthine oxidase inhibition of IC50= 93.1 µg/mL-1 [14]. A. marmelos is perennial plant that using fruit for medicinal purpose. Composing of phytochemicals such as coumarin, eugenol, tannins and mucilage [15]. By FRAP assay, the 95% ethanol extract and aqueous extract of A. marmelos fruit had 111.61 ± 0.59 Fe2+ and TEAC value = 39.04 ± 0.23 mg trolox equivalent and 95% ethanol extract had 27.35 ± 2.54 mg trolox equivalent. By ABTS assay, aqueous extract and ethanol extract of A.marmelos fruit had EC50 = 36.40 ± 1.23 µg/mL and 68.07 ± 5.23 µg/mL respectively [16]. Moreover, there is highly phenolic of 83.8± 37.6 mg/GAE per 100 ml [17]. C. sappan is perennial plant that using core for medicinal purpose. Composing of phytochemicals such as flavonoid and sterol. By infusion method, aqueous extract of C. sappan had total phenolic of 147.02 ± 0.63 mgGAE/g. By DPPH assay, aqueous extract of C. sappan had total phenolic of 33.91 ± 1.50 % [18]. P. interruptum is climber plant [19]. The 80% ethanol extract of P. interruptum had total phenolic 7.3 ± 0.8 mg GAE/g. By DPPH assay, the 80% ethanol extract of P. interruptum had IC50=138.7 ± 2.2 µg/mL and lipid peroxidation inhibition of IC50=38.7 ± 0.1 µg/mL [20]. C. buchanani was used boiled vine to for muscle pain relief. The vine of C. buchanani composes of phytochemicals such as buchananine, cardenolides and cardenolide glycoside. As far as we know, no previous report of antioxidant in C. buchanani but there are various pharmacology effect such as chondro protective activities, anti-inflammatory activities, analgesic activity and hepatoprotective activity [21]. Free radical is single electron in atom or molecule, unstable and rapidly react with biomolecules. Free radical in form of ROS and RNS such as super oxide anion, hydroxyl radicals nitric oxide, nitrogen dioxide which born from oxygen using in organism. Quantity of free radical that imbalance with antioxidant bring about to oxidative stress and causing diseases such as cancer, heart disease, Alzheimer, diabetes and neurotoxin. Naturally, organism can produce antioxidant that control quantity of free radical in the body for reducing destructive effects of free radical. For protecting body from free radical, eating antioxidant food such as vegetables, fruits and medicinal plants for increase antioxidant is necessary [22]. Although there are some previous reports about antioxidant in medicinal plants of Ko-Klan remedies, but there is no previous research in each formulas from Ko-Klan remedies. The aims of this research were investigated to phytochemical constituents and antioxidant activities from various formulas by water infusion extraction.

Materials and methods

Collection of plant materials The 1st remedy composed with Mallotus repandus (stem): Rhinacanthus nasutus (whole): Elephantopus scaber (whole): Aegle marmelos (fruit); 1: 1:1:1 (w:w:w:w). The 2nd remedy composes with Mallotus repandus (stem): Rhinacanthus nasutus (whole): Elephantopus scaber (whole): Aegle marmelos (fruit); 5:2.5:1.5:1.5 (w:w:w:w). The 3rd remedy composed with Mallotus repandus (stem): Rhinacanthus nasutus (whole): Elephantopus scabe (whole): Ceasalpinia sappan (core): Cryptolepis buchanani (stem): Piper interruptum (stem); 2:1:1:2:2:2 (w:w:w:w). R. nasutus and E. scaber were collected from Maha Sarakham Province, northeastern Thailand. The specimens were identified and deposited at the Faculty of Medicine, Mahasarakham University, Thailand (code; R. nasutus:MSU. MED; RN0001/KM and E. scaber: MSU.MED; ES0001/KM). Remaining plants were purchased from Thong In pharmacy Co.,Ltd. All of the fresh materials were cleaned and dried at 50°C for 48 h in a hot air oven and then powdered.

Preparation of extracts The aqueous infusion extraction was prepared using by boiled water at 80 °C for 20 min (1:200 w/v). The residue powder was excluded by using filter papers. The filtrate was evaporated using by a rotary evaporator (Heidolph Laborota 4000, Germany) and freeze-dried to obtain dark brown extract. The extracts were kept in the fridge at -4 °C until be used.

Total phenolic content assay Total phenolic content was determined according to a modified procedure.[9] Sample (100 µL) will be oxidized with 500 µL of 0.2 N Folin-Ciocalteu’s reagent and neutralized by adding 400 µL of 7.5% Na2CO3. The absorbance measured at 765 nm after mixed and incubated in room temperature for 30 min. The results were expressed as gallic acid equivalents (mgGE/gExt).

Total flavonoid content assay Flavonoid content was estimated using the aluminum chloride colorimetric method .[10] The extracts from recipe will be mixed with 100 µL of 5% aluminum chloride (w/v), 400 µL of 2.5% NaNO2 After 5 min, 500 µL of 5% AlCl3. The mixture will be allowed to stand at room temperature for 10 min. ICoFAB2019 Proceedings | 141

The solution was mixed 2,000 µL distilled water. The results was measured at 415 nm. The TFC was calculated from a standard quercetin equivalent (mgQE/gExt).

DPPH free radical scavenging activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacities of wheat extracts were estimated by the reduction of the reaction color between DPPH solution and sample extracts as previously described by prior method. DPPH was dissolved in ethanol to a 0.039 mg/mL. The recipe of extract at various concentrations was diluted with distilled water to get sample solution. 100 µL of the sample solution following which 900 µL DPPH (0.1 mM) working solution. After a 30 min reaction kept in the dark at ambient temperature then absorbance of the solution was measured at 515 nm. In this study, will be used Trolox and ascorbic acid as standard substances. Blanks were run in each assay. DPPH radical ability was expressed as IC50 (mg/mL) and the inhibition percentage calculated using the following formula: DPPH scavenging activity (%) = (A0-A1)/ A0 x 100 where A0 is the absorbance of the control and A1 is the absorbance of the sample.

ABTS+ radical scavenging activity In ABTS assay, the recipe of extract will be allowed to react with ABTS+, a model stable free radical derived from 2,2- azinobis ( 3- ethylvenzothiazolin- 6- sulphonic acid) ( ABTS+) assay was performed.[12] The ABTS+ (900 µL) was added to the extracts (100 µL) and thoroughly mixed. The mixture was held at room temperature for 6 min, and absorbance was immediately measured at 734 nm. Trolox and ascorbic acid solution in 80% ethanol was prepared and assayed under the same conditions. ABTS scavenging ability was expressed as IC50 (mg/mL) and the inhibition percentage calculated using the following formula: ABTS scavenging activity (%) = (A0-A1)/ A0 x 100 where A0 is the absorbance of the control and A1 is the absorbance of the sample.

Statistical analysis All assays were expressed as mean± standard deviation (SD) from five separate experiments (n = 5). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. Differences at p < 0.05 were considered to be significant.

Results and discussion

Total phenolic compounds and total flavonoid contents The results showed that the extracts from all of Ko-Klan remedy had both TPC and TFC. The TPC was found that the 1st remedy (12.7927±0.0762 mgGE/gExt) was higher than the 3rd remedy and the 2nd remedy (8.3655±0.0800 and 7.6846±0.0789 mgGE/gExt), respectively. Moreover, TFC from 1st remedy (40.7925±0.5372 mgQE/gExt) had still more content than the 3rd remedy and the 2nd remedy (20.5432± 0.4095 and 17.9524±0.2972 mgQE/gExt), respectively. Phenolics were the main antioxidant components, and their total contents were directly proporty to their antioxidant activity [23]. According to Phuaklee P. et al.(2015), studied on 95% methanol extract and aqueous extract of Aegle marmelos, state that extracts had high total phenolic content 83.8± 37.6 mg/GAE per 100 ml. Furthermore, Taokaenchan N. et al. (2017) demonstrated that aqueous extract of Caesalpinia sappan by infusion methode had total phenolic content 147.02 ± 0.63 mgGAE/g. Moreover, this study is consistent with Klinthong et al. (2015) on Piper interruptum states that 80% ethanol extract of plant had 7.3 ± 0.8 mg GAE/g of total phenolic content. Nevertheless, this research had study on high electron solvent and necessary to study on other solvent including type and quantity of total phenolic content in all of Ko-Klan remedies. (As shown in Table 1)

DPPH free radical scavenging activity rd The result showed that the 3 remedy (IC50 = 0.0122±0.00005) was exerted on free radical nd st scavenging activity higher than 2 remedy and 1 remedy (IC50 =0.0153±0.00009, 0.0204±0.00018 mg/mL), respectively. The 3rd and 2nd remedy were more potent on DPPH scavenging activity than the standard controls, ascorbic acid (IC50 = 0.0162±0.00029 mg/mL), and Trolox (IC50 = 0.0444±0.00075 mg/mL). The result was consistented with a research of Wonglom et al (2016), revealed that methanol extract of Mallotus repandus had IC50 = 24.45 µg/mL by DPPH assay. Furthermore, according to the study of Teansuwan et al. (2015), by DPPH assay, ethanol extract of Rhinacanthus nasutus had IC50= 55.56±7.71 µg/mL. This result related well with study of Chan et al. (2017) reported that by DPPH assay, 70% ethanol extract of Elephantopus scaber with separated extraction by ESEAF 1,000 µg/mL found that IC50 = 69.70±0.01 µg/mL. There is Superoxide Anion Radical Scavenging Activity (SOD) IC50 3.79±0.16 µg/mL consistent with Pongpiriyadacha et al. (2009) which study ethanol extract of elephantopus scaber by 3 hours ICoFAB2019 Proceedings | 142

of 80 °C reflux, SC50= 12.4 µg/ml-1. Moreover, the research had shown xanthine oxidase enzyme inhibitory activity IC50= 93.1 µg/ml-1. A similar conclusion was reached by Taokaenchan et al. (2017), aqueous extract of Caesalpinia sappan by infusion had 33.91 ± 1.50 % when examined by DDPH assay. Therefore, This result related well with studies of Klinthong et al. (2015)’s research on Piper interruptum Opiz states that by DPPH assay, 80% ethanol extract of Piper interruptum Opiz had IC50=138.7 ± 2.2 µg/mL and lipid peroxidation inhibitory activity IC50=38.7 ± 0.1 µg/mL. (As shown in Table 1)

2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+ ) activity + nd rd st In the study, using ABTS assay, the 2 , the 3 and 1 remedy (IC50 =0.0022±0.00002, 0.0037± 0.00003, and 0.0040±0.00003 mg/mL), had more potent on free radical scavenging activity than ascorbic acid (IC50 = 0.0099±0.00022 mg/mL) and trolox (IC50 = 0.0230±0.00035 mg/mL), as the standard controls. Overall these findings are in accordance with findings reported by Abdullakasim et al. (2007), studied 95% ethanol and aqueous extracts of Aegle marmelos fruit by ABTS assay, EC50 = 36.40 ± 1.23 µg/mL. (As shown in Table 1)

Table 1 Total phenolic (TPC), flavonoid contents (TFC), DPPH and ABTS radical scavenging activities of various formula from Ko-Klan remedies by aqueous infusion preparation method

TPC TFC DPPH ABTS Samples (mgGE/gEtx) (mgQE/gEtx) (IC50 mg/ml) (IC50 mg/ml) 1st Remedy 13.7473±0.0949a 40.7925±0.5372a 0.015±0.00002c 0.004±0.00002c

2nd Remedy 11.1438±0.0875c 17.9524±0.2972c 0.014±0.00004b 0.002±0.00001a

3rd Remedy 12.1120±0.1441b 20.5432±0.4095b 0.011±0.00004a 0.003±0.00003b

d d Ascorbic acid - - 0.016±0.00029 0.010±0.00022 e e Trolox - - 0.044±0.00075 0.023±0.00035 TPC was measured with gallic acid equivalents (mgGE/gExt). TFC was measured with quercetin equivalent + (mgQE/gExt). Antioxidant activities showed IC50 of different extracts from recipe. DPPH and ABTS radical scavenging activity were used ascorbic acid and trolox as standard substances. Different letters indicated significantly different at p < 0.05.

Conclusions

The all of formulas from Ko-Klan recipe ingredient with phenolic compound and flavonoid contents. Moreover, the recipe had more effect on free radical scavenging activity. Furthermore, isolation and active compound(s) were evaluated. The pharmaceutical preliminary was confirmed usage indication in Thai traditional medicine. However, any sign or symptoms were clarified in next study. Koklan remedies can be developed as healthy drink. So, the study of proportion of herbal ingredients and flavor are necessary.

Acknowledgements

The study was supported by Faculty of Medicine, Mahasarakham University, Maha sarakham, Thailand.

References

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[4] Wu-Yang H., Yi-Zhong C. and Yanbo Z. “Natural Phenolic Compounds From Medicinal Herbs and Dietary Plants: Potential Use for Cancer Prevention”. Nutrition and Cancer 2010; 62(1): 1-20 [5] Sriset Y., Chatuphonprasert W. and Jarukamjorn K. “Quantitative Determination of Bergenin in Mallotusrepandus (Willd.) Muell. Arg. Stem Extract by Reverse Phase-High Performance Liquid Chromatography”. Isan Journal of Pharmaceutical Sciences 2018; 14(1): 67-74 [6] Wonglom P., Putthabun K .and Sarapark N. “Antioxidant in Mallotusrepandus (Willd.) Muell, Tinospora crispa (L.) and Azadirachta indica A.Juss. extracts”. [Thai traditional medicine research department]. Ubonrachathani; Rajabat Ubonrachathani University; 2559. [7] Teansuwan N., Sripanidkulchai B. and Jaipakdee N. “Effect of four herb extracts on melanin synthesis”. Isan Journal of Pharmaceutical Sciences 2016; 11: 33-42 [8] Bunyapraphatsara N. and Chokechaicharoenpon O. “Thai traditional medicinal plant vol.2”. Bangkok: Citizen; 2541. [9] Wutthithammaveth W. “Thai medicinal plants encyclopedia and Thai pharmaceutical principle”. Bangkok: OdianStore; 2540 [10] Chatterjee M. and Mukherjee A. “Elephantopus scaber L: 1. AN OVERVIEW”. Indian J.L.Sci 2014; 4 (1): 51-54 [11] Farha A.K. and Remani P.R. “Phytopharmacological Profile of Elephantopus scaber”. Pharmacologia 2014; 2014: 272-85 [12] Wang J.J., Li P., Guo Z., Baosai L. Edward J K. and Chunlin L. “Bioactivities of Compounds from Elephantopus scaber, an Ethnomedicinal Plant from Southwest China” Evidence-based Complementary and Alternative Medicine (EVID-BASED COMPL ALT) 2014; 2014(4): 7 [13] Chan C.K., Tan L. T.H., Andy S.N., Kamarudin M.N.A. Goh B.H. and Kadir H.A. “Anti- neuroinflammatory Activity of Elephantopus scaber L. via Activation of Nrf2/HO-1 Signaling and Inhibition of p38 MAPK Pathway in LPS-Induced Microglia BV-2 Cells” Front Pharmacol 2017; 8 : 397 [14] Sheeba K.O, Wills P.J, Latha B.K, Rajalekshmy R, Latha M.R. “Antioxidant and antihepatotoxic efficacy of methanolic extract of Elephantopus scaber Linn in Wistar rats”. Asian Pacific Journal of Tropical Disease 2012: 904-8. [15] Pongpiriyadacha, Y., P. Nuansrithong and N. Sirintharawech. “Antioxidant activity and xanthine oxidase inhibitor from thai medicinal plants used for tonic and longevity”. Proceedings of the 47th Kasetsart University Annual Conference, March 17-20, 2009, Bangkok Thailand, pp: 1-9. [16] Phuaklee P., Dechayont B., CHUNTHORNG-ORN J. and Ittharat A. “Anti-allergic activity Anti- inflammatory and Antioxidant activity of Aegle marmelos fruit”. Thammasat Medical Journal 2018; 18: 349-57 [17] Abdullakasim P, Songchitsomboon S, Techagumpuch M, Balee N, Swatsitang P, Sungpuag P. “Antioxidant capacity, total phenolics and sugar content of selected Thai health beverages”. Int J Food Sci Nutr 2007;58:77-85. [18] Taokaenchan N., Areesrisom P., Tangtragoon T., Kongbuntad W., Tarachai Y. and Kawaree R. “Effect of Dying temperature to Antioxidant property and Nutritivevalue of Caesalpinia sappan L. tea”. 8th Academically Conference of Plant Genetic Conservation Project; 29 November – 1 December 2017; Saraburi: Thailand 2017. Pages 165-170 [19] Pooma R. and Suddee S. “Thai Plant Names by Tem Samitinan Additional edits 2014”. Bangkok: Forest Herbaruim Public Service National Park Service; 2014. [20] S. Klinthong, R. Khammanit, S. Phornchirasilp2, R. Temsiririrkkul and N. Siriwatanametanon. “In vivo anti-inflammatory and in vitro antioxidant activities of a Thai traditional formula, Rid-si-duang- ma-ha-kan, for hemorrhoid treatment”. Mahidol Univ J Pharm Sci 2015; 42 (3), 144-152 [21] Sriset Y., Chatuphonprasert W. and Jarukamjorn K. “Pharmacological Activities of Cryptolepis dubia (Burm.f.) M.R.Almeida”. Isan Journal of Pharmaceutical Sciences 2017; 13(1): 1-10 [22] Painupong A. “Free Radicals and Anti-oxidantsin Human Health”. PKRU SciTechJournal 2017; 1(2): 20-27 [23] Liu SC, Lin JT, Wang CK, et al. Antioxidant properties of various solvent extracts from lychee (Litchi chinenesis sonn.)flowers. Food Chem 2009;114:577e81

doi:10.14457/MSU.res.2019.25 ICoFAB2019 Proceedings | 144

Plant Diversity in Burapha University, Sa Kaeo Campus

Chakkrapong Rattamanee*, Sirichet Rattanachittawat and Paitoon Kaewhom

Faculty of Agricultural Technology, Burapha University Sa Kaeo Campus, Sa Kaeo 27160, Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Plant diversity in Burapha University, Sa Kaeo campus was investigated from June 2016–June 2019. Field expedition and specimen collection was done and deposited at the herbarium of the Faculty of Agricultural Technology. 400 plant species from 271 genera 98 families were identified. Three species were pteridophytes, one species was gymnosperm, and 396 species were angiosperms. Flowering plants were categorized as 7 species in 7 genera 3 families, Monocots 106 species in 58 genera 22 families and 283 species in 201 genera 69 families. Fabaceae has the greatest number of species among those families.

Keywords: Biodiversity, Conservation, Sa Kaeo, Species, Dipterocarp forest

Introduction

Deciduous dipterocarp forest or dried dipterocarp forest covered 80 percent of the forest area in northeastern Thailand spreads to central and eastern Thailand including Sa Kaeo province in which the elevation is lower than 1,000 meters above sea level, dry and shallow sandy soil. Plant species which are common in this kind of forest, are e.g. Buchanania lanzan, Dipterocarpus intricatus, D. tuberculatus, Shorea obtusa, S. siamensis, Terminalia alata, Gardenia saxatilis and Vietnamosasa pusilla [1]. More than 80 percent of the area of Burapha University, Sa Kaeo campus was still covered by the deciduous dipterocarp forest called ‘Khok Pa Pek’. This 2-square-kilometers forest locates at 13°44' N latitude and 102°17' E longitude in Watana Nakorn district, Sa Kaeo province. The elevation ranges from 70-90 meters above sea level. Forest deterioration has been remaining because of the development of the campus, however the plant diversity in this area has not been considered. Thus, the aim of this study was to explore the plant diversity in Burapha University, Sa Kaeo campus. Hopefully, the information from this report will be realized by the authority of the University, then plan to protect the plant diversity before the deforestation. The list of plant species found in the area are presented in this paper as well.

Materials and methods

Plant diversity was investigated along the trails in Khok Pa Pek forest in Burapha University, Sa Kaeo campus since June 2016-June 2019. Plant habitat and habit were recorded, photographs were taken, specimen were collected and deposited at the Herbarium of the Faculty of Agricultural Technology, Burapha University Sa Kaeo campus. Morphological characters were studied and identified to species using many volumes of the Flora of Thailand. Plant specimens were compared to the deposited specimen in Bangkok Forest Herbarium (BKF) as well. Scientific names and vernacular names were applied following Office of the Forest Herbarium [2] and www.theplantlist.org.

Results and discussion

Plants 400 species of 271 genera 98 families were identified from the forest in Burapha University, Sa Kaeo campus. It is approximately 4% comparing to the estimation of total plant species in Thailand [3]. Three species were pteridophytes, one species was gymnosperm, and 396 species were flowering plants (Table 1). Even though most area of forest in the campus is the deciduous dipterocarp forest and it sometimes is burnt by fire from human but the pteridophytes can be found. Isoetes coromandelina L.f. grows near the seasonal canal in the rainy season. Lygodium flexuosum (L.) Sw. and Helminthostachys zeylanica (L.) Hook. also occur near the seasonal swamp in the forest. The only one gymnosperm found in this forest was Cycas siamensis Miq. It survives from wildfire by the thick-bark underground stem. ICoFAB2019 Proceedings | 145

Flowering plants found in the forest of Burapha University, Sa Kaeo campus were categorized as Magnoliids 7 species in 7 genera 3 families, Monocots 106 species in 58 genera 22 families and Eudicots 283 species in 201 genera 69 families (Table 1) according to ‘An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV’ [4]. Fabaceae has the greatest number of species among those flowering plant families.

Table 1 Plant species list in Khok Pa Pek, Burapha University Sa Kaeo Campus.

No. Family Classification Scientific name Plant habit Local name 1. Eudicots Asystasia gangetica (L.) T.Anderson Herb baya บาหยา 2. Acanthaceae Eudicots Barleria strigosa Willd. Undershrub sang korani สังกรณี 3. Acanthaceae Eudicots Dyschoriste erecta (Burm.f.) Kuntze Shrub ya sam chan หญา้ สามช้นั 4. Acanthaceae Eudicots Justicia diffusa Willd. Herb khiang phra เขียงพร้า 5. Acanthaceae Eudicots Ruellia repens L. Exotic Herb toi ting lueai ต้อยต่ิงเล้ือย 6. Acanthaceae Eudicots Ruellia tuberosa L. Exotic Herb toi ting ต้อยต่ิง 7. Acanthaceae Eudicots laurifolia Lindl. Climber rang chuet รางจืด 8. Acanthaceae Eudicots Thunbergia similis Craib Climber chingcho noi จิงจ้อน้อย 9. Amaranthaceae Monocots Gomphrena celosioides Mart. Exotic Herb ban mairu roi pa บานไม่รู้โรย ป่า 10. Amaryllidaceae Monocots Crinum wattii Baker Herb bua bok บัวบก 11. Anacardiaceae Eudicots Buchanania lanzan Spreng. Tree ma muang maeng wan มะม่วงแมงวัน 12. Anacardiaceae Eudicots Buchanania siamensis Miq. Tree thanon chai ธนนไชย 13. Anacardiaceae Eudicots Lannea coromandelica (Houtt.) Merr. Tree kok kan กอกกัน 14. Magnolids Ellipeiopsis ferruginea (Buch.-Ham. ex Shrub nom maeo pa นมแมวป่า Hook.f. & Thomson) R.E.Fr. 15. Annonaceae Magnolids Miliusa velutina (Dunal) Hook.f. & Tree khang hua mu ขางหัวหมู Thomson 16. Annonaceae Magnolids Uvaria dulcis Dunal Climber nom wua นมวัว 17. Annonaceae Magnolids Blume Climber phi phuan พีพวน 18. Eudicots Aganonerion polymorphum Pierre ex Spire Climber som lom ส้มลม 19. Apocynaceae Eudicots marginatum (Roxb.) Climber mok khruea โมกเครือ D.J.Middleton 20. Apocynaceae Eudicots Calotropis gigantea (L.) Dryand. Exotic Shrub rak รัก 21. Apocynaceae Eudicots Ceropegia sootepensis Craib Herbaceous wan sam phi nong ว่านสามพี่ Climber น้อง 22. Apocynaceae Eudicots Cryptolepis sinensis (Lour.) Merrill Climber yan khi phueng ยา่ นข้ีผ้ึง 23. Apocynaceae Eudicots Dischidia major (Vahl) Merr. Creeping Herb chuk rohini จุกโรหินี 24. Apocynaceae Eudicots Heterostemma siamicum Craib Climber khruea khao khom noi เครือ เขาขมน้อย 25. Apocynaceae Eudicots Holarrhena curtisii King & Gamble Shrub phut thung พุดทุ่ง 26. Apocynaceae Eudicots Holarrhena pubescens Wall. ex G.Don Tree mok yai โมกใหญ่ 27. Apocynaceae Eudicots kerrii Craib Climber tang ต้าง 28. Apocynaceae Eudicots Hoya pachyclada Kerr Climber tang yai ต้างใหญ่ 29. Apocynaceae Eudicots Ichnocarpus frutescens (L.) W.T.Aiton Climber khruea pla song daeng เครือปลาสงแดง 30. Apocynaceae Eudicots Lygisma inflexum (Costantin) Kerr Climber ko tian คอเทียน 31. Apocynaceae Eudicots Marsdenia glabra Costantin Climber phak saeo ผักแส้ว 32. Apocynaceae Eudicots Marsdenia tenacissima (Roxb.) Moon Climber khruea thai song khaw เครือ ไทสงขาว 33. Apocynaceae Eudicots Oxystelma esculentum (L.f.) Sm. Climber chamuk pla lot จมูกปลาหลด 34. Apocynaceae Eudicots Spirolobium cambodianum Baill. Shrub phet cha hueng เพชรหึง 35. Apocynaceae Eudicots juventas (Lour.) Merr. Climber thao prasong เถาประสงค์ 36. Apocynaceae Eudicots Toxocarpus villosus (Blume) Decne. Climber thao wan daeng เถาวัลย์แดง 37. Apocynaceae Eudicots Wrightia pubescens R.Br. Shrubby Tree mok โมก 38. Araceae Monocots Amorphophallus napiger Gagnep. Herb buk บุก 39. Araceae Monocots Amorphophallus paeoniifolius (Dennst.) Herb buk บุก Nicolson 40. Araceae Monocots Amorphophallus parvulus Gagnep. Herb buk i lok บุกอีลอก 41. Araceae Monocots Pseudodracontium macrophyllum Gagnep. Herb i lok อีลอก ex Serebryanyi 42. Araceae Monocots Scindapsus officinalis (Roxb.) Schott Climber khruea ngu khiao เครืองูเขียว 43. Araceae Monocots Typhonium flagelliforme (Lodd.) Blume Herb taphit kap yao ตะพิดกาบยาว 44. Arecaceae Monocots Phoenix paludosa Roxb. Palm peng thale เป้งทะเล 45. Aristolochiaceae Magnolids Aristolochia pothieri Pierre ex Lecomte Climber krachao thung thong กระเช้า ถุงทอง 46. Asparagaceae Monocots Asparagus racemosus Willd. Climber sam sip สามสิบ 47. Asteraceae Eudicots Ageratum conyzoides L. Herb sap raeng sap ka สาบแร้งสาบ กา 48. Asteraceae Eudicots Bidens pilosa L. Exotic Herb dao krachai taiwan ดาวกระจายไต้หวัน 49. Asteraceae Eudicots Chromolaena odorata (L.) R.M.King & Exotic Herb sap suea สาบเสือ H.Rob. 50. Asteraceae Eudicots Eclipta prostrata (L.) L. Herb kameng กะเม็ง ICoFAB2019 Proceedings | 146

51. Asteraceae Eudicots Praxelis clematidea (Griseb.) R.M.King & Exotic Herb sap muang สาบม่วง H.Rob. 52. Asteraceae Eudicots Sphagneticola trilobata (L.) Pruski Exotic kradum thong lueai กระดุม Herbaceous ทองเล้ือย Climber 53. Asteraceae Eudicots Synedrella nodiflora (L.) Gaertn. Herb phak khraet ผักแครด 54. Bignoniaceae Eudicots Dolichandrone serrulata (Wall. ex DC.) Tree khae na แคนา Seem. 55. Bignoniaceae Eudicots Millingtonia hortensis L.f. Tree pip ปีบ 56. Boraginaceae Eudicots Heliotropium indicum L. Herb ya nguang chang หญ้างวงช้าง 57. Boraginaceae Eudicots Heliotropium strigosum Willd. Herb ya nok yung หญ้านกยูง 58. Burmanniaceae Monocots Burmannia coelestis D.Don Saprophytic Herb sarat chanthon สรัสจันทร 59. Burmanniaceae Monocots Burmannia wallichii (Miers) Hook.f. Saprophytic Herb khao kam noi ข้าวก่าน้อย 60. Eudicots subulatum Guillaumin Tree ma kok kluean มะกอกเกล้ือน 61. Eudicots Lobelia alsinoides Lam. Herb sadao din สะเดาดิน 62. Campanulaceae Eudicots Lobelia thorelii E.Wimm. Herb phak lueam phua ผักลืมผัว 63. Cannabaceae Eudicots Trema orientalis (L.) Blume Shrubby Tree po ปอ 64. Capparaceae Eudicots Capparis flavicans Kurz Shrub nam ko kai หนามเกาะไก่ 65. Capparaceae Eudicots Capparis micracantha DC. Shrubby Tree chingchi ชิงชี่ 66. Celastraceae Eudicots Celastrus paniculatus Willd. Climber mak taek หมากแตก 67. Celastraceae Eudicots Siphonodon celastrineus Griff. Tree ma duk มะดูก 68. Cleomaceae Eudicots Cleome gynandra L. Herb phak sian ผกั เส้ียน 69. Cleomaceae Eudicots Cleome rutidosperma DC. Herb phak sian khon ผกั เส้ียนขน 70. Cleomaceae Eudicots Cleome viscosa L. Herb phak sian phi ผกั เส้ียนผี 71. Clusiaceae Eudicots Garcinia cowa Roxb. ex Choisy Shrubby Tree cha muang ชะมวง 72. Colchicaceae Monocots Gloriosa superba L. Herbaceous dong dueng ดองดึง Climber 73. Eudicots quadrangulare Kurz Tree sakae na สะแกนา 74. Combretaceae Eudicots Getonia floribunda Roxb. Climber ting tang ต่ิงตง่ั 75. Combretaceae Eudicots Terminalia alata B.Heyne ex Roth Tree rok fa รกฟ้า 76. Combretaceae Eudicots Terminalia calamansanay (Blanco) Rolfe Tree sakuni สกุณี 77. Commelinaceae Monocots Cyanotis axillaris (L.) D.Don ex Sweet Herb phak plap na ผักปลาบนา 78. Commelinaceae Monocots Cyanotis cristata (L.) D.Don Herb ya hua rak noi หญ้าหัวรากน้อย 79. Commelinaceae Monocots Murdannia gigantea (Vahl) G.Brückn. Herb ya ngon ngueak หญ้าหงอน เงือก 80. Convolvulaceae Eudicots Argyreia lanceolata Choisy Climber thao kradueng chang เถา กระดึงช้าง 81. Convolvulaceae Eudicots Argyreia mekongensis Gagnep. & Courchet Woody Climber phung mu พุงหมู 82. Convolvulaceae Eudicots Argyreia mollis (Burm.f.) Choisy Climber khruea phu ngoen เครือพูเงิน 83. Convolvulaceae Eudicots Argyreia osyrensis (Roth) Choisy Climber hun หุน 84. Convolvulaceae Eudicots Argyreia versicolor (Kerr) Staples & Woody Climber thao kradueng chang เถา Traiperm กระดึงช้าง 85. Convolvulaceae Eudicots Ipomoea aquatica Forssk. Creeping Herb phak bung ผักบุ้ง 86. Convolvulaceae Eudicots Ipomoea biflora (L.) Pers. Herbaceous phak bung song dok ผักบุ้ง Climber สองดอก 87. Convolvulaceae Eudicots Ipomoea nil (L.) Roth Herbaceous wan phak bung ว่านผักบุ้ง Climber 88. Convolvulaceae Eudicots Ipomoea obscura (L.) Ker Gawl. Herbaceous sa uek สะอึก Climber 89. Convolvulaceae Eudicots Jacquemontia paniculata (Burm.f.) Hallier Herbaceous chingcho phi จ้ิงจอ้ ผี f. Climber 90. Convolvulaceae Eudicots Merremia cissoides (Lam.) Hallier f. Herbaceous sa uek สะอึก Climber 91. Convolvulaceae Eudicots Merremia hederacea (Burm.f.) Hallier f. Herbaceous thao sa uek เถาสะอึก Climber 92. Convolvulaceae Eudicots Merremia hirta (L.) Merr. Herbaceous chingcho nuan จิงจ้อนวล Climber 93. Convolvulaceae Eudicots Merremia umbellata (L.) Hallier f. Climber chingcho khao จิงจ้อขาว 94. Convolvulaceae Eudicots Operculina petaloidea (Choisy) Ooststr. Herbaceous ban bai บานบ่าย Climber 95. Cornaceae Eudicots Alangium salviifolium (L.f.) Wangerin Shrubby Tree pru ปรู๋ subsp. hexapetalum (Lam.) Wangerin 96. Costaceae Monocots Cheilocostus speciosus (J.Koenig.) Herb ueang mai na เอ้ืองหมายนา C.D.Specht 97. Cucurbitaceae Eudicots Coccinia grandis (L.) Voigt Herbaceous tam lueng ตาลึง Climber 98. Cucurbitaceae Eudicots Gymnopetalum scabrum (Lour.) W.J. de Herbaceous khi ka khao ข้ีกาแดง Wilde & Duyfjes Climber 99. Cucurbitaceae Eudicots Solena heterophylla Lour. Herbaceous tam lueng tua phu ตาลึงตัวผู้ Climber 100. Cycadaceae Gymnosperms Cycas siamensis Miq. Shrub prong pa ปรงป่า 101. Cyperaceae Monocots Cyperus compactus Retz. Herb ya bai khom หญ้าใบคม 102. Cyperaceae Monocots Cyperus iria L. Herb ya kok sai หญ้ากกทราย 103. Cyperaceae Monocots Cyperus procerus Rottb. Herb ya takrap หญ้าตะกรับ 104. Cyperaceae Monocots Cyperus rotundus L. Herb ya haeo mu หญ้าแห้วหมู 105. Cyperaceae Monocots Diplacrum caricinum R.Br. Herb kok chai luk lai กกชายลูกลาย 106. Cyperaceae Monocots Fimbristylis insignis Thwaites Herb kok kandan กกกันดาร 107. Cyperaceae Monocots Fimbristylis ovata (Burm.f.) J. Kern Herb ya kuk mu หญ้ากุกหมู ICoFAB2019 Proceedings | 147

108. Cyperaceae Monocots Fimbristylis quinquangularis (Vahl) Kunth Herb ya rat khiat หญ้ารัดเขียด 109. Cyperaceae Monocots Fimbristylis tetragona R.Br. Herb kok kan dok กกก้านดอก 110. Cyperaceae Monocots Fuirena ciliaris (L.) Roxb. Herb ya khom bang klom หญ้าคม บางกลม 111. Cyperaceae Monocots Fuirena umbellata Rottb. Herb ya sam khom หญ้าสามคม 112. Cyperaceae Monocots Kyllinga brevifolia Rottb. Herb ya dok khao หญ้าดอกขาว 113. Cyperaceae Monocots Lipocarpha chinensis (Osbeck) J. Kern Herb ya hon ngueak หญ้าหอนเงือก 114. Cyperaceae Monocots Pycreus pumilus (L.) Nees Herb kok khi ma กกข้ีหมา 115. Cyperaceae Monocots Rhynchospora hookeri Boeckeler Herb kok khang nam กกข้างน้า 116. Cyperaceae Monocots Rhynchospora longisetis R.Br. Herb kok cho nam tan กกช่อน้า ตาล 117. Cyperaceae Monocots Scleria levis Retz. Herb ya sam khom หญ้าสามคม 118. Cyperaceae Monocots Scleria terrestris (L.) Fassett Herb ya khom bang khao หญ้าคม บางเขา 119. Dilleniaceae Eudicots Dillenia hookeri Pierre Shrub san din ส้านดิน 120. Dilleniaceae Eudicots Dillenia ovata (Blume) Hoogland Tree san bai lek ส้านใบเล็ก 121. Dilleniaceae Eudicots Dillenia parviflora Griff. Tree san hing ส้านห่ิง 122. Dilleniaceae Eudicots Dillenia pentagyna Roxb. Tree san chang ส้านช้าง 123. Dilleniaceae Eudicots Tetracera loureiri (Finet & Gagnep.) Pierre Climber rotsukhon รสสุคนธ์ ex Craib 124. Dioscoreaceae Monocots Dioscorea alata L. Herbaceous man sao มันเสา Climber 125. Dioscoreaceae Monocots Dioscorea brevipetiolata Prain & Burkill Herbaceous man thian มันเทียน Climber 126. Dioscoreaceae Monocots Dioscorea depauperata Prain & Burkill Herbaceous sa man สามัน Climber 127. Dioscoreaceae Monocots Dioscorea glabra Roxb. Herbaceous man nang non มันนางนอน Climber 128. Dioscoreaceae Monocots Dioscorea hispida Dennst. var. hispida Herbaceous kloi กลอย Climber 129. Dioscoreaceae Monocots Dioscorea pentaphylla L. Herbaceous man on มันอ้อน Climber 130. Dioscoreaceae Monocots Dioscorea pierrei Prain & Burkill Herbaceous man i mo มันอีโม่ Climber 131. Eudicots Dipterocarpus intricartus Dyer Tree sa baeng สะแบง 132. Dipterocarpaceae Eudicots Dipterocarpus tuberculatus Roxb. Tree kung กุง 133. Dipterocarpaceae Eudicots Shorea obtusa Wall. Tree teng เต็ง 134. Dipterocarpaceae Eudicots Shorea siamensis Miq. Tree rang รัง 135. Droseraceae Eudicots Drosera burmanni Vahl Insectivorous chok bo wai จอกบ่วาย Herb 136. Droseraceae Eudicots Drosera indica L. Insectivorous ya nam khang หญา้ น้า ค้าง Herb 137. Ebenaceae Eudicots Diospyros rhodocalyx Kurz Shrubby Tree tako na ตะโกนา 138. Eriocaulaceae Monocots Eriocaulon echinulatum Mart. Herb kradum nam กระดุมหนาม 139. Eriocaulaceae Monocots Eriocaulon infirmum Steud. Herb kradum thao กระดุมเทา 140. Eriocaulaceae Monocots Eriocaulon laoense Moldenke Herb kradum lao กระดุมลาว 141. Eriocaulaceae Monocots Eriocaulon quinquangulare L. subsp. Herb kradum bai daeng กระดุม longibracteatum Praj. & J. Parn. ใบแดง 142. Eriocaulaceae Monocots Eriocaulon setaceum L. Herb sarai hua mai khit สาหร่ายหัว ไม้ขีด 143. Eriocaulaceae Monocots Eriocaulon xeranthemum Mart. Herb kradum chio กระดุมจิว๋ 144. Eriocaulaceae Monocots Eriocaulon zollingerianum KÖrn. Herb chuk nok yung จุกนกยูง 145. Eudicots Cnesmone laotica (Gagnep.) Croizat Climber tarang tang kwang ตะรังตังกวาง 146. Euphorbiaceae Eudicots Euphorbia hirta L. Herb nam nom ratchasi น้า นมราชสีห์ 147. Euphorbiaceae Eudicots Suregada multiflorum (A.Juss.) Baill. Tree khan thong phayabat ขันทองพยาบาท 148. Euphorbiaceae Eudicots Thyrsanthera suborbicularis Pierre ex Herb po ka pla ปอกะปลา Gagnep. 149. Fabaceae Eudicots Acacia harmandiana (Pierre) Gagnep. Tree kra thin phi man กระถินพิมาน 150. Fabaceae Eudicots Acacia megaladena Desv. var. indo- Climber khi raet ข้ีแรด chinensis I.C.Nielsen 151. Fabaceae Eudicots Aeschynomene indica L. Undershrub sano hang kai โสนหางไก่ 152. Fabaceae Eudicots Afzelia xylocarpa (Kurz) Craib Tree ma kha mong มะค่าโมง 153. Fabaceae Eudicots Albizia lebbeck (L.) Benth. Tree phruek พฤกษ์ 154. Fabaceae Eudicots Albizia lebbekoides (DC.) Benth. Tree khang คาง 155. Fabaceae Eudicots Alysicarpus vaginalis (L.) DC. Herb thua lisong na ถว่ั ลิสงนา 156. Fabaceae Eudicots Bauhinia acuminata L. Shrub ka long กาหลง 157. Fabaceae Eudicots Phanera bracteata Benth. Climber siao khruea เส้ียวเครือ 158. Fabaceae Eudicots Bauhinia saccocalyx Pierre Shrubby Tree som siao ส้มเส้ียว 159. Fabaceae Eudicots Butea monosperma (Lam.) Taub. Tree thong kwao ทองกวาว 160. Fabaceae Eudicots Cajanus scarabaeoides (L.) Thouars Herbaceous khi non thao เถาข้ีหนอน Climber 161. Fabaceae Eudicots Cassia bakeriana Craib Tree kanlapa phruek กัลปพฤกษ์ 162. Fabaceae Eudicots Cassia fistula L. Tree khun คูน 163. Fabaceae Eudicots Cassia grandis L.f. Exotic Tree kanla phruek กาฬพฤกษ์ 164. Fabaceae Eudicots Centrosema pubescens Benth. Exotic Climber thua lai ถว่ั ลาย 165. Fabaceae Eudicots Chamaecrista mimosoides (L.) Greene Undershrub sano noi โสนน้อย ICoFAB2019 Proceedings | 148

166. Fabaceae Eudicots Crotalaria montana Heyne ex Roth Herb ya hang khang หญ้าหางค่าง 167. Fabaceae Eudicots Crotalaria sessiliflora L. Herb phuang khon พวงขน 168. Fabaceae Eudicots Crotalaria spectabilis Roth. Herb ma hing men มะห่ิงเม่น 169. Fabaceae Eudicots Dalbergia cochinchinensis Pierre Tree phayung พะยูง 170. Fabaceae Eudicots Dalbergia nigrescens Kurz Tree cha nuan ฉนวน 171. Fabaceae Eudicots Delonix regia (Bojer ex Hook.) Raf. Tree hang nok yung farang หาง นกยูงฝร่ัง 172. Fabaceae Eudicots Dendrolobium lanceolatum (Dunn) Shrub kraduk ueng กระดูกอึ่ง Schindl. 173. Fabaceae Eudicots Derris scandens (Roxb.) Benth. Climber thao wan priang เถาวัลย์เปรียง 174. Fabaceae Eudicots Desmodium heterocarpon (L.) DC. Herb khang khanna ขางคันนา 175. Fabaceae Eudicots Desmodium styracifolium (Osbeck) Merr. Herb phi suea nam ผีเส้ือน้ า 176. Fabaceae Eudicots Desmodium triflorum (L.) DC. Trailing Herb ya klet hoi หญ้าเกล็ดหอย 177. Fabaceae Eudicots Dunbaria bella Prain Climber thao khrang เถาคร่ัง 178. Fabaceae Eudicots Dunbaria fusca (Wall.) Kurz Climber thua pak nok muang ถว่ั ปาก นกม่วง 179. Fabaceae Eudicots Dunbaria glandulosa (Dalzell & A.Gibson) Climber thua khruea ถว่ั เครือ Prain 180. Fabaceae Eudicots Eriosema chinense Vogel Herb haeo pradu แห้วประดู่ 181. Fabaceae Eudicots Flemingia involucrata Benth. Shrub sakae saeng สะแกแสง 182. Fabaceae Eudicots Flemingia stricta Roxb. Shrub ka sam pik กาสามปีก 183. Fabaceae Eudicots Leucaena leucocephala (Lam.) de Wit Shrubby Tree kra thin กระถิน 184. Fabaceae Eudicots Macroptilium atropurpureum (DC.) Urb. Herb thua seratro ถว่ั เซราโตร 185. Fabaceae Eudicots Macroptilium lathyroides (L.) Urb. Herb thua phi ถว่ั ผี 186. Fabaceae Eudicots Millettia brandisiana Kurz Tree kra phi chan กระพ้ีจน่ั 187. Fabaceae Eudicots Mimosa pudica L. Exotic Herb maiyarap ไมยราพ 188. Fabaceae Eudicots Mimosa diplotricha C.Wright ex Sauvalle Exotic Herb maiyarap nam ไมยราพหนาม 189. Fabaceae Eudicots Peltophorum dasyrachis (Miq.) Kurz Tree a rang อะราง 190. Fabaceae Eudicots Phyllodium pulchellum (L.) Desv. Shrub klet pla chon เกล็ดปลาช่อน 191. Fabaceae Eudicots Pterocarpus macrocarpus Kurz Tree pradu pa ประดู่ป่า 192. Fabaceae Eudicots Pueraria phaseoloides (Roxb.) Benth. Climber thua sian pa ถว่ั เส้ียนป่า 193. Fabaceae Eudicots Senna garrettiana (Craib) H.S.Irwin & Tree samae san แสมสาร Barneby 194. Fabaceae Eudicots Senna occidentalis (L.) Link Exotic chumhet lek ชุมเห็ดเล็ก Undershrub 195. Fabaceae Eudicots Senna siamea (Lam.) H.S.Irwin & Barneby Tree khi lek ข้ีเหลก็ 196. Fabaceae Eudicots Sindora siamensis Teijsm & Miq. Tree ma kha tae มะค่าแต้ 197. Fabaceae Eudicots Spatholobus parviflorus (DC.) Kuntze Climber thao phan sai เถาพันซ้าย 198. Fabaceae Eudicots Tephrosia purpurea (L.) Pers. Herb khram pa ครามป่า 199. Fabaceae Eudicots Tephrosia vestita Vogel Undershrub thua nok yan ถว่ั โหนกยาน 200. Fabaceae Eudicots Uraria crinata (L.) Desv. ex DC. Undershrub hang ma chok หางหมาจอก 201. Fabaceae Eudicots Uraria lagopodioides (L.) DC. Undershrub hang krarok หางกระรอก 202. Fabaceae Eudicots Uraria rotundata Craib Undershrub hang krarok หางกระรอก 203. Fabaceae Eudicots Xylia xylocarpa (Roxb.) Taub. Tree daeng แดงนา 204. Flacourtiaceae Eudicots Flacourtia indica (Burm.f.) Merr. Shrubby Tree mak ben หมากเบน 205. Gentianaceae Eudicots Canscora alata (Roth) Wall. Herb pang pae lek ปังแปเล็ก 206. Gentianaceae Eudicots Cracosna carinata (Dop) Thiv Herb sam yot สามยอด 207. Gentianaceae Eudicots Fagraea fragrans Roxb. Tree kan krao กันเกรา 208. Hydrocharitaceae Monocots Ottelia alismoides (L.) Pers. Aquatic Herb santawa สันตะวาใบพาย 209. Hydroleaceae Eudicots Hydrolea zeylanica (L.) Vahl Herb di ian ดีเอี่ยน 210. Hypericaceae Eudicots Cratoxylum cochinchinense (Lour.) Blume Tree tio kliang ต้ิวเกล้ียง 211. Hypericaceae Eudicots Cratoxylum formosum (Jack) Dyer Tree tio khao ต้ิวขาว 212. Hypoxidaceae Monocots Curculigo orchioides Gaertn. Herb wan phrao ว่านพร้าว 213. Isoetaceae Pteridophytes Isoetes coromandelina L.f. Fern kra thiam na กระเทียมนา 214. Lamiaceae Eudicots Gmelina paniculata H.R. Fletcher Climber chong maeo ช้องแมว 215. Lamiaceae Eudicots Hyptis suaveolens (L.) Poit. Shrub maeng lak kha แมงลักคา 216. Lamiaceae Eudicots Leucas lavandulifolia Sm. Herb ya prik bai khaep หญ้าปริกใบ แคบ 217. Lamiaceae Eudicots Platostoma cochinchinense (Lour.) Herb hang suea หางเสือ AJ.Paton 218. Lamiaceae Eudicots Premna herbacea Roxb. Undershrub kha pia num ขาเปี๋ยนุ่ม 219. Lamiaceae Eudicots Rotheca serrata (L.) Steane & Mabb. Undershrub akkhi thawan อัคคีทวาร 220. Magnolids Cassytha filiformis L. Parasitic sangwan phra in สังวาลพระ herbaceous อินทร์ climber 221. Lauraceae Magnolids Litsea glutinosa (Lour.) C.B.Rob. Tree mi men หมีเหม็น 222. Lecythidaceae Eudicots Careya arborea Roxb. Tree kradon กระโดน 223. Lentibulariaceae Eudicots Utricularia aurea Lour. Aquatic Herb sarai khao niao สาหร่ายข้าว เหนียว 224. Lentibulariaceae Eudicots Utricularia minutissima Vahl Herb thip keson ทิพเกสร 225. Linderniaceae Eudicots Lindernia aculeata (Bonati) T.Yamaz. Herb khem daeng เข็มแดง 226. Linderniaceae Eudicots Lindernia antipoda (L.) Alston Herb mak lin nam khang หมากล้ิน น้า ค้าง 227. Linderniaceae Eudicots Lindernia cambodgiana (Bonati) Philcox Herb ya khamen หญ้าเขมร 228. Linderniaceae Eudicots Lindernia ciliata (Colsm.) Pennell Herb phak i hae ผักอีแฮ ICoFAB2019 Proceedings | 149

229. Linderniaceae Eudicots Lindernia crustacea (L.) F.Muell. Herb ya kap hoi tua mia หญ้า กาบหอยตัวเมีย 230. Linderniaceae Eudicots Lindernia kerrii T.Yamaz. Herb phak chi mo ผักชีหมอ 231. Linderniaceae Eudicots Lindernia micrantha D.Don Herb ya hom keao หญ้าหอมแก้ว 232. Linderniaceae Eudicots Lindernia pierreanoides T.Yamaz. Herb phak bia sai ผกั เบ้ียทราย 233. Linderniaceae Eudicots Lindernia pusilla (Willd.) Bold Herb ya phu si หญ้าพูสี 234. Loganiaceae Eudicots Strychnos nux-blanda Hill Shrubby Tree tumka khao ตูมกาขาว 235. Loranthaceae Eudicots Dendrophthoe pentandra (L.) Miq. Parasitic Shrub kafak ma muang กาฝากมะม่วง 236. Lygodiaceae Pteridophytes Lygodium flexuosum (L.) Sw. Climbing Fern moi mae mai หมอยแม่หม้าย 237. Eudicots floribunda Jack Tree ta baek na ตะแบกนา 238. Lythraceae Eudicots Lagerstroemia loudonii Teijsm. & Binn. Tree salao เสลา 239. Lythraceae Eudicots Lagerstroemia macrocarpa Wall. Tree inthanin bok อินทนิลบก 240. Lythraceae Eudicots Lagerstroemia speciosa (L.) Pers. Tree inthanin nam อินทนิลน้า 241. Eudicots Abelmoschus moschatus Medik. subsp. Herb Som chaba โสมชบา tuberosus (Span.) Borss.Waalk. 242. Malvaceae Eudicots Bombax insigne Wall. Tree ngio pa ง้ิวป่า 243. Malvaceae Eudicots Corchorus olitorius L. Herb kra chao กระเจา 244. Malvaceae Eudicots Decaschistia crotonifolia Wight & Arn. Shrub hua kai ok yai หัวไก่โอก 245. Malvaceae Eudicots Grewia abutilifolia Vent. ex Juss. Shrub khao chi ข้าวจี่ 246. Malvaceae Eudicots Grewia eriocarpa Juss. Tree khi thao ข้ีเถา้ 247. Malvaceae Eudicots Helicteres angustifolia L. Shrub khi tun ข้ีตุ่น 248. Malvaceae Eudicots Helicteres hirsuta Lour. Shrub po tao hai ปอเต่าไห้ 249. Malvaceae Eudicots Helicteres lanceolata A.DC. Shrub khao chi lek ข้าวจี่เล็ก 250. Malvaceae Eudicots Helicteres elongata Wall. ex Bojer Shrub khi on ข้ีอน้ 251. Malvaceae Eudicots Hibiscus glanduliferus Craib Shrub po tom ปอต่อม 252. Malvaceae Eudicots Malachra capitata (L.) L. Undershrub po khan ปอคัน 253. Malvaceae Eudicots Melochia corchorifolia L. Undershrub seng lek เส้งเล็ก 254. Malvaceae Eudicots Urena rigida Wall. ex Mast. Undershrub khi on ข้ีอน้ 255. Malvaceae Eudicots Sida cordifolia L. Undershrub ya khat bai pom หญ้าขัดใบ ป้อม 256. Melastomataceae Eudicots Memecylon edule Roxb. Shrubby Tree phlong mueat พลองเหมือด 257. Melastomataceae Eudicots Osbeckia nepalensis Hook.f. Shrub en a nam เอนอา้ น้า 258. Meliaceae Eudicots Azadirachta indica A.Juss. Tree sadao สะเดา 259. Menispermaceae Eudicots Stephania pierrei Diels Climber bua bok บัวบก 260. Molluginaceae Eudicots Mollugo pentaphylla L. Herb ya khai hao หญ้าไข่เหา 261. Moraceae Eudicots Artocarpus lacucha Buch.-Ham. Tree hat หาด 262. Moraceae Eudicots Ficus hispida L.f. Shrubby Tree ma duea plong มะเดื่อปล้อง 263. Mulpighiaceae Eudicots Aspidopterys tomentosa (Blume) A.Juss. Climber kai kom khruea ก่ายกอมเครือ 264. Eudicots Syzygium cumini (L.) Skeels Tree wa หว้า 265. Ochnaceae Eudicots Ochna integerrima (Lour.) Merr. Shrubby Tree chang nao ช้างน้าว 266. Eudicots Anacolosa ilicoides Mast. Shrubby Tree ko sae ก่อแซะ 267. Olacaceae Eudicots psittacorum (Willd.) Vahl Climber nam chai khrai น้า ใจใคร่ 268. Oleaceae Eudicots Jusminum anodontum Gagnep. Climber sai kai ไส้ไก่ 269. Onagraceae Eudicots Ludwigia hyssopifolia (G.Don) Exell Herb thian na เทียนนา 270. Ophioglossaceae Pteridophytes Helminthostachys zeylanica (L.) Hook. Terrestrial Fern phak nok yung ผักนกยูง 271. Eudicots Melientha suavis Pierre Shrubby Tree phak wan pa ผักหวานป่า 272. Orchidaceae Monocots Dendrobium venustum Teijsm. & Binn. Epiphytic Orchid ueang khao niao ling เอ้ือง ข้าวเหนียวลิง 273. Orchidaceae Monocots Eulophia andamanensis Rchb.f. Terrestrial Orchid mu kling หมูกล้ิง 274. Orchidaceae Monocots Eulophia graminea Lindl. Terrestrial Orchid hua khao tom หัวข้าวต้ม 275. Orchidaceae Monocots Eulophia herbacea Lindl. Terrestrial Orchid ueang maeng mum เอ้ืองแมง มุม 276. Orchidaceae Monocots Eulophia macrobulbon (E.C.Parish & Terrestrial Orchid wan ueng ว่านอึ่ง Rchb.f.) Hook.f. 277. Orchidaceae Monocots Eulophia promensis Lindl. Terrestrial Orchid wan dok lueng ว่านดอกเหลือง 278. Orchidaceae Monocots Geodorum recurvum (Roxb.) Alston Terrestrial Orchid wan nang tam ว่านนางตาม 279. Orchidaceae Monocots Geodorum terrestre (L.) Garay Terrestrial Orchid wan chung nang ว่านจูงนาง 280. Orchidaceae Monocots Habenaria anomaliflora Kurzweil & Terrestrial Orchid ua kam ma yi อ้วั กา มะหยี่ Chantanaorr. 281. Orchidaceae Monocots Habenaria commelinifolia (Roxb.) Wall. ex Terrestrial Orchid chuang kham dok chao จวง Lindl. คาดอกขาว 282. Orchidaceae Monocots Habenaria hosseusii Schltr. Terrestrial Orchid nang ua khang yao นางอ้วั คาง ยาว 283. Orchidaceae Monocots Nervilia crociformis (Zoll. & Moritzi) Terrestrial Orchid ueang din bai bua เอ้ืองดินใบ Seidenf. บัว 284. Orchidaceae Monocots Nervilia mekongensis S.W.Gale, Schuit. & Terrestrial Orchid wan phaen din yen me Suddee kong ว่านแผ่นดินเย็นแม่โขง 285. Orobanchaceae Eudicots Aeginetia indica L. Parasitic Herb dok din daeng ดอกดินแดง 286. Orobanchaceae Eudicots Aeginetia pedunculata Wall. Parasitic Herb dok din ดอกดิน 287. Orobanchaceae Eudicots Buchnera cruciata Buch.-Ham. ex D.Don Herb ya khao kam หญ้าข้าวก่า 288. Orobanchaceae Eudicots Centranthera cochinchinensis (Lour.) Herb ya khrang khon หญา้ คร่ังขน Merr. 289. Orobanchaceae Eudicots Centranthera tranquebarica (Spreng.) Herb ya khom lueang หญ้าโคม Merr. เหลือง 290. Orobanchaceae Eudicots Sopubia fastigiata Bonati Herb hang ma chok หางหมาจอก ICoFAB2019 Proceedings | 150

291. Oxalidaceae Eudicots Biophytum sensitivum (L.) DC Herb kra thuep yop กระทืบยอบ 292. Passifloraceae Eudicots Adenia viridiflora Craib Climber phak sap ผักสาบ 293. Passifloraceae Eudicots Passiflora foetida L. Exotic Climber ka thok rok กะทกรก 294. Pedaliaceae Eudicots Sesamum indicum L. Exotic Herb nga งา 295. Phyllanthaceae Eudicots Antidesma ghaesembilla Gaertn. Tree mao khai pla เม่าไข่ปลา 296. Phyllanthaceae Eudicots Bridelia harmandii Gagnep. Shrub sam sa tia ซา ซาเต้ีย 297. Phyllanthaceae Eudicots Bridelia retusa (L.) A.Juss. Tree teng nam เต็งหนาม 298. Phyllanthaceae Eudicots Bridelia stipularis (L.) Blume Scandent Shrub ma ka khruea มะกาเครือ 299. Phyllanthaceae Eudicots Flueggea virosa (Roxb. ex Willd.) Voigt Shrub kang pla khao ก้างปลาขาว 300. Phyllanthaceae Eudicots Glochidion coccineum (Buch.-Ham.) Müll. Shrubby Tree ka nam กาน้า 301. Phyllanthaceae Eudicots Phyllanthus amarus Schumach. & Thonn. Herb luk tai bai ลูกใต้ใบ 302. Phyllanthaceae Eudicots Phyllanthus emblica L. Shrubby Tree ma kham pom มะขามป้อม 303. Phyllanthaceae Eudicots Phyllanthus virgatus G.Forst. Herb khang amphai ขางอาไพ 304. Phyllanthaceae Eudicots (L.) Merr. Shrub phak wan ban ผักหวานบ้าน 305. Plantaginaceae Eudicots Adenosma indianum (Lour.) Merr. Herb kratai cham กระต่ายจาม 306. Plantaginaceae Eudicots Limnophila aromatica (Lam.) Merr. Herb phak kha yaeng ผักแขยง 307. Plantaginaceae Eudicots Limnophila chinensis (Osbeck) Merr. Herb phak kha yaeng ผักแขยง 308. Plantaginaceae Eudicots Limnophila geoffrayi Bonati Herb phak kha yaeng ผักแขยง 309. Plantaginaceae Eudicots Limnophila indica (L.) Druce Aquatic Herb sarai chat สาหร่ายฉัตร 310. Plantaginaceae Eudicots Limnophila poilanei T.Yamaz. Herb nang ubon นางอุบล 311. Poaceae Monocots Bambusa bambos (L.) Voss Bamboo phai pa ไผ่ป่า 312. Poaceae Monocots Brachiaria mutica (Forssk.) Stapf Exotic Grass ya khon หญ้าขน 313. Poaceae Monocots Chloris barbata Sw. Exotic Grass ya rang nok หญ้ารังนก 314. Poaceae Monocots Chrysopogon aciculatus (Retz.) Trin. Grass ya chao chu หญ้าเจ้าชู้ 315. Poaceae Monocots Chrysopogon nemoralis (Balansa) Holttum Grass faek don แฝกดอน 316. Poaceae Monocots Echinochloa (L.) Link Grass ya khao nok หญ้าข้าวนก 317. Poaceae Monocots Imperata cylindrica (L.) Raeusch Grass ya kha หญ้าคา 318. Poaceae Monocots Melinis repens (Willd.) Zizka Grass ya dok chomphu หญ้าดอก ชมพู 319. Poaceae Monocots Ophiuros exaltatus (L.) Kuntze Grass ya kha yong หญ้าโขย่ง 320. Poaceae Monocots Oryza meyeriana (Zoll. & Moritzi) Baill. Grass khao pa ข้าวป่า var. granulata (Nees & Arn. ex Watt) Duist. 321. Poaceae Monocots Oryza rufipogon Griff. Grass ya khao phi หญ้าข้าวผี 322. Poaceae Monocots Pennisetum pedicellatum Trin. Exotic Grass ya kha chon chop หญ้าขจรจบ 323. Poaceae Monocots Pennisetum polystachion (L.) Schult. Exotic Grass ya kha chon chop lek หญ้า ขจรจบเล็ก 324. Poaceae Monocots Saccharum procerum Roxb. Grass ya khamong หญ้าโขมง 325. Poaceae Monocots Saccharum spontaneum L. Grass lao เลา 326. Poaceae Monocots Setaria parviflora (Poir.) M.Kerguelen Grass ya hang ma chok หญ้าหางหมา จอก 327. Poaceae Monocots Vietnamosasa ciliata (A.Camus) Bamboo chot โจด T.Q.Nguyen 328. Poaceae Monocots Vietnamosasa pusilla (A.Chev. & Bamboo phek เพ็ก A.Camus) T.Q.Nguyen 329. Polygalaceae Eudicots Polygala chinensis L. Herb kham tia คา เต้ีย 330. Polygalaceae Eudicots Polygala triflora L. Herb kham tia คา เต้ีย 331. Polygalaceae Eudicots Salomonia longiciliata Kurz Herb niam ton pik เนียมต้นปีก 332. Pontederiaceae Monocots Monochoria hastata (L.) Solms Aquatic Herb phak top thai ผักตบไทย 333. Pontederiaceae Monocots Monochoria vaginalis (Burm.f.) C.Presl ex Aquatic Herb kha khiat ขาเขียด Kunth 334. Portulacaceae Eudicots Portulaca oleracea L. Herb phak bia yai ผกั เบ้ียใหญ่ 335. Potamogetonaceae Monocots Potamogeton nodosus Poir. Aquatic Herb nae pak pet แหนปากเป็ด 336. Eudicots cambodiana Pierre Shrubby Tree ta khrong ตะครอง 337. Rhamnaceae Eudicots (L.) Mill. Climber lep yiao หนามเล็บเหยี่ยว 338. Eudicots Neolamarckia cadamba (Roxb.) Bosser Tree taku ตะกู 339. Rubiaceae Eudicots Canthium berberidifolium Geddes Shrub ngiang duk เงี่ยงดุก 340. Rubiaceae Eudicots Catunaregam tomentosa (Blume ex DC.) Shrubby Tree nam thaeng หนามแท่ง Tirveng. 341. Rubiaceae Eudicots Dioecrescis erythroclada (Kurz) Tirveng. Shrubby Tree ma khang daeng มะคังแดง 342. Rubiaceae Eudicots Gardenia obtusifolia Roxb. ex Hook.f. Shrubby Tree kramop กระมอบ 343. Rubiaceae Eudicots Gardenia saxatilis Geddes Shrub khoi khok ข่อยโคก 344. Rubiaceae Eudicots Haldina cordifolia (Roxb.) Ridsdale Tree khwao ขว้าว 345. Rubiaceae Eudicots Oldenlandia diffusa (Willd.) Roxb. Herb hom chaeo na โหมแจ่วนา 346. Rubiaceae Eudicots Hedyotis ovatifolia Cav. Herb phak khang khao ผักค้างคาว 347. Rubiaceae Eudicots Hymenodictyon orixense (Roxb.) Mabb. Tree som kop ส้มกบ 348. Rubiaceae Eudicots Ixora nigricans R.Br. ex Wight & Arn. Shrub khem nam เข็มน้า 349. Rubiaceae Eudicots Knoxia roxburghii (Spreng.) M.A.Rau Exotic Herb ya khamen หญ้าเขมร 350. Rubiaceae Eudicots Mitragyna diversifolia (Wall. ex G.Don) Shrubby Tree kra thum na กระทุ่มนา Havil. 351. Rubiaceae Eudicots Morinda citrifolia L. Shrubby Tree yo ban ยอบ้าน 352. Rubiaceae Eudicots Morinda coreia Ham. Shrubby Tree yo pa ยอป่า 353. Rubiaceae Eudicots Oldenlandia pterita (Blume) Miq. Herb ya phong phot khao หญ้าพง พดเขา 354. Rubiaceae Eudicots Paedaria linearis Hook.f. Climber tot mu tot ma ตดหมูตดหมา ICoFAB2019 Proceedings | 151

355. Rubiaceae Eudicots Pavetta indica L. Shrub khem pa เข็มป่า 356. Rubiaceae Eudicots Tamilnadia uliginosa (Retz.) Tirveng. & Shrubby Tree talum phuk ตะลุมพุก Sastre 357. Rutaceae Eudicots Aegle marmelos (L.) Corrêa Tree ma tum มะตูม 358. Rutaceae Eudicots Citrus × aurantiifolia (Christm.) Swingle Exotic Shrubby ma nao มะนาว Tree 359. Rutaceae Eudicots Citrus hystrix DC. Shrubby Tree ma krut มะกรูด 360. Rutaceae Eudicots Glycosmis pentaphylla (Retz.) DC. Shrubby Tree khoei tai เขยตาย 361. Rutaceae Eudicots perforata (Blanco) Merr. Scandent Shrub khontha คนทา 362. Sapindaceae Eudicots Allophyllus cobbe (L.) Raeusch. Shrub to sai ต่อไส้ 363. Sapindaceae Eudicots Lepisanthes rubiginosa (Roxb.) Leenh. Shrubby Tree ma huat มะหวด 364. Sapindaceae Eudicots Schleichera oleosa (Lour.) Merr. Tree ta khro ตะคร้อ 365. Scrophulariaceae Eudicots Scoparia dulcis L. Exotic Herb krot nam กรดน้ า 366. Smilacaceae Monocots Smilax ovalifolia Roxb. Ex D.Don Climber thao wan yang เถาวลั ยย์ ้งั 367. Smilacaceae Monocots Smilax verticalis Gagnep. Shrub khruea dao เครือด่าว 368. Solanaceae Eudicots Physalis angulata L. Exotic Herb thong theng โทงเทง 369. Solanaceae Eudicots Solanum stramoniifolium Jacq. Exotic ma uek มะอึก Undershrub 370. Stemonaceae Monocots Stemona aphylla Craib Herbaceous non tai yak หนอนตายหยาก Climber 371. Stemonaceae Monocots Stemona involuta Inthachub Herbaceous khruea pung dok san เครือปุ Climber งดอกส้ัน 372. Stemonaceae Monocots Stemona tuberosa Lour Herbaceous non tai yak หนอนตายหยาก Climber 373. Taccaceae Monocots Tacca leontopetaloides (L.) Kuntze Herb thao yai mom ท้าวยายม่อม 374. Thymelaeaceae Eudicots Aquilaria crassna Pierre ex Lecomte Tree kritsana กฤษณา 375. Eudicots Colona auriculata (Desf.) Craib Shrub po phran ปอพราน 376. Tiliaceae Eudicots Microcos tomentosa Sm. Tree phlap phla พลับพลา 377. Urticaceae Eudicots Acalypha indica L. Herb tamyae maeo ตาแยแมว 378. Verbenaceae Eudicots Phyla nudiflora (L.) Greene Exotic Creeping ya klet pla หญ้าเกล็ดปลา Herb 379. Verbenaceae Eudicots Stachytarpheta jamaicensis (L.) Vahl Herb phan ngu khiao พันงูเขียว 380. Vitaceae Eudicots Ampelocissus martini Planch. Woody Climber khruea i koi เครืออีโก่ย 381. Vitaceae Eudicots Cissus repanda Vahl Climber thao wan pun เถาวัลย์ปูน 382. Vitaceae Eudicots guineensis G. Don Shrub kradang nga daeng กระดังงา แดง 383. Vitaceae Eudicots Leea rubra Blume ex Spreng. Shrub katang bai daeng กะตังใบแดง 384. Vitaceae Eudicots Parthenocissus quinquefolia (L.) Planch. Exotic Climber thao khan daeng เถาคันแดง 385. Xyridaceae Monocots Xyris indica L. Herb ya kli klak หญา้ ข้ีกลาก 386. Xyridaceae Monocots Xyris pauciflora Willd. Herb ya bua หญ้าบัว 387. Zingiberaceae Monocots Boesenbergia rotunda (L.) Mansf. Herb krachai กระชาย 388. Zingiberaceae Monocots Curcuma alismatifolia Gagnep. Herb prathum ma ปทุมมา 389. Zingiberaceae Monocots Curcuma angustifolia Roxb. Herb krachiao กระเจียว 390. Zingiberaceae Monocots Curcuma harmandii Gagnep. Herb cho morakot ช่อมรกต 391. Zingiberaceae Monocots Curcuma parviflora Wall. Herb krachiao khao กระเจียวขาว 392. Zingiberaceae Monocots Curcuma singularis Gagnep. Herb krachiao khok กระเจียวโคก 393. Zingiberaceae Monocots Globba albiflora Ridl. Herb hong hoen หงส์เหิน 394. Zingiberaceae Monocots Globba annamensis Gagnep. Herb kha ling ข่าลิง 395. Zingiberaceae Monocots Kaempferia marginata Carey ex Roscoe Herb pro เปราะ 396. Zingiberaceae Monocots Stahlianthus pedicellatus Chaveer. & Herb wan phet phraiwan ว่านเพชร Mokkamul ไพรวัลย์ 397. Zingiberaceae Monocots Zingiber barbatum Wall. Herb khing khon nu ขิงขนหนู 398. Zingiberaceae Monocots Zingiber gramineum Noronha ex Blume Herb phlai nok ไพลนก 399. Zingiberaceae Monocots Zingiber junceum Gagnep. Herb khing kaeng ขิงแคง 400. Zingiberaceae Monocots Zingiber zerumbet (L.) Roscoe ex Sm. Herb krathue กระทือ

Conclusions

Plant diversity in Burapha University, Sa Kaeo campus was investigated. 400 species from 271 genera 98 families were identified.

Acknowledgements

This research was funded by the research grant of Burapha University via the Research Council of Thailand (Grant code 2560A10802046, Grant contract no. 192/2560). The authors were very grateful to Dr.Phongsak Phonsena, Dr.Somran Suddee, Dr.Phanom Sutthisaksopon, Dr.Paweena Traiperm, Dr.Sawai Mattapha and Mr.Phattaravee Prommanut who kindly help us to identified some species.

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References

[1] Santisuk T. Forest of Thailand. Department of National Parks, Wildlife, and Plant Conservation, 2012, 124 p. (in Thai). [2] Office of the Forest Herbarium. Tem Smitinand’s Thai Plant Names, revised edition 2014. Office of the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Bangkok. 2014, 827 p. [3] Clark R., Wearn J., Simpson D.A., Abstracts from 16th Flora of Thailand Conference 2014, Thai Forest Bulletin (BOT); 2014, 42: 105-152. [4] The Angiosperm Phylogeny Group, M. W. Chase, M. J. M. Christenhusz, M. F. Fay, J. W. Byng, W. S. Judd, D. E. Soltis, D. J. Mabberley, A. N. Sennikov, P. S. Soltis, P. F. Stevens. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society, Volume 181, Issue 1, May 2016, Pages 1–20. 2016, DOI: 10.1111/boj.12385. doi:10.14457/MSU.res.2019.26 ICoFAB2019 Proceedings | 153

Parasitic Infection in Common Lowland Frog (Hoplobatrachus rugulosus Wiegmann) and Disease Treatment

Panarat Phadee1* and Phongsri Julawong2

1Program in Fisheries, Department of Agricultural Technology, Faculty of Technology, Mahasarakham University, Maha Sarakham, 44150 Thailand 2Division of Fisheries, Roi-et College of Agriculture and Technology, Roi-et, 45170 Thailand

*Corresponding author’s e-mail: [email protected]

Abstract:

Parasitic infection in common lowland frog (Hoplobatrachus rugulosus Wiegmann) in Maha Sarakham and Roi-et Provinces was studied. The samples of the present study were collected from 3 stages of frog (Tadpole, young and adult frog), totally 833 frogs. The results revealed that all stages were affected by parasites throughout the year, in which 91.72% evidently. Both ectoparasite and endoparasite were found to affect the frogs at 9.89 and 90.11%, respectively. The 2 genera of ectoparasite, belonging to ciliate protozoa including Epistylis and Acineta, while endoparasite especially intestinal parasite found 2 genera of ciliate protozoa including Opalina and Balantidium and 4 genera of parasitic rotifer including Monostyla, Lecane, Philodina and Bracheonus. The finding shows that Opalina and Balantidium were the highest prevalence and abundance in each stage. Sodium chloride, potassium permanganate and formalin were used as chemicals to treat ectoparasite and applied to control the parasites in tadpole and young frog. The results showed that 0.5-1% of sodium chloride could reduce number of parasites, showing 3-4% of tadpole and young frog died within 1 hour. Potassium permanganate and formalin could be effective to control the parasite at 10 ppm and 50-60 ppm, while the concentration at 30-40 ppm showed high survival rate after treament. For endoparasite treatment, 3 internal anti-parasite drugs including Albendazole, Metronidazole and Piperazine were used to treat Opalina sp. and Balantidium in tadpole and young frog via oral administration. This study found that Metronidazole showed high effectiveness to treat the parasites whereas another drugs were not effective to treat parasite.

Keyword: Common Lowland Frog, Ectoparasite, Endoparasite and Parasitic prevention

Introduction

Common lowland frog (Hoplobatrachus rugulosus Wiegmann), is an eatable frog and is one of an economically amphibians in Thailand. The frog production has widely supplied from both natural water resources and cultured frog farms especially in the northeastern and northern part. People can eat all stages (tadpole, young frog and adult frog) of this animal. Recently, frog culture has been popular in order to low cost production, short time raring and high price per unit [1]. However, an intensive farming, as high density commonly causes poor environment, animal stress and finally disease induction that can affected by bacteria, parasite, fungi, viruses and worms [2], [3]. Parasitic diseases of frog have a superior position and have received a significant attention in Thailand, one of tropical country. Away from their direct damage effect on frog tissues, parasitic agents may act as stress factors rendering the frog more susceptible to other diseases [4]. Also the drastic indirect effect played by frog parasites; their retardation of frog growth with combination of frog mortality constitute the most economical impact concerning frog production. Parasitic infection in frog could normally found both external and internal infection at all stages, the common and serious parasitic diseases in aquaculture. Ectoparasites of frog could be considered as one of the most prevalent causes of diseases affecting skin and gills cause of gill inflammation and distortion of normal anatomy which impairing their respiratory foundation. It is the primary site of nitrogenous waste excretion and plays an important roles in ionic balance [5], [2] in skin causing irritation, inflammation and loss of the surface epithelium which this in turn open the way for secondary invaders [6]. In addition, endoparasites had been some reports of helminthic parasites recovered from frogs. Some nematodes [7], [8], [9], trematodes and cestode [10] had been described from the intestine of several species of frogs. The treatment of parasitic infection in frog is usually applied chemicals and drugs that may toxic to frogs. Fortunately, it usually takes higher concentration of the chemicals and drug to harm the frogs than it does to harm the pathogen. Chemical treatment may be linked to side effects such as toxic stress using high ICoFAB2019 Proceedings | 154

concentration [11] However, the chemo therapeutants are still necessary to test in controlled laboratory studies. Therefore, in the present study aimed to investigate the prevalence of parasitic diseases of cultured frog and determined the effects of some chemicals and drugs for treatment both ectoparasites and ecdoparasites on infecting frogs as eradicate of parasitic infection.

Materials and Methods

Sampling sites and frog collection The 3 stages of diseased frog (tadpole, young frog; 25-45 days old and adult frog; market size) were collected from 3 farms in Nadoon District (15º40´18´´N, 103º12´41´´E; elevation of 174 m) and 1 farm in Muang district (16º11´48´´N, 103º16´08´´E; elevation of 146 m), Maha Sarakham province and 1 farm from Tawatburi district (16º02´47´´N, 103º43´33´´E; elevation of 145 m), Roi-Et province. Affected frogs, tadpole, young frog and adult frog were sampled monthly for 1 year (12 times), totally 320 tadpoles, 330 young frogs and 183 adult frogs were sampled for parasitic examination.

Parasitic examination Diseased frogs were collected and kept in plastic containers, then transported back to the laboratory. The frogs in the laboratory were placed in the glass tanks with tap water and holding substrates for young and adult frogs. Clinical signs of skin, appendage and external organs of each specimen were firstly examined by the naked eyes for detection of any macroscopically visible lesions. Samples of mucus were scraped gently from the external organs and wound, then spread on a clean slide and freshly examined under phase contrast microscopic examination for ectoparasite. Affected frogs were euthanized with sodium pentobarbital and dissected to open body cavity, the intestine liver and stomach were removed and placed in slide glass containing a physiological solution and examined endoparasites using a compound microscope at 40, 100 and 400x magnifications. Parasitic genera were identified on the basis of their morphological characteristics according to Sirikanjana [12] Chatmongkolkul et al. [13] Purivirojkul [14] May [15] Keppeler et al. [16] Hossack et al. [17] and Comas et al. [18]. Prevalence and mean abundance were calculated according to Bush et al. [19].

Parasitic treatments

Experimental design There was 2 experiments including bath challenge of 3 chemicals (sodium chloride, formalin and potassium permanganate) to control ectoparasite and oral administration of 3 anti-parasitic drugs (albendazole, piperazine and metronidazole) to control endoparasite. The experimental design was a completely randomized block with 6, 9 and 9 treatments for sodium chloride, formalin and potassium permanganate, and three replicates with 10 frogs each of tadpole and young frog. In vivo tests consisted of therapeutic baths of 8 days with various sodium chloride, formalin and potassium permanganate concentrations (Table 1). Therapeutic baths were performed in 10 L glass aquaria, with a static water system, at 25ºC and without aeration. Therapeutic oral administration using 3 anti-parasitic drugs with 7 treatments of albendazole, piperazine and metronidazole, and three replicates with 10 frog each of tadpole and young frog (Table 1) were carried out in 10 L glass aquaria, with a static water system and without aeration.

Diseased frogs: maintenance and monitoring The 2 stages of frog (tadpole and young frog with average weight of 1.12±3.55 and 5.38±4.25 g, respectively), infested naturally with parasites or showing parasitic infection signs in plastic (PE) earthen pond from commercial frog farm in Nadoon District, Maha Sarakham Province, were used to infest experimental frog. The stock of infested frogs was held in two 250 L tanks for use in trial treatment. In addition, the tank of un-infested frog was held in 250 L tanks to provide frog for un-infested control treatments and for periodic addition to the infested stock. Parasites were monitored by microscopic examination of both external and internal organs. Frogs were euthanized with sodium pentobarbital. In tadpole, gill arch was removed from each frog and mucus was scraped from the total left-hand side and caudal fin, and in young frog the scraping was along the skin and appendages. The tissue samples were placed on microscope slides, covered with a coverslip and examined at 40-400 magnification to search for ectoparasites. Endoparasites were examined from intestine, liver, stomach and body cavity. A total of parasites were found in the external and internal organs of frogs, with a prevalence of 100%, and mean abundance of 137.5 ± 17.2. The remaining frogs were subjected to food deprivation for 24 h prior to the start of therapeutic baths and oral administration for gastrointestinal emptying.

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Treatment of frog parasites To test the efficiency of 3 chemicals on natural infested frogs, with the treatment concentrations present in Table 1. The experimental frogs were test in 3 chemicals for 8 days baths and feed daily, in order to collect the gill, appendage, fin, mucus and skin, one frog of each replicate was daily used to evaluate the efficacy of the parasitic treatments at day 0, 2, 4, 6 and 8. Samples were scraped gently, then spread on a clean slide and freshly examined under phase contrast microscopic examination for ectoparasite. After the therapeutic baths, the remaining frogs were kept for a recovery period of 8 days, with clean water. At the end of this period, the parasitic load of the frog, the occurrence of mortality were also evaluated. To test the efficiency of 3 anti-parasitic drugs on natural infested frogs by fed with floating commercial feed mixed with the drugs using the concentrations present in Table 1. The experimental frogs were fed 2 times a day (at 8.00 and 16.00) for 7 days, in order to collect the internal organs, one frog of each replicate was daily used to evaluate the efficacy of the parasitic treatments at day 0, 2, 4, 6 and 8. Samples were euthanized with sodium pentobarbital and dissected to open body cavity, the intestine liver and stomach were removed and placed in slide glass containing a physiological solution and examined endoparasites using a compound microscope. After the therapeutic feeds, the remaining frogs were kept for a recovery period of 7 days, with clean water. At the end of this period, the parasitic load of the frog, the occurrence of mortality were also evaluated.

Statistical analysis Data were expressed as mean of 3 replications ± s.d. (standard deviation). All statistical analyses were conducted using one-way ANOVA. Differences among groups were compared using Duncan’s Multiple Range test. A p-value of less than 0.05 was taken to indicate statistical significance.

Table 1 Treatment concentrations of anti-parasitic chemicals and drugs for bath and oral administration Challenges

Anti-parasitic chemicals Concentrations (% and ppm) and drugs T1 T2 T3 T4 T5 T6 T7 T8 T9

Bath challenge Sodium chloride: NaCl (%) 0 0.5 1 1.5 2 3 4 - - Formalin (ppm) 0 10 20 30 40 50 60 70 80 Potassium permanganate: 0 0.5 1 2 3 4 6 8 10 KMnO4 (ppm) Oral administration Albendazole (ppm) 0 12.5 25 50 100 200 400 - - Piperazine (ppm) 0 12.5 25 50 100 200 400 - - Metronidazole (ppm) 0 12.5 25 50 100 200 400 - -

Results and Discussions

The distribution and prevalence of parasitic infection of frog Inspection a total of 833 frogs, including tadpole, young frog and adult frog 320, 330 and 183 frogs, respectively, were collected from Maha Sarakham and Roi-Et Provinces throughout the year. The results revealed that all stages were affected by parasites throughout the year, in which 91.72% evidently. Both ectoparasite and endoparasite were found to affect the frogs at 9.89 and 90.11%, respectively (Figure 1). Disease frogs from Nadoon 1, Nadoon 2, Nadoon 3, Muang 1 and Tawatburi 1 were collected and found in 200, 202, 191, 65 and 175 infested frogs with infection rates 24.68, 24.4, 22.58, 8.13 and 20.21%, respectively. The distribution and prevalence of the parasites revealed infection with 2 genera of ectoparasite, belonging to ciliate protozoa including Epistylis and Acineta, while endoparasite especially intestinal parasite found 2 genera of ciliate protozoa including Opalina and Balantidium and 4 genera of parasitic rotifer including Lecane, Monostyla, Bracheonus and Philodina with prevalence 8.88, 8.4, 86.55, 59.06, 5.16, 4.32, 3.72 and 1.92%, respectively (Table 2). Mixed infection with more than 2 parasitic genera among ectoparasies, endoparasites or both were commonly occurred. The rotifer is zooplankton that can found in natural water resources but some of rotifer could become pathogenic parasites to caused damage of various kind of host such as the genera Brachionus, Volvox, Lecane, Monostyla and Philodina บ[15], [16], [20]. This finding shows that Opalina was the highest prevalence and abundance in each stage, followed by Balantidium, Epistylis, Acineta, Lecane, Monostyla, Bracheonus and Philodina, respectively. Except the genus Acineta could found only in tadpole. Frog infected with ciliate protozoa Epistylis and Acineta showed ICoFAB2019 Proceedings | 156

slimy pale skin with sever blood spots scattered on the body especially at the base of appendage and skin. While the endoparasites especially Opalina and Balantidium, the frogs became skinny, lean, loss of appetite or anorexia and apathy. In severe case there was no food in the intestine and large amount of parasites were found, however, these was not caused to be death. Concerning seasonal prevalence in investigated that highest infection rate with Opalina and Balantidium was recovered in October and September followed by April, May, March and June. The lowest infection was November, December, January and February, respectively. The mixed infection of the parasites was recorded in rainy season prevalence of 53.7%. There was found that farm management system affected to parasitic infection, especially water quality management. Cysts of these ciliates protozoa are passed in faecal material but they are difficult to detect and identify. In contrast, trophozoites present in colonic content can be differentiated on the basis of their morphological characteristics. It would therefore be better to collect samples of at postmortem rather than faecal samples to screen for enteric ciliates in frogs. The examination of postmortem samples would also facilitate the detection of endocommensal opalinids present within the colon and rectum of amphibian hosts [21]. Similar to Sririkanont [22] reported that parasitic infection of frog in the southern part of Thailand found 3 genera of Opalina sp., Protoopalina sp. and Balantidium sp. On the other hand, stocking density and water quality management was influence to parasitic infection. However, Goldberg et al. [23] report that the endoparasites in ranid frogs from Papua New Guinea were found 1 species of Cestoda, 3 species of Digenea, 18 species of Nematoda, 2 species of Acanthocephala and 1 species of Pentastomida. Other hosts are listed in Bursey et al. [24] and include frogs, lizards, and a mammal from Australia. Rhabdias australiensis was described by Moravec and Sey [25] from Rana daemeli collected in Queensland, Australia. Papua New Guinea is a new locality record, and New Britain is a new island record. Seuratascaris numidica is known from a variety of anurans from Europe, the Orient, and Australia [26].

Parasitic infection of frog in various Parasitic infected organ of frogs (%) stages

10%

23% Tadpole 38% Ectoparasite Young frog Endoparasite Adult frog 39% 90%

Figure 1 Parasitic infection and distribution of frog in Maha Sarakham and Roi-et province

Table 2 Parasitic infection, distribution, prevalence and mean abundance of various stages of frog

Frog Isolated frogs/ Infected Total Parasitic genera Prevalence Mean Infected frogs parasites (%) abundance habitats ±SD All stages 833 / skin 74 1464 Epistylis 8.88 19.78 ± 2.85 55 1045 Acineta 8.4 19.00 ± 3.35 intestine 721 36458 Opalina 86.55 50.01 ± 2.74 492 14465 Balantidium 59.06 29.40 ± 3.18 43 625 Lecane 5.16 14.53 ± 4.18 36 358 Monostyla 4.32 9.94 ± 2.81 31 298 Bracheonus 3.72 9.32 ± 3.36 16 143 Philodina 1.92 8.94 ± 2.45

Effect of 3 chemicals on ectoparasitic treatment in frog. Chemicals are routinely used in aquaculture for treating parasitic and fungal infections of aquatic animals. This study was intended to assess the effect of 3 of the commonly used chemicals to control parasitic infection in common lowland frog. Ectoparasitic treatments with sodium chloride of frogs in this study shown at the concentration of 0.5-1% could totally eliminate number of parasites at day 8 and 4-6, respectively in both tadpole and young ICoFAB2019 Proceedings | 157

frog, at concentration of 1-1.5% parasites were eliminated at day 4 but at 1.5% tadpole and young frog were died at day 4 and 6 post exposure. While at concentration 3-4% tadpole was eradicated parasite at day 2 and died within 3 days, and young frog was eradicated at day 4 and died at day 5-6 (Table 3). The prevalence of replicates of control was 60 to 100%, while in treatments 2-7, the prevalence was reduced to zero on skin and gills. In the analysis performed per infected organ, the highest prevalence was found on skin 46%, appendage 24% and gills 30%. The concentrations 0.5-1% was effective for the control of parasites. Vargas et al. [27] used sodium chloride a dose of 3% sodium chloride for 10 min reduced the prevalence of ciliate protozoa fron 46 to 2%. The action mechanism of sodium chloride, which eliminates protozoa and bacteria [28] controlled by an osmotic pressure difference. Color changes also occurred in fish exposed to doses of sodium chloride treatments at different doses, observed as darkening of the skin in response to the treatments. Nouh and Selim [29] presented similar results in a study by exposing to O. niloticus (25 ppm) of formalin, with organisms presenting a darkening of the skin among other symptoms. The change in color can have various causes, including the presence of toxic substances in wastewater [30], [31], [32], such as chlorine. However, the sodium chloride application could reduce the mortality then mantain the high survival rate of the juvenile that infected by Argulus for early days of infection. On the other hand, highest dose of sodium chloride give more protection for fish that attacked by parasite that is showed by highest survival rate and salt could improve the osmoregulation of fish and probably inhibit the parasites [33] Formalin is an effective chemotherapeutic agent used to treat various external parasitic infections in fish and other aquatic animals. The concentrations typically used for prolonged bath treatment are 25-500 ppm [34], [35], whereas the concentration suggested for short-term bath treatment is 250 ppm for duration of not more than 60 min [36]. However, in this study, formalin was proven effective against ectoparasites; Epistylis and Acineta, natural infested in tadpole and young frog found that at the concentration of 30-40 ppm was not effective post exposure from 1-8 days but shown high survival rate of both tadpole and young frog. While at the concentration of 50-60 ppm parasite was reduced and eradicated in young frog but tadpoles were died at day 8 and 6. At concentration of 70-80 ppm could control the parasites at day 2 but after day 3 and 4 all tadpole were died. The survival of the parasites was very high at the suggested range of concentration and duration of the treatment (Table 4). Therefore formalin is considered a suitable treatment for ectoparasites of frog at 50-60 ppm for long bath but at 70-80 ppm may use for short period or less that 36 h. In addition, Rowland et al. [37] suggested that the used of formalin 30 ppm could control ichthyophthiriosis, but at 20 ppm remained infested with theronts and trophonts on day 17 and survival at both concentrations was 100%. The action mechanism of formalin is alkylation of chemical groups of proteins, and nucleic acids. Alkylating agents are generally attached to the methyl or ethyl group of proteins and DNA, translating these molecules as nonfunctional and cause the death of the microorganism [38]. As for possible damage, observations of the gills in organisms exposed to formaldehyde treatments showed inflammation in the gill lamellae. Formalin is used as chemotherapeutic agent and may cause damage to branchial tissue because these are substances that have a toxic effect on the fish or others as observed by Mert et al. [39]. Potassium permanganate (KMnO4) has been tested as a anti-parasite and fungicide on several species of aquatic animals. Effective dosages have been found to vary with the fish species tested. In this study the effect of KMnO4 to the ectoparasites of frogs found that at concentration of 10 ppm could control the parasite at day 1-5 after that the parasites could be increased, the other concentration were reduced number of parasite but increased later days (Table 5). KMnO4 is an oxidizing agent that has been used for many years in aquaculture. As an oxidizer, it is able to chemically "burn up" organic material. This includes undesirable organic matter such as bacteria, parasites, and fungus, as well as desirable material such as gill tissue and mucus [40]. Because the chemical cannot distinguish between desirable and undesirable organic matter, it is up to the individual to use the chemical in a manner that results in maximum benefit and minimum harm to treated aquatic animals. KMnO4 can be administered at a concentration of 2 ppm as a long-term bath (four- hour minimum) in fresh water or salt water systems. KMnO4 is also reasonably safe to use in recirculating systems and has minimal impact on biofilters when used at 2 ppm. Treated water should retain the purple coloration for at least four hours. If a total application of 6 ppm KMnO4 does not result in maintenance of the purple color for at least four hours, the system should be cleaned [34]. Most of the organisms that are treated with KMnO4 thrive in an organically rich environment; therefore, improved sanitation can have a tremendous impact on treatment efficacy. Potassium permanganate can also be used as a short-term bath at concentrations of 10 mg/L for 30 minutes. At this concentration, careful observation of fish is mandatory to avoid mortality [41].

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Table 3 Number of ectoparasite survived in the Sodium chloride (NaCl) treatment of tadpole and young frog

NaCl Days post exposure of tadpole NaCl Days post exposure of young frog (%) 0 2 4 6 8 (%) 0 2 4 6 8 0 ++++ ++++ ++++ +++ ++++ 0 ++ ++ ++ + ++ 0.5 +++ +++ ++ + - 0.5 ++ ++ + + - 1 +++ ++ - D D 1 ++ ++ + - - 1.5 +++ ++ D D D 1.5 ++ ++ + - D 2 +++ + D D D 2 ++ + - D D 3 +++ - D D D 3 + + - D D 0 +++ - D D D 0 ++ + - D D Number of parasite: ++++ = >50 (Most); +++ = 31-50 (More); ++ = 11-30 (Moderate); + = 1-10 (low); - = 0 (Non) and D= Death

Table 4 Number of ectoparasite survived in the formalin treatment of tadpole and young frog

Formalin Days post exposure of tadpole Formalin Days post exposure of young frog (ppm) 0 2 4 6 8 (ppm) 0 2 4 6 8 0 ++++ +++ ++++ ++++ ++++ 0 ++ ++ + ++ ++ 10 ++++ +++ +++ ++++ +++ 10 ++ ++ + ++ + 20 +++ +++ ++ +++ ++ 20 + + + + + 30 ++++ +++ +++ - - 30 + ++ + + + 40 ++++ +++ + - - 40 ++ + + - - 50 +++ ++ ++ - D 50 ++ + + - - 60 +++ + - D D 60 ++ + - - D 70 +++ - D D D 70 + - - D D 80 ++++ D D D D 80 ++ - - D D Number of parasite: ++++ = >50 (Most); +++ = 31-50 (More); ++ = 11-30 (Moderate); + = 1-10 (low); - = 0 (Non) and D= Death

Table 5 Number of ectoparasite survived in the potassium permanganate (KMnO4) treatment of tadpole and young frog

KMnO4 Days post exposure of tadpole KMnO4 Days post exposure of young frog (ppm) 0 2 4 6 8 (ppm) 0 2 4 6 8 0 ++++ +++ ++++ ++++ +++ 0 ++ ++ ++ ++ +++ 0.5 +++ +++ +++ +++ ++ 0.5 ++ ++ ++ ++ ++ 1 +++ +++ +++ +++ ++ 1 ++ ++ ++ ++ + 2 +++ +++ +++ +++ +++ 2 + ++ + + - 3 ++++ +++ +++ ++ ++ 3 ++ ++ + ++ + 4 ++++ +++ ++ + + 4 ++ ++ + + + 6 +++ ++ ++ ++ ++ 6 ++ ++ + + - 8 +++ +++ ++ ++ - 8 ++ ++ + + + 10 +++ ++ ++ + - 10 + ++ + - - Number of parasite: ++++ = >50 (Most); +++ = 31-50 (More); ++ = 11-30 (Moderate); + = 1-10 (low) and 0 = 0 (Non)

Metronidazole, albendazole and piperazine are a commercially available antibiotic used to eliminate anaerobic bacteria and protozoans from human and animal hosts. We hypothesized that administration of metronidazole to an common lowland frog host in culture frog farm would result in the clearance of its ectoparasites population, especially Opalina and balentedium. In present study, oral administration with albendazole and piperazine feed to control internal parasite found that these anti-parasitic drugs could not reduce Opalina and balentedium in the intestine as same as control groups in both tadpole and young frog (Table 6-7). However, Albendazole and piperazine are anti- anthelmintic or anti-worm medication. It prevents newly hatched insect larvae (worms) from growing or multiplying in your body and used to treat certain infections caused by worms such as pork tapeworm and dog tapeworm. Therefore, these could not effective to ciliate protozoa in frog, Metronidazole could effective to Opalina and balentedium in the intestine at 100-200 ppm with feed that was reduce number of parasites and eradicated within day 6 and 4, respectively, while 400 ppm was also reduced the parasite and eradicated but frog were less uptake in both stages. The concentration of ICoFAB2019 Proceedings | 159

12.5 and 25 ppm were not effective to the parasites, but at 50 ppm could reduce the number of parasites (Table 8). Metronidazole is a versatile drug that is bacteriocidal, trichomonacidal, and amoebicidal. The termicidal, when used medically, refers to something that kills rather than suppresses (static) the organisms it targets. The cidal action against susceptible bacteria appears to disrupt DNA and nucleic acid synthesis in the bacteria. Metronidazole is especially effective against anaerobes (bacteria that live in the absence of oxygen) and is considered to be one of the drugs of choice for anaerobic infections. The mode of antiprotozoan action is unknown [42]. However, Nickol and Tufts [6] reported that 10 ppm of metronidazole injected orally by micropipette into posterior oropharynx could effective to control Opalinids in juvenile Woodhouse’s toads (Bufo woodhousii). Moreover, these suggested that the treating juvenile B. woodhousii with a single oral dose of metronidazole results in rapid, reliable, and well-tolerated clearance of opalinids from the gastrointestinal tract. While, Kolmstetter et al. [43] indicated that a metronidazole dosage of 20 mg/kg PO q 48 hr should be adequate for the treatment of most anaerobic infections in yellow rat snakes (Elaphe obsoleta quadrivitatta) by injection. The most recent study was performed on red rat snakes and found that an oral dose of metronidazole at 50 mg/kg every forty-eight hours was more than enough to control susceptible anaerobic bacteria and protozoa [42]. Jacobson and Kollias [44] reported deaths in indigo snakes at dosages above 100 mg/kg; however, dosages at 40 mg/kg have been given safely. However, in this study showing high dosage to treat the parasites that may cause by the given method that need to get high concentration.

Table 6 Number of endoparasite survived in the Albendazole (Alben.) treatment of tadpole and young frog

Alben. Days post exposure of tadpole Alben. Days post exposure of young frog (ppm) 0 2 4 6 8 (ppm) 0 2 4 6 8 0 ++++ ++++ ++++ ++++ ++++ 0 ++++ ++++ ++++ ++++ ++++ 12.5 ++++ ++++ ++++ ++++ ++++ 12.5 ++++ ++++ ++++ ++++ ++++ 25 ++++ ++++ ++++ ++++ ++++ 25 ++++ ++++ ++++ ++++ ++++ 50 ++++ ++++ ++++ ++++ ++++ 50 ++++ ++++ ++++ ++++ ++++ 100 ++++ ++++ ++++ ++++ ++++ 100 ++++ ++++ ++++ ++++ ++++ 200 ++++ ++++ ++++ ++++ ++++ 200 ++++ ++++ ++++ ++++ ++++ 400 ++++ +++ ++++ +++ +++ 400 ++++ +++ ++++ +++ +++ Number of parasite: ++++ = >101 (Most); +++ = 51-100 (More); ++ = 21-50 (Moderate) and + = 1-20 (low)

Table 7 Number of endoparasite survived in the Piperazine (Piper.) treatment of tadpole and young frog

Piper. Days post exposure of tadpole Piper. Days post exposure of young frog (ppm) 0 2 4 6 8 (ppm) 0 2 4 6 8 0 ++++ ++++ ++++ ++++ ++++ 0 ++++ ++++ ++++ ++++ ++++ 12.5 ++++ ++++ ++++ ++++ ++++ 12.5 ++++ ++++ ++++ ++++ ++++ 25 ++++ ++++ ++++ ++++ ++++ 25 ++++ ++++ ++++ +++ ++++ 50 ++++ ++++ ++++ ++++ ++++ 50 ++++ ++++ ++++ ++++ +++ 100 ++++ ++++ ++++ ++++ ++++ 100 ++++ ++++ ++++ +++ ++++ 200 ++++ ++++ ++++ ++++ ++++ 200 ++++ ++++ ++++ ++++ +++ 400 ++++ ++++ ++++ +++ +++ 400 ++++ ++++ ++++ +++ +++ Number of parasite: ++++ = >101 (Most); +++ = 51-100 (More); ++ = 21-50 (Moderate) and + = 1-20 (low)

Table 8 Number of endoparasite survived in the Metronidazole (Metron.) treatment of tadpole and young frog

Metron. Days post exposure of tadpole Metron. Days post exposure of young frog (ppm) 0 2 4 6 8 (ppm) 0 2 4 6 8 0 ++++ ++++ ++++ ++++ ++++ 0 ++++ ++++ ++++ ++++ +++ 12.5 ++++ ++++ ++++ ++++ +++ 12.5 ++++ ++++ ++++ ++++ +++ 25 ++++ ++++ ++++ +++ +++ 25 ++++ ++++ ++++ +++ +++ 50 ++++ +++ ++ ++ + 50 ++++ +++ ++ ++ + 100 ++++ ++ + - - 100 ++++ ++ ++ - - 200 ++++ ++ - - - 200 ++++ + + - - 400 ++++ ++ - - - 400 ++++ + - - - Number of parasite: ++++ = >101 (Most); +++ = 51-100 (More); ++ = 21-50 (Moderate) and + = 1-20 (low)

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Conclusions

It is concluded that parasitic infection of common lowland frog from plastic-earthen ponds could occur throughout the year, in which 91.72% evidently. There was found ectoparasite and endoparasite at 9.89 and 90.11%, respectively, according to water quality and farm management. The ectoparasite, belonging to ciliate protozoa including Epistylis and Acineta, while endoparasite found 2 genera of ciliate protozoa including Opalina and Balantidium and 4 genera of parasitic rotifer including Monostyla, Lecane, Philodina and Bracheonus. Ectoparasite treatment challenge using NaCl, KMnO4 and formalin to control the parasites in tadpole and young frog showed that 0.5-1%, 10 and 30-50 ppm were effective to control the disease. More higher concentrations may damaged and caused of death. Endoparasite treatment using 3 internal anti-parasite drugs: Albendazole, Piperazine and Metronidazole to tadpole and young frog with oral administration found that Albendazole, Piperazine could not effective to the parasites, only metronidazole showed effective to treat the parasites at 100 ppm and above concentration could eradicated parasite but the frog was less of apatite.

Acknowledgements

This work as financially supported by the Research Grant of Mahasarakham University and frog farmers in Maha Sarakham and Roi-Et province.

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