ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Preface The Faculty of Technology, Mahasarakham University (MSU), Thailand initiated the 1st International Postgraduate Symposium on Food, Agriculture and Biotechnology in 2014 (IPSFAB 2014) with the main aims to provide a stage for Thai and international postgraduates to present their research at the international stage. In 2018, the symposium had been renamed as ‘International Conference on Food, Agriculture and Biotechnology (ICoFAB)’ to include postgraduates, researchers and lecturers as primary participants and this has continued so till this day.
Our conferences in the past six years (2014 – 2019) have been successful and continued to grow in terms of turn outs and partnerships with international institutes. We have been honored by distinguished scientific committees who kindly have contributed to our Proceedings continually, audiences and renowed keynote/invited speakers from the fields of Food, Agriculture and Biotechnology from around the world. Every year, the potential academic networks or research collaborations amongst Thai and international researchers have been developed.
Due to the unprecedented coronavirus pandemic around the globe in 2020, our conference has been transformed to the virtual online format to encourage continuous sharing of research knowledge and close academic networking in spite of social physical distancing.
We hope that the 7th ICoFAB2020 virtual conference would bring you fruitful discussions and future research collaborative networks amongst national and international researchers while you can stay safe at home or at workplace during this difficult time.
(Assoc. Prof. Dr. Anuchita Moongngarm) Dean of the Faculty of Technology Mahasarakham University
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Organizing Committee 1. Assoc. Prof. Dr. Anuchita Moongngarm 2. Asst. Prof. Dr. Sirirat Deeseenthum 3. Asst. Prof. Dr. Khanitta Pengmeesri 4. Asst. Prof. Dr. Manatchaya Sungsriin 5. Asst. Prof. Dr. Chakrapong Chaikong 6. Asst. Prof. Dr. Pariyaporn Itsaranuwat 7. Asst. Prof. Dr. Duljira sukboonyasatit 8. Asst. Prof. Dr. Waranyoo Kaewduangta 9. Asst. Prof. Dr. Eakapol Wangkahart 10. Mr. Pongthep Charoensak
MSU Editorial Board for Proceeding 1. Asst. Prof. Dr. Sirirat Deeseenthum 2. Asst. Prof. Dr. Kedsirin Sakwiwatkul 3. Asst. Prof. Dr. Luchai Butkhup 4. Asst. Prof. Dr. Eakapol Wangkahart 5. Asst.Prof. Dr. Ruchuon Wanna 6. Dr. Wanida Chuenta 7. Asst. Prof. Dr. Wasan Duangkhamchan 8. Asst. Prof. Dr. Pheeraya Chottanom 9. Asst. Prof. Dr. Panarat Phadee 10. Assoc. Prof. Dr. Maratree Plainsirichai 11. Assoc. Prof. Prasit Chutichudech 12. Assoc. Prof. Dr. Anut Chantiratikul Associate Editor of Journal of sustainability science and management 1. Assoc. Prof. Dr. Anuchita Moongngarm 2. Asst. Prof. Dr. Vijitra Luang-In 3. Asst. Prof. Dr. Ruchuon Wanna 4. Asst. Prof. Dr. Eakapol Wangkahart 5. Assoc. Prof. Dr. Anut Chantiratikul
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Editor of Science Technology and Engineering Journal Assoc. Prof. Dr. Sirithon Siriamornpun
Scientific Committee 1. Prof. Yongqi Shao, Zhejiang University, China 2. Prof. He Yueqiu, Yunnan Agricultural University, China 3. Prof. He Chaoxing, Institute of Vegetable and Flowers. Chines Academy of Agricultural Sciences, China 4. Prof. Ping Zhang, Xishuangbanna Tropical Botanical Garden, China 5. Prof. Jun Zou, College of Fisheries and Life Science, Shanghai Ocean University, China 6. Assoc. Prof. Dr. Bei Wang, College of Fishery, Guangdong Ocean University, China 7. Dr. Song Xiaoming, Zhejiang Academy of Medical Science, China 8. Prof. Dr. Cao Liting, Zhejiang Academy of Medical Science, China 9. Prof. Dr. Li Yutao, Zhejiang Academy of Medical Science, China 10. Chen Jian, Zhejiang Academy of Medical Science, China 11. Asst. Prof. Dr. R. Ranjith Kumar, Kongunadu Arts and Science College, India 12. Assoc. Prof. Dr. V. Selvi, Kongunadu Arts and Science College, India 13. Asst. Prof. Dr. Narendhirakannan R.T., Kongunadu Arts and Science College, India 14. Dr. Indira A. Jayraaj, Kongunadu Arts and Science College, India 15. Dr. K. Kalaivani, Kongunadu Arts and Science College, India 16. Dr. K. Surekha, Kongunadu Arts and Science College, India 17. Asst. Prof. Dr. K. Vel Murugan, Kongunadu Arts and Science College, India 18. Dr. Parasuraman Aiya Subramani, Centre for fish immunology, Vels institute for science, technology and advanced studies, India 19. Asst. Prof. Dr. V. Meiyalagan, Kongunadu Arts and Science College, India 20. Prof. Hanny Wijaya, IPB, Indonesia 21. Assoc. Prof. Suprayogi, Jederal Soedieman University, Indonesia 22. Dr. Condro Wibowo, Jederal Soedieman University, Indonesia 23. Prof. Satoru Kondo, Chiba University, Japan 24. Asst. Prof. Dr. Abdulhadi Albaser, University of Sebha, Libya 25. Assoc. Prof. Dr. Khamsah Suryati Mohd, Universiti Sultan Zainal Abidin, Terengganu, Malaysia 26. Assoc. Prof. Dr. Nyuk Ling Ma, Universiti Malaysia Terengganu, Malaysia 27. Dr. Nurul Huda Abd Kadir, Universiti Malaysia Terengganu, Malaysia 28. Asst. Prof. Dr. Sinouvassane Djearamane, Universiti Tunku Abdul Rahman, Malaysia 29. Dr. Mohd Naqiuddin Husri, Malaysian Palm Oil Board, Malaysia
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
30. Dr. Nor Omaima Harun, Faculty of Science and Marine Environment, University of Malaysia teregganu, Malaysia 31. Dr. Steve Bird, School of Science, Faculty of Science and Engineering, University of Waikato, New Zealand 32. Dr. Miguel Bao, Department of Seafood Safety and Health, Contaminants and Biohazards, Institute of Marine Research, Norway 33. Dr. Ong Yek Cheng, National University of Singapore, Singapore 34. Assoc. Prof. Dr. Ko Tung Chang, National Pingtung University of Science and Technology, Taiwan 35. Asst. Prof. Dr. Benjawan Chutichudet, Mahasarakham University, Thailand 36. Asst. Prof. Dr. Waranyoo Kaewduangta, Mahasarakham University, Thailand 37. Asst.Prof.Dr. Pongsak Khunrae, Microbiology Department, King Mongkut's University of Technology Thonburi, Thailand 38. Assoc. Prof. Dr. Pawinee Chaiprasert, School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Thailand 39. Asst.Prof.Dr. Thanaset Senawong, Faculty of Technology, Khon Kaen University, Thailand 40. Asst. Prof. Dr. Vichai Leelavatcharamas, Faculty of Technology, Khon Kaen University, Thailand 41. Asst. Prof. Dr. Rujikan Nasanit, Biotechnology Department, Silpakorn University, Thailand 42. Dr. Supaporn Chunchom, Rajamangala University of Technology Isan, Thailand, Thailand 43. Asst.Prof.Dr. Noppakun Pakdeenarong, Faculty of Science, Mahasarakham University, Thailand 44. Asst. Prof. Dr. Saranyu Khammuang, Faculty of Science, Mahasarakham University, Thailand 45. Asst. Prof. Dr. Rakrudee Sarnthima, Faculty of Science, Mahasarakham University, Thailand 46. Assoc. Prof. Dr. Ratchaneewan Aunpad, Faculty of Allied Health Science, Thammasat University, Thailand 47. Dr. Adisak Romsang, Faculty of Science, Mahidol University, Thailand 48. Asst. Prof. Dr. Surachai Rattanasuk, Science and Technology Department, Roi Et Rajabhat University, Thailand 49. Asst. Prof. Dr. Rossaporn Jiamjariyatam, Faculty of Science, Srinakharinwirot University, Thailand 50. Asst. Prof. Dr. Duangjai Pisuttharachai, King Mongkut’s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Thailand 51. Dr. Supachai Suttijalean, Faculty of Technology, Mahasarakham University, Thailand 52. Mr. Pattana Pasorn, Walairukhavej Botanical Research Institute Mahasarakham University, Thailand 53. Dr. Ahmed Attaya, Department of Biological sciences/University of Massachusetts, Lowell, Massachusetts, USA 54. Dr. Pipat Piewngam, National Institutes of Health, USA
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Contents
Full paper Page Development of Kombucha and its functional property from agricultural waste 1 (fermented tea broth) Sawarin Wispen
Effect of background color and nitrite stress on cooked shrimp color of pacific 10 white shrimp (Litopenaeus vannamei) Kritch Poomkaew
Effect of Gelation Addition on Physico-Chemical Characteristics 16 of Bastard Oleaster Gummy Jelly Krittalak Pasakawee
Effect of Pectic Oligosaccharides from Fruit Peels as Prebiotic in Animal Feed 23 Pornpan Saenphoom
Factors affecting the implementation of GAP among banana (Gros Michel) 29 growers in Ban Lat district, Phetchaburi province, Thailand Almerice Enold
In Vitro Cytotoxicity of Cordyceps militaris Extracts on Different Human Cancer 38 Cell Lines Jitsuda Kullawat
In vitro digestibility of fishmeal reduction diet in combination with protease 44 enzyme by Nile tilapia (Oreochromis niloticus) digestive enzyme Pornpot Putnuan
Investigation the Physical, Mechanical Properties of Edible Film from Riceberry 51 flour Walaiporn Hemso
Method Validation for Quantitative Determination of Gallic Acid from Acacia 59 concinna (Willd.) D.C. Chiramet Auranwiwat Phytochemicals and Antioxidation of Fractionated Sugarcane Extracts: Suphanburi 65 50 Variety Phongsathorn Motham Preparation and characterization of water hyacinth cellulose/keratose composite 74 films Patcharida Chaosri
Pro-Inflammation Cytokine Secretion of Peripheral Blood Mononuclear Cells by 83 Edible Mushroom
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Sinee Siricoon
Quality Characteristics of Reduced-Fat Vienna Sausage Using Rice Flour and 90 Skim Milk Powder Mixture as a Fat Replacer Wichanard Sakunwiwat
Rice Straw Hydrolysate as a Promising Culture Medium for Astaxanthin 100 Production by the Red Yeast Xanthophyllomyces dendrorhous Pasinee Phoproek The effect of drying with controlling relative humidity of drying air on the color 111 and texture of mango sheet products Kwanchanok Prachunchonakorn
The Effect of Temperature Humidity Index (THI) on Egg Production in Pradu- 119 Hangdum Chaing Mai High Egg Production Strain Chickens Kamonnate Pimrueng
The Nutritive Value and Bioactive Compounds of Alfalfa (Medicago sativa) 127 Grown at Burapha University, Sa Kaeo Campus Supreena Srisaikham
Neuroblastoma Cell-Line Toxicity due to Increased Alzheimer’s-Related Proteins 135 and Cell Death Subsequent to High-Dose Palmitic Acid Exposure Phansa Phitthayaphong
Effects of Mash Feed, Sinking Feed and Floating Feed on Growth Performance, 143 Feed Utilization and Hematology of Nile Tilapia (Oreochromis niloticus) Muhammad Kamruzzaman
ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
ICoFAB2020 doi: 10.14457/MSU.res.2020.1 International Conference on Food, Agriculture and Biotechnology
DEVELOPMENT OF KOMBUCHA AND ITS FUNCTIONAL PROPERTY FROM AGRICULTURAL WASTE (FERMENTED TEA BROTH)
SAWARIN WISPEN1, CHALAT SANTIVRANGKANA1, PIMPINAN SOMSONG2, PIMSIRI TIYAYON2 AND WARANGKANA SRICHAMNONG1,*
1Food Science Unit, Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand 2School of Agricultural Resources, Chulalongkorn University, Bangkok, Thailand
*Corresponding author: [email protected]
Abstract:
The aim of this project was to develop a functional product from fermented tea broth which is a by-product of fermented tea (Miang). The broth that was from the 15th day fermented tea was used. The broth was boiled with the additional of 10% sugar for brix adjustment to make original and pineapple kombucha. Original fermented broth kombucha were produced form mixing with water and fermented broth (1:1.5) and add (1:1) pineapple for pineapple favor. After that, they were mixed with scoby and 3% of previous kombucha which produced from fermentation of Komagataeibacter saccharivorans, Zygosaccharomyces bailii, Dekkera bruxellensis. The analysis of antioxidant activity, total phenolic content and flavonoids content were performed at interval of 0, 1, 3, 5, 7, 9 and 11 day. The result showed that total phenolic of both original and pineapple kombucha were reduced. Total phenolic contents of original kombucha was in ranged of 32.1 - 42.05 µmol GAE/ 100 mL. Total phenolic contents of pineapple favour was in ranged of 23.1 ± 0.67 - 29.57 ± 1.19 µmol GAE/ 100 mL. While antioxidant (FRAP & ORAC) slightly increased in both batched. Antioxidant activity on FRAP of original kombucha was 311,527.8 ± 38,978.9 µmol TE / 100 mL. Antioxidant activity on ORAC of original kombucha and pineapple favor were highest at day 7 (620,442.1 ± 79,695.4 and 506,842 ± 212,017.1 µmol TE / 100 mL, respectively). Total flavonoids were decreased from 0th day fermented tea kombucha as it formed dimer and complex structure (10.15 – 6.92 µmol CE / 100 mL). Therefore, per serving size the developed kombuchas contained high amount of both phenolics and flavonoids. Fermented broth can be developed into Kombucha that containing functional property compounds.
Keywords: Fermented tea; Kombucha; Antioxidant; Value added
Introduction
Fermented tea (Miang) is an ethnic fermented tea leaf which made form Assam tea or (Camellia sinensis var.assamica). It is commonly grown in the mountainous areas of Nan, Chiang Rai, Chiang Mai, Phayao and Phrae in northern parts of Thailand. Based on the number of tea plantation areas, Chiang Mai is the largest area for tea plantation; nevertheless, the largest of Miang production is established in Nan. Miang is frequently consumed after meals like a snack. Moreover, Miang is commonly served at ceremonial occasions by the northern Thai people such as wedding ceremony, a celebration party to a funeral (Khanongnuch et al., 2017; Somsong et al., 2018). Miang is often consumed during working hours as consumers claim that it helps refilling strength required during working activities.
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Fermented broth is a liquid by-product from fermented tea (Miang) production and is usually discarded. Estimate of the broth was 2,500 mL per 1 kilogram of fermented tea production that was throw away, leading to environmental pollution. For example, air pollution and water pollution when they were thrown away in river and household area. In addition, leaching for bioactive compounds into fermented broth occur during fermentation process. Total phenolic content in fermented water was 1,450.86 mg/100 mL (Phromrukachat et al., 2010). Furthermore, local people produce Miang for a living, it can be value added product by using fermented broth. They will increase income, reduce waste and save environment. Therefore, kombucha can be that value added product. It is fermented beverage because of the health benefits. This product has slightly acidic, carbonated, and sweet taste. Fermentation process is formed the symbiosis of bacteria and yeasts as kombucha culture. The mainly common substrate is black tea or green tea (Sinir et al., 2019). Komagataeibacter spp. and Acetobacter spp. and Lactobacillus spp., among bacteria; Zygosaccharomyces spp. and Brettanomyces spp. are the most abundant yeasts (Marsh et al., 2014; Fessard et al., 2016). During fermentation acetic acid bacteria produced the cellulose pellicle layer and biofilm (Vitas et al., 2018). Therefore, kombucha have been reported as beneficial health-promoting effects, such as prevention of cancer, high blood pressure, and improving digestion function (Tu et al., 2019). Thus, the aim of this study was to develop a healthy product from fermented tea broth and kombucha was chosen because the physical appearance is similar to Miang while containing the health benefit compounds.
Materials and methods
Fermented broth preparation
Fresh mature tea leaves (leaves 4-6 from top) were randomly handpicked from Assam tea trees in October 2019 at Ban Si Na Pan located in Nan province. Fermented broth was made in the lab by fermenting of tea leave around 15 days by starter culture from the same area. Mature tea leaves were steamed at 100°C for 1 hour and cooled. Then, steam leaves were packed in containers and covered with white clothes under aerobic conditions for 7 days. Next, the steamed tea leaves which was covered with mature fungal growth are washed with water. After that, 1,000 gram of fungal growth tea leaves, 8 gram of salt, 8 mL of starter and 2,500 mL of DI water were fermented under anaerobic condition for 15 days. This broth was used to make kombucha.
Sample preparation
Kombucha scoby and previous kombucha was purchased from kombucha DIY company which produced from fermentation of Komagataeibacter saccharivorans, Zygosaccharomyces bailii, Dekkera bruxellensis.
Fermented broth Kombucha Fermented broth was diluted with water in ratio at 1:1.5 and add 10% w/v of sucrose. Then, they were boiled until sucrose well dissolved and pasteurized at 73°C 15 min in water bath. After cooling down to room temperature, the solution was poured into a glass jar and were inoculated with 2.5% (w/v) or 13 gram/500 mL and 3% of previous kombucha. The fermentation was controlled at 37°C in incubator for 11 day or until biofilm was visible.
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Fermented broth with pineapple kombucha Fermented broth was similar to original favour with the addition of pineapple at ratio at 1:1.5 and add pineapple juice. pH and total soluble (TSS)
Changes in pH of kombucha was measured using a pH meter (F20, Mettler Toledo, Bangkok, Thailand). Total soluble solid was measured using a hand refractometer (brix N1, Atago, Tokyo, Japan) at 25ºC, respectively
Estimation of total phenolic content (TPC)
The total phenolic content of the samples determined that the method base on Somsong et al. (2017). In brief, samples were diluted 10 times with distilled water and a 150 µL volume was further diluted with 2.4 mL distilled water. This was followed by adding 150 µL of Folin- Ciocalteu reagent (2 N) diluted with distilled water (1:10). The solutions were mixed well for 2 min before adding 7.5% (w/v) of 300 µL NaCO3 to account for an ascorbic acid correction and mixed again. After incubation for 2 h at room temperature in the dark, the absorbance at 765 nm was determined microplate reader. Gallic acid concentration used to establish the standard curve ranged from 0.01–0.1 mg/mL. The results were expressed as mg gallic acid equivalent/100 mL (mg GAE/100ml).
Estimation of the antioxidant activity (AA)
Antioxidant activity was determined using two assays, FRAP and ORAC. The antioxidant activity of fermented broth samples determined using FRAP assay and ORAC assay from Somsong et al. (2017). The working FRAP reagent prepared by mixing 300 mM acetate buffer, pH 3.6, with 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM of hydrochloric acid and 20 mM of FeCl3·6H2O at a ratio 10:1:1. The acetate buffer (pH 3.6) prepared using 3.1 g sodium acetate trihydrate and 16 mL acetic acid in 1 L distilled water. After that, Samples absorbance was then read at 593 nm after 30 min. A standard curve used concentrations between 100 – 600 μM L−1 of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (trolox). For ORAC assay, the reaction carried out in 75 mM phosphate buffer (pH 7.0), and the final reaction mixture was 1 mL. The samples or standard (20 µL) and fluorescein (160 µL; 120 mM solutions were preincubated for 15 minutes at 37°C; then 2,2′-azobis-(2- amidinopropane) dihydrochloride (AAPH) solution (20 µL; 480 mM, final concentration) will be added rapidly. The fluorescence (485) recorded every minute for 80 minutes in fluorescence spectrophotometer. A blank (PBS) will using methanol instead of the antioxidant solution. After that, Samples absorbance was then read at 485 nm at 37°C and a standard curve used concentrations between 5 – 50 µM Trolox. These two assays were performed using microplate reader and the results of antioxidant activities were expressed in μM Trolox/100 ml.
Estimation of total flavonoid content (TFC)
The flavonoids content will be determined by aluminium trichloride method which modified from Ahlem Rebaya et al. (2015). Catechin within the range of 0.01-0.1 mg mL-1 was used as a standard. A volume of 25 µL of standard were added to 15 μL of a 5% NaNO2 solution. The
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology mixture was allowed to stand for 6 min, then 30 μL of aluminium trichloride (10%) were added and incubated for 5 min, will be followed by the addition of 150 µL of NaOH (1M). After 15 min of incubation the mixture turned to pink and the absorbance were measured at 510 nm using microplate reader. The total flavonoids content was expressed as g E catechin mg/100 mL.
Statistical analysis
Analysis experiments were done at least in triplicate. Data was processed by a statistical software (IBM SPSS Statistics for Windows Version 18.0, IBM, Armonk, New York, U.S.A.) and presented as mean and standard deviation. Differences between mean values were analyzed by one-way Analysis of Variance and distinguished by Duncan’s New Multiple Range Test and the confidence level of 95% (p≤0.05).
Results and Discussion
Comparison of pH and total soluble (TSS) between fermentation days of fermented broth kombucha (original)
The results of pH and total soluble at interval of 0,1,3,5,7,9,11 day in original kombucha are shown in Figure 1. The changes of pH values and total soluble during fermented broth of Miang fermentation with kombucha was rapidly decreased. The decrease in pH value attributed to the production of acids during fermentation. pH values started at 4.12 ± 0.07 then 4.07 ± 0.05, 3.87 ± 0.08, 3.49 ± 0.13, 3.26 ± 0.10, 3.12 ± 0.07 and 3.00 ± 0.07 respectively. However, pH values at 0, 3, 5, 7, 9, 11 had a statistically significant decrease (p ≤ 0.05). There are two stages which are alcoholic and acetic acid fermentation (Li et al., 2015). In alcoholic fermentation stage, yeast hydrolyze sucrose into glucose and fructose, formerly the ethanol is produced that total soluble solid was decreased. After that stage, it is acetic acid fermentation, the acetic acid bacteria use glucose to produce glucuronic acid and transform ethanol into acetic acid (Ayed et al., 2016). Therefore, TSS are decrease between 10 significant different at day 0, 7, 9 and 11.
4.6 12 a a a a c d a 4.2 a e 8
b
Brix) ° 3.8 pH
pH c total soluble 4 d 3.4 e ( Total soluble f 3.0 0 0 1 3 5 7 9 11 Fermentation day
Figure 1: pH and total soluble of different fermentation day in fermented broth kombucha (original) during day 0 to day 11 ( = pH, = TSS).
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Comparison of total phenolic, antioxidant activity (FRAP, ORAC) and total flavonoid of different fermentation day in fermented broth kombucha (original)
The change in TPC value of original kombucha with fermentation day was oscillate during fermentation day (Figure 2a). The value of TPC was decrease significantly at day 3. After fermentation at day 5, the TPC was increased and the highest at day 5. Then, it was slightly decreased at day 7, 9 and slightly increase at day 11. However, they were not significantly. The oscillation of TPC might be caused by some complex phenolic compounds. They might be degraded under the acidic environment or enzymatic by bacteria or yeast in SCOBY. In particular, Saccharomyces sp, Lactobacillus sp., and Acetobacter sp. which have ability to excreted tannase, an enzyme that could degrade tannins. (Naland, 2004; Dwiputri & Feroniasanti, 2019).
50 14 a ab ab ab ab a
) ab 12 40 b a a 10 b b b 30 8 b
20 6 4 10
TFC (mg CE/100ml) (mg TFC 2
TPC (µmol GAE/100ml 0 0 0 1 3 5 7 9 11 0 1 3 5 7 9 11 Fermentation day Fermentation day
400,000 800,000 ab a abc a a a 350,000 bc 700,000 a a abc c abc a 300,000 600,000 a 250,000 500,000 200,000 400,000 150,000 300,000 100,000 200,000
50,000 100,000 FRAP (µmol TE/100ml) (µmol FRAP
- - ORAC (µmol TE/100ml) (µmol ORAC 0 1 3 5 7 9 11 0 1 3 5 7 9 11 Fermentation day Fermentation day
Figure 2: Total phenolic content (a), Total flavonoid (b), FRAP (c), and ORAC values (d) of original kombucha in different fermentation day.
TFC of original kombucha was slightly decreased (Figure2b). The TPC varied from 10.47 ± 1.18 to 10.47 ± 1.18 mg CE/100 mL. TFC value was tuned point at day 5 (8.13 ± 0.68 mg CE/100 mL) significantly. Consequently, the first day is the day that was reported to contain highest catechin in fermented tea leaves (Sampanvejsobha et al., 2013; Phromrukachat et al., 2010). After that, during fermentation of kombucha, the catechins formed dimeric catechins called theaflavins, larger compouednds called proanthocyandins, and very large oligomers and polymers called thearubigens. The chemical structures of these flavonoid oligomers in tea are very complex and have yet to be entirely characterized. (Dwyer & Peterson, 2013; Subramanian et al., 1999)
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
However, the antioxidant activity was slightly increase (Figure 2c and 2d). They were a fluctuation of antioxidant activity occur during fermentation day. The content of antioxidant activity was decreased at day 3 (271,250.00 ± 46,550.73 µmol TE /100 mL, 563,320.26 ± 125,456.15 µmol TE /100 mL on FRAP and ORAC, respectively). After day 5 of fermentation the antioxidant activity was slightly increased and the highest content at day 9 (314,305.56 ± 15,426.5273 µmol TE /100 mL) significantly on FRAP. For the highest content of ORAC was 620,442.09 ± 79,695.4173 µmol TE /100 mL at Day 7 then the fermentation period of ORAC was insignificantly. The fluctuation of antioxidant might be due to the activity of the microorganism during fermentation process and the stability of some antioxidant compounds . Especially, the microorganisms have the competence to degrade polyphenols compound (Dwiputri & Feroniasanti, 2019). Zhao & Shah (2016) reported that after fermentation, TPC was increase in phenolic acids, derivatives, and flavan-3-ols and decrease in flavonols (Zhao & Shah, 2016). Therefore, in current study, we found that TPC was increase but total flavonoid was decease.
Comparison of pH and total soluble (TSS) between fermentation days of pineapple favor kombucha
4.4 20 a a 4.2 a 16 a 4.0 a b a 3.8 b 12 b pH b pH 3.6 b 8
b solubleTotal total soluble 3.4 b b 4 3.2
3.0 0 0 1 3 5 7 9 11 fermentation day Figure 3: Comparison of pH and total soluble solid of different fermentation day in pineapple favor kombucha ( = pH, = TSS).
The results of pH and total soluble at interval of 0, 1, 3, 5, 7, 9, 11 day in pineapple favor kombucha are shown in Figure 3. The changes of pH values and total soluble solid during fermentation was rapidly decreased. The decrease in pH value is attributed to the production of acids during fermentation. pH values started at 3.95 ± 0.01 then 3.88 ± 0.00, 3.75 ± 0.00, 3.44 ± 0.01, 3.29 ± 0.01, 3.23 ± 0.01 and 3.17 ± 0.00, respectively. However, pH values was changed value significantly at day 5. Therefore, TSS was decreased 16.18 ± 1.67 brix to 7.56 ± 0.64 brix and total soluble value was significantly at day 5 (12.62 ± 0.60 brix). Several authors have the same behavior (Chakravorty et al., 2016; Sun et al., 2015).
Comparison of total phenolic, antioxidant activity (FRAP, ORAC) and total flavonoild of different fermentation day in pineapple favor kombucha
The change in TPC value of pineapple favor kombucha with fermentation day was slightly increased during fermentation day (Figure 4a). The value of TPC was increased significantly
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
(29.57 ± 1.20 µmol GAE/100 mL) at day 11. Likewise, other researchers found rather same results. After fermentation, TPC value was increased (Chakravorty et al., 2016; Jayabalan et
35 6 ab a a ab 30 ab ab ab a 5 b ab 25 4 bc 20 c 3 c 15 c 10 2
5 1
TFC (mg CE/100ml) (mg TFC TPC (µmol GAE/100ml) (µmol TPC 0 0 0 1 3 5 7 9 11 0 1 3 5 7 9 11 Fermentation day Fermentation day
180,000 a 900,000 160,000 a a 800,000 ab a a a ab 140,000 a a 700,000 120,000 600,000 ab 100,000 500,000 b 80,000 400,000 b b 60,000 300,000 40,000 200,000
20,000 100,000 ORAC (µmol TE/100ml (µmol ORAC FRAP (µmol TE/100mol (µmol FRAP - - 0 1 3 5 7 9 11 0 1 3 5 7 9 11 Fermentation day Fermentation day al., 2014). Phenolic compounds were increased such as gallic acid, m-coumaric acid and Resveratrolthat they were produced form microbiological (Amorim et al., 2018).
Figure 4: Total phenolic content (a), Total flavonoid (b), FRAP (c), and ORAC values (d) of pineapple favor kombucha in different fermentation day
TFC of original kombucha was slightly decrease (Figure 4b). The TPC varied from 4.83 ± 0.34 to 2.12 ± 0.31 mg CE/100 mL. TFC value was tuned point at day 5 (3.27 ± 0.18 mg CE/100 mL) significantly and other turning point was significantly at day 7 (2.66 ± 0.81 mg CE/100 mL). Then, values were stable. In the same way, Gaggìa et al. (2019) had total flavonoid content decrease that found catechins were decreased. However, the antioxidant activity of FRAP was slightly increased from the Figure 4c. The highest content of FRAP was 139,814.81 ± 12,633.80 µmol TE /100 mL at Day 7 then the fermentation period of FRAP was insignificantly. The researcher had report antioxidant activity of FRAP in pineapple was no significant change (Fessard et al., 2016). For the antioxidant activity of ORAC during fermentation day was oscillate that shown the figure 4d. The content of antioxidant activity was decreased significantly at day 1 (83,390.72b ± 39,232.59 µmol TE /100 mL on ORAC, respectively). In current study, day 3 of fermentation the antioxidant activity was increased and the highest content (594,714.96 ± 116,029.61 µmol TE /100 mL) significantly. After that, the antioxidant activity content of ORAC was slightly decreased and significantly at day 9 to be stable. The antioxidant activity of ORAC was slightly increased from the Figure 4c. The fluctuation of antioxidant might be due to the activity of the microorganism during fermentation process and the constancy of some antioxidant
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology compounds. Especially, the microorganisms may be used to perform mild or strong, slow or rapid food fermentation that could be ability to modulate antioxidant capacity and enzyme activities. Antioxidant activity of ORAC in pineapple juice was occur that fermented juice is lower than initial stage (Fessard et al., 2016; Septembre-Malaterre et al., 2018). Finally, original favor fermented broth kombucha had higher content of total phenolic, antioxidant activity (FRAP, ORAC) and total flavonoild than pineapple favor fermented broth kombucha. Similarly, total soluble of original favor fermented broth kombucha had lower than pineapple favor fermented broth kombucha that is good for health. Moreover, original favor fermented broth kombucha used by-product to add value. On the other hand, pineapple favor fermented broth kombucha used pineapple juice that is normal product.
Conclusion
Kombucha beverages were successfully produced by using fermented tea broth (Miang) that is waste of Miang fermentation. They are suitable for health that have higher significantly total phenolic and antioxidant than day 0. The most suitable period fermentation of original fermented tea kombucha is day 5 because total phenolic, antioxidant activities are highest than other days. Likewise, pH at day 5 is suitable for people as it is either too bitter or sour. For pineapple favor fermented broth kombucha is day 3 since antioxidant activity on ORAC is higher than other days. Nevertheless, fermented broth kombucha should evaluated for sensory evaluation to confirm characteristic that suitable for customers. Moreover, various reasons may contributed to the changed these results such as climate, season, and species. In terms of selecting high antioxidant tea or pineapple, attention needs to be paid to plantation, season and species.
References
Abdeltaif, S., SirElkhatim, K., & Hassan, A. (2018). Estimation of Phenolic and Flavonoid Compounds and Antioxidant Activity of Spent Coffee and Black Tea (Processing) Waste for Potential Recovery and Reuse in Sudan. Recycling, 3(2). Amorim, J. C., Piccoli, R. H., & Duarte, W. F. J. F. R. I. (2018). Probiotic potential of yeasts isolated from pineapple and their use in the elaboration of potentially functional fermented beverages. 107, 518-527. Ayed, L., Abid, S. B. & Hamdi, M. (2017). Development of a beverage from red grape juice fermented with the Kombucha consortium. Annals of microbiology, 67(1), 111-121. Chakravorty, S., Bhattacharya, S., Chatzinotas, A., Chakraborty, W., Bhattacharya, D., & Gachhui, R. J. I. J. o. F. M. (2016). Kombucha tea fermentation: Microbial and biochemical dynamics. 220, 63-72. Dwiputri, M. C. & Feroniasanti, Y. L. (2019). Effect of Fermentation to Total Titrable Acids, Flavonoid and Antioxidant Activity of Butterfly Pea Kombucha. Paper presented at the Journal of Physics: Conference Series. Dwyer, J. T. & Peterson, J. J. T. A. j. o. c. n. (2013). Tea and flavonoids: where we are, where to go next. 98(6), 1611S-1618S. Fessard, A., Bourdon, E., Payet, B., & Remize, F. (2016). Identification, stress tolerance, and antioxidant activity of lactic acid bacteria isolated from tropically grown fruits and leaves. Canadian journal of Microbiology, 62(7), 550-561. Gaggìa, F., Baffoni, L., Galiano, M., Nielsen, D. S., Jakobsen, R. R., Castro-Mejía, J. L., Dinelli, G. (2019). Kombucha beverage from green, black and rooibos teas: a
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comparative study looking at microbiology, chemistry and antioxidant activity. Nutrients, 11(1), 1. Jayabalan, R., Malbaša, R. V., Lončar, E. S., Vitas, J. S., Sathishkumar, M. J. C. R. i. F. S., & Safety, F. (2014). A review on kombucha tea—microbiology, composition, fermentation, beneficial effects, toxicity, and tea fungus. 13(4), 538-550. Jayasekera, S., Molan, A. L., Garg, M., & Moughan, P. J. (2011). Variation in antioxidant potential and total polyphenol content of fresh and fully-fermented Sri Lankan tea. Food Chemistry, 125(2), 536-541. Marsh, A. J., O'Sullivan, O., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food microbiology, 38, 171-178. Naland, H. (2004). Kombucha Teh Ajaib Pencegah dan Penyembuh Aneka Penyakit. Jakarta: PT. Agromedia Pustaka. Rebaya, A., Belghith, S. I., Baghdikian, B., Leddet, V. M., Mabrouki, F., Olivier, E., Ayadi, M. T. (2014). Total phenolic, total flavonoid, tannin content, and antioxidant capacity of Halimium halimifolium (Cistaceae). Journal of applied pharmaceutical science, 5(1), 52-57. Septembre-Malaterre, A., Remize, F., & Poucheret, P. (2018). Fruits and vegetables, as a source of nutritional compounds and phytochemicals: Changes in bioactive compounds during lactic fermentation. Food Research International, 104, 86-99. Sinir, G. Ö., Tamer, C. E., & Suna, S. (2019). Kombucha Tea: A Promising Fermented Functional Beverage. In Fermented Beverages (pp. 401-432): Elsevier. Somsong, P., Tiyayon, P., & Srichamnong, W. (2017). Antioxidant of green tea and pickle tea product, miang, from northern Thailand. Paper presented at the IV Asia Symposium on Quality Management in Postharvest Systems 1210. Subramanian, N., Venkatesh, P., Ganguli, S., Sinkar, V. P. J. J. o. a., & chemistry, f. (1999). Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins . 47(7), 2571-2578. Sun, T.-Y., Li, J.-S., & Chen, C. (2015). Effects of blending wheatgrass juice on enhancing phenolic compounds and antioxidant activities of traditional kombucha beverage. Journal of Food and Drug Analysis, 23(4), 709-718. Tu, C., Tang, S., Azi, F., Hu, W., & Dong, M. (2019). Use of kombucha consortium to transform soy whey into a novel functional beverage. Journal of Functional Foods, 52, 81-89. Yao, L. H., Jiang, Y. M., Caffin, N., D'Arcy, B., Datta, N., Liu, X., Xu, Y. (2006). Phenolic compounds in tea from Australian supermarkets. Food Chemistry, 96(4), 614-620. Zhao, D., & Shah, N. P. (2016). Lactic acid bacterial fermentation modified phenolic composition in tea extracts and enhanced their antioxidant activity and cellular uptake of phenolic compounds following in vitro digestion. Journal of Functional Foods, 20, 182-194.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.2 International Conference on Food, Agriculture and Biotechnology
EFFECT OF BACKGROUND COLOR AND NITRITE STRESS ON COOKED SHRIMP COLOR OF PACIFIC WHITE SHRIMP (Litopenaeus vannamei)
POOMKAEW KRITCH1, ORAPINT JINTASATAPORN1, SRINOY CHUMKAM2
1Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand 2Faculty of Agricultural Technology,Valaya Alongkorn Rajabhat University, Pathum Thani, Thailand
*Corresponding author :[email protected]
Abstract:
The effect of background color and nitrite stress on cooked shrimp color of pacific white shrimp (litopenaeus vannamei) was conducted. Two factors of A: back ground color, black and white and B: no nitrite stress and with nitrite stress condition at 10 ppm were assigned in Factorial 2X3 in complete randomized designed (Factorial 2X3 in CRD) then 6 treatments and 3 replicates were studied. Shrimp with average weight of 7.43-8.21 gr/ind were exposure in treatment condition of no nitrite stress for 1 hr and with nitrite stress at 10 ppm for 1 hr and 24 hr then sampling for boiled and conducted color measurement by colorimeter and salmofan to compare the red intensity of cooked shrimp color. The results showed that the black background condition could promote the cooked shrimp color both in normal condition and under nitrite stress condition better than white background condition. The nitrite stress condition at 10 ppm could enhance the cooked shrimp color in short term periods of 1 hr. better than normal condition. For long term exposure to nitrite stress condition at 10 ppm for 24 hr., the stress condition reduced the redness of cooked shrimp color and reduced the carotenoid accumulation in shrimp.
Keywords: Cooked shrimp color; Background color: Nitrite stress
Introduction
White shrimp (Litopenaeus vannamei) are economically important aquatic animals and are widely cultivated in Latin America. North America and some countries in Asia. For Thailand, the introduction of white shrimp to culture since 1998 brought continuous income into the country for a high value. The trend of the white shrimp culture industry grows dramatically. However, shrimp feed is one of the factors that are variable for shrimp production and environment and considered as the main cost of culturing white shrimp (Valencia Castañeda, et al., 2018). The color of the shrimp after boiled is considered one of the main qualities that consumers and business people pay more attention to the price of white shrimp. The consumers in China and the European zone gives the high price of shrimp according to dark red after boiled compare to shrimp with light color. Hence, the red intensity of cooked shrimp color considers as an opportunity to increase revenue and increase business competition (Parisenti et al., 2011) During shrimp culture many factors effect on shrimp color especially water quality and disease infection. Ammonia and nitrite are widely present as a common toxic substance in aquatic systems. Nitrite is not only a toxic intermediate produced during ammonia nitrification
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology but also a product of denitrification of nitrate by bacteria during nitrogen cycle (Tomasso, 2012). Nitrite concentrations in coastal seawater are approximately 10–15 nM (0.14–0.21 NO2–N μg/L) (Kieber Seaton, 1995). However, in shrimp culture especially in closed system and biofloc system, nitrite concentration increases in middle and late-stage cultures. The nitrite concentration can reach 1.43 mM (20 NO2–N mg/L) and seriously induces shrimp stress and effects the health of farmed animals (Tacon et al., 2002). Focusing on the behavior of shrimp, it is known that shrimp has ability to change their appearance to blend in with background color by moving the pigments in chromatophores in epidermal layers beneath the exoskeleton (Robison Charlton, 1973). The movements of pigments in live shrimp have a direct effect on the color of cooked shrimp. This study intends to investigate the effect of background color and nitrite on the visual appearance of cooked shrimp color.
Materials and methods
Experimental animals
Five hundred white shrimp (Litopenaeus vannamei) with average weight of 7.43-8.21 gr/ind obtained from private farm at Nakorn Pathom, Thailand. Shrimp were culture in earth pond and fed diet without carotenoid supplementation for 2 months before move to acclimated in net cage of 3X4X1.5 m for 7 days before start the trial. Commercial feed (Grobest103S), protein 38% lipid 7% fiber <3% moisture <12% was applied to shrimp 2 times a day during acclimated periods.
Nitrite stress condition
The nitrite stress condition was set up by adding sodium nitrite 4.92 g in 100L water.
Experimental designed
The research was designed in the Factorial 2X3 in Complete Randomized Design (Factorial 2X3 in CRD). Two factors that influence on cooked shrimp color was assigned. Factor A was tank background color of black and white. Factor B was nitrite stress condition of no nitrite stress condition and nitrite stress condition at 10 ppm. Hence, the trial was 6 treatments and 3 replicates.
T1: No Nitrite stress for 1hr in black background T2: Nitrite stress for 1 hr. in black background T3: Nitrite stress for 24 hr. in black background T4: No Nitrite stress for 1 hr. in white background T5: Nitrite stress for 1 hr. in white background T6: Nitrite stress for 24 hr. in white background
Experimental conditions
Shrimp with average weight of 7.43-8.21 gr/ind were randomly distributed into black and white tanks of each of 100 litter capacity that contain 12 ppt saline water. Shrimp were stocked at 25 ind/tank for 24 hr. During this time, shrimp was starvation, no feed was applied to shrimp. For 24 hr of nitrite stress condition, shrimp were sampling for boiled to determine the effect of background and nitrite stress condition on cooked shrimp color.
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Data Collection: Cooked Shrimp Color Measurement
At the end of nitrite stress condition for 1 and 24 hr, ten shrimp from each treatment and replicate were sampling at each period for cooked and study on the color measurement. Cold shocked by ice was done immediately then boiled in 8% saltwater for 2 minute. The Salmon fan were used to measure shrimp color and graded score by colorimeter NH-1, measured on the first abdomen of shrimp compare to the fan. The C.I.E. LAB system (L*, a*, b*) refer to Brightness : L*, Red coloration :a*, Yellow coloration:b*. The total carotenoids (TC) was determined by weight sample then add extract solution of HEAT (Hexane:Acetone:Ethanol: Toluene 10:7:6:7) after that sonicate in cool and dark condition. Supernatant was recorded OD at 470 nm. The amount of total carotenoids was calculated.
Statistical data analysis
All the data from the experiment were subjected to analysis in Factorial 2x2 in CRD by statistical software. Duncan's Multiple Range Test was applied to compare the difference between experimental groups (Steel Torrie, 1980).
Results and Discussion
The effect of background color and nitrite stress on cooked shrimp color of Pacific white shrimp (litopenaeus vannamei) was presented in Table 1.
Table 1: Color measurements of cooked shrimp (Litopenaeus vannamei) after stocked in black and white background under nitrite stress condition at different periods
Treatment Background Nitrite stress L* a* b* carotenoid T1 Black No Nitrite 1 hr 53.79±4.13b 17.24±2.16b 18.69±1.63bc 1.501±0.09a
T2 Nitrite 1 hr 55.22±0.91c 24.77±2.82a 23.02±0.79a 1.08±0.20b
T3 Nitrite 24 hr 46.80±1.59b 17.24±0.28b 20.62±1.28ab 0.84±0.03b
T4 White No Nitrite 1 hr 62.08±2.73a 10.96±2.66c 12.23±3.52e 0.97±0.28b
T5 Nitrite 1 hr 61.14±0.60a 17.15±1.25b 16.36±2.01cd 0.83±0.26b
T6 Nitrite 24 hr 51.99±2.73b 12.23±1.90c 14.77±1.93de 0.79±0.15b
P-value background <0.001 <0.001 <0.001 0.010 P-value nitrite <0.001 <0.001 0.013 0.007 interaction 0.535 0.560 0.938 0.138 Note: Mean with different superscript letters a,b,c in the same column indicates significantly differences(P<0.05) L* refer to Brightness, a* refer to Red coloration, b* refer to Yellow coloration
Shrimp stocked in black and white background for 1and 24 hrs. The results showed that the black background decreased the brightness (p<0.05) but increased the redness (a*) and
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology yellowness (b*) of cooked shrimp (p<0.05) including promoting the accumulation of carotenoid in shrimp better than the white background (p<0.05). Hence, the results implied that the background color of tank could reduce the stress of shrimp then maintain carotenoid accumulated in shrimp (p<0.05). Under the nitrite stress condition at 10 ppm for 1 and 24 hr, the cooked shrimp color showed significantly increased (p<0.05) the redness, yellowness after immerge in nitrite for 1 hr compare to no nitrite stress for 1 hr and nitrite stress for 24 hr. The black background demonstrated the better enhancement of red color than the white background condition at every period. Moreover, the combination effects of black background and nitrite stress condition on enhancing cooked shrimp color resulting in increasing the Salmofan score was shown in Figure 1.
Black background
Normal condition: Nitrite Stress at 10 ppm Nitrite Stress at 10 ppm for 24 No Nitrite Stress for 1 hr. for 1 hr. hr.
White background
Normal condition: Nitrite Stress at 10 ppm for Nitrite Stress at 10 ppm for 24 No Nitrite Stress for 1 hr. 1 hr. hr.
Figure 1: Cooked shrimp (Litopenaeus vannamei) color after stocked in black and white background under nitrite stress condition at 10 ppm for 1 hrs.
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Carotenoid accumulation in cooked shrimp stock in black background was higher than in white background and decreased after nitrite stress condition in every period. These could imply that shrimp color decreased because carotenoid has been used as antioxidant for reducing free radicals that induced by nitrite stress condition for long time exposure to nitrite for 24 hr then carotenoid accumulation was not enough for color expression. In particular, astaxanthin, which is the carotenoid form that can express the high red color intensity in cooked shrimp and has powerful antioxidant properties (Kuhn et al., 2010). Astaxanthin was utilized very fast for scavenging free radicals resulting in a lower level of redness. According to the literature review, shrimp quite be the nocturnal animal, the black background could maintain the redness of cooked shrimp better than the white background due to the reduction of stress. In addition, it is known that shrimp are capable on changing their appearance to blend with background color, through movement of pigment in chromatophores at epidermal layer beneath the exoskeleton (Fingerman, 1965; Robison Charlton, 1973). Morphological and physiological color changes have been described in crustaceans relating to slow and rapid changes resulting from environmental or hormonal factors (Rao, 1985; Melville-Smith et al., 2003). The movements of pigments in live shrimp may have a direct effect on the color of cooked shrimp (Chien Jeng, 1992). Nitrite was found to induce shrimp stress, causing many free radicals then shrimp use carotenoid as an antioxidant to eliminate free radicals that occur (Barbieri, et al. 2016)., hence, the carotenoids move to the epidermal layer beneath the exoskeleton that cause the increasing of redness and yellowness and effect on decreasing carotenoids accumulation.
Conclusion
The black background condition could promote the cooked shrimp color both in normal condition and under nitrite stress condition better than white background condition. The nitrite stress condition at 10 ppm could enhance the cooked shrimp color in short term periods of 1 hr. better than normal condition. For long term exposure to nitrite stress condition at 10 ppm for 24 hr, the stress condition reduced the redness of cooked shrimp color and reduced the carotenoid accumulation in shrimp.
Acknowledgements
The author would like to thank The Nutrition and Aquafeed Laboratory, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand for facility and financial support.
References
Barbieri, E., Bondioli, A. C. V., de Melo, C. B., & Henriques, M. B. (2016). Nitrite toxicity to Litopenaeus schmitti (Burkenroad, 1936, Crustacea) at different salinity levels. Aquaculture research, 47(4), 1260-1268. Chien, Y. H., and Jeng, S. C. (1992). Pigmentation of kuruma prawn, Penaeus japonicus Bate, by various pigment sources and levels and feeding regimes. Aquaculture, 102(4), 333- 346. Fingerman, M. (1965). Chromatophores. Physiol. Rev. 45, 296–339. Kieber, R. J. P. Seaton (1995). Determination of subnanomolar concentrations of nitrite in natural waters. Analytical Chemistry 67(18):3261-3264
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Kuhn, D. D., Smith, S. A., Boardman, G. D., Angier, M. W., Marsh, L., Flick Jr, G. (2010). Chronic toxicity of nitrate to Pacific white shrimp, Litopenaeus vannamei: impacts on survival, growth, antennae length, and pathology. Aquaculture, 309(1-4), 109-114. Melville-Smith, R., Cheng, Y.W. Thompson, A.W. (2003). Factors affecting colour change in ‘white’ western rock lobsters, Panulirus cygnus. Journal of Experimental Marine Biology and Ecology. 291, 111–129 Parisenti, J., L.H. Beirão, M. Maraschin E. R. Oliveira. (2010). Pigmentation and carotenoid content of shrimp fed with Haematococcus pluvialis and soy lecithin. Aquaculture Nutrition 17(2):e530 - e535 Ramírez‐Rochín, J., Frías‐Espericueta, M. G., Fierro‐Sañudo, J. F., Alarcón‐Silvas, S. G., Fregoso‐López, M. G., & Páez‐Osuna, F. (2017). Acute toxicity of nitrite on white shrimp Litopenaeus vannamei (Boone) juveniles in low‐salinity water. Aquaculture research, 48(5), 2337-2343. Rao, K.R., 1985. Pigmentary effectors, in integuments, pigments and hormonal processes. In: Bliss, D.E., Mantel, L.H. (Eds.), The Biology of Crustacea, Vol. 9. Academic Press, New York, pp. 395–462. Robison Jr,W.G. Charlton, J.S. (1973). Microtubules, microfilaments, and pigmentmovement in the chromatophores of Palaemonetes vulgaris (Crustacea). J. Experimental Zoology 186, 279–304. Tacon, A.G.J., J.J. Cody , L.D. Conquest, S. Divakaran, I.P. Forster O.E. Decamp. (2002). Effect of culture system on the nutrition and growth performance of Pacific white shrimp, Litopenaeus vannamei (Boone) fed different diets. Aquaculture Nutrition 8;121^137 Tomasso, J. R. (2012). Environmental nitrite and aquaculture : a perspective. Aquaculture international, 20(6), 1107-1116. Steel, R. G. D. J.H. Torrie. 1980. Principles and Procedures of Statistics: a biometeric approach (2nd Ed.) Mc Growwhill: New York. Valencia-Castañeda, G., Vanegas-Pérez, R. C., Frías-Espericueta, M. G., Chávez-Sánchez, M. C., Ramírez-Rochín, J., Páez-Osuna, F. (2018). Comparison of four treatments to evaluate acute toxicity of nitrite in shrimp Litopenaeus vannamei postlarvae: Influence of feeding and the renewal water. Aquaculture, 491, 375-380.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.3 International Conference on Food, Agriculture and Biotechnology
EFFECT OF GELATION ADDITION ON PHYSICO-CHEMICAL CHARACTERISTICS OF BASTARD OLEASTER GUMMY JELLY
PANIDA BANJONGSINSIRI*, NOWWAPAN DONRUNG AND KRITTALAK PASAKAWEE*
The Expert Centre of Innovative Health Food (InnoFood), Thailand Institute of Scientific and Technological Research (TISTR) 35 Moo 3 Technopolis, Khlong Ha, Khlong Luang, Pathum Thani 12120, Thailand
*Corresponding author: [email protected], [email protected]
Abstract:
Gummy jelly, a gelling-based confectionary product, was prepared by the combination of sugar, gelling agent, and other components. The objective of this present studies was to investigate the effect of different levels of gelation on physico-chemical characteristics of fruit gummy jelly product prepared from Bastard oleaster fruits. Bastard oleaster juice was mixed with water, sugar, citric acid, and the varied concentration of gelatin (at 8%, 9%, and 10% by weight of totally ingredients), then formed in molds and placed at 25C for 2-4 hours until it dried. The physico-chemical characteristics of gummy jelly products were analyzed. It was found that pH value, total soluble solid, total acidity, and springiness of gummy jelly products showed statistically significant increase as the increasing of gelatin addition (p≤0.05), in the following ranges, 3.51-3.66, 66.4-69.8Brix, 1.19-1.65% ,8.41-9.19 N, respectively. In contrast, the moisture content decreased from 7.35% to 6.33% with the loss of vitamin C content, 6.88-4.79 mg ascorbic acid/100 g gummy product (p≤0.05). There were no significant differences in hardness, cohesiveness, gumminess, and chewiness, except of springiness among three gelatin concentrations of gummy jelly samples. The results revealed that gelatin addition was effective in improving the physico-chemical texture characteristics of gummy jelly products.
Keywords: Bastard oleaster fruit; Fruit gummy jelly; Physico-chemical characteristic
Introduction
Bastard oleaster (Elaeagnus latifolia L.) is one of an endemic fruit plant cultivated in the upper- north and north-eastern part of Thailand. The fruits are attractive in varied size from small to large with a fresh color, while the mature fruits had dark red, red, orange-red and yellow colors with sourness and sweetness taste (Yingthongchai & Sirikhum, 2008). It is also reported to be a source of vitamins, minerals, essential fatty acids and other phytochemical compounds, especially phenolic acids and flavonoids (Panja et al., 2014; Patel, 2015). Seal (2012) found the high mineral quantities containing in its fruit such as potassium of 13580 mg/kg and calcium of 5860 mg/kg, and sodium of 965 mg/kg. Fruits also exhibited the high content of vitamin A, C, and total phenolic acid at 621.37-626.17 mg/100 g, 2.37-17.26 mg/100 g, and 2.32-3.81 mg/g, (Yingthongchai & Sirikhum, 2008). The bastard oleaster fruits can be consumed in the form of food and drink products, such as jam, leather, salsa, wine, and cookies (Patel, 2015). The shorter shelf life and perishable nature of fruits causes obstacle on its optimum utilization. Hence, the suitable preservation processes can be effective way for maintaining its taste and other nutrients.
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Gummy jelly, a gelled confectionary product, is obtained from the mixing process of concentrated sugar solution, gelling agent, and other ingredients such as acids, coloring and flavouring agents. The melted mixture is shaped into the molds of different types, shapes, and sizes. The drying step aims to reduce a moisture content of product and increase its solid content (Delgado & Banon, 2015). Generally, the drying period range from 24 hrs more. This product can be prepared by the traditional processes or using a modern confectionary machine. Gummy jelly products are characterized by a texture in terms of hardness and chewiness incorporated by sugar, gelatin, or starch based gels (Burey et al., 2009; Marfil et al., 2012; Meesang et al., 2003; Delgado & Banon, 2015). The majority of confectionary gel formation contains sucrose, glucose syrup, gelatin, starch, water, including food acids (Burey et al., 2009). According to Meesang et al. (2003), it has been reported that the viscosity and texture models of gummy jelly depended on the gelatin content, sucrose/glucose syrup ratio, and citric acid content resulted in the acceptability score. Jiamjariyatam (2018) found that the interaction of gelatin (9, 12, and 15%) and isomaltulose (20, 30, 40, 50, and 100%) concentration affected aw, pH value, including sensory texture quality of gummy jelly. Regarding the gummy jelly development, gelatin is a simple component added for its gel formative structure. Gelatin can form junction zones by its helix structure and develop the gelling network. Several publications reported that the amount of gelatin affected the texture characteristics of gummy jelly products. The hardness, cohesiveness, gumminess, and chewiness of gummy jelly products increased with the increasing of gelatin content (Thanomwong, 2008; Tiampakdee, 2006; Meesang et al., 2003). This objective was to study the effect of gelatin addition in gummy jelly products produced from Bastard oleaster pulp. The physico-chemical criteria qualities of gummy jelly, especially texture profile were investigated among each treatment.
Materials and Methods
Bastard oleaster fruits
The fresh Bastard oleaster (Elaeagnus latifolia L.) fruits was collected during November, 2018 from Nong Khai province, Thailand. It was washed, kept in a vacuum bag, and frozen at -18°C to -20°C in a freezer until using. Then, Bastard oleaster fruits were thawed and depulped to achieve the clear juice. The physico-chemical characteristics of Bastard oleaster juice such as pH value, total soluble solid content, total acidity, and total ascorbic acid content were analyzed.
The Bastard oleaster fruit gummy jelly preparation
The preparation of Bastard oleaster fruit gummy jelly was conducted following as: Bastard oleaster juice (50 g) was mixed with water (40 mL), sugar (36 g), glucose syrup (50 mL), and gelatin 250 bloom with the different concentration (at 8%, 9%, and 10% by weight of totally composition) at 70-80C. The gelatin solution was prepared by dissolving in warm water at the ratio of gelatin and water of 1:1 (w/w). The mixture was heated until 65°Brix and cooled to 55- 60C. After that, the mixed syrup was subsequently added glycerin (4 g), citric acid (1.9 g) and apple flavor (0.10 g) then formed in casing molds into a circle shape. The mold was placed at 25-30C for 2-4 hours.
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology
Physico-chemical analysis
Sample was blended to measure the water activity using a water activity analyzer (4 TE, Agua lab, UK) and moisture content using an infrared moisture analyzer (IR-35, Denver instrument, USA). Sample (5 g) was homogenized with distilled water (20 mL) at 5,000 rpm for 30 seconds. The sample solution was filtered through Whatman paper No.4 and taken the clear solution for analysis. The total soluble solid content (TSS) and pH value were determined using a digital hand - held pocket refractometer (PAL-1 and PAL-3, Atago, Japan) and a pH meter (Seven easy, Mettler Toledo, Switzerland), respectively. The content of total titratable acidity (TTA) was analyzed by the titrimetric method No.942.15 (AOAC, 2000) using an auto-titrator (DL53, Mettler-Toledo, Switzerland). The data was computed and reported as the percentage of citric acid content. The content of vitamin C was conducted by the titrimetric method No.967.21 (AOAC, 2020) using an auto-titrator, in which of visual reduction of 2,6 dichloro- phenolindophenol dye was measured and expressed as mg of ascorbic acid (AA) per 100 g sample. Color value was measured using a Minolta chromameter (CR-400, Konica Minolta Inc., Japan). The chromameter was calibrated by a white color standard (Y=86.80, x=0.3196, y=0.3372) using the Illuminant D65 light source with 2 degree standard observer. The colorimetric data was operated as CIE Lab scale in triplicated measurement and displayed the average data in forms of L value (Lightness, 0-100), a value ((-) green to (+) red), and b value ((-) blue to (+) yellow) value. Texture quality was evaluated by measuring the texture profile analysis (TPA) test with a Texture Analyzer (TA500, Lloyd instrument, UK). The gummy jelly sample was compressed twice with a compression plate probe and a 5-kg load cell. A probe penetrated to a depth of 50% distance at a test speed of 30.0 mm./min and a trigger point of 0.05 N. Five pieces of sample were tested for each treatment. The Hardness1 and Hardness2 were expressed as maximum force (N) achieve at the first bite and the second bite, respectively. Springiness related to the distance (mm) that a sample recovers during the time that elapses between the end of the first bite and the start of the second bite. Cohesiveness was calculated as ratio of area of second compression cycle to that of area of first compression cycle. Gumminess is defined as the multiplication of product hardness and cohesiveness and Chewiness is defined as the product of hardness x cohesiveness x springiness.
Statistical analysis
Data were analyzed by the analysis of variance procedure (ANOVA) using a completely randomized design (CRD) with the addition of gelatin in fruit gummy jelly preparation. Mean separation was calculated according to the Duncan’s New Multiple’s Range Test (DMRT) at the 95% confidence level. The T-test differential mean between two samples was analyzed at the 95% confidence level. The results were presented as mean and standard deviation. All statistical analysis was performed with SPSS.
Results and Discussion
The changing of gummy jelly texture can be observed by texture profile analysis (TPA), in which the deformation of different descriptors is under a force, a distance, and the time deformation. Texture characteristics of gummy product made from Bastard oleaster fruits were shown in Table 1. There were no significant differences in hardness, cohesiveness, gumminess, and chewiness (p>0.05) except for a statistically significant difference in springiness (p≤0.05).
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The Hardness1, a maximum force (N), was achieved from the first compression force test and the Hardness2 was also obtained from the second bite, respectively. In this study, gummy jelly samples from Bastard oleaster fruit with 8-9% gelatin addition showed 5.96-6.41 N of Hardness1 with the 30-40% reduction of Hardness2 (3.66-4.11 N). This also seems to indicate that Bastard oleaster gummy jelly remained a strong formation after the first bite. The averages of springiness showed an increased springiness from 8.41 mm to 9.19 mm when the gelatin content was added from 8% to 9% (p≤0.05). The springiness means elasticity or property of resisting deformation. This finding is agreed with the reports of Thanomwong (2008) suggesting that the springiness, hardness and chewiness intensity scores of lemongrass flavour gummy jelly increased with the predictive regression models when the amount of gelatin increased from 7% to 8%. The increase in hardness, springiness, gumminess, and chewiness through the gelation addition can be explained that the simultaneous action of gelling agent as gelatin on gelation system containing sugars, water, and food acids resulting in the chewy gel formation (Burey et al., 2009; Delgado & Banon, 2014). Gelatin provides elasticity through a sol-gel transition with a gelling network junction zones by the weak hydrogen bonds (Zainol et al., 2020) resulting the hardness of gelatin-based gummy confection increased (Marfil et al., 2012; Jiamjariyatam, 2018; Zainol et al., 2020).
Table 1: Texture of Bastard Oleaster fruit gummy jelly with the different contents of gelatin
Texture 8% Gelatin 9% Gelatin 10% Gelatin Hardness1ns (N) 6.16±1.34 5.96±0.90 6.41±0.94 Hardness2ns (N) 3.66±1.08 4.11±0.43 4.11±0.96 Cohesivenessns 0.25±0.11 0.21±0.04 0.18±0.07 Springiness (mm) 8.41±0.45a 9.19±0.06b 9.19±018b Gumminessns (N) 1.20±0.53 1.24±0.11 1.44±0.75 Chewinessns (N.mm) 11.04±4.99 11.38±1.05 12.19±6.71 Note: nsno significant difference values (p>0.05) a-cThe superscript lowercase in the same row are significantly different (p≤0.05)
The physic-chemical [rp[erties of Bastard oleaster fruit gummy were presented in Table 2. The pH value, total acidity, total soluble solid content, and moisture content of all three gummy samples were in the range of 3.51-3.66, 1.19-1.65% 66.4-69.8°Brix, 6.33-7.35%, respectively. The results of pH value, total soluble solid (TSS), and total acidity of gummy product showed significant increase because of the increasing of gelatin content (p≤0.05). In contrast, the moisture content and the content of vitamin C decreased while the amount of gelatin was added. There were no significant differences in water activity within the range of 0.73-0.74. In the gelled gummy systems, water acts as a plasticizer to aid gelation of gelatin formation (Siegwein, 2010). Gummy preparation is a varied combination of water, sugars and gelling agents presenting in various viscosity and gel formation ability. Gelatin has a high affinity for water (the value of bound water 0.44 g/g dry material) in sucrose/starch/gelatin system (Burey et al., 2009). Burey et al. (2009) suggested that the higher of solid content in gelatin solution, the gel formation will set faster affecting by gel concentration. Then, the free water move from gummy composition and evaporate during drying stage causing decreases a moisture content and increases a solid content linked to the texture changing from soft to hard shape (Delgado & Banon, 2014). Regarding results in Table 2, it also showed the increasing of TSS with decreasing of moisture content in Bastard oleaster gummy jelly with addition of gelatin powder. The increasing of gelatin content; as a result, pH value of products considerably increased (p≤0.05). This finding is in agreement with the report of Jiamjariyatam (2018). There is a possibility that gelatin derived from collagen, which is a protein and the subunit of gelatinis
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology amino acid causing the high pH value. (Jiamjariyatam, 2018). The increasing of total acidity in gummy jelly product was found in the 10% and 9% gelatin addition treatments using a shorter cooking time than 8% gelatin addition. It is possible to observe the different in moisture content of each gummy jelly product. In addition, the content of vitamin C in the 10% gelatin formulation would rather lose after a longer drying period than that of the 8% and 9% gelatin formulas.
Table 2: Physico-chemical characteristics of Bastard oleaster fruit gummy jelly with the different contents of gelatin
Physico-chemical characteristics 8% Gelatin 9% Gelatin 10% Gelatin pH value 3.51±0.01a 3.59±0.01b 3.66±0.01c Total soluble solid (°Brix) 66.40±0.17a 68.30±0.17b 69.80±0.17c Total acidity as citric acid (%) 1.19±0.07a 1.49±0.11b 1.65±0.15b Moisture content (%) 7.35±0.12c 6.62±0.18b 6.33±0.07a Water activityns 0.73±0.01 0.73±0.01 0.74±0.01 Color: L* value 29.53±0.04a 30.81±0.26b 32.51±0.39c a* valuens 20.91±0.06 20.78±0.28 20.60±0.53 b* value 10.87±0.10b 11.68±0.21c 8.44±0.32a Vitamin C (mg AA/100 g sample) 6.88±0.63b 6.67±0.36b 4.79±0.36a Note: nsno significant difference values (p>0.05) a-cThe superscript lowercase in the same row are significantly different (p≤0.05)
Moreover, the addition of gelatin in gummy products had affected L* value and b* value (p ≤ 0.05). The results showed that the increasing of gelatin content in gummy resulted the L* value increased but a* value decreased. It is possible that gelatin solution has a light yellow causing the color changing in gummy products. In addition, the acidifying or pH regulating agent such as citric acid, which might affect jelly gelation depending on the isoelectric point (pI) of gelatin types used (Burey et al., 2009). Table 3 showed the physico-chemical qualities of Bastard oleaster juice, Bastard oleaster fruit gummy jelly, and the commercial gummy jelly products. The developed fruit gummy jelly product with 9% gelatin addition which was selected using sensory evaluation results (it was not shown in this report) had the characteristics following Thai Community Product Standard (TCPS) of dried jelly (TCPS.520/2003). The results of citric acid and vitamin C contents showed that Bastard oleaster gummy jelly product had the significant lower than raw material (p≤0.05). It is possible that it could be lost by the heating and air sensitivity during the cooking and air drying processes. The developed fruit gummy jelly had a higher water activity than that of the commercial gummy jelly products (p≤0.05). Wills (1990) reported that jelly confectionary products contained a high moisture (18-22%) showed the water activity in a range of 0.65-0.75. Barbosa-Cánovas et al. (2020) reported that the commercial gummies and jellies varied in water content of 11-22% and water activity also widely ranged between 0.51 and 0.79. Additionally, the solid content and total acidity in the commercial gummy jelly products showed higher than of the Bastard oleaster gummy jelly (p≤0.05). It might be from the different composition of each product. The commercial gummy jelly product may be contain a higher sugar compositions such as sucrose and glucose syrup and content of acid addition such as ascorbic acid, citric acid and sugar.
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Table 3: Physico-chemical characteristics of Bastard oleaster juice, Bastard oleaster fruit gummy jelly, and the commercial gummy jelly product.
Physico-chemical characteristics Bastard Bastard oleaster Commercial oleaster juice fruit gummy jelly Gummy jelly pH value 2.97±0.01B 3.59±0.01a,A 2.92±0.01b Total acidity as citric acid (%) 3.62±0.03A 1.49±0.11b,B 2.05±0.08a Total soluble solid (°Brix) 7.73±0.06B 68.30±0.17b,A 74.50±0.06a Vitamin C (mg AA/100 g sample) 13.25±0.05A 6.67±0.36B - Water activity - 0.73±0.01a 0.67±0.01b Note: - = no analysis a-bThe superscript lowercases in the same row are significantly different between Bastard oleaster fruit gummy jelly and the commercial gummy jelly product (p≤0.05) A-BThe superscript uppercases in the same row are significantly different between Bastard oleaster juice and Bastard loeaster fruit gummy jelly product (p≤0.05)
Conclusion
The investigation of development on gummy jelly product from Bastard oleaster fruit based on gelatin addition has been presented. The increased gelatin levels were significantly affected reducing the moisture content, vitamin C, and a* value but increasing the total acidity, solid content, and L* value including springiness. This findings can be concluded that the addition of gelatin in the range of 8-10% in Bastard oleaster gummy jelly product resulted in soft and elastic gummy gel. Its texture is softer than that of the commercial gummy jelly products. It is potential to apply Bastard oleaster fruits in food and drink products. For further experiments, functionality studies of products should be more conducted such as sensory evaluation, biochemical active components with their biological activities.
Acknowledgement
The authors are grateful thank Thailand Institute of Scientific and Technological Research (TISTR) for providing facilities and funding support.
References
AOAC, Association of Analytical Communities. (2000). Official Methods of Analysis (17th ed.). Gaithersberg, MD, USA. Barbosa-Cánovas, G.V., Fontana Jr., A.T., Schmidt, S.J., & Labuza, T.P. (2020). Water activity in foods: Fundamental and applications. Technology & Engineering, Wiley Publicher, 640 p. Burey, P., Bhandari, B.R., Rutgers, R.P.G., Halley, P.J., & Torley, P.J. (2009). Confectionery gels: a review on formulation, rheological and structural aspects. International Journal of Food Properties, 12, 176–210. Delgado, P. & Bañón, S. (2015). Determining the minimum drying time of gummy confections based on their mechanical properties. CyTA – Journal of Food, 13(3), 329-335. Jiamjariyatam, R. (2018). Influence of gelatin and isomaltulose on gummy jelly properties. International Food Research Journal, 25(2), 776-783.
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Marfil, P. H. M., Anhê, A. C. B. M., & Telis, V. R. N. (2012). Texture and microstructure of gelatin/corn starch-based gummy confections. Food Biophysics, 7, 236–243. http://doi. org/10.1007/s11483-012-9262-3 Meesang, S., Wuttijumnong, P., Pongsawatmanit, R., & Chenputhi, S. (2003). Effect of gelatin sucrose/glucose syrup ratio and citric acid on physical properties and sensory quality of gummy jelly product. Kasetsart University, Bangkok, 20-27. (in Thai) Panja, S., Chaudhuri, D., Ghate, N.B., & Minh, H.L. (2014). In vitro assessment of phytochemicals, antioxidant and DNA protective potential of wild edible fruit of Elaeagnus latifolia Linn. Fruits, 69(4), 303-314. Patel, S. (2015). Plant genus Elaeagnus: underutilized lycopene and linoleic acid reserve with permaculture potential. Fruits, 70(4), 191-199. Seal, T. (2012). Evaluation of nutritional potential of wild edible plants traditionally used by the Tribal prople of Meghalaya state in India. American Journal of Plant Nutrition and Fertilization Technology, 2(1), 19-26. Siegwein, A.M. (2010). The effect of soy protein isolate addition on the physic-chemical properties of gummi confections. M.Sc. dissertation in Food Science and Nutrition, The Ohio State University, United State. Thanomwong, C. (2008). Effect of gelatin and citric acid on sensory qualities of lemongrass flavored gummy jelly. Available online http://www.conference.phuket.psu.ac.th/ proceedings/psu_open_week_2008/data/sci/1_31.pdf (in Thai) Tiampakdee, A. (2006). Effect of pH, gelatin, pectin, sugars, and fruits juices on texture of gummy jelly. M.Sc. dissertation in Food Science and Technology, Chiang Mai University, Thailand. TISI, Thai Industrial Standards Institute. (2003). Thai Community Product Standard of dried jelly: TCPS.520/2003. Bangkok, Thailand. Wills, D. (1998). Water activity and its importance in making candy. The Manufacturing Confectioner, 71-74. Yingthongchai, P., & Sirikhum, P. (2008). Genetic diversity of Elaeagnus litolia Linn.: morphological and physico-chemical characters of fruits. Agricultural Science Journal, 39(3(Suppl.)), 118-121. Zainol, M.K., Che-Esa, N.S., Azlin-Hasim, S., Zamri, A.I., Mohd Zin, Z., & Abdul Majid, H.A. (2020). The ramification of Arabic gum and gelatine incorporation on the physicochemical properties of Belimbing Buluh (Averhoa belimbi) fruits pastilles. Food Research, 4(2), 532-538.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.4 International Conference on Food, Agriculture and Biotechnology
EFFECT OF PECTIC OLIGOSACCHARIDES FROM FRUIT PEELS AS PREBIOTIC IN ANIMAL FEED
PORNPAN SAENPHOOM*, PATTARAPORN POOMMARIN, SUPHAVADEE CHIMTONG, WASUNAN NIMANONG, SUDARAT ARTKIDKAN
Faculty of Animal Science and Agricultural Technology, Silpakorn University, Phetchaburi, 76120, Thailand
*Corresponding author: [email protected]
Abstract:
The objective of this study was to improve fruit peels (pomelo peel, lime peel and mango peel) with enzyme (Hemicell®) as prebiotics in animal feed. There were 2 experiments designed in completely randomized design (CRD). In the experiment 1, it consisted of 6 treatments with 3 types of fruit peels (pomelo peel, lime peel and mango peel) and 2 levels of enzymes (0 and 1% (w/w)) with 3 replications. The samples were taken to measure chemical compositions, reducing sugar content and oligosaccharides analysis. The results were showed that chemical compositions were significantly different among treatments (P<0.01). Enzyme treated lime peel had higher ash, crude protein and ether extract content but lower cellulose than other treatments (P<0.01), 4.98, 6.40, 8.87% and 9.92%, respectively. Enzyme treated and untreated mango peel contained higher gross energy than other treatments (P<0.01), 4,100.54 and 4,069.14 kcal/kg, respectively. In addition, reducing sugar content was significantly different among treatments (P<0.05). Enzyme treated mango peel contained higher reducing sugar content than other treatments (P<0.05). There were 42.04, 28.86, 177.06, 77.28, 37.09 and 265.91 mg/g, respectively. Oligosaccharides analysis by Thin layer chromatography method showed that all treatments releasing oligosaccharides. In experiment 2, it was examined prebiotic properties (concentration of sugar 1.5 mg/ml), the results showed that glucose product from all treatments could increase growth of Lactobacillus plantarum but enzyme treated lime peel could decrease growth of Escherichai coli. In conclusion, lime peel treated enzyme can be used as prebiotics as it could increase probiotics and decrease growth of pathogenic bacteria.
Keywords: Fruit peels; Enzyme; Oligosaccharides; Prebiotics
Introduction
Pectin is one of the most complex carbohydrates and mostly found in the plant cell wall. The pectin is broken by enzyme and possible to release pectic oligosaccharides (POS). POS works as prebiotics because its increase the growth of Lactobacilli and Bifidobacteria and decrease the growth of Escherichai coli. Source of POS mainly obtained from the peels of citrus, apple and sugar beet pulp obtained from agricultural by-products (Baldassarre et al., 2018). Thailand is one of the most abundant sources of tropical fruits and mainly export in the world. Therefore, agricultural by-products are available and low price such as pineapple, orange and mango peel. The pectin of citrus peel contains 35% from pomelo peel, 32% from lime peel and 21% from mango peel (Gullon et al., 2013; Huang et al., 2014; Gragasin et al., 2014). Thus, the aim of this study was to improve fruit peels (pomelo peel, lime peel and mango peel) with enzyme (Hemicell®) as prebiotics in animal feed.
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Materials and Methods
Preparation of enzyme treated fruit peel
Fruit peels (pomelo, lime and mango) were obtained from the market at Cha-am city. The peels were cut and dried at 60oC for 1-2 days in hot air oven. After that, the peels were grind to be the size of 1×1 mm. This experiment consisted of 6 treatments and 3 replicates. The fruit peels were treated with commercial enzyme (Hemicell@, endo-1,4 β-mannanase) at 2 levels (0 and 1% w/w) dissolved with phosphate buffer (pH 7). The peels were incubated at 55oC for 6 hours. Then they were centrifuged with speed of 10,000 rpm for 10 mins. The supernatant sample was collected for reducing sugar and oligosaccharides analysis. While, the solid sample was dried in hot air oven at 60oC for 24 hours and stored in a refrigerator for proximate analysis.
Determination of reducing sugar
Approximately 0.5 ml of the supernatant was transferred into sample tube in which 0.5 ml of dinitrosalicylic acid (DNS) reagent was added into each tube. The mixture was boiled for 10 mins and left to be cool by immersing the sample tube into cold water immediately. The absorbance was read at 540 nm with distilled water as blank (Miller, 1959). Different concentrations of glucose (0.15, 0.20, 0.25 and 0.30 mg/ml) were prepared using similar procedure described above to develop a glucose standard curve for the above assay.
Determination of oligosaccharides
Oligosaccharides analysis was performed using thin-layer chromatography (TLC) described by Srinang et al. (2008). The supernatant sample was spotted near the bottom of silica gel plate (Merck & Co., Inc. art. No.1.05554 size 20×20 cm). Then, the TLC plate was placed in a shallow pool of a solvent (2-propanol: ammonium hydroxide: distilled water, 7:1: 2) in a developing chamber so that only the very bottom of the plate was in contact with the liquid. This liquid would act as the mobile phase which would slowly rise up the TLC plate by capillary action. After thoroughly drying the TLC plate with 10% (v/v) sulfuric acid in ethanol solution in an operating hood, and heated at 100oC until dry. The spot on TLC plate was compared with pectin.
Determination of chemical compositions
Dried solid samples from centrifuge were analyzed for dry matter (DM), crude protein (CP), ash, crude fiber (CF), ether extract (EE) (AOAC, 1990), neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) (Goering and Van Soest, 1970). Gross energy was determined using bomb calorimeter (CAL 2K, South Africa).
Prebiotic properties
The glucose products of treated fruit peel were used to evaluate its ability to support growth of beneficial microbes (Lactobacillus plantarum) and pathogenic microbes (Escherichia coli). The concentration of glucose used was 0.5, 1.0, 1.5 and 2.0 mg/ml respectively. The L. plantarum and E. coli were inoculated into nutrient broth and incubated at 37oC. The growth of L. plantarum was measured at 0, 6, 12, 18, 25 and 48 hrs while the growth of E. coli was
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology measured at 0, 3, 6, 9, 12, 15 and 18 hrs by spectrophotometer and reading absorbance at 600 nm.
Statistical analysis
The data were analyzed the variance procedures by using SAS software (SAS, 1998). A significance level of P < 0.05 was used to differentiate between means.
Results and Discussion
Chemical compositions
Chemical compositions of untreated and enzyme treated fruit peels are shown Table 1. The results showed that chemical compositions were significantly different among treatments (P<0.01). Enzyme treated lime peel had higher ash, crude protein and ether extract content but lower cellulose than other treatments (P<0.01). They were 4.98, 6.40, 8.87% and 9.92%, respectively. Enzyme treated and untreated mango peel consisted of higher gross energy than other treatments (P<0.01). The gross energy value was 4,100.54 and 4,069.14 kcal/kg, respectively. However, enzyme treated fruit peels increased CP and decreased CF content indicating that the enzyme (Hemicell@) is crude enzyme and effective in breaking down fiber in fruit peels. The above results were in agreement with Khanongnuch et al. (2006) who reported that enzyme treated copra meal reduced crude fiber up to 14%. Similarly, previous studies found that enzyme treated (Hemicell@) reduced hemicellulose in taro leaves and cellulose in coconut meal in comparison to untreated enzyme. They were 14.55 vs 15.15% and 26.33 vs 39.57% respectively (Saenphoom et al., 2016; Saenphoom et al., 2020).
Reducing sugar
Reducing sugars content of untreated and enzyme treated fruit peels are shown in Table 1. The results showed that enzyme treated mango peel had higher reducing sugar content than other treatment (P<0.05). They were 42.04, 28.86, 177.06, 77.28, 37.09 and 265.91 mg/g, respectively because mango peel had higher soluble dietary fiber (19.45%) than pomelo peel (6.43%) and lime peel (19.20%) (Figuerola et al., 2005; Arumugam & Manikandan, 2011). In addition, enzyme treated fruit peels had higher reducing sugar content than untreated (Figure. 2) due to the activity of enzyme. The enzyme can break down fiber into monosaccharide sugars. Similarly, Saenphoom et al. (2020) found that enzyme treated (Hemicell@) can reduce the increase of reducing sugar in coconut meal comparing to untreated enzyme. They were 0.71 vs 3.79 mg/g, respectively.
Oligosaccharides
Oligosaccharides of untreated and enzyme treated fruit peels were shown in Figure 1. Glucose products was found in both untreated and treated enzyme fruit peels (row 2 to 7) and lighter than pectin molecule. The glucose products might be as galacturonic acid and pectic oligosaccharides (POS). Similarly, Saenphoom & Chimtong (2014) reported that oligosaccharides content was not significantly different between untreated and treated enzyme tea leaves.
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Table 1: Chemical compositions and reducing sugar of untreated and enzyme treated fruit peels (% on dry matter basis).
Treatments Items SEM T1 T2 T3 T4 T5 T6 Dry matter (%) 94.99c 95.79b 97.11a 94.29d 95.69b 97.04a 0.22 Moisture (%) 5.01b 4.21c 2.89d 5.71a 4.31c 2.96d 0.02 Ash (%) 3.56d 4.85b 2.75f 4.31c 4.98a 3.29e 0.01 Crude protein (%) 3.83e 5.93b 5.26c 4.69d 6.40a 5.45c 0.03 Ether extract (%) 6.67b 6.65b 8.71a 8.10a 8.87a 8.59a 1.87 NDF (%) 67.74a 37.54f 40.40e 60.18b 44.70c 42.27d 0.13 ADF (%) 39.30a 25.14c 25.98c 37.70a 26.83b 26.44c 0.55 ADL (%) 29.61a 3.62f 8.31d 27.07b 16.05c 6.17e 0.07 Hemicellulose (%) 29.06a 12.85f 14.63e 21.98b 18.75c 16.87d 0.03 Cellulose (%) 9.76c 21.52a 17.67b 10.63c 10.78c 20.27a 0.16 Gross energy 3,680.72b 3,626.10b 4,069.14a 3,729.20b 3,518.01b 4,100.54a 33.05 (kcal/kg) Reducing sugar 42.04d 28.86f 177.06b 77.28c 37.09e 265.91a 0.38 (mg/g) a,b,c,d,e,f Means with different superscripts in row are significantly different )P<0.01(, T1= Untreated pomelo peel, T2= Untreated lime peel, T3= Untreated mango peel, T4= Enzyme treated pomelo peel, T5= Enzyme treated lime peel, T6= Enzyme treated mango peel, NDF =Neutral detergent fiber, ADF =Acid detergent fiber, ADL =Acid detergent lignin, SEM= Standard error of mean
Pectin 1 2 3 4 5 6 7
Figure 1: Oligosaccharides of untreated and enzyme treated fruit peels Row 1 = Pectin, Row 2 = Untreated pomelo peel, Row 3= Untreated lime peel, Row 4 = Untreated mango peel, Row 5 = Enzyme treated pomelo peel, Row 6= Enzyme treated lime peel, Row 7 = Enzyme treated mango peel.
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Prebiotics properties
The glucose products from all treatments increased growth of L. plantarum. However, enzyme treated lime peel decreased growth of E. coli at the concentration of 1.5 mg/ml (Figure 2). Li et al. (2013) reported that POS of lime peel decreased growth of E. coli. at prolong, stationary and lag phases.
Figure 2: Microbial growth of untreated and enzyme treated fruit peels.
Conclusion
Enzyme hydrolysis effectively breaks down cell wall of fruit peels and releases reducing sugar and oligosaccharides. Moreover, glucose product from all treatments increase growth of L. plantarum but enzyme treated lime peel decrease growth of E.coli. In conclusion, lime peel treated enzyme can be used as prebiotics because it could increase probiotics and decrease growth of pathogenic bacteria.
References
AOAC. (1990). Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists, Arlington, VA Arumugam, R., & Manikandan, M. (2011). Fermentation of pretreated hydrolyzates of banana and mango fruit wastes for ethanol production. Asian Journal of Experimental Biological Sciences, 2(2), 246-256. Baldassarre, S., Neha, B., Sandra, V.R., Winnie, D., Miranda, M., Stefano, S & Kathy, E., (2018). Continuus production of pectic oligosaccharides from onion skin with an enzyme member reactor. Food Chemistry, 267, 101-110. Figuerola, F., Hurtado, M.L., Estévez, A.M., Chiffelle, I., & Asenjo, F. (2005). Fibre concentrates from apple pomace and citrus peel as potential fibre sources for food enrichment. Food Chemistry, 91, 395-401. Goering and Van Soest. (1970). Determination of neutral detergent fiber, hemicellulose, cellulose, and lignin in breads. Bureau of Nutritional Science, Canada, 56(5), 437-441. Gragasin, M. C. B., Ligisan, A. R., Torres R. C., & Estrella. R. (2014). Utilization of mango peels as source of pectin. Philippine enter for Postharvest Development and Mechanizaton, 4(1), 2243-8483.
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Gullón, B., Gómez, B., Martínez-Sabajanes, M., Yáñez, R., Parajó, J.C., & Alonso. J.L. (2013). Pectic oligosaccharides: Manufacture and functional properties. Trends in food science & technology, 30(2), 153-161. Huang, R., Cao, M., Guo, H., Qi, W., Su, R. & He, Z. (2014). Enhanced ethanol production from pomelo peel waste by integrated hydrothermal treatment multienzyme formulation and fed-batch operation. Journal of agricultural and food chemistry, 62(20), 4643-4651. Khanongnuch, C., Sa-nguansook, C., & Lumyoung, S. (2006). Nutritive quality of β- mannanase treated copra meal in broiler diets and effectiveness on some fecal bacteria. International Journal of Poultry Science,5(11), 1087-1091. Li, S., Li, T., Zhu, R., Wang, N., Song, Y., Wang, S., & Guo, M. (2013). Antibacterial action of pectic oligosaccharides. International Journal of Food Properties,16(3), 706-712. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426-428. Saenphoom, P., & Chimtong, S. (2014). Effect of enzyme treated copra meal on nutritive value, reducing sugar and oligosaccharides as prebiotics. The Standard International Journal, 18-21. Saenphoom, P., Chimtong, S. Poomparin, P., Nimanong, W., Somsub, P., & Kheawkasem, P. (2020). Improvement of coconut meal and sugar palm peel using enzyme as prebiotics in animal feed. Khon Khaen Agricuture Journal, 48(1), 621-628. Saenphoom, P., Chimtong, S., Phiphatkitphaisan, S., & Somsri, S. (2016). Improvement of taro leaves using pre-treated enzyme as prebiotics in animal feed. Agriculture and Agricultural Science Procedia,11, 65-70. SAS. (1998). User’s Guide: Statistics, Version 9.2 Edition, SAS Inst, Cary, N.C. Srinang, T., Tachaapaikoon, C., Kyu, K.L & Ratanakhanokchai, K. (2008). Fermented products of Bacillus sp. Strain TW-1 grown on corn hull medium under limited oxygen condition. KMUTT Research and Development Journal, 31(2), 291-304.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.6 International Conference on Food, Agriculture and Biotechnology
FACTORS AFFECTING THE IMPLEMENTATION OF GAP AMONG BANANA (GROS MICHEL) GROWERS IN BAN LAT DISTRICT, PHETCHABURI PROVINCE, THAILAND
ALMERICE ENOLD, MANA KANJANAMANEESATHIAN, ALISA K. YOSHIDA*
Faculty of Animal Science and Agricultural Technology, Silpakorn University Phetchaburi IT Campus, Cha-Am, Phetchaburi 76120, Thailand
*Corresponding author: [email protected]
Abstract:
Banana (Gros Michel) or Kluai Hom Thong (Musa acuminata, AAA Group) is an economic crop in Thailand and it is an important product to export under the standard of good agricultural practice (GAP). This study aims to investigate factors affecting the GAP implementation among banana (Gros Michel) growers who adopt GAP for practice in Ban lat district, Phetchaburi province. This study revealed that most of farmers encountered water scarcity but they realized the importance of recording the data during the GAP practice. Gender, number of family members and farming organization membership were the factors highly impacting the implementation of GAP among these banana growers. The results from this study should direct the staffs in the relevant organizations to focus on these three key issues to improve growing banana with GAP certification.
Keywords: Gros Michel banana; Good agricultural practice; Ban Lat; Phetchaburi
Introduction
Gros Michel banana or Kluai Hom Thong (Musa acuminata, AAA Group) is an economic crop in Thailand. It is widely grown in several provinces including Saraburi, Pathum Thani, Phetchaburi and Chumphon. In Phetchaburi, Ban Lat district is famous for banana (Gros Michel). The banana from this district has been exported to Japan, Hong Kong and Singapore under management of Ban Lat Agricultural Cooperative since 1999. At present, about 12 tons of banana (Gros Michel) have been exported to Japan every week, with the prospect of the increased demand in the future. To prepare for this expectation, the Cooperative and the banana growers have to collaborate to produce banana (Gros Michel) based on GAP standard. This standard is globally accepted as the minimum requirement which guarantees that the produces are certified for export (Amekawa, 2010). However, Thai banana (Gros Michel) growers are still having problem implementing GAP due to issues such as lack of technical knowledge and experience in practicing GAP. GAP adoption is the issue which have been studied in various crops and locations in Thailand. The studies indicated that factors influencing the implementation of GAP in each region are diverse. For example, Fakkhong & Suwanmaneepong (2017) reported that level of education, land ownership and membership to the farming organizations significantly influenced GAP implementation for rice production in the eastern part of Bangkok. Suwanmaneepong et al. (2016) found that farming experience and participating in GAP training positively related to the implementation of GAP by fruit growers in Rayong province. At Chumphon province, Pongvinyoo et al. (2014) found that self-confidence of the growers positively affected GAP practice, while farming experiences had negative impact to the farmers’ understanding of GAP among coffee growers. The diversity of crops, locations and
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology growers requires that to promote GAP successfully, it is very important to understand the socio- economic characteristics of the growers in each specific area. Thai grower, like growers in other developing countries, are recalcitrant to change and this situation is a challenge for agricultural officers to promote GAP practice. On top of the behavioral issue, knowledge gap hindered the growers to implement GAP in the past as well (Amekawa, 2010). The purpose of this study aims to study the factors affecting the GAP implementation among banana (Gros Michel) growers which have never been investigated before.
Materials and Methods
The study area and sample size
The study was carried out in Ban Lat district, Phetchaburi province, located in the western region of Thailand. Ban Lat district covers 29831.36 hectare, in which 16328.32 hectare is an area use for agricultural production (http://banlat.phetchaburi.doae.go.th/, 2019). Within this area, 1287.84 hectare have been used to grow banana (Gros Michel). There are 69 banana growers, who were registered and practiced GAP under DOAE supervision in the years 2019-2020. They were subjected to the questionnaire and this study was conducted from January to March 2020 using semi-structured questionnaires.
Data analysis
The primary data was collected to identify the main variables that influenced the implementation of GAP. Descriptive statistics, including frequency distribution, percentages, means, standard deviations, multiple regressions and correlation coefficients, were used for statistical analysis. Some characteristics of farmers were selected as independent variables, while dependent variable of GAP implementation was a practice score that was the cumulative total of GAP practices applied in producing banana (Gros Michel). The levels of GAP practice are as follow: 0.00-1.49 None 1.50-2.50 Low 2.51-3.50 Moderate 3.51-4.00 High
Results and Discussion
Characteristics of banana (Gros Michel) growers in the study area
The socio-economics characteristics of the respondents are shown (Table 1). The respondents were almost equal between male (57.8%) and female (42.2%). Most (91%) of their ages were 41 years to over 60 years old. This is consistent with a report of DOAE (2017) that indicated that the age of the head of households belonged to the old age group. Forty seven percent of the respondents graduated from primary school or lower, which the remaining received education at the higher levels.
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Table 1: Socio-economic characteristics of respondents (n=69).
Attributes Characteristics Frequency Percentage Gender Male 27 42.2 Female 37 57.8 Age of farmer 20-30 years 0 0 31-40 years 6 9 41-50 years 23 34.3 51-60 years 29 43.3 > 60 years 9 13.4 Education level Lower than primary school 2 2.9 Primary school 30 44.1 Junior secondary school 6 8.8 Senior secondary school 20 29.4 Bachelor’s degree 6 8.8 Master’s degree 4 5.9 Doctoral degree 0 0 The number of family members 1-3 persons 34 53.1
4-6 persons 24 37.5 > 6 persons 6 9.4 The number of family laborers 1 person 8 14.8 2 persons 25 46.3 3 persons 12 22.2 > 3 persons 9 16.7 Farming experience < 10 years 12 20.7 10-20 years 20 34.5 > 20 years 26 44.8 Membership of farming Yes 37 66.1 organization No 19 33.9 Cultivated area < 1.6 hectare 36 54.5 1.6-3.2 hectare 22 33.3 > 3.2 hectare 8 12.1 Land ownership status Owner 30 56.6 Rent 23 43.4 Financial support Government project 3 4.7 Bank 7 10.9 Own funds 54 84.4 Income/year <100,000 Baht 34 52.3 100,001-200,000 Baht 22 33.8 200,001-300,000 Baht 8 12.3 300,001-400,000 Baht 1 1.5 400,001-500,000 Baht 0 0 >500,000 Baht 0 0 GAP training/year At least 1 time 16 23.5 (>2 times) 37 54.4 Never 15 22.1
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The family was characterized as small-sized (1-3 persons) (53.1%) with about two persons (46.3%) involved in farming activities. About 44.8% had farming experience of more than 20 years, which invariably positively impacted agricultural productivity (Anigbogu et al., 2015). The long experience in farming by these farmers might influence and strengthen their perceptions about certain farming practices such as applications of fertilizers and pest control measures (Farouque, 2007). About 66.1% were members of farming organizations. This helps them to manage their farm with modern technologies and integrated financial services. Most respondents owned less than 1.6 hectare (54.5%). Growers who had land ownership (56.6%) were more than those who rented the land (43.4%). Most (84.4%) of the respondents used their own funds to manage their farm and about 52.3% had income less than 100,000 Baht per year. Most respondents (77.9%) indicated that they attended GAP training programs organized by DOAE at least once a year.
GAP perception among Gros Michel banana farmers
The banana (Gros Michel) growers primarily received GAP information from the agricultural extension officers (59.4%) and made their decision to practice GAP in growing banana. This makes them qualify to receive GAP certificate. The remaining respondents included growers who received information about GAP from TV (11.6%), social media (8.7%), friends (8.7%), radio (5.8%), newspaper (2.9%) and family member (2.9%). The market, which is sensitive to quality and the customers who prepare to pay at the higher prices for environment and health, should advocate the adoption of GAP. Berdegué et al. (2003) reported that the growers were motivated to adopt GAP when the increased prices were expected from the exported product. This is consistent to our study as the approximately 28% of the growers adopted the GAP in producing banana based on customer preference, product price (25.4%), concern about health (19.4%) and care of the environment (14.9%). Hobbs (2003) also stated that the farmers should understand more about agricultural standard, they will possibly improve farming practices to gain access to a market which can offer high price. Hobbs (2003) work corresponded with our study that about 51.4% of the farmers tended to replace conventional farming with GAP when under market offered high price, about 24.2% were responsive to high demands, about 22.7% were sensitive to high prices, and about 4.5% were responsive to access to multiple sale channels. The hindrance for the growers to adopt GAP included water source and availability (32.1%), usage of chemical substances (30.4%), farming practices before harvest (12.5%), data collection (8.9%), farm location (7.1%), practices during harvest and post-harvest (5.4%), personal hygiene (3.6%), and transportation was not their constraint. Source and availability of water for irrigation was the major constraint because the growers in Phetchaburi accessed to irrigation canals in which the water may be contaminated with hazardous or prohibited substances. Growers were comfortable with delivering the banana because the Ban Lat Agricultural Cooperatives provided pick-up service at their farms. This service is one of the reasons that most of the growers (55.3%) have sold their banana to Ban Lat Agricultural Cooperative (Table 2).
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Table 2: GAP Gros Michel banana grower’s opinions.
GAP Gros Michel banana grower’ s opinions Frequency Percentage Where do you get the information about GAP? TV 8 11.6 Radio 4 5.8 Social Media 6 8.7 Newspaper 2 2.9 Friend 6 8.7 Agricultural officer 41 59.4 Family member 2 2.9 Why do you prefer to use the GAP system? Customer preference 19 28.4 Community agreement 5 7.5 Concern about health 13 19.4 Product price 17 25.4 Care to environment 10 14.9 Other 3 4.5
High price 15 22.7 No effect to the environment 16 24.2 High demand 16 24.2 No or less deleterious effect to consumer’s health 9 13.6 Good production 7 10.6 Other 3 4.5 What are the major constraints for you in the GAP regulation? Water source 18 32.1
Agricultural chemical 17 30.4 Harvest and post-harvest 3 5.4 Personal hygiene 2 3.6 Farm location 4 7.1 Production management before harvest 7 12.5 GAP Gros Michel banana grower’ s opinions Frequency Percentage Data collection 5 8.9 Where do you sell the product? Local market 17 36.2
Supplier 26 55.3 Own shop 4 8.5
GAP Implementation level of Gros Michel banana farmers
Most growers in the study area implemented GAP on their farms at a moderate level (Table 3). Most (54.5%) of the respondents were growers who owned a small farm (cultivated area <1.6 hectare). Some growers had found it very difficult to comply with GAP rules and standards, which contributed to the low proportion of farmers practicing GAP. In addition, about 54.4% of the respondents had participated in GAP training only twice and this might have impacted
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology on their capability to implement GAP. The growers had the lowest GAP issue (2.81%) regarding water sources although they had difficulty to access to clean water for irrigation. This is because they could prepare their own water reservoir in the farm, the operation that increases the cost of production. On the other hand, the farmers highly understood about the significance of recording the farming practices (3.16%). This is because the local GAP extension officers facilitate this step by providing the forms for the growers in the area. The officers also visited the growers once a month, an action that establishes the understanding between these two stakeholders. In contrast, coffee growers in Chumphon province had the least understanding about the significance of recording data of the farm practices in GAP (Pongvinyoo et al., 2014).
Factors influencing the implementation of GAP among farmers in the study area
Multiple regression was employed to investigate factors influencing the implementation of GAP practices among growers. The results revealed an F-ratio of 43.831 which was not significant. However, an R-squared value of 0.998 indicated that the eleven variables explained 99.8% of the implementation of GAP by growers. These variables, including gender, age, education level, the number of family members, the number of family laborers, farming experience, membership of a farmer organization, cultivated area, land ownership status, GAP training and financial support, were not statistically significant to the implementation of GAP. However, the result of Pearson correlation coefficient showed that gender, the number of family labour and the farming organization membership were highly and positively significant to GAP implementation (Table 4). As banana (Gros Michel) is the horticultural product that requires carefully handling, human labour is very important. The extra labour from the other family members should play an important role in the activities that requires labour for GAP implementation.
Table 3: GAP implementation level of the Gros Michel banana farmers.
GAP GAP practical level (%) Average Practical Implementation practical S. D level items None Low Moderate High score Water source 19.1 10.6 40.4 29.8 2.81 0.16 Moderate Cultivation site 24.4 - 44.4 31.1 2.82 0.17 Moderate Use of hazardous agricultural 22.7 4.5 31.8 40.9 2.91 0.18 Moderate substances Product storage and 22.0 2.4 43.9 31.7 2.85 0.17 Moderate on-site transportation Disease and pest-free 7.1 9.5 45.2 38.1 3.14 0.13 Moderate production Management of 7.0 11.6 46.5 34.9 3.09 0.13 Moderate quality production Harvesting and post- 9.5 2.4 54.8 33.3 3.12 0.13 Moderate harvest handling Data recording 7.0 9.3 44.2 39.5 3.16 0.13 Moderate Overall 2.80 1.08 Moderate
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In addition, grower’s membership to the agricultural organizations significantly influenced to growers implementing GAP. Most of banana (Gros Michel) growers were the member of the Ban Lat Agricultural Cooperative. This facilitates them to access to an agricultural extension officers who can provide knowledge about banana and GAP practices. Khaengkhan & Khumsoonthon (2016) suggested that forming grower groups could contribute to the growers to get higher standards. Fakkhong & Suwanmaneepong (2017) also reported that membership to the farming organizations impact significantly and positively to the growers in implementing GAP. Sriwichailamphan et al. (2008) revealed that age, farm size, and contract situation (market assessment) influenced the pineapple growers to understand GAP. Mankeb et al. (2013) also showed that the grower’s understanding to GAP was influenced by age, farming experience and education. Salakpetch (2007) indicated that level of farmer’s education and GAP extension services were the important factors to improve the grower’s GAP understanding. Pongvinyoo et al. (2014) reported that farming experience had negative impact and cultivated area had the positive impact on the perception of GAP understanding among coffee growers in Chumphon. Ganpat et al. (2014) indicated that the level of compliance with GAP was directly related to farming experience. Suwanmaneepong et al. (2016) demonstrated that farming experience had a positive relationship to GAP implementation by fruit farmers in Rayong province, Thailand These past studies indicated that to promote GAP it is very important to understand the socio-economic characteristics of the farmers in each specific area. Thai farmers’ adherence to conventional farming methods was the challenge for extension institutions in promoting the standard GAP procedure. Table 4: Multiple regression and Pearson correlation coefficient results.
Pearson Multiple Regressiona Sig Correlation B SE Beta t Sig (Constant) -5.969 1.147 -5.206 0.121 Gender -0.806 0.156 -0.408 -5.154 0.122 -0.494* 0.043 Age 1.167 0.250 0.647 4.662 0.135 0.300 0.159 Education 0.193 0.109 0.254 1.773 0.327 0.126 0.341 The number of family member 1.060 0.334 0.398 3.175 0.194 0.222 0.233 The number of family labor 0.092 0.089 0.095 1.039 0.488 0.646** 0.009 Farming experience -0.462 0.145 -0.384 -3.177 0.194 0.200 0.256 Belong to farmer organization 1.491 0.192 0.654 7.775 0.081 0.570* 0.021 Cultivate area 0.194 0.151 0.151 1.284 0.421 0.322 0.142 Land owner 0.370 0.246 0.187 1.504 0.374 -0.165 0.296 GAP training 0.637 0.140 0.354 4.541 0.138 0.300 0.159 Financial support 0.437 0.070 0.380 6.211 0.102 0.000 0.500 F ratio 43.831 R squared 0.998 Adjusted R squared 0.975 * Correlation is significant at 0.05 level, ** Correlation is significant at 0.01 level.
Conclusion
Good Agricultural Practice (GAP) for banana (Gros Michel) has facilitated the growers to export the product overseas. This can be achieved by the active collaboration between the
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology growers and the agricultural extension officers. GAP implementation will bring about the access to market which the customers who prepare to pay high price for quality. This study revealed that most of growers had difficulty to access to clean water for irrigation. They coped with this problem by having water reservoir in their own farm. The growers did practice data recording in the farming practices as required by GAP. The factors influencing implementation of GAP among banana (Gros Michel) growers in Ban Lat district, Phetchaburi province were gender, number of family members and farming organization membership. This study provided the information for the agricultural extensionists to focus their effort to the relevant target growers to improve banana farming practices in accordance with the GAP.
Acknowledgement
The first author would like to express his heartfelt gratitude to the Thailand International Cooperation Agency (TICA), Ministry of Foreign Affairs of the Kingdom of Thailand, for providing him a scholarship to study for the Master of Science Degree in Bioscience for Sustainable Agriculture at the Faculty of Animal Science and Agricultural Technology, Silpakorn University, Phetchaburi IT Campus, Cha-Am, Phetchaburi, Thailand
References
Amekawa, Y. (2010). Rethinking Sustainable Agriculture in Thailand: a Governance Perspective. Journal of Sustainable Agriculture, 34(4), 389-416. Anigbogu, T. U., Agbasi, O. E., & Okoli, I. M. (2015). Socioeconomic Factors Influencing Agricultural Production among Cooperative Farmers in Anambra State, Nigeria. Berdegué, J.A., Balsevich, F., Flores, L., & Reardon, T. (2003). The Rise of Supermarkets in Central America: Implications for Private Standards for Quality and Safety of Fresh Fruits and Vegetables. The USAID-RAISE/SPS project, Michigan State University, East Lansing, Michigan. DOAE. (2017). Farmers registration database. Center of Information Technology and Communication, Department of Agricultural Extension, 31 pages. Fakkhong, S., & Suwanmaneepong, S. (2017). The implementation of Good Agricultural Practice among Rice Farmers in Eastern Region of Bangkok, Thailand. International Journal of Agricultural Technology, 13(7.3), 2509-2522. Farouque, M. (2007). Farmers' Perception of Integrated Soil Fertility and Nutrient Management for Sustainable Crop Production: A Study of Rural Areas in Bangladesh. Journal of Agricultural Education, 48(3), 111-122. Ganpat, W., Badrie, N., Walter, S., Roberts, L., Nandlal, J., & Smith, N. (2014). Compliance with Good Agricultural Practices (GAPs) by State-registered and Non-registered Vegetable Farmers in Trinidad, West Indies. Food Security, 6, 61-69. Hobbs, J.E. (2003). Incentive for the Adoption of Good Agricultural Practices, Background Paper for the FAO Expert Consultation on a Good Agricultural Practices Approach. Rome, Italy, 10-12 November, 2003. Khaengkhan, P., & Khumsoonthon, J. (2016). Learning and Acceptance using GAP Rice Production. Journal of Science and Technology Mahasarakham University, 35(1), 133- 140. Mankeb, P., Limunggura, T., In-go, A., & Chulilung, P. (2013). Adoption of Good Agricultural Practices by Durian Farmers in Koh Samui District, Surat Thani Province, Thaila nd eastern and the southern parts of Thailand. Society for Social Management System (SSMS) 2013 conference, 2-4 December 2013, Sydney, Australia.
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Pongvinyoo, P., Yamao, M., & Hosono, K. (2014). Factors Affecting the Implementation of Good Agricultural Practices (GAP) among Coffee Farmers in Chumphon Province, Thailand. American Journal of Rural Development, 2(2), 34-39. Salakpetch, S. (2007). Quality Management System: Good Agricultural Practices (GAP) for On-farm Production in Thailand. Food and Fertilizer Technology Center, 91-98. Sriwichailamphan, T., Sriboonchitta, S., Wiboonpongse, A., & Chaovanapoonphol, Y. (2008). Factors Affecting Good Agricultural Practice in Pineapple Farming in Thailand. Acta Horticulturae, 794, 325-334. Suwanmaneepong, S., Kullachai, P., & Fakkhong, S. (2016). An Investigation of Factors Influencing the Implementation of GAP among Fruit Farmers in Rayong Province, Thailand. International Journal of Agricultural Technology, 12(7.2), 1745-1757.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.7 International Conference on Food, Agriculture and Biotechnology
IN VITRO CYTOTOXICITY OF CORDYCEPS MILITARIS EXTRACTS ON DIFFERENT HUMAN CANCER CELL LINES
KULLAWAT JITSUDA1, CHOTTANOM PHEERAYA2, BUTKHUP LUCHAI1*
1Natural Antioxidant Innovation Research Unit (NAIRU), Department of Biotechnology, Faculty of Technology, Mahasarakham University, Maha Sarakham 44150, Thailand. 2Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University, Maha Sarakham 44150, Thailand.
*Corresponding author: [email protected]
Abstract:
This study was conducted to evaluate the in vitro cytotoxicity of C. militaris extracts against cancer cells. The cytotoxic activity was tested using Resazurin reduction microplate assay (REMA). The in vitro assay for the C. militaris extracts was measured using three human cancer cells: Human hepatocellular carcinoma (HepG2), Human breast adenocarcinoma (MCF-7), and Cervical adenocarcinoma (HeLa). The REMA assay indicated that Ethyl alcohol (EtOH) and Ethyl acetate (EtOAc) extract of C. militaris exhibited significant cytotoxicity on MCF-7, HepG-2 and HeLa cancer cells in the dose-dependent manner. C. militaris extracted with EtOAc exhibited stronger cytotoxicity on MCF-7 than HepG2 and HeLa. The results obtained indicated that the probable use of the C. militaris extract in preparing recipes for cancer-related ailments. Further studies will identify bioactive compounds and elucidate more detailed molecular mechanisms of cell death.
Keywords: Codyceps militaris; Cordycepin; Adenosine; Cytotoxicity
Introduction
C. militaris is a medicinal mushroom that has been widely used in Asia for the treatment of various diseases. This fungus is an herbal drug known for its use as a source of therapeutic bioactive compounds (Wasser & Weis, 1999; Smith et al., 2002). It has been widely used as a folk tonic food in Asia extensively (Ying et al., 1987). Cordyceps is relatively considered to be a non-toxic medicinal mushroom, besides a few negative published data (Shrestha et al., 1987). The main bioactive components that possess medicinal property include cordycepin (3'- deoxyadenosine) and adenosine (Cunningham et al., 1950). These compounds have pharmacological actions, including anti-cancer (De Silva et al., 2012), anti-tumor, antioxidant (Ramesh et al., 2012), anti-hyperlipidemia (Guo et al., 2010) and anti-fungus (Kim et al., 2003). However, in C. militaris more nucleosides than 10 types which are related to the function of the central nervous system (Das et al., 2010). Apart from being used in usual surgical methods, radiotherapy, and chemotherapy, herbal medicine can also be used as the primary complementary and alternative medicines for treating various cancers, including colorectal cancer (Zhai et al., 2013; Ling et al., 2014; Lee et al., 2015). Cordycepin is one of the cytotoxic analogs of nucleoside, first tested as a chemotherapy agent (Jeong et al., 2011; Tuli et al., 2013; Yoon et al., 2018). In the present study, we investigated the cytotoxic effect of C. militaris extract with EtOH and EtOAc on Human breast adenocarcinoma MCF-7, Human hepatocellular carcinoma HepG2 and Cervix adenocarcinoma HeLa cells death.
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Materials and Methods
Chemicals and materials
Resazurin sodium salt was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethyl alcohol (EtOH) (AR grade) and Ethyl acetate (EtOAc) (AR grade) were purchased from BDH (Poole, UK). Dulbecco's minimum essential medium, Fetal bovine serum (FBS), 100X Penicillin/Streptomycin solution, 0.25 % Trypsin-EDTA and the other cell culture reagents were purchased from GIBCO®, Invitrogen Corporation (Carlsbad, CA, USA).
Culture condition of C. militaris
The healthy fruiting body of C. militaris was obtained from the Lungyood farm Saraburi Province, Thailand was the culture at 20˚C in dark for 7 days on potato dextrose agar (PDA). Mycelium was cultured continuously in liquid medium at 20˚C for 21 days, so as to obtain mycelium pellets. The basic medium for the fruiting body of C. militaris was used by modification from Lungyood Chaemprasert farm and sterilized by autoclaving at 121 °C for 30 min. The seed starter culture with 5 mL into bottle culture and incubated in the dark at 22˚C for mycelium stage, after 14 days, controlled with a 14 h light/10 h dark cycle at 18 °C for stimulation stage. Fruiting body stage controlled with a 12 h light cycle at 22 °C. Sixty-day- old were harvested and dried at 50 °C for the next analysis
Preparation of the extract
The powder of C. militaris were extracted using 1000 mL of solvent (EtOH and EtOAc) in a flask, incubated at 37°C for 48 h using at 200 rpm. After that, separating the clear parts using a centrifuge at 10,000 rpm for 30 min and re-extraction. Collect all clear supernatant and a centrifuge at 10,000 rpm for 30 min. Next step, filtered using Filter Paper, Whatman filter No. 4 and then evaporated the solvent by evaporator at 42 °C. Freeze-dried and store the crude extract at -20 °C for experimentation.
Human Cancer Cell Lines and Culture
Human breast adenocarcinoma MCF-7 (ATCC® HTB-22™), Human hepatocellular carcinoma HepG2 (ATCC® HB-8065™) and Cervical adenocarcinoma HeLa (ATCC® CCL- 2™) cell line was obtained from the American Type Culture Collection (ATCC, Manassasa, VA, USA). Cancer cells were maintained in DMEM medium contemning with 4 mM L- glutamine, 10% FBS and 1% Penicillin/Streptomycin solution incubated at 37˚C in a humidified incubator with 5% CO2 atmosphere and the new medium culture was replaced every 3 days. Trypsinization with 0.25% trypsin‑EDTA and the fresh DMEM was renewed every 3 day.
Cell viability
Cell viability for cancer cells was detected using REMA is carried out according to the Kuete et al. (2013). Cancer cells (5×103 cells/well) were added to 96-well plates for 24 h at 37˚C in an incubator. The medium was discarded, and cancer cells were exposed to a crude extract of different concentrations (final concentration 0-500 µg/mL) dissolved in the DMEM media at 37˚C for 24 h. Resazurin reagent final concentration 0.001% w/v added to replace media and incubated for 1 h. Product of resazurin reduction measured at excitation wavelength of 530 nm
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology and an emission wavelength of 590 nm by a microplate reader (Synergy 4HT Microplate Reader). Each bar represents the mean ± SEM from three experiments. The viability was evaluated based when compared to the control that not treated with the crude extract. Maximal cancer cell killing effect (Emax) and half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) according to the National Cancer Institute guideline (Boyd, 1997).
Statistical analysis
Repeat all 3 tests to find mean and standard deviation using One-way ANOVA and Duncan's Multiple Range Test with SPSS version 19.0 (IBM, Armonk, NY, US), considering the differences. Significantly when p <0.05
Results and Discussion
Effect of Cordyceps militaris crude extract on the survival of cancer cells
The fruiting body of C. militaris was grown for 60 daysharvested and dried at 50 ˚C in an incubator readilyfor extraction with different solvents. Evaporated and freeze-dried crude extract, followed dissolved crude extract at 10 mg/mL in the DMEM media to determine the C. militaris crude extract on the survival of MCF-7, HepG2 and HeLa cancer cells using Resazurin reduction assay. The results clearly demonstrated that C. militaris extract from EtOH and EtOAc showed the highest inhibitory effect on the survival of cancer cells effectively after treated with differenced solvent extract of C. militaris in a dose-dependent manner, except the extract with EtOH on HeLa cell, shown as the Emax and IC50. The C. militaris extract with EtOAc show the highest inhibited the survival effect on MCF-7 after incubate for 48 h (Figure 1A-B) (IC50 = 33.64±0.78 µg/mL) (Table 1) followed extract with EtOAc for 24 h (IC50 = 112.95±6.01 µg/mL) and extract with EtOH incubate for 48 h (IC50 = 112.35±3.04 µg/mL), respectively. In liver HepG2 (Figure 1C-D) and cervical HeLa cancer cell (Figure 1E-F), found that C. militaris form differenced extract affect the cell viability that significantly in a dose- dependent manner. C. militaris extract with EtOAc show the highest on Cytotoxicity HepG2 (IC50 = 63.99±13.01 µg/mL) (Table 2) and HeLa (IC50 = 92.24±0.58 µg/mL) (Table 3) after incubate for 48 h, respectively. C. militaris extract with EtOAc inhibited the survival of cancer cells than EtOH extract. However, MCF-7 cells are more sensitive to inhibited the survival of C. militaris extract with EtOAc than HepG2 and HeLa. From previous research reports Asatiani et al. (2018) C. militaris extract with EtOAc and chloroform extract appeared to be the most active showing the most profound decrease in cell viability on HPAF-II, HCT116, PC3 and T47D, which is in line with our research that EtOAc has the best activity to inhibit cancer cells. C. militaris extract affects the cytotoxicity on cancer cells, including lung carcinoma, B16 melanoma, lymphocytic, prostate (PC3), breast (MCF-7), hepatocellular (HepG2, Hep3B) and colorectal (HT-29 and HCT116) cells (Nakamura et al., 2015); (Huo et al., 2017). C. militaris extract with 50% ethanol exhibited significant MCF-7 cell inhibitory effects on human breast MCF-7 cancer cells (Lee et al., 2019).
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Table 1: IC50 of C. militaris extract on MCF-7
MCF-7 cancer cell Treatment Incubation time Emax (%) IC50 (µg/mL)
EtOH 24 h 88.42±0.38c 236.10±6.79c 48 h 87.73±0.60c 112.35±3.04b EtOAc 24 h 91.21±0.71b 112.95±6.01b 48 h 93.00±0.26a 33.64±0.78a Each value is the mean ± SEM of three experiments. Small letters indicate significant differences in the column (p < 0.05).
Table 2: IC50 of C. militaris extract on HepG2
HepG2 cancer cell Treatment Incubation time Emax (%) IC50 (µg/mL)
EtOH 24 h 88.50±0.59c 174.80±4.10c 48 h 87.43±0.11d 135.60±3.54b EtOAc 24 h 92.48±0.13b 134.20±2.69b 48 h 95.05±0.07a 63.99±13.01a Each value is the mean ± SEM of three experiments. Small letters indicate significant differences in the column (p < 0.05).
Table 3: IC50 of C. militaris extract on HeLa
HeLa cancer cell Treatment Incubation time Emax (%) IC50 (µg/mL)
EtOH 24 h 21.31±1.15c >500 48 h 39.59±0.83b >500 EtOAc 24 h 90.83±1.18a 237.25±9.69a 48 h 92.24±0.58a 161.90±3.96a Each value is the mean ± SEM of three experiments. Small letters indicate significant differences in the column (p < 0.05).
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Figure 1: Effect of C. militaris from different solvent extract on survival of cancer cell, 1A-B; MCF-7 cancer cell, 1C-D; HepG2 cancer cell and 1E-F; HeLa cancer cell, each value is the mean ± SEM of three experiments. p< 0.05, compared to control.
Conclusion
In this study, the C. militaris extract with EtOAc affected the highest inhibition of the survival of MCF-7, HepG2, and HeLa cancer cells. In further research will be studied on the mechanisms of cytotoxicity of the other cancer cells. Data of study can be used for developing cancer drugs.
References
Asatiani, M. D., Sharvit, L., G.S., B., J.S.L., C., Elisashvili, V., & S.P., W. (2018). Cytotoxic Activity of Medicinal Mushroom Extracts on Human Cancer Cells. Journal of Biotechnology and Biomedical Engineering, 1, 1–7.
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Huo, X., Liu, C., Bai, X., Li, W., Li, J., Hu, X., & Cao, L. (2017). Aqueous extract of: Cordyceps sinensis potentiates the antitumor effect of DDP and attenuates therapy- associated toxicity in non-small cell lung cancer via IκBα/NFκB and AKT/MMP2/MMP9 pathways. RSC Advances, 7(60), 37743–37754. Jeong, J. W., Jin, C. Y., Park, C., Hong, S. H., Kim, G. Y., Jeong, Y. K., Lee, J. D., Yoo, Y. H., & Choi, Y. H. (2011). Induction of apoptosis by cordycepin via reactive oxygen species generation in human leukemia cells. Toxicology in Vitro, 25(4), 817–824. Kuete, V., Voukeng, I. K., Tsobou, R., Mbaveng, A. T., Wiench, B., Beng, V. P., & Efferth, T. (2013). Cytotoxicity of Elaoephorbia drupifera and other Cameroonian medicinal plants against drug sensitive and multidrug resistant cancer cells. BMC Complementary and Alternative Medicine, 13(1), 1. Lee, D., Lee, W. Y., Jung, K., Kwon, Y. S., Kim, D., Hwang, G. S., Kim, C. E., Lee, S., & Kang, K. S. (2019). The inhibitory effect of cordycepin on the proliferation of MCF-7 breast cancer cells, and its mechanism: An investigation using network pharmacology- based analysis. Biomolecules, 9(9). Lee, H. H., Lee, S., Lee, K., Shin, Y. S., Kang, H., & Cho, H. (2015). Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis. DARU, Journal of Pharmaceutical Sciences, 23(1), 1–8. Ling, C. Q., Yue, X. Q., & Ling, C. (2014). Three advantages of using traditional Chinese medicine to prevent and treat tumor. Journal of Integrative Medicine, 12(4), 331–335. Nakamura, K., Shinozuka, K., & Yoshikawa, N. (2015). Anticancer and antimetastatic effects of cordycepin, an active component of Cordyceps sinensis. Journal of Pharmacological Sciences, 127(1), 53–56. Tuli, H. S., Sharma, A. K., Sandhu, S. S., & Kashyap, D. (2013). Cordycepin: A bioactive metabolite with therapeutic potential. Life Sciences, 93(23), 863–869. Yoon, S. Y., Park, S. J., & Park, Y. J. (2018). The anticancer properties of cordycepin and their underlying mechanisms. International Journal of Molecular Sciences, 19(10). Zhai, X. F., Chen, Z., Li, B., Shen, F., Fan, J., Zhou, W. P., Yang, Y. K., Xu, J., Qin, X., Li, L. Q., & Ling, C. Q. (2013). Traditional herbal medicine in preventing recurrence after resection of small hepatocellular carcinoma: A multicenter randomized controlled trial. Journal of Chinese Integrative Medicine, 11(2), 90–100.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.8 International Conference on Food, Agriculture and Biotechnology
IN VITRO DIGESTIBILITY OF FISHMEAL REDUCTION DIET IN COMBINATION WITH PROTEASE ENZYME BY NILE TILAPIA (Oreochromis niloticus) DIGESTIVE ENZYME
PORNPOT PUTNUAN1, ORAPINT JINTASATAPORN1, SRINOY CHUMKAM2
1Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand 2Faculty of Agricultural Technology, Valaya Alongkorn Rajabhat University under the Royal Patronage, Pathum Thani, Thailand
*Corresponding author: [email protected]
Abstract:
The in vitro digestibility of fishmeal reduction diet in combination with protease enzyme by using Nile tilapia (Oreochromis niloticus) digestive enzyme was conducted and assigned in Factorial 223 in Complete Randomized Design (Factorial 223 in CRD). Factor A was fishmeal levels of fishmeal 5% and fishmeal 0%. Factor B was protease enzyme supplementation of no protease enzyme supplementation and with protease enzyme supplementation at 100 unit/Kg of feed. Factor C was Tilapia digestive enzyme of no Tilapia digestive enzyme supplementation, enzyme from Tilapia liver, enzyme from Tilapia intestine . Hence, the trial was conducted on 12 treatments and 3 replicates. The results showed that showed the high ability (p<0.05) of intestinal enzyme on protein, carbohydrate and phosphorus digestibility. The protease enzyme supplementation at 100 unit/Kg of feed showed no improving (p>0.05) of protein, carbohydrate and phosphorus digestibility. The zero fishmeal diet exhibited the better protein digestibility (p<0.05) than diet of fishmeal 5% but showed no significantly differences (p>0.05) on carbohydrate and phosphorus digestibility.
Keywords: in vitro digestibility; Exogenous protease; Tilapia digestive enzyme; Fishmeal reduction diet
Introduction
Digestion is a complex process in animals for maintenance their growth. The study on feed digestibility most focusing on in vivo digestibility in animal body which is complicate and costly. Many researchers work on the in vitro methodologies for predicting the potential bioavailability of a given nutrient or chemical substance. Moreover, some researchers work for a better understanding of the different processes, interactions and factors affecting the hydrolysis of protein, lipid and carbohydrate in feeds. The simulation of aquatic animal digestion is based on the methodologies that already developed for terrestrial animals and humans. In the study of fish, most publication have focused on salmonids, rainbow trout, gilthead seabream (Sparus aurata), bluefin tuna (Tunnus thynnus), common carp (Cyprinus carpio) and turbot (Psetta maxima) (Gomes et al., 1994). The studies in fish to date have been concentrated on the nutritional quality of protein-rich feed ingredients in particular fishmeal and also have focused on the development of methods to assess carbohydrate hydrolysis (Cousin et al., 1996; Omondi Stark, 1996; Simon, 2009). A few studies have been conducted on the in vitro digestive evaluation of lipids (Koven et al., 1997). The objective of this study was focus on the ability of Nile tilapia digest enzyme for digestion the diet composed of fishmeal, shrimp meal and animal protein compared to the digestion of fishmeal reduction diet
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology with increasing plant protein like corn gluten meal and investigated the ability of exogenous protease enzyme adding in fish diet for enhance feed protein digestion.
Materials and Methods
The in vitro digestibility of fishmeal reduction diet supplemented protease enzyme by Nile tilapia (O. niloticus) digestive enzyme was conducted. The mash feeds were incubated with Nile tilapia digestive enzyme from liver and intestine after that analysis the products liberated from the diets. The modified in vitro digestibility method of Rungruangsak-Torrissen, et al. (2002) was applied in this study.
Experimental diets
Two isonitrogeneous diets of 33.5% protein, isolipidic diets of 8.5% lipid of Tilapia feed were formulated. The material compositions presented in Table 1.
Table 1: Material compositions of Tilapia diets
Materials (%) Diet A Diet B Fishmeal 5.0 0.0 Marine protein 2.0 0.0 Animal protein 15.0 10.0 Plant protein 41.0 56.0 -Soybean meal 36.0 36.0 -Corn protein 5.0 20.0 Cassava chip 31.1 26.1 Oil 4.0 6.0 Premix 1.9 1.9 Total 100.0 100.0
Experimental designed
To compare the digestibility of experimental diets from Nile tilapia digestive enzyme, the in vitro digestibility was designed in the Factorial 2X2X3 in Complete Randomized Design (Factorial 2x2X3 in CRD). Three factors of fishmeal levels, protease enzyme supplementation and Tilapia digestive enzyme were assigned. Factor A was fishmeal levels of 5%Fishmeal (5%FM) and 0%Fishmeal (0%FM). Factor B was protease enzyme supplementation of no protease enzyme supplementation (0protease) and with protease enzyme supplementation 100 unit/Kg of feed (100protease). Factor C was Tilapia digestive enzyme of no Tilapia digestive enzyme supplementation (no Tilapia enzyme), Tilapia liver enzyme, Tilapia intestinal enzyme. Hence, the trial was conducted on 12 treatments, as following:
Treatments T1-5%FM-0proteas-no Tilapia Enzyme T2-5%FM-0proteas-Tilapia liver Enzyme T3-5%FM-0proteas-Tilapia intestinal Enzyme T4-5%FM-100proteas-no Tilapia Enzyme T5-5%FM-100proteas-Tilapia liver Enzyme T6-5%FM-100proteas-Tilapia intestinal Enzyme
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T7-0%FM-0proteas- noTilapia Enzyme T8-0%FM-0proteas-Tilapia liver Enzyme T9-0%FM-0proteas-Tilapia intestinal Enzyme T10-0%FM-100proteas-noTilapia Enzyme T11-0%FM-100proteas-Tilapia liver Enzyme T12-0%FM-100proteas-Tilapia intestinal Enzyme
In vitro digestibility of diets
The in vitro digestibility of experimental diets was conducted by using Nile tilapia enzyme from live and intestine. Three replicates of each treatment diet were conducted. The experimental diets of fishmeal 5% in T1-T6 and fishmeal 0% in T7-T12 were incubated with and without adding Nile tilapia digestive enzyme for investigated the digestibility of all feed materials in diet of fishmeal 5% with 41.0% plant protein and zero fishmeal with 56% plant protein. The experimental diets without protease enzyme (0 unit/Kg of feed) with fishmeal 5% in T1-T3 and with fishmeal 0% in T7-T9 compare to diets of protease enzyme 100 unit/Kg of feed with fishmeal 5% in T4-T6 and with fishmeal 0% in T10-T12 were incubated with and without adding Nile tilapia digestive enzyme for investigated the efficacy of exogenous protease enzyme supplementation incorporation with Nile tilapia digestive enzyme for digesting all feed materials in diet.
Enzyme extraction Twelve Nile tilapia with average weight of 58.65±4.75 g/fish was collected the whole digestive tract then separated liver and intestine for enzyme extraction. Each digestive organ was homogenized on ice in Tris HCl pH 7.4 (1:3 w/v) using a micro-homogenizer (THP- 220, OMNI International, USA). The homogenate was centrifuged at 10,000g for 20 min at 4 oC and then supernatant was collected and kept at -20oC until use for in vitro digestibility study. The total protein concentration of crude enzyme extract was determined according to standard method of Lowry et al. (1951) using bovine serum albumin as standard protein.
In vitro digestibility condition for nutrient digestion The experimental diets were crushed to fine particle for study in vitro digestibility. Each sample was weighed then applied into phosphate buffer pH 8. Antibiotic was applied for control the microorganism. The mixture was pretreatment by curing for 1 h at room temperature 25 oC after that the reaction was stopped by immerged at 100 oC for 20 minutes and leave it cool down. The solution was divided into 3 parts. Part 1: Collected the sample of each treatment (0hr.) stored at -20 °c for to be control Part 2: The solution without adding any Nile tilapia enzyme and incubate for 16 h (16 h) Part 3: The solution adding Nile tilapia digestive enzyme from liver or intestine and then incubate for 16 h.
Determination of protein digestibility The in vitro digestibility of protein was studied by measuring the reactive amino group using the Ninhydrin assay. A solution of undigested control (0 h) and the digested mixture (16 h) were mixed with cd-ninhydrin reagent then incubated at 84 ๐C for 5 minutes and suddenly cool down on ice. The mixture was measured at 507 nm and calculated the amino acid concentration by using tyrosine as a standard. The in vitro digestibility of protein was expressed as mg Tyrosine/mg sample (Eid Matty, 1989).
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Determination of carbohydrate digestibility The in vitro digestibility of carbohydrate was studied by measuring the increasing of reducing sugar using a colorimetric method with 3,5- dinitrosalicylic acid (DNS). The digested mixture was added DNS then incubate at room temperature (25oC) after that the reaction was stopped by immerged at 100 oC for 5 minutes and cool down. The mixture was measured absorbance at 540 nm and compare with maltose standard curve. The in vitro digestibility of carbohydrate was expressed as mg maltose/ mg sample (Bedford Classen., 1993; Miller, 1959).
In vitro digestibility condition for phosphorus digestion The in vitro digestibility condition for phosphorus digestion was carried out follow the in vitro digestibility condition for protein and carbohydrate digestion. The experimental diets were crushed and weighed. Acetate buffer pH 5.0 was used for incubated the diets. Antibiotic was applied and the mixture was pretreatment for 1 h at room temperature (25 oC) then the reaction was stopped by immerge at 100 oC for 20 minutes and leave it cool down. The solution was divided into 3 parts of control (0 h), incubated solution without adding any Nile tilapia enzyme supplementation (16 h) and solution adding Nile tilapia digestive enzyme from liver or intestine then incubate for 16 hour (16 h). Determination of phosphorus digestibility was conducted by measuring the orthophosphate liberated from diets. The assay mixture of acetone, H2SO4, (NH4)2MoO4 2:1:1 was prepared. A solution of undigested control (0 h) and the digested mixture (16 h) were mixed with assay mixture and citric acid then measured at 355 nm. The orthophosphate liberated from diets was calculated by comparing to orthophosphate-phosphorus standard curve. The in vitro digestibility of phosphorus was expressed as mg phosphate-phosphorus/mg sample.
Statistical data analysis
All data means from the experiment were subjected to analysis in Factorial 2x2X3 in CRD by statistical software. Duncan's Multiple Range Test was applied to compare the difference between experimental groups (Steel Torrie, 1980).
Results and Discussion
Protein and amino acid digestibility
The results of in vitro digestibility of protein to amino acid of fishmeal reduction diet in combination with protease enzyme by using Nile tilapia (O. niloticus) digestive enzyme was presented in Table 1. The result showed that diet of zero fishmeal with plant protein 56% (corn protein 20%) had higher protein digestibility (p<0.05) in term of amino acid liberated from the diet than diet of fishmeal 5% with plant protein 41% (corn protein 5%) which compose of cassava chip 31%. The fiber in cassava chip may confound the protein digestion of diet. Focusing on the efficacy of exogenous protease enzyme supplementation, there were not significantly differences (p>0.05) on the amount of the amino acid liberated from diet with and without exogenous protease enzyme supplementation due to the high digestibility of corn protein in the diet of zero fishmeal. Comparing the efficacy of enzyme from Nile tilapia digestive organ, liver and intestine, the results showed that Nile tilapia intestinal enzyme exhibited the high efficacy (p<0.05) to liberate amino acid from fish feed than activity of enzyme from the liver and exogenous protease enzyme supplementation in fish diet. These
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology indicated that exogenous protease enzyme and liver protease enzymes have lower ability to digest protein to amino acids due to the digestive protease enzymes from fish liver including pancreatic enzyme most are endoprotease like trypsin, chymotrypsin that can hydrolyze protein to peptide and release a little amino acid which different from protease enzymes from intestinal epithelial that are exoprotease such as aminopeptidase, dipeptidase, tripeptidase which digest peptide to amino acid (Lim and Webster, 2006). The diet of T12 0%FM-100proteas-Tilapia intestinal enzyme was the highest protein digestibility (p<0.05) follow by T6 5%FM- 100proteas-Tilapia intestinal enzyme, T9 0%FM-0proteas-Tilapia intestinal enzyme and T3 5%FM-0proteas-Tilapia intestinal enzyme, respectively. The protein digestion from diets of fishmeal 5% and 0% both without and with exogenous protease enzyme supplementation including without Nile Tilapia enzyme supplementation were in the same range. It is indicated that the exogenous protease enzyme supplementation in the diet at 100 unit/Kg of feed had low ability to digest protein the diets compare to enzyme from liver and intestine of Nile tilapia . There are many factor related to the low protein digestibility of exogenous protease enzyme supplementation such as the low dosage of protease enzyme enzyme supplementation in the diet, loose of enzyme activity during extruded feed processing, quality of enzyme, etc. Therefore, diet of zero fishmeal with high plant protein of soybean meal and corn protein with protease enzyme was the high protein digestibility diet for Nile tilapia.
Carbohydrate digestibility
In vitro digestibility of carbohydrate in experimental diet by using Nile tilapia digestive enzyme was conducted. The results in Table 1 demonstrated that carbohydrate digestibility in term of reducing sugar content (the sum of glucose, xylose, maltose, and mannose) obtained from diet of fishmeal 5% and 0% showed no significantly differences (p>0.05) and also exogenous protease enzyme supplementation in the diets showed no significantly differences (p>0.05) on the carbohydrate digestibility of diets. The intestinal enzyme of Nile tilapia exhibited the high ability on the carbohydrate digestibility (p<0.05) than liver enzyme and native carbohydrase enzymes in the diets. Nile tilapia is omnivorous fish that has high ability to digest both plant and animal materials. Moreover, Tilapia has long intestine 5-15 times of body length, hence, Tilapia has high ability to digest or ferment fiber in their long intestine to be sugar and energy (Lim Webster, 2006).
Phosphorus digestibility
The in vitro digestibility of phosphorus in experimental diets by using Nile tilapia digestive enzyme was presented in Table 1. The results showed no significantly differences (p>0.05) on phosphate-phosphorus obtained from diet of fishmeal 5% and 0%. The diets without and with exogenous protease enzyme supplementation also showed no significantly differences (p>0.05) on the phosphorus digestibility. The intestinal enzyme of Nile tilapia exhibited the high ability on the phosphorus digestibility (p<0.05) than liver enzyme and native phytase enzymes in the diets. Nile tilapia is omnivorous fish that has a little cellulase and phytase enzyme activity in digestive tract and has long intestine for digest or ferment fiber by water bone bacteria that release fibrolytic enzyme or carbohydrase including phytase to digest fiber and phytate in plant materials then releasing sugar and phosphorus (Lim Webster, 2006).
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Table 1: Amino acids liberated from in vitro digestion of Nile tilapia experimental diets
Amino acids Reducing sugar Phosphate Treatments (g/100gfeed) (g/100gfeed) (g/100gfeed) T1-5%FM-0proteas-no Tilapia Enzyme 1.11 d 5.44ab 0.015 d T2-5%FM-0proteas-Tilapia liver Enzyme 6.26 c 4.73b 0.031 bcd T3-5%FM-0proteas-Tilapia intestinal Enzyme 10.93 b 6.18 ab 0.050 abc T4-5%FM-100proteas-no Tilapia Enzyme 1.32 d 5.65 ab 0.021 cd T5-5%FM-100proteas-Tilapia liver Enzyme 7.10 c 5.52 ab 0.045 abc T6-5%FM-100proteas-Tilapia intestinal Enzyme 11.37 ab 8.52 a 0.064 a T7-0%FM-0proteas- noTilapia Enzyme 1.70 d 6.30 ab 0.036 abcd T8-0%FM-0proteas-Tilapia liver Enzyme 7.83 c 5.73 ab 0.050 abc T9-0%FM-0proteas-Tilapia intestinal Enzyme 11.36 ab 6.54 ab 0.052 ab T10-0%FM-100proteas-noTilapia Enzyme 1.71 d 7.48 ab 0.036 abcd T11-0%FM-100proteas-Tilapia liver Enzyme 7.86 c 6.15 ab 0.056 ab T12-0%FM-100proteas-Tilapia intestinal Enzyme 12.72 a 8.68 a 0.057 ab p-Value FM 0.007 0.191 0.066 p-Value Protease Enzyme 0.116 0.060 0.156 p-Value Tilapia Enzyme <0.001 0.043 0.001 p-Value interaction FM*Protease 0.964 0.909 0.487 p-Value interaction FM*Tilapia enzyme 0.662 0.762 0.250 p-Value interaction Protease*Tilapia Enzyme 0.568 0.465 0.819 p-Value interaction 0.503 0.886 0.983 FM*Protease* Tilapia Enzyme Note : Data with superscript letters a,b,c in the same column indicates significantly difference (P<0.05)
Conclusion
The in vitro digestibility of fishmeal reduction with protease enzyme supplementation by using Nile tilapia digestive enzyme extract showed the high ability (p<0.05) of intestinal enzyme on protein, carbohydrate and phosphorus digestibility. The protease enzyme supplementation at 100 unit/Kg of feed showed no improvement (p>0.05) of protein, carbohydrate and phosphorus digestibility. The zero fishmeal diet exhibited the better protein digestibility (p<0.05) than diet of fishmeal 5% but showed no significantly differences (p>0.05) on carbohydrate and phosphorus digestibility.
Acknowledgements
The author would like to thank the Nutrition and Aquafeed Laboratory, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand for facility and financial support.
References
Bedford M.R H.L. Classen. (1993). An in vitro assay for prediction of broiler intestinal viscosity and growth when fed rye-based diets in the presence of exogenous enzymes. Poult Sci. 72(1):137-43.
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Cousin M., G. Cuzon, J. Guillaume. (1996). Digestibility of starch in Penaeus vannamei: in vivo and in vitro study on eight samples of various origin. Aquaculture 140: 361–372. Eid A.E. A.J. Matty. (1989). A Simple In Vitro Method for Measuring Protein Digestibility. Aquaculture 79: 111-119. Gomes, E.F., P. Rema, S.J. Kaushik. (1994). Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchus mykiss): Digestibility and growth performance. Aquaculture 130: 177-186 Koven, W.M., R.J. Henderson J.R. Sargent. (1997). Lipid digestion in turbot (Scophthalmus maximus): In-vivo and in-vitro studies of the lipolytic activity in various segments of the digestive tract. Aquaculture. 151: 155–171 Lim, C.E. C.D. Webster. (2006). Tilapia Biology, Culture, and Nutrition. Food Products Press, New York Miller, K. (1959). Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. 31: 426-428. Omondi, J.G. J.R. Stark. (1996). In vitro carbohydrate digestibility tests in the Indian white shrimp, Penaeus indicus. Aquaculture. 139: 315–328 Rungruangsak-Torrissen K, A. Rustad, J. Sunde, S.A. Eiane, H.B. Jensen, J. Opstvedt, E. Nygard, T.A. Samuelsen, H. Mundheim, U. Luzzana, G. Venturini. (2002). In-vitro digestibility based on fish crude enzyme extract for prediction of feed quality in growth trials. Journal of the Science of Food and Agriculture. 82: 644-654 Simon, C.J. (2009). Identification of digestible carbohydrate sources for inclusion in formulated diets for juvenile spiny lobsters, Jasus edwardsii. Aquaculture. 290: 275– 282. Steel, R. G. D. J.H. Torrie. (1980). Principles and Procedures of Statistics: a biometeric approach (2nd Ed.) Mc Growwhill: New York.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.9 International Conference on Food, Agriculture and Biotechnology
INVESTIGATION THE PHYSICAL, MECHANICAL PROPERTIES OF EDIBLE FILM FROM RICEBERRY FLOUR
HEMSO WALAIPORN*, SUTTIVATTANAVET WANNEE PLOYPETCHARA THONGKORN
Expert Center of Innovative Health Food, Thailand Institute of Scientific and Technological Research 35 Mu 3 Technopolis, Tambon Khlong Ha, Amphoe Khlong Luang, Pathum Thani 12120, Thailand
*Corresponding author email: [email protected]
Abstract:
Edible film samples containing riceberry flour at 15, 20, 25 and 30% (w/w) with adding glycerol as a plasticizer were extruded. The film properties were investigated. The results showed that the thickness and opacity of film samples decreased while moisture content, aw, and water solubility increased with an increase in the concentration of glycerol. The colors (L, a*, b*) values of the films were changed with glycerol concentration. The mechanical properties in terms of tensile strength and load at the maximum of the film decreased but elongation at break increased with an increase in the concentration of glycerol. Adding glycerol as a plasticizer affected the physical and mechanical properties of riceberry film depending the concentration of glycerol.
Keywords: Riceberry, Film, Glycerol, Physical Properties, Mechanical Properties
Introduction
Plastic packaging materials such as polyethylene are commonly used in the food industry due to low cost, high strength, and convenience for the production process however polyethylene is non-degradable packaging and becomes the main problem for the environment (Debiagi et al., 2014). Biofilms substituted conventional plastic packaging were interesting because of reducing environmental pollution problems and expanding the shelf life of food products with inhibiting microorganisms' growth (Campos-Requena et al., 2017). Presently, the environmental pollution is the major problems for several organizations to concern especially the food packaging produced from the synthetic materials being substituted by the biodegradable materials. Therefore, using biodegradable or edible film is an alternative material to reduce environmental problems (Noiduang et al., 2015) Edible film and coating production are increasing for use in the food industries especially fresh food products due to an important part of extending the shelf life according to inhibition moisture transfer, inhibition oxidation occurrence, and retardation respiration. Edible film and coating might be composed of polysaccharides, protein, or lipids (Tassavil et al., 2009). Riceberry (Oryza Sativa), a deep purple grain rice variety of Thailand, is a crossbreed of Hom Nil rice and Khao Dawk Mali 105 (Sivamaruthi et al., 2018). Riceberry contains many health beneficial compounds such as poly-phenols compounds, anthocyanin, vitamin E, and Gamma-Oryzanol, that compounds act as antioxidants and reduce risk of diseases such as cancer, cardiovascular heart attack, and diabetes (Settapramote et al., 2018). Riceberry flour is a by-product (broken rice) obtained from the milling process, and main component of rice berry flour is starch comprising amylose and amylopectin. Therefore, the starch edible films produced by different amylose-amylopectin content showed different properties such as glass
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology transition temperature, tensile strength, elongation at break, permeability, and morphology of films (Forsell et al., 2002; Wittaya, 2012 and Menzel, 2014). Most edible film produced from starch such as tapioca and rice were studied due to the amylose-amylopectin composition affecting the gel network formation and the film strength (Imprapai et al., 2010). Moreover, the concentration of starch is an important factor affecting mechanical and physical properties as well as suitability for applied in the food industry (Wu et al., 2002). Water is the well-known plasticizer for a starch edible film, but water is easy to evaporate resulting in the brittleness of film. Most researchers used glycerol as a plasticizer. The concentration of glycerol around 15-30% could change starch to thermoplastic starch and increase gelatinization temperature since the molecular weight of glycerol was higher than water. Therefore, it could not penetrate between starch granules and increase free space of the amorphous matrix (Belgacem and Gandini, 2008). Thus, the objective of this research is to investigate the physical and mechanical properties of edible film from riceberry flour.
Materials and Methods
Material
Riceberry was purchased from Pathumthani (Rice wholesaler) and the rice was dried at 50±3°C for 8 h. Then, the rice was milled into riceberry flour at 100 mesh particle size.
Film Preparation
Film samples were prepared by mixing riceberry flour with 15, 20, 25, and 30% glycerol as plasticizer (w/w of starch weight) and adjusted moisture content as 30% (Table 1). After that, the samples were extruded with die and screw as 0.3 mm width and 285 mm length, respectively. The temperature used at barrel 1 and barrel 2 as 110 and 110°C, respectively. The extruded films were dried at 50±3°C in a circulated air oven (Binder Model KMF240, Germany) for 24 h. All film samples were kept in plastic bags at 23±3°C and 50% relative humidity (RH) for not less than 40 h before measurement (Chanpord et al., 2015)
Table 1: Mixed components for extrusion
Condition Rice Berry Flour (g) Glycerol (g) Water (g) RB15 100 15 30 RB20 100 20 29 RB25 100 25 28 RB30 100 30 27
Film Thickness The thickness of the film was measured at 5 different positions with a Mitutoyo Electronic digital micrometre, Japan). The averaged thickness was calculated (Domene-López et al., 2019).
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Moisture content and Solubility
The moisture content of the film was determined by cutting the film into 1x1 cm. Then, the film was dried at 110°C in an oven (Binder Model ED Series, Germany) until constant weight. The moisture content was calculated using the equation (1):
Moisture content (%) = ((m0 - m1)/m0) x100 (1) where m0 and m1 were the weight before and after drying, respectively The water solubility of the films was measured by placing the dried films in 10 mL tubes and filling with 9 mL of distilled water. The sample stored at 25°C for 24 h before the films were taken out and dried again at 110°C for 5 h. Water solubility was calculated using (Equation (2)):
Water solubility (%) = ((m0 – mf)/m0) x100 (2) where mf was the weight of final dried film (Domene-López et al.2019).
Color
Color of the film was measured on 5 cm x 5 cm. A CIE colorimeter (Hunter Associates Laboratory, Inc., VA. USA) was used to determine the film L*, a* and b* color value (L* = 0 (black) to 100 (white); a* = -60 (green) to +60 (red); and b* = -60 (blue) to +60 (yellow)) (Bourtoom, 2008).
Opacity
The opacity of the films was determined using the UV VIS spectrophotometer 200V (Hitachi, Japan) at a wavelength of 600 nm. The opacity of the film was calculated following equation:
Opacity =Abs600/x (3) where x is the thickness of the film (mm) and Abs600 is the absorbance of the film measured at 600 nm (Domene-López et al. 2019).
Mechanical properties
Mechanical properties of the film (tensile strength, maximum load, and percentage of elongation at break, E) were measured following to ASTM D822 Standard test (ASTM) with LLOYD TA plus Material Tester. The film of each formulation was cutting into 100X15 mm. Mechanical properties were recorded during extension at 50 mm/min, with an initial distance between the grips of 50 mm (Zhong & Xia, 2008).
Statistical analysis
All the samples were analyzed statistically using SPSS version 17. Different means were investigated by ANOVA and Duncan’s multiple range tests at a level of significance of 0.05.
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Results and Discussion
Thickness and opacity
The thickness of riceberry film was decreased with increasing concentration of glycerol (Figure 1). The thickness would be related to the solid content of total mass in the film forming suspension depending on the reordering of molecular chain form more compact matrix in composite starch (Apriyana et al. 2016; Hafnimardiyanti and Armin 2016; Nindjin, Beyrer, and Amani 2015). The films could increase solid concentration in the film-forming dispersion and would become thicker due to the increased total residual mass in the film (Apriyana et al. 2016), this studies showed that the increase in glycerol concentration in the film extrusion would decrease the riceberry flour compositon in the system and would decrease the total solid content in the same weight resulting in a decrease in film thickness.
Figure 1: Thickness and Opacity of riceberry film.
The opacity is a conversion value of transparency. The opacity of the riceberry film showed decreased with increasing concentration of glycerol (Figure1) and opacity also had a positive relationship with thickness as similar to Galdeano et al. (2013) that the higher thickness was observed in the higher opacity values in oat starch film. The low glycerol concentration will cause higher opacity due to no other components between the polymer chains and enabling less incident light to pass through the film (Galdeano et al. 2013). Like this study, the higher glycerol concentration would make the thinner film and decrease the opacity of the film. Transparency is an important characteristic of food packaging; the lower in transparency could protect food from the light and would be higher in quality in food packaging film (Saberi et al., 2016). However, the low opacity or high transparency of the film still helps the food products to be easily visible (Galdeano et al. 2013).
Color
The change in color properties of the film was investigated by colorimeter. All of the film samples showed a positive value of L*, a*, and b*(Table 2). Glycerol content affected the color of the film especially 30% glycerol plasticized film showed the highest a* and b* value. It
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology indicated that the film color would be reddish and yellowish. The L* value decreased while a* and b* values increased with an increase in glycerol concentration from 15% to 30%. At low glycerol concentration, amylose chains in starch film system would form crystalline resulting in an opaque film (Fig. 1) while an increase in glycerol concentration would decrease L* value and less opacity due to glycerol acting as a plasticizer and decreasing crystalline in the system (Theamdee and Pansaeng, 2019). Moreover, the hydrophilic and water-holding properties of glycerol (Apriyana et al. 2016) would make it easily evaporated and maintain the riceberry color to remain with the film. However, the film colour was not clearly changed in the sample of 20% and 25% glycerol plasticized film.
Table 2: Color properties of riceberry flour film.
Glycerol (%) L* a* b* 15 30.24±0.53c 7.28±0.18ab 0.78±0.21a 20 28.69±1.16b 8.10±1.53b 1.83±0.41b 25 27.17±0.19a 6.72±0.28ab 0.40±0.11a 30 28.53±0.598b 11.03±0.79c 3.54±0.60c *Different letters in each column indicates a difference in glycerol concentration, the significant difference at p0.05.
Moisture content, water solubility and Aw
The moisture content of the film increased with an increase in glycerol concentration as the same trends with water solubility and aw (Table 3). The increase in glycerol concentration could increase moisture content in the system due to a hydrophilic property of starch and glycerol acted as a water holding agent (Apriyana et al. 2016). Moreover, hydroxyl group of glycerol interacted with water by hydrogen bond and would be as a plasticizer, the higher level of plasticizer would increase the film’s moisture affinity (Mali et al. 2005).
Table 3: Moisture content, water solubility, and aw of riceberry flour film.
Glycerol (%) Moisture content (%) Solubility aw 15 12.89±1.86a 33.57±0.97a 0.51±0.00a 20 13.93±0.66a 33.76±2.01a 0.51±0.00a 25 14.25±0.43a 36.38±1.26b 0.51±0.00a 30 17.53±0.66b 38.78±2.71b 0.52±0.00b *Different letters in each column indicates a difference in glycerol concentration, the significant difference at p0.05.
The high water solubility in edible products would be advantageous ability during cooking (Laohakunjit and Noomhorm 2004). The water solubility increased with increasing glycerol content similar results to Laohakunjit and Noomhorm (2004) who reported the highest water solubility of the film showed with the highest glycerol addition, and Hafnimardiyanti and Armin (2016) whose results showed the increasing water solubility trends with increasing plasticizer content. The solubility of the edible film is strongly influenced by the hydrophilic property of edible compounds and humidity of the starch film which plasticizer increased space between starch chains attributed to water facilitating migration into film matrix (Hafnimardiyanti and Armin 2016). The film with 30% glycerol addition showed the highest aw might affect the plasticizing and hygroscopic properties of glycerol which increased mobility region on the film and increased the water up take (Theamdee and Pansaeng, 2019).
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However, the film in this study had aw around 0.51-0.52 which would be safe from microbial (aw range is optimized for microorganism around 0.70-0.99 (Theamdee and Pansaeng, 2019)).
Mechanical properties
The mechanical properties of the film were depending on re-arrangement and orientation of polymer network that related to inter and intra-molecular interaction in the system and also water induced crystallization or retrogradation of the film (Saberi et al. 2017). The tensile strength and maximum load of the riceberry film decreased while elongation at break increased with increased glycerol concentration (Figure 2). The decrease in tensile strength and increase in elongation with increase plasticizer concentration might be due to plasticizer inhibited strong intermolecular interaction between starch chains and formed hydrogen bonds between plasticizer and starch molecule as a result of increase free volume and decrease mechanical resistant (Apriyana et al. 2016; Bourtoom 2008; Osés et al. 2009). The increase in elongation at break of the film might relate to moisture content as the result in Table 3 and Figure 2 that showed the same trends as increasing elongation at break and moisture content with increasing plasticizer concentration. These results might because glycerol plasticized film have high hygroscopic character and reduced forces between the adjacent macromolecules according to increase stretch ability (Bourtoom 2008). Moreover, the lowest tensile strength and the maximum load of the film showed the highest aw. Mali et al. (2005) reported the stress and Young’s modulus decrease with increase RH; at high RH would be high equilibrium moisture content to exert a plasticizing effect to enhance mobility, increase free volume, and increase flexibility. Thus, glycerol could act as a plasticizer, and the concentration of glycerol was important factor that affected the properties of riceberry flour film. The major of interaction between glycerol and starch in the system might relate to hydrogen bonding between functional groups of both starch and the glycerol as a resulting in an increase in the proportion of glycerol which have more interactions occur between their functional groups (Ajiya et al., 2017)
Conclusion
The properties of extruded riceberry flour film plasticized with glycerol (15, 20, 25, and 30%) were investigated for thickness, opacity, color, moisture content, water solubility, aw, and mechanical properties. The results of the experiments showed that film thickness decreased with increasing concentration of glycerol, and opacity had a positive relationship with thickness. Moisture content, water solubility, and aw increased with an increasing in concentration of glycerol. In the mechanical properties of the film, the tensile strength and maximum load of the riceberry flour film decreased, while the elongation at break increased with increasing glycerol concentration. At high glycerol concentration, glycerol had a good ability to act as a plasticizer in riceberry flour film system. Glycerol could affect the physical and mechanical properties of the riceberry flour film based on the concentration of glycerol due to the hydroxyl groups of glycerol formed hydrogen bonds hydrophilic property of glycerol and interacted with the riceberry flour film matrix. As with the extrusion temperature might result in the reduction of substances antioxidants. However, other factors such as extrusion conditions for produced riceberry flour film and antioxidant effect have to study in the future.
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Figure 2: Mechanical properties of riceberry flour film.
Acknowledgment
The authors are grateful thank Thailand Institute of Scientific and Technological Research (TISTR) for providing facilities and funding support.
References
Apriyana, Wuri, Crescentiana Dewi Poeloengasih, Hernawan, Septi Nur Hayati, and Yudi Pranoto. (2016). Mechanical and Microstructural Properties of Sugar Palm (Arenga Pinnata Merr.) Starch Film: Effect of Aging. AIP Conference Proceedings 1755(1):150003.
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Bourtoom, Thawien. (2008). Plasticizer Effect on the Properties of Biodegradable Blend Film from Rice Starch-Chitosan. Chanpord, W., Laohakunjit, N. and Kerdchoechuen, O., (2015), Development of Films for Suppression of Bad Breath by Extrusion Technique. Agricultural Sci. J. 46(3), (Suppl.): 349-352. Domene-López, Daniel, Juan Carlos García-Quesada, Ignacio Martin-Gullon, and Mercedes G. Montalbán. (2019). Influence of Starch Composition and Molecular Weight on Physicochemical Properties of Biodegradable Films. Polymers 11(7). Galdeano, Melicia Cintia, Allan Eduardo Wilhelm, Suzana Mali, and Maria Victória Eiras Grossmann. (2013). Influence of Thickness on Properties of Plasticized Oat Starch Films. Brazilian Archives of Biology and Technology 56(4):637–44. Hafnimardiyanti, H., and M. I. Armin. (2016). “Effect of Plasticizer on Physical and Mechanical Charateristics of Edible Film from Mocaf Flour. Der Pharmacia Lettre, 2016, 8 (19):301-3088. Laohakunjit, Natta, and Athapol Noomhorm. (2004). Effect of Plasticizers on Mechanical and Barrier Properties of Rice Starch Film. Starch - Stärke 56(8):348–56. Mali, S., L. S. Sakanaka, F. Yamashita, and M. V. E. Grossmann. (2005). Water Sorption and Mechanical Properties of Cassava Starch Films and Their Relation to Plasticizing Effect.” Carbohydrate Polymers 60(3):283–89. Nindjin, C., M. Beyrer, and G. N. Amani. (2015). Effects of Sucrose and Vegetable Oil on Properties of Native Cassava ( Manihot Esculenta Crantz) Starch-Based Edible Films. African Journal of Food, Agriculture, Nutrition and Development 15(2):9905-9921– 9921. Osés, Javier, Idoya Fernández-Pan, Mauricio Mendoza, and Juan I. Maté. (2009). Stability of the Mechanical Properties of Edible Films Based on Whey Protein Isolate during Storage at Different Relative Humidity. Food Hydrocolloids 23(1):125–31. Saberi, Bahareh, Suwimol Chockchaisawasdee, John B. Golding, Christopher J. Scarlett, and Costas E. Stathopoulos. (2017). Physical and Mechanical Properties of a New Edible Film Made of Pea Starch and Guar Gum as Affected by Glycols, Sugars and Polyols . International Journal of Biological Macromolecules 104:345–59. Settapramote, Natwalinkhol, Thunnop Laokuldilok, Dheerawan Boonyawan, and Niramon Utama-ang. 2018. “Physiochemical, Antioxidant Activities and Anthocyanin of Riceberry Rice from Different Locations in Thailand.” 6:84–94. Sivamaruthi, Bhagavathi, Periyanaina Kesika, and Chaiyasut. (2018). Anthocyanins in Thai Rice Varieties: Distribution and Pharmacological Significance. International Food Research Journal 25(5): 2024–2032. Zhong, Qiu-Ping, and Wen-Shui Xia. (2008). Physicochemical Properties of Edible and Preservative Films from Chitosan/Cassava Starch/Gelatin Blend Plasticized with Glycerol. Food Technology and Biotechnology 46(3).
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ICoFAB2020 doi: 10.14457/MSU.res.2020.10 International Conference on Food, Agriculture and Biotechnology
METHOD VALIDATION FOR QUANTITATIVE DETERMINATION OF GALLIC ACID FROM ACACIA CONCINNA (WILLD.) D.C.
PATTRA AHMADI PIRSHAHID*, CHIRAMET AURANWIWAT, WIRIYAPORN SUMSAKUL, SINEE SIRICOON, WARAPORN SORNDECH
Expert Center of Innovative Health Food, Thailand Institute of Scientific and Technological Research, Pathumthani 12120, Thailand
*Corresponding author: [email protected]
Abstract:
Gallic acid is one of the most antioxidative components found in Acacia genus. Acacia concinna (Willd.) D.C. belongs to Acacia genus and Fabaceae family, this genus has been used as traditional medicine. The development and validation of RP-HPLC method to determine gallic acid from the ethanolic extract of Acacia concinna. The chromatographic condition was performed on Symmetry Shield RP18 (4.6 x 250 mm) with ACN and 0.5% H3PO4 as mobile phases, flow rate 1.0 mL/min and detection at 272 nm. The quantitative results using external standard, standard gallic acid showed the linear range between 5-50 µg/mL while LOD and LOQ exhibited the values at 0.87 and 2.65 µg/mL, respectively. The accuracy and precision also examined. In the real sample, the ethanolic extract of A. concinna consisted of gallic acid 1.042 %w/w.
Keywords: Gallic acid; Acacia concinna; Method validation
Introduction
Acacia concinna (Willd.) D.C. is a member of Fabaceae family which found in tropical rainforests area in Southern Asia and locally name as “Som-Poi”. This plant has used for traditional medicine to treat the skin deceases (Sekie et al., 1999). In some area, the pods of this plant have been used for washing hair and promoting hair growth (Sekie et al., 1999; Tezuka et al., 2000). The report on phytochemical analysis of this plant found the various groups of secondary metabolites including flavonoids (Tezuka et al., 2000), lactam (Sekine et al., 1989), saponins (Gafur et al., 1997; Kiuchi et al., 1997) and terpenoids (Sekie et al., 1997 and Anjaneyulu et al., 1999). These isolated compounds exhibited the arachidonate 5- lipoxygenase activity and cytotoxicity (Sekie et al., 1999; Tezuka et al., 2000). Gallic acid is of the phenolic compounds has been isolated from the plant in Acacia genus (Khalia et al., 1989; Salam et al., 2011) and exhibited interesting biological activity such as antioxidant, anti- inflammatory, antitumor and anti-obesity (Thompson et al., 2013; Kim et al., 2019). In this study, we reported the resulted in the method validation of gallic acid to determine quantitative amount in real sample. The linear range, LOD, LOQ, accuracy and precision of this method also investigated and applied to measure in the ethanolic extract of A. concinna.
Materials and Methods
General Experimental Procedure
The RP-HPLC was performed on Water 600 controller, equipped with a Water 486 detector, USA using Symmetry Shield RP18 column (4.6 x 250 mm, 5 µm), injection loop 20 µL, flow
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology rate 1 mL/min and detection wavelength at 272 nm at 30 C. A binary gradient elution system consisted of acetonitrile (A) and 0.5% H3PO4 (B) and using a gradient program: 0-5 min, 0% A, 5-6 min, 30% A, 6-20, 30% A, 21-30 min, 90% A, 31-40 min, 90% A. The ultrasonic bath was Bandelin Sonorex, Banoelin. The acetonitrile and MeOH were purchased in HPLC grade from RCI Lab Scan, Thailand while phosphoric acid was obtained from Merck, Germany. Gallic acid standard compound was purchased from Sigma, Switzerland.
Plant materials and extraction
The pods of A. concinna were collected from Chiang Mai Province in 2014. The pods of A. concinna (1.0 Kg) were chopped to small pieces then extracted with hexanes (3L x 1). The solution was filtered, and the residue was extract with 95% EtOH (3L x 6). The removal of solvent under reduce pressure provided the ethanolic extract as a dark brown gum.
Preparation of standard Stock Solution
Accurately weighed 10 mg of standard gallic acid then transferred to a 50 mL volumetric flask and dissolved in MeOH 20 mL. The stock solution was sonicated for 15 minutes and adjusted with MeOH to 50 mL. The stock solution has concentration 200 µg/mL.
Method validation
The method was validated and evaluated as per ICH guidelines.11 The parameters lists were specificity, linearity and range, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy.
Specificity
The method’s ability established the specific for the target compounds which showed good separation without the effect from matrix. The specific of the method was carried out by a comparison of retention time of sample with the standard.
Linearity and Range
Preparation of standard solution 5, 10, 15, 20, 25 and 50 µg/mL from stock solution into volumetric flask and adjusted with MeOH. The linearity was plotted from the peak area of standard solution and concentration.
Limit of detection (LOD) and limit of quantitation (LOQ)
The values of LOD and LOQ were calculated from the calibration curve using the formula as LOD = 3.3SD or 3.3δ/S while LOQ = 10SD or 10δ/S
Precision
Precision studies were performed on the analysis of six times of the samples which injected as the same condition as the standard gallic acid. The results were expressed in terms of percent relative standard deviation (% RSD).
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Accuracy
Accuracy of this method was investigated the recovery experiments by addition the standard compounds of gallic acid into sample solution. All samples were analyzed in triplicate and recovery was calculated.
Determination of gallic acid in 95% EtOH extract of A. concinna
Accurately weighed 100 mg of 95% EtOH extract of A. concinna then transferred to a 100 mL volumetric flask and dissolved in MeOH 20 mL. The sample solution was sonicated for 15 minutes and adjusted with MeOH to 50 mL.
Results and Discussion
Specificity
The specificity of the method exhibited good separation of the standard gallic acid and sample solution. The chromatogram illustrated clear peak and did not affect from the other matrix. A standard gallic acid and the extract showed peak at retention time 13.2 minutes (Figure 1).
(a)
(b)
Figure 1: The specificity test of standard gallic acid (a) and 95% EtOH extract of A. concinna (b)
Linearity and Range
The caliblation curve for standard gallic acid presented a good correlation coeffient (R2 = 0.9998) with the formula y = 72.519x + 33.542 in the scope of concentration between 5-50 µg/mL (Figure 2).
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4000 y = 72.519x + 33.542 3500 R² = 0.9998 3000 2500 2000
1500 Peak area Peak 1000 500 0 0 10 20 30 40 50 60 Concentration (µg/mL)
Figure 2: The calibration curve of standard gallic acid
Accuracy and precision
The accuracy tests were performed by the calculation of percent recovery by adding three different concentrations of the standard solution mixed with the real sample. The results showed the percent recovery between 98.667-100.139 % (Table 1). The precision of multiple sampling measurements showed the express of agreement of resulted under prescribed conditions. The relative standard deviation (%RSD) of the measurement established the precision were 0.299-1.754% as shown in Table 1.
Table 1: The summary of method validation data in the tests of accuracy and precision
Sample %Recovery %RSD Std. gallic acid 10 ppm - 1.754 Sample + std. gallic acid 10 ppm 99.896 0.564 Sample + std. gallic acid 20 ppm 100.139 0.884 Sample + std. gallic acid 30 ppm 98.667 0.299
Determination of gallic acid in 95% EtOH extract of A. concinna
The crude extract of A. concinna was measured with the optimized condition and method’s validated. A comparison of peak area of standard compound with extract of A. concinna found gallic acid contained 10.996 µg/mL or 1.042 %w/w in sample The method validation of RP-HPLC to determine gallic acid with the optimized condition using Symmetry Shield RP18 column (4.6 x 250 mm, 5 µm), injection loop 20 µL, flow rate 1 mL/min and detection wavelength at 272 nm at 30 C. Mobile phase were ACN and 0.5% H3PO4. This method has been validated in specificity which exhibited good separation in chromatogram without the effect from matrix. The extract and standard gallic acid were eluted at 13.2 minutes supported this peak was gallic acid. Range of this method over 5-50 µg/mL established a good linear relationship between concentration and peak area (R2 = 0.9998). LOD and LOQ of this method were calculated from the calibration curve found in the concentration of 0.87 and 2.65 µg/mL, respectively. After the standard gallic acid was added in the extract solution, the results showed the acceptable of %recovery in the range of 98.667-
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100.139 %. Furthermore, the precision of the method found the %RSD between 0.299-1.754% which suitable for method to measure the quantitation. In the real sample, six ample of 95% EtOH extract solution of A. concinna were measured in the aforementioned method. Thus, the extract contained gallic acid in the value of 10.996 µg/mL or 1.042 %w/w from a comparison of peak area with the calibration curve.
Conclusion
The validated method to determine gallic acid in A. concinna (Willd.) D.C. has been developed using RP-HPLC. The method is specificity, good linearity and range, accurate and precise to quantitative detection of gallic acid. This phenolic compound is interesting for health industry and this method can apply to search for the other sources of gallic acid in the nature. Acknowledgements
We would to thanks Mrs. Pattra Ahmadi Pirshahid for special guidance. Thanks Thailand Institute of Scientific and Technological Research (TISTR) for supporting.
References
Anjaneyulu, A.S.R., Bapuji, M., Ramachandra Row, L. Sree, A. (1979). Structure of Acacigenin-B, A Novel Triterpene Ester Isolated from Acacia concinna. Phytochemistry, 18, 463-466. Gafur, M.A., Obata, T., Kiuchi, F. Tsuda, Y. (1997). Acacia concinna Sponins. I. Structures of Prasapogenols, Concinnosides A-F, Isolated from the Alkaline Hydrolysate of the Highly Polar Saponin Fraction. Chem. Pharm. Bull., 45, 620-625. ICH. Text on Validation of Analytical Procedures. Volume 60: FDA; 1995 Khalia, S.A., Yagi, S.M., Khristova, P. Duddek, H. (1989). (+)-Catechin-5-galloy Ester as a Novel Natural Polyphenol from the Bark of Acacia nilotica of Sudanese Origin. Planta Medica, 55, 556-558. Kim, H.S., Moon, J.H., Kim, Y.M. Huh, J.Y. (2019). Epigallocatechin Exerts Anti-Obesity Effect in Brown Adipose Tissue. Chem. Biodiversity, 16, e1900347. Kiuchi, F., Gafur, M.A., Obata, T., Tachibana, A. Tsuda, Y. (1997). Acacia concinna Sponins. II. Structures of Monoterpenoid Glycosides in the Alkaline Hydrolysate of Saponin Fraction. Chem. Pharm. Bull., 45, 807-812. Salam, M.M., Davidorf, F.H. Abdel-Rahman, M.H. (2011). In vitro anti-uveal melanoma activity of phenolic compounds from the Egyptian medicinal plant. Fitoterapia, 82, 1279-1284. Sekine, T., Arita, J., Saito, K., Ikegami, F., Okonogi, S. Murakoshi, I. (1989). (+)- Acacialactam, a New Seven-Membered Lactam from the Seeds of Acacia concinna. Chem. Pharm. Bull., 37, 3164-3165. Sekine, T., Fukasawa, N., Ikegami, F., Saito, K., Fujil, Y. Murakoshi, I. (1997). Structure and Synthesis of a New Monoterpenoidal Carboxamide from the Seeds of the Thai Medical Plant Acacia concinna. Chem. Pharm. Bull. 45, 148-151. Tezuka, Y., Honda, K., Banskota, A.H., Thet, M.M. Kadota, S. (2000). Kinmoonosides A- C, Three New Cytotoxicity Spaonins from the Fruits of Acacaia concinna, a Medicinal Plant Collected Myanmar. J. Nat. Prod., 63, 1658-1664. Thompson, M.A. Collins, P.B. (2013). Handbook on Gallic Acid: Natural Occurrences, Antioxidant Properties and Health Implications chapter Antioxidant, Antitumoral and
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Anti-Inflammatory Activities of Gallic Acid. 4th ed. New York: Nova Science Publishers.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.11 International Conference on Food, Agriculture and Biotechnology
PHYTOCHEMICALS AND ANTIOXIDATION OF FRACTIONATED SUGARCANE EXTRACTS: SUPHANBURI 50 VARIETY
PHONGSATHORN MOTHAM, ANSAYA THONPHO, PRASONG SRIHANAM*
Department of Chemistry, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand
*Corresponding author: [email protected]; [email protected]
Abstract:
The objective of this work is to fractionate the Suphanburi 50 sugarcane extract using silica gel column chromatography and screen its phytochemical contents and antioxidation in each fraction. The phytochemicals including total phenolic, flavonoids, saponin, proanthocyanidin and condensed-tannin were varied by the fractions depending on the eluting solvents. Moreover, the antioxidation of the fractionated extracts were also varied following the eluting solvents. All phytochemicals were positively corelated to all antioxidation methods, but in variable values. The obtained results indicated that the sugarcane, Suphanburi 50 variety is a natural good source of phytochemicals and exposed antioxidant activity which would be supported health benefit.
Keywords: Sugarcane; Suphanburi 50; Fractionation; Phytochemical; Antioxidation
Introduction
The medicinal plants including vegetables, fruits, herbs and cereals, have been known as the important sources of secondary metabolites known as phytochemicals (Farhadi et al., 2016). They huge groups such as quinines, phenolics, phytosterols, tannins, flavonoids, alkaloids and saponins (King & Young, 1999; Farhadi et al., 2016). These substances are popularly used for health promotion, personalized treatment and disease prevention worldwide (Sen & Chakraborty, 2017). This was according they have various biological activities such as antioxidant, antibacterial, anti-inflammatory, anti-diabetic and anti-aging (Ignea et al., 2013; Guetat et al., 2017; El‐Gawad et al., 2019; Elshamy et al., 2019). They were proved already for their safety without side effects compared with synthetic substances and effective in medicinal and nutritional applications (Saucedo-Pompa et al., 2018). Many studies have demonstrated that they significantly prevent some diseases, and reduce some effects of reactions (Meng et al., 2012). 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 north-eastern area. The sugarcane composed high content of sucrose. Therefore, the main application of sugarcane is sugar production. However, phytochemicals in sugarcane have also been studied and reported (Duarte-Almeida et al., 2007; Feng et al., 2014; Naowaset & Srihanam, 2017). Maha Sarakham Province, the central of the north-eastern Thailand, have different varieties of sugarcane planted in the Agricultural Research and Development Centre. As literature reviews, the information about phytochemicals in sugarcane, especially in Suphanburi 50 variety never been reported. Therefore, the goal of this work is to prepare crude extract of the sugarcane for screening their phytochemicals and antioxidation. The crude extract was then partial purified by fractionation throughout silica gel column and eluted by various solvent systems. The
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology fractionated extracts were then determined for phytochemicals and antioxidation as comparison with the crude extract.
Materials and Methods
Materials
The sugarcane samples in this work is Suphanburi 50, a popular variety for fresh juice drinking. They were kindly supplied from Agricultural Research and Development Center Mahasarakham, Maha Sarakham, Thailand. The sugarcane samples were cut all leaves, crushed juice and dried in an oven at 60 °C for 18 h. The dried sugarcanes were grinded and kept in a seal bag at room temperature.
Preparation of crude extract
The 1 g of dried sugarcane was weighed and 25 mL of ethanol was then added into the sugarcane samples. 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.
Fractionation of the crude extract
The crude extract was loaded on a 60 cm × 4.5 cm i.d. glass column packed with silica gel (60- 200 mesh). The column was then eluted with the different polarity of solvent mixtures at a flow rate of 1.0 mL/min. The fractions were eluted by starting with ethyl acetate/methanol in the following ratios successively: 100:0, 75:25, 50:50, 25:75 and 0:100. After that 10 mL of each fraction is collected continuously. The absorbance of each tube was measured at 280 nm using a UV–V is spectrophotometer to identify each fraction. Sub-fractions were grouped and pooled before concentration using rotary vacuum evaporator. The obtained residues were dissolved in methanol and stored at - 4 C̊ until analysis.
Total phenolic content
The total phenolic content )TPC( was determined using a modified colorimetric method of Škerget et al. (2005) . A 1 mL of crude solution was mixed with 5 mL of 10% Folin-Cioca lteu 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 1h .Then, the mixture was measured at 765 nm using UV-Vis spectrophotometer .Gallic acid was used as standard and results were expressed as mg GAE/ g DW. Total flavonoid content
The total flavonoid content (TFC) of the bagasse extract was measured using a modified previous method of Jia et al. (1999). 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
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology spectrophotometer. Quercetin was used as standard and results were expressed as mg QE/g DW.
Total saponin content
The total saponin content (TSC) was determined following the method of Hiai et al. (1976). Briefly, A 250 μL of standard solution or methanolic extracts 250 μL of 8% vanillin-etha nol solution were mixed. A 2.5 mL of concentrated H2SO4 (72%) was then added with the mixture and stand in an ice water bath. The mixture solution was warmed at 60C for 15 min, and then cooled in ice-cold water to room temperature. The reaction mixture was measured at 560 nm using a UV-Vis spectrophotometer against a blank. The aescin was used as standard and results were expressed as mg AES/g DW.
Total proanthocyanidin content
The total proanthocyanidin content (TPAC) was analyzed via the procedure of Li et al. (2006). Each 200 μL methanolic extract solution and 1.5 mL of 4% vanillin-ethanol solution was mixed together before adding 750 μL concentrated HCl. After left for 15 min, the mixture was measured at 500 nm using a UV-Vis spectrophotometer. The catechin was used as standard and results were reported as mg CE/g DW.
Total condensed-tannins content
Total condensed-tannins content (CDT) of bagasse extracts was investigated following the modified methods of Chupin et al. (2013). A 0.5 mL of extract was mixed with 4% vanillin- methanol and 1.5 mL of 3 M HCl. The mixture was then stand in dark at room temperature for 15 min before measuring the absorbance at 500 nm. The catechin was used as standard and results were expressed as milligrams mg CE/g DW.
DPPH assay
DPPH assay was carried out to measure the free radical scavenging activity following previous method of Thaipong et al. (2006). 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 equation 1.
DPPH radical scavenging (%) = [(A0–As)/A0] 100 (1) 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 TE/g DW.
ABTS assay
The ABTS assay was performed following the method of Trolox equivalent antioxidant capacity (TEAC) by Berg et al. (1999). 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
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology 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).
Ferric reducing antioxidant potential (FRAP) assay
The FRAP assay was conducted according to previous method (Berg et al., 1999). 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 mM FeSO4/ g DW.
Cupric reducing antioxidant capacity
The cupric reducing antioxidant capacity (CUPRAC) was described by Apak et al. (2004). A -2 -3 500 μL of 10 M CuCl2 solution was mixed with 500 μL 7.5 x 10 M neocuproine solution in ethanol and acetate buffer at pH 7.0. The methanolic extract or standard (x μL) and H2O [(550 - x) μL] were added to the mixture solution. The absorbance was recorded at 450 nm after incubation for 30 min at room temperature using a UV-Vis spectrophotometer. The results were expressed as mg TE/g DW.
Statistical analysis
Results are expressed as the mean ± standard deviation (SD). Determination of correlation (Pearson correlation coefficient, r) on phytochemical and antioxidation was analysed using SPSS software for Windows (version 19).
Results and Discussion
Phytochemical contents
Table 1 showed phytochemical contents found in crude and fractionated extracts. The results indicated that the fractionated extracts found higher contents of TPC, TFC and TSC than crude extract, while the SF2M75 (eluted by ethyl acetate/methanol at 25:75 (v/v)) has higher TPAC than crude extract. In case of CDT, the SF2M75 and SF3M50 (eluted by ethyl acetate/methanol at 50:50 (v/v)) have higher CDT than the crude extract. Beside the fractionated extracts, the SF2M75 showed the highest phytochemical contents than other. Among the phytochemicals, TSC is predominant substance, following TFC and TPC, respectively. The results indicated that almost tested phytochemicals found highly after fractionation. This mean that silica gel column could be used to separate some impurity in the crude extract. The CDT found the lowest content in crude extract, but it found in higher content than TPAC in the fractionated fractions. Moreover, the SF4M75 and SF5M100 not detected of the TPAC. In general, the obtained phytochemical contents varied by the eluting fractions. This may be involved the chemical
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology structure of each substances as well as the variable polarity of mobile phase that used for the fractionation of the crude extract (Zahradníková et al., 2008; Naowaset & Srihanam, 2017). Furthermore, the types and contents of phytochemicals were differed by various factors including instrument, method and procedures analysis (Berli et al., 2012; Feng et al., 2014; Antoniolli et al., 2015).
Table 1: Phytochemical contents in crude and fractionated extracts.
TPC TFC TSC TPAC CDT Samples a (mg GAEa) (mg QE ) (mg AESa) (mg CEa) (mg CEa) Crude 6.640 ± 0.000 4.524 ± 0.081 30.228 ± 0.051 2.030 ± 0.275 1.537 ± 0.696 SF1M0 - - - - - SF2M25 0.520 ± 37.563 69.681 ± 0.818 195.238 ± 2.182 4.022 ± 0.077 13.018 ± 0.215 SF3M50 17.646 ± 1.138 11.575 ± 0.161 49.048 ± 3.299 0.178 ± 0.038 1.778 ± 0.077 SF4M75 10.682 ± 1.217 11.097 ± 0.061 68.907 ± 2.968 ND 0.798 ± 0.133 SF5M100 14.213 ± 0.578 10.230 ± 0.278 176.927 ± 1.427 ND 0.400 ± 0.133 a/g DW ND = not detected
Antioxidation test
The antioxidation of crude and fractionated extracts was shown in Table 2. The results found that the fractionated extracts by methanol 25% (SF2M25) and 50% (v/v) (SF3M50) had higher antioxidation than the crude extract in every tested method. Besides the fractionated extracts, the SF2M25 has the most power antioxidation, and then SF3M50. The SF4M75 and SF5M100 showed lower antioxidation power than the crude extract by DPPH assay, but in higher power by ABTS, FRAP and CUPRAC assays. The radical scavenging activities of the SF2M25 were higher than the crude extract for DPPH and ABTS about 18 folds while metal reducing power activities for FRAP and CUPRAC were about 55 and 30 folds, respectively. The results indicated that the extracts preferred to act as reducing power than radical scavenging. This might be according to the sugarcane extract composed high content of ortho-dihydroxyl polyphenols such as flavonoids and saponin which could be interacted well with Fe2+ via coordinate linkages (Andjelkovic et al., 2006; Moran et al., 1997). Moreover, phenolic compounds which composed high hydroxyl groups are good antioxidant (Xia et al., 2011; Visioli et al., 2011; Guendez et al., 2005; Kim et al., 2006; Katalinić et al., 2010).
Correlation analysis
The correlation extinction between phytochemicals and antioxidant activity of the extracts was shown in Table 3. The TPC has high positively correlated all substances, except TSC. Moreover, it showed high positively correlated to every antioxidation assays. TFC showed similar trend of correlation like the TPC and TPAC, but has slightly higher reducing power than the TPC and the same value to TPAC. Surprise, TSC indicated moderated value of positive correlation to all antioxidation assays while CDT showed high positively correlated to all antioxidation assays, except ABTS. This indicated that all tested substances have synergistic effect on oxidants. From the results, most of phytochemicals showed preferably on reducing power than scavenging activity.
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Table2 : Antioxidation of crude and fractionated extracts.
Samples DPPH ABTS FRAP CUPRAC (IC50 mg/mL) (IC50 mg/mL) (µM Fe2+/g DW) (mg TE/g DW) Crude 19.819 ± 0.094 3.501 ± 0.330 3.511 ± 0.439 1.185 ± 0.016 SF1M0 ND ND ND ND
SF2M25 1.327 ± 0.028 0.212 ± 0.004 167.052 ± 1.228 31.963 ± 2.135
SF3M50 11.215 ± 0.054 1.857 ± 0.022 21.071 ± 0.614 4.972 ± 0.025 SF4M75 14.914 ± 0.083 8.770 ± 0.176 5.034 ± 0.294 1.393 ± 0.092
SF5M100 16.366 ± 0.119 4.515 ± 0.042 11.000 ± 0.291 3.041 ± 0.035
ND = not detected
Table 3: Correlation extinction (r) of phytochemical contents and antioxidation of sugarcane extracts.
List TPC TFC TSC TPAC CDT DPPH ABTS FRAP CUPRAC
TPC 1 0.971** 0.608* 0.976** 0.979** -0.970** -0.805** 0.986** 0.987**
TFC 1 0.638* 0.999** 0.996** -0.953** -0.650* 0.997** 0.994**
TSC 1 0.633* 0.591* -0.422 -0.416 0.631* 0.633* List TPC TFC TSC TPAC CDT DPPH ABTS FRAP CUPRAC
TPAC 1 0.998** -0.959** -0.672* 0.999** 0.995**
CDT 1 -0.973** -0.687* 0.998** 0.995** DPPH 1 0.758** -0.966** -0.964**
ABTS 1 -0.708* -0.718**
FRAP 1 .997**
CUPRAC 1 ** Correlation is significant at the 0.01 level (2-tailed).
* Correlation is significant at the 0.05 level (2-tailed). Conclusion
Fractionation by silica gel column chromatography could be used for partial purification of the crude extract. The fraction eluted by the mixture of ethyl acetate/methanol at 75:25 ratio obtained the highest phytochemical contents. The highest substance found in the Suphanburi
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50 sugarcane extract was TSC, and then TFC and TPC respectively. The phytochemical contents varied by the eluting solvent. All fractionated extracts showed almost higher antioxidation than the crude extract, except SF1M0. The phytochemical contents were positively correlated to all antioxidation assays with higher reducing power than free radical scavenging. This work suggested that the Suphanburi 50 variety of sugarcane is a good source of phytochemicals, expressed high antioxidant potential. This indicated that the extract of this sugarcane might be used as human health supplement.
References
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Guetat, A., Al‐Ghamdi, F.A., & Osman, A.K. (2017). The genus Artemisia L .in the northern region of Saudi Arabia :Essential oil variability and antibacterial activities .Natural Product Research, 31)5 :(598‐603. Hiai, S., Oura, H., & Nakajima, T. (1976). Color reaction of some sapogenins and saponins with vanillin and sulfuric acid. Planta Medica, 29(02), 116-122. Ignea, C., Dorobanţu, C.M., Mintoff, C.P., Branza-Nichita, N., Ladomery, M.R., Kefalas, P., & Chedea, V.S. (2013). Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts .Food Chemistry 141)4(, 3967- 3976 . Jia, Z., Tang, M.C., & Wu, J.M. (1999). The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64(4), 555-559. Katalinić, V., Možina, S.S., Skroza, D., Generalić, I., Abramovič, H., Miloš, M., & Boban, M. (2010). Polyphenolic profile, antioxidant properties and antimicrobial activity of grape skin extracts of 14 Vitis vinifera varieties grown in Dalmatia (Croatia). Food Chemistry, 119(2), 715-723. Kim, S.-Y., Jeong, S.-M., Park, W.-P., Nam, K.C., Ahn, D.U., & Lee, S.-C. (2006). Effect of heating conditions of grape seeds on the antioxidant activity of grape seed extracts. Food Chemistry, 97(3), 472-479. King, A., & G. Young. (1999). Characteristics and occurrences of phenolic phytochemicals . Journal of the American Dietetic Association, 99)9 :(213-218. Li, Y., Guo, C., Yang, J., Wei, J., Xu, J., & Cheng, S. (2006). Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chemistry, 96(2), 254-260. Meng, J.F., Fang, Y.L., Qin, M.Y., Zhuang, X.F., & Zhang, Z.W. (2012). Varietal differences among the phenolic profiles and antioxidant properties of four cultivars of spine grape (Vitis davidii Foex) in Chongyi County (China). Food Chemistry, 134(4), 2049-2056. Moran, F.J., Klucas, R.V., Grayer, R.J., Abian, J., & Becana, M. (1997). Complexes of iron with phenolic compounds from soybean nodules and other legume tissue: prooxidant and antioxidant properties. Free Radical Biology Medicine, 22, 861-870. Naowaset, D. & Srihanam, P. (2017). Phytochemical contents and antioxidant activity of partially purified sugarcane extract by silica gel column. Journal of Science and Technology MSU, Special issue, 444.453- Saucedo-Pompa, S., Torres-Castillo, J.A., Castro-Lόpez, C., Rojas, R., Sánchez-Alejo, E.J., Ngangyo-Heya, M., & Martínez-Ávila, G.C.G. (2018). Moringa plants: Bioactive compounds and promising applications in food products. Food Research International, 111, 438-450. Sen, S., & Chakraborty, R. (2016). Revival, modernization and integration of Indian traditional herbal medicine in clinical practice: importance, challenges and future. Journal of Traditional and Complementary Medicine, 7, 234-244. Škerget, M., Kotnik, P., Hadolin, M., Hraš, A.R., Simonic, M., & Knez, Ž. (2005). Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chemistry, 89, 191-198. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., & Byrne, D.H. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 19, 669- 675. Visioli, F., Lastra, C.A., Andres-Lacueva, C., Aviram, M., Calhau, C., & Cassano, A. (2011). Polyphenols and human health: a prospectus. Critrical Review on Food Science, 51, 524-546.
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Xia, D., Wu, X., Shi, J., Yang, Q., & Zhang, Y. (2011). Phenolic compounds from the edible seeds extract of Chinese Mei (Prunus mume Sieb. et Zucc) and their antimicrobia l activity. LWT - Food Science and Technology, 44(1), 347-349. Zahradníková, L., Schmidt, Š., Sékelyová, Z., & Sekretár, S. (2008). Fractionation and identification of some phenolics extracted from evening primrose seed meal. Czech Journal of Food Sciences, 26, 58-64.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.12 International Conference on Food, Agriculture and Biotechnology
PREPARATION AND CHARACTERIZATION OF WATER HYACINTH CELLULOSE/KERATOSE COMPOSITE FILMS
PATCHARIDA CHAOSRI, PRASONG SRIHANAM*
Department of Chemistry, Faculty of Science, Mahasarakham University, Maha Sarakham 44150, Thailand
*Corresponding Author: [email protected]
Abstract:
This research aimed to extract cellulose from water hyacinth for preparation of composite films with different ratios of hair keratose. The keratose film has more transparency than other films. All films were then investigated their morphology, secondary structure, and thermal properties using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) pectrophotometer, and Thermogravimetric analyzer (TGA), respectively. The results found that the native keratose film has smooth surfaces without phase separation, while the cellulose film has rough and some fibers appeared on its surface. The cellulose/keratose composite films have specific absorption peaks of each polymer functional groups. The secondary structure of the keratose film has a β-pleated sheet resulting in the fragile texture and brittle. Adding cellulose increased flexibility and thermal properties of the keratose film. This was due to the keratose and cellulose formed interaction via functional groups. The result suggested that cellulose and keratose are well compatible together. The finding results might be advantaged to use as basic information to prepare for the cellulose/keratose composite films obtaining desire properties for applications.
Keywords: Film; Cellulose; Keratose; Secondary structure; Thermal property
Introduction
The plastics from petroleum sources are gradually caused by environmental pollution. The people around the world starting to protect this problem in many ways including plastics reducing use (Pico & Barceló, 2019). Development and discovery of biodegradable polymers have been a pervasive interest to replace the plastic from petroleum sources (Liu et al., 2018). Biodegradable polymers are plastics in which could be degraded by the action of living organisms in the environment, such as fungus, bacteria, or various processes in living organisms or environments. In recent year, user-friendly and eco-friendly biopolymer-based materials have been widely proposed, especially for agricultural and marine originated sources of raw materials for biopolymers (Rajabinejad et al., 2018; Brodin et al., 2017; Bertolino et al., 2016; Sagnelli et al., 2016; Kai et al., 2016). Keratose is a structural protein in which the form of keratin obtained by oxidizing extraction (Wang et al., 2017; Rajabinejad et al., 2018). It is water-soluble. The extracted keratoses degrade relatively fast in vivo and have a higher molecular weight in comparison to other types of keratin. The keratoses are highly promising candidates for encapsulation of chemicals for cosmetic, pharmaceutical and biomedical applications due to their non-toxicity, biocompatibility, biodegradability, and non-immunogenicity (Rajabinejad et al., 2018). Cellulose is one type of carbohydrate. It is a homopolymer of glucose linked together via β-1,4-glycosidic bonds (Ul-Islam et al., 2012). The structure of cellulose is a mixture of hemicellulose (20-30%) and lignin (15-30%) to form the complexation structure (Lee et al.,
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2008). Recently, the cellulose is widely applied in various industries such as food (Klemm et al., 2005), pharmacy (Shokri & Adibkia, 2013), pulp and paper (Singh et al., 2016), water pollution treatment (Peng et al., 2019), or wine and beer (Chakraborty et al., 2016). The important source of cellulose is the cell wall of the plant (Bledzki & Gassan, 1999). In this work, the cellulose was extracted from water hyacinth, a fresh plant that rapidly grown and widely spread around the world (Wissel et al., 2008). The water hyacinth is a virulent cause of water pollution (Sundari & Ramesh, 2012). (Mochochoko et al., 2013; Juárez-Luna et al., 2019). However, it composed of a high content of cellulose (Zhu et al., 2009). The goal of this work is to extract keratose from human hair and cellulose from the water hyacinth for the preparation of composite films. The films were then characterized and discussed to assess the possible way for the application of both biodegradable materials.
Materials and methods
Preparation of water hyacinth cellulose
The water hyacinth samples were collected in the Mahasarakham University then washed with tap water before cutting into small pieces. The samples were dried in an oven for 24 h and then crushed into powder. The cellulose was extracted according to previously reported (Wissel et al., 2008) with some modifications. The 10 g of water hyacinth powder was digested by 100 mL 5% NaOH (w/v) with stirring and warming for 3 h. The mixture was then washed with distilled water until neutral. After dried at 90°C for 24 h, the dried sample was then bleached by 5% NaOCl (w/v) at room temperature for 24 h. The bleached samples were washed to neutral and dried at 90 °C for 24 h. Finally, the samples were hydrolyzed by 5% H2SO4 at 60 °C for 8 h to obtain the cellulose solution. The cellulose solution was then stirred, washed with distilled water, and filtered before use.
Preparation of keratose solution
Human hair was collected from student volunteer in Mahasarakham University, Maha Sarakham Province, Thailand. The hair was warmed to 40C before immersing in n-hexane for 12 h to remove some lipids. Hexane, NaOH, peracetic acid, and Tris (hydroxymethyl) aminomethane used for the oxidizing extraction, and ethyl acetate used for the microparticles process, were purchased from Sigma-Aldrich®. Keratose was obtained from human hair by using oxidizing extraction followed the method previously reported (Rajabinejad et al., 2018) with some modification. The hair samples were extracted with peracetic acid with the ratio of hair: oxidizing solution of 1:30 (w/v). The mixture of hair and oxidizing reagent was left in a thermostatic water bath for 24 h at 25°C. The hair was washed to remove unreacted reagent, washed with distilled water and dried at 90°C for 1 h. The hair was then vigorously shaken in TRIS 1 M at 37°C for 2 h. The resulting solution of keratoses was filtered with a dialysis membrane (MW cut off = 3 kDa), neutralized with sodium hydroxide, and dialyzed for 3 days against distilled water. At the end of the dialysis, the solution was centrifuged to remove insoluble hair. The concentration of extracted keratose was checked by the evaporation technique and adjusted to 1.5% (w/v) with distilled water.
Preparation of cellulose/keratose composite films
The cellulose (CE) and keratose (KE) solutions with different ratios (4:0, 3:1, 1:1, 1:3, and 0:4) were prepared and stirred homogeneously for 30 min. The mixture was then cast onto a 4.5 cm
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology diameter petri dish followed by solvent evaporation at room temperature for 24 h. The films were peeled off. The obtained films were placed 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.
Secondary structure investigation
The secondary structure of films was analyzed by Fourier transform infrared (FTIR) spectroscopy (Perkin Elmer-Spectrum Gx, USA) in the spectral region of 4000-400 cm-1 at 4 cm-1 spectral resolution and 32 scans with air as reference. Before analysis, the KBr disk method was used for sample preparation.
Thermal property analysis
A thermogravimetric analyzer (TGA) (SDTQ600, TA-Instrument Co. Ltd., New Castle, DE, USA) was used for thermal stability investigation of the microparticles both with and without sample drug. Simply, 3-5 mg microparticle was heated from 50-1000C with 20 C/min of heating rate under nitrogen atmosphere.
Results
Transparency of films
Figure 1 shows the transparency of all films. The results indicated that the cellulose (EC) (Figure 1a) had white, dense, and lower transparency than brown keratose (KE) film (Figure 1b). The transparency and color of CE/KE composite films varied following the ratio of native polymers. All composites films have higher transparency than CE, but in lower than KE film.
Morphology of films
Figures 2 and 3 show morphology both surface (a) and cross-section (b) of films. At low magnification (Figure 2), the native CE film has a rough surface according to the random woven of fiber with clearly observed at high magnification (Figure 3). However, the CE film formed dense texture without separation as revealing by cross-section (b). The KE film has dense in texture without cracks. At high magnification, film still has a dense texture with many pores in small sizes. Considering the CE/KE composite films, the morphology of each ratio varied by the content of the polymer. At high content of cellulose (3:1), the film has a rough surface and appears fibers woven together. Increasing magnification, the texture found various sizes of fibers at the edge of film. At the equal ratio (1:1), the composite film has a smooth surface but in lower than the KE film. Moreover, the texture of the film has a small size of particles embedded in its surface. At high content of keratose (1:3), the morphology of film was similar but smoother than the 1:1 ratio. However, the film has a crack in texture at high magnification (Figure 3).
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Figure 1: Transparency of films; CE (a), KE (b), CE/KE composite at 3:1 (c), 1:1 (d) and 1:3 (e) ratios.
Figure 2: SEM micrographs of surface (I) and cross-section (II) of films; CE (a), KE (b), CE/KE composite at 3:1 (c), 1:1 (d) and 1:3 (e) using 200X magnification.
Figure 3: SEM micrographs of surface (I) and cross-section (II) of films; CE (a), KE (b), CE/KE composite at 3:1 (c), 1:1 (d) and 1:3 (e) using 2000X magnification. Secondary structure of films
Figure 4 shows FTIR spectra of the films. The CE film indicated the absorption peaks at 3339, 2899, 1680-1519 , 1057 cm-1 (Figure 4a) while the absorption peaks of KE film appeared at 3205, 1678 -1519, 1042 cm-1 (Figure 4e). The CE/KE composite films (Figure 4b,c,d) showed variable absorption peaks depending on the content of cellulose or keratose.
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Figure 4: FTIR spectra of of films; CE (a), KE (e), CE/KE composite at 3:1 (b), 1:1 (c) and 1:3 (d).
Thermal property of films
Figure 5 shows the TG curves of films. All prepared films have decomposition peaks of more than 3 points. The first point is less than 100 C, second is a range 150-300 C and the third is a range of 300-400 C. The maximum temperature of decomposition rate (Td, max) of films was clearly observed by DTG curves as shown in Figure 6.
Figure 5: TG curves of films; CE (a), KE (e), CE/KE composite at 3:1 (b), 1:1 (c) and 1:3 (d).
Discussion and Conclusion
Water hyacinth is a water pollutant and caused a big problem with water pollution worldwide. It composed of the high content of cellulose which has been interested in applications. The cellulose showed good properties such as high strength, heat durability, and biodegradability. Moreover, it could be mixed with other polymers to prove some properties including poly (vinylidene fluoride) or PVDF (Zhang, 2012), silk fibroin (Zhou, 2013), and chitosan (Abdul Khalil et al., 2016). SEM images indicated that the texture of cellulose film linked together without phase separation. This morphology was similar to the previous report (Qua et al., 2011). However, the consistency of fiber was lower than the amino acid components in keratose. The homogeneous size of amino acids in keratose supported the transparency and
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology smooth surfaces of the film. Considering the CE/KE composite films, the difference types and ratios used were the main factor on the film morphology.
Figure 6: DTG curves of films; CE (a), KE (e), CE/KE composite at 3:1 (b), 1:1 (c) and 1:3 (d).
With FTIR spectra, cellulose film showed peaks of hydroxyl group1 (-OH) at 3700- 3000 cm-, carbonyl group (C=O) at 1700-1800 cm-1 (Karatzos et al., 2012), and the methyl -1 group (-CH2) at 2900-2800 cm (Fan et al., 2012). Furthermore, the peaks of hemicellulose at 1735 cm-1 and lignin at 1240 cm-1 (Sun et al., 2020) were also observed. The keratose film showed absorption peaks of the amide group (R-COONH-R), which the main bonding of amino acids. Amide I (1700-1600 cm-1) is the responsible peak of the carbonyl group (-CO-), amide II (1600-1500 cm-1) is responsible peak of amine group (-NH-) and methyl group (-CH-), and amide III (1300-1150 cm-1) is a responsible peak of -CN- stretching, plane -NH-, -C-C- and - CO- stretching (Sharma et al., 2017). The absorption peak at 1040 cm-1 confirmed cysteic acid group of the oxidized keratin (keratose) (Pakkaner et al., 2019). The absorption peaks from FTIR spectra indicated that the keratose film has a β-sheet structure. This film has rapidly brittle and very hard to manual. The high content of hydroxyl groups in cellulose supported the hydrophilic property of the film. This helped to increase the flexibility of the film. The mixture of cellulose and keratose resulted to change of the absorption peaks of the main functional groups. However, they still appeared in all of the main groups which varied by the ratio of the polymer. The keratose supported the strength of cellulose film while cellulose helped to decrease the brittle of keratose. This suggested that cellulose and keratose were good compatibilities via interaction formation such as hydrogen bonds, hydrophobic interaction, and van der valve force. Films showed decomposition peaks at least 3 points. At lower 100 °C is caused by water evaporation in polymer molecules (Dou et al., 2019). The temperatures in the range of 150-300 °C are the decomposition point of hemicellulose and lignin and 300-400 °C are the decomposition of cellulose chain. The keratose showed a decomposition point in the range of 250-300 °C (Rajabinejad et al., 2018). The composite films showed variable decomposition points depending on the ratio used as like as the thermal property. Cellulose content helped to increase the thermal property of the films. However, the highest Td, max found in the composite film at 1:1 ratio. This result confirmed the compatibility of the cellulose and keratose. In conclusion, the properties of films depended on many factors such as type of polymer, the mixed ratio as well as the preparation methods. The results obtained from this
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology work suggested that the cellulose could be extracted from the wastewater hyacinth. This cellulose could also be used as a biodegradable polymer for blending with keratose or other materials for value-added and replaced of non-degradable polymer like synthetic plastics.
Acknowledgement
This research was financially supported by Faculty of Science, Mahasarakham University (Grant year 2020).
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Liu, R., Dai, L., Zou, Z. & Si, C. (2018). Drug-loaded poly (L-lactide)/lignin stereocomplex film for enhancing stability and sustained release of trans-resveratrol. International Journal of Biological Macromolecule, 119, 1129-1136. Mochochoko, T., Oluwafemi, O.S., Jumbam, D.N. & Songca. S.P. (2013). Green synthesis of silver nanoparticles using cellulose extracted from an aquatic weed; water hyacinth. Carbohydrate Polymers, 98, 290-294. Pakkaner, E., Yalçın, D., Uysal, B. & Top, A. (2019). Self-assembly behavior of the keratose proteins extracted from oxidized Ovis aries wool fibers. International Journal of Biological Macromolecules, 125, 1008-1015. Peng, B., Yao, Z., Wang, X., Crombeen, M., Gsweene, D. & Tam, K.C. (2019). Cellulose- based materials in wastewater treatment of petroleum industry. Green Energy & Environment, 5, 37-49. Picó, Y. & Barceló, D. (2019). Analysis and prevention of microplastics pollution in water: current perspectives and future directions. ACS, 4, 6709-6719. Qua, E.H., Hornsby, P.R., Sharma, H.S.S. & Lyons, G. (2011). Preparation and characterisation of cellulose nanofibres. Journal of Materials Science, 46, 6029-6045. Rajabinejad, H., Patrucco, A., Caringella, R., Montarsolo, A., Zoccola, M. & Davide Pozzo, P. (2018). Preparation of keratin-based microcapsules for encapsulation of hydrophilic molecules. Ultrasonics Sonochemistry, 40, 527-532. Rajabinejad, H., Patrucco, A., Montarsolo, A., Rovero, G. & Tonin, C. (2018). Physicochemical properties of keratin extracted from wool by various methods. Textile Research Journal, 88, 2018. Sagnelli, D., Hebelstrup, K.H., Leroy, E., Rolland-Sabaté, A., Guilois, S., Kirkensgaard, J.J.K., Mortensen, K., Lourdin, D. & Blennow A. (2016). Plant-crafted starches for bioplastics production. Carbohydrate Polymers. 152, 398-408. Sharma, S., Gupta, A., Chik, S.M.S.T., Kee, C.G., Mistry, B.M., Kim, D.H. & Sharma, G. (2017). Characterization of keratin microparticles from feather biomass with potent antioxidant and anticancer activities. International Journal of Biological Macromolecules, 104, 189-196. Shokri, J. & Adibkia, K. (2013). Application of cellulose and cellulose derivatives in pharmaceutical industries. Cellulose - medical, pharmaceutical and electronic applications, In Tech, pp. 47. Singh, S., Singh, V.K., Aamir, M., Dubey, M.K., Patel, J.S., Upadhyay, R.S. & Gupta, V.K. (2016). Cellulase in Pulp and Paper Industry. New and Future Developments in Microbial Biotechnology and Bioengineering, pp. 152-162. Sundari, M.T. & Ramesh, A. (2012). Isolation and characterization of cellulose nanofibers from the aquatic weed water hyacinth-Eichhornia crassipes. Carbohydrate Polymers, 87, 1701-1705. Sun, D., Onyianta, A.J., O’Rourke, D., Perrin, G., Popescu, C.-M., Saw, L.H., Cai, Z. & Dorris, M. (2020). A process for deriving high quality cellulose nanofibrils from water hyacinth invasive species. Cellulose, 27, 3727-3740. Ul-Islam, M., T. Khan, & J.K. Park. (2012). Water holding and release properties of bacterial cellulose obtained by in situ and ex situ modification. Carbohydrate Polymers. 88(2), 596-603. Wang, J., Hao, S., Luo, T., Zhou, T., Yang, X. & Wang, B. (2017). Keratose/poly (vinyl alcohol) blended nanofibers: Fabrication and biocompatibility assessment. Mater. Sci. Eng. C 72, 212-219. Wissel, H., Mayr, C. & Lücke, A. (2008). A new approach for the isolation of cellulose from aquatic plant tissue and freshwater sediments for stable isotope analysis. Organic Geochemistry, 39, 1545-1561.
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Zhang, X., Feng, J., Liu, X. & Zhu, J. (2012). Preparation and characterization of regenerated cellulose/poly (vinylidene fluoride) (PVDF) blend films. Carbohydrate Polymers, 89(1), 67-71. Zhou, L., Wang, Q., Wen, J., Chen, X. & Shao, Z. (2013). Preparation and characterization of transparent silk fibroin/cellulose blend films. Polymer, 54(18), 5035-5042. Zhu, Y.L., Zayed A.M., Qian, J.H., de Souza, M. & Terry, N. (1999). Phytoaccumulation of trace elements by wetland plants: II. Water hyacinth. Journal of Environmental Quality, 28, 339e344.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.13 International Conference on Food, Agriculture and Biotechnology
PRO-INFLAMMATION CYTOKINE SECRETION OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY EDIBLE MUSHROOM
PATTRA AHMADI PIRSHAHID1, SINEE SIRICOON1, CHIRAMET AURANWIWAT1, WIRIYAPORN SUMSAKUL1, WARAPORN SORNDECH1, THONGKORN PLOYPETCHARA1, KWANCHAI RATTANAMANEE2
1Expert Centre of Innovative Health Food, Thailand Institute of Scientific and Technological Research, Pathum Thani 12120, Thailand 2Naresuan University, Phitsanulok 65000, Thailand
*Corresponding author: [email protected]
Abstract:
Inflammation is a natural process of the innate immune system that associated with the increase in the level of proinflammatory cytokines including tumor necrosis factor- alpha ( TNF- α) , interleukin 1β ( IL- 1β) and interleukin 6 ( IL- 6) . Prolonged inflammation, known as chronic inflammation, related to many chronic diseases such as autoimmune diseases, wound healing, rheumatoid arthritis and cardiovascular disease. The present study aims to investigate the effect of mushroom extracts on the cytokines secretion from lipopolysaccharide ( LPS) stimulated peripheral blood mononuclear cells (PBMCs) involved in immune regulation. The secretion of TNF- α, IL- 1β and IL- 6 was measured by ELISA. The ethanol extract of all mushroom significantly reduced the production of TNF- α. Furthermore, Lentinula edodes, Isaria tennipes, Pleurotus osttreatus and Lentinus squarrosulas Mont. were decreased IL- 1β level and Pleurotus osttreatus, Isaria tennipes and Lentinus squarrosulas Mont. were significantly suppressed IL- 6 secretion in LPS- treated PBMCs. The application of these extracts showed important anti- inflammatory activity which involved in the treatment and prevention of inflammation and associated diseases.
Keywords: Cytokine; Anti-inflammatory activity; ELISA; Mushroom
Introduction
Inflammation is a complex biological process of the body that response to pathogens, damaged cells, irradiation, toxins, heat, or any other cause (Elsayed et al., 2014). It is a protective attempt that is characterized by pain, redness, heat, swelling, disturbance of function and inflammation causes fever (Javed et al., 2019). The releasing of pro-inflammatory cytokines is occurred after the inflammatory response. Cytokine is a polypeptide which is created and secreted by body cells. It plays an important role in both non-specific and specific immunity. For specific immunity, cytokines are released after T lymphocyte and most of the non-specific immunity cytokines are secreted from mononuclear phagocyte but it also stimulated by T lymphocyte . Hence, pro-inflammatory cytokines are secreted in conditions that stimulate the immune system, such as infectious and most of the cytokines that are released are interleukins (ILs), tumor necrosis factors (TNFs), and interferon (IFN)-γ (Luo & Zheng, 2016). Nowadays, several studies which are related to anti-inflammatory activity of various medicinal plant and fungi are reported. Mushroom is one of the most common fungi grown on every continent except Antarctica and grows throughout the year (Bellettini et al. 2019). This plant is the conspicuous
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology umbrella-shaped fruiting body (sporophore) of certain fungi belonging to Basidiomycetes and Ascomycetes. Several bioactive compositions and nutritional values were found in mushrooms and therapeutic benefits of mushrooms specific bioactive nutraceuticals have been investigated. The antitumor, immunomodulatory and anti-inflammatory activities of mushrooms extracts were the most investigated health benefits by researchers (Ma et al. 2018). Several studies reported the bioactive properties of mushroom extracts such as anti-inflammatoryactivities (Choi et al. 2014; Han et al. 2013; Taofiq et al. 2015), antitumour/anticancer (Sliva et al. 2102; Zong et al. 2012; Carocho M & Ferreira. 2013), antioxidant (Ferreira et al. 2009; Heleno et al. 2015; Mu et al. 2012), immunomodulatory (Yamanaka et al. 2012; Han et al. 2011; Enshasy & Hatti-Kaul.2013), antibacterial and antiviral (Adotey et al. 2011; Alves et al. 2012; Alves et al. 2013). The aim of the present study was to investigate the in vitro pro-Inflammation cytokine secretion of mushroom. In order to contribute the knowledge concerning benefit of mushroom extracts on medical purpose as well as for economical purpose in the future.
Materials and Methods
Mushroom extraction
The plant samples were washed with fresh water, sliced and crushed. Cleaned mushroom samples were transferred to oven (MEMMERT) for drying. The 100 g of dried samples were macerated into 95% ethanol at room temperature. Then the extracts were filtered using the filter paper, and the filtrate was furthered for evaporation to remove the solvent through a rotary evaporator. The residual crude distilled extracts were freeze dried and weighed. The samples were stored at -20°C for further study.
Isolation of murine PBMC
The peripheral blood mononuclear cells (PBMC) of mice were isolated from EDTA-treated venous blood by Ficoll density gradient centrifugation method. The sample was diluted 1:1 with sterile phosphate-buffered saline (PBS), layered over Ficoll-Hypaque (Sigma-Aldrich, U.S.A.), and centrifuged at 400xg for 15 min at room temperature. The supernatant was removed and the pellet was resuspended in 6 ml of PBS. The mixture was centrifuged at 400xg for 10 min at room temperature and the supernatant was removed by pipetting. Finally, the cell pellet was resuspended in RPMI 1640 (Gibco Laboratories, Grand Island, N.Y., U.S.A.), supplemented with 1% antibiotic-antimycotic (Invitrogen, U.S.A.), 2 mM glutamine (Invitrogen, U.S.A.), and 10% fetal bovine serum (Invitrogen, U.S.A.).
Culture of murine PBMC
PBMCs were cultured in RPMI 1640, supplemented with 1% antibiotic-antimycotic, 2 mM glutamine, and 10% fetal bovine serum at 37°C with 5% CO2. Cell count and viability were measured by trypan blue exclusion assay.
Measurement of Pro-inflammatory cytokine
To determine the effect of the mushroom extracts in vitro, PBMCs were seeded into a 24-well 6 plate with the density of 1×10 cells/well and cultured at 37°C with 5% CO2 for 24 h. On the next day, when the cells reach confluence, the cells were treated with mushroom extracts at concentrations of 0.001, 0.01, 0.1, 1 mg/ml and dexamethasone (1 µM) with another two more
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology wells left untreated. After 2 h, all treated wells and an untreated well were supplemented with Lipopolysaccharide (LPS; 10 nM) to encourage inflammation. An untreated well without LPS remained as the control group. Cultures were incubated in a humidified atmosphere of 37°C with 5% CO2 overnight. The supernatant was collected for quantitative analysis of proinflammatory cytokines (IL-1β, IL-6 and TNF- α) using an enzyme linked immunosorbent assay kit with a specific antibody to each cytokine according to the manufacturer’s protocol (BioLegend, Canada). Briefly, supernatants from treated samples and control were added to each well of 96-well plate coated with the capture antibody against the respective cytokine provided in the kit and all incubation steps were performed at room temperature. The optical density at 450 nm was measured with microplate reader Greiner bio-one, Germany). Consequently, the graph of the level of cytokines against concentrations of treatment was plotted.
Results and Discussion
Extraction yield
The percentage of mushroom yields in ethanolic extract were shown in Table 1.
Table 1: The percentage of ethanolic extract content of the mushroom extract.
Plant name Amount of Amount of extracts % yield extracts (g) obtained (g) Lentinula edodes 100 23.67 23.67 Pleurotus osttreatus 100 7.44 7.44 Isaria tennipes 100 11.73 11.73 Lentinus squarrosulas Mont. 100 14.2 14.2 Agaricus Blazei Murrill 100 21.34 21.34
Measurement of Pro-inflammatory cytokine
To assess the pro-inflammatory effects of mushroom extracts. Cytokine expression of LPS- stimulated PBMC was measured by ELISA method. LPS treatment alone significantly released IL-1β, IL-6 and TNF- α in PBMCs. The effect of mushroom extracts on the production of IL- 1β, IL-6 and TNF- α were studied at different concentration (0.001, 0.01, 0.1 and 1 mg/ml). The results showed that all mushroom extracts were significant reduction in TNF- α at concentration 0.1 and 1 mg/ml (Figure 1A). Lentinula edodes and Isaria tennipes were slightly suppressed production of IL-1β at concentration 0.1 and 1 mg/ml, while Pleurotus osttreatus and Lentinus squarrosulas Mont. were significantly decreased the secretion of IL-1β at concentration 0.001, 0.1 and 1 mg/ml (Figure 1B). Furthermore, IL-6 secretion reduced after treat with Pleurotus osttreatus, Isaria tennipes and Lentinus squarrosulas Mont. at all concentration compared to control culture (Figure 1C).
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(A)
(B)
(C)
Figure 1: Production of TNF- α (A), IL-1β (B) and IL-6 (C) were measured in the supernatant of LPS-stimulated PBMC by ELISA with specific antibodies to each cytokine. M4 = Lentinula edodes, M5= Pleurotus osttreatus, M6 = Isaria tennipes, M7 = Lentinus squarrosulas Mont., M8 = Agaricus Blazei Murrill, LPS = Lipopolysaccharide, dex = dexamethasone. This study evaluated the production of pro- inflammatory cytokines including IL- 1β, IL-6 and TNF- α by murine PBMC after in vitro treat with mushroom extracts. It is well known
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology that LPS activates the signaling pathway such as NF- kB via the stimulation of Toll- like receptor 4 ( TLR4) ( Muniandy et al. 2018) which can induce inflammatory response that is characterized by increased pro- inflammatory cytokines ( Lu et al. 2018) . Cytokines and mediators are essential for the maintenance of optimum inner environment of our system from injurious agents and the pro-inflammatory play a key role in inflammation by its chemotactic and vasoactivator properties. IL- 1β, IL- 6 and TNF- α are pro- inflammatory mainly released by macrophages, and involved in inflammatory processes in many diseases such as rheumatoid arthritis ( Lee et al. 2012; Assaf et al. 2016) . In this study, we found that all mushroom extracts significantly decreased TNF- 훼 level, Lentinula edodes, Isaria tennipes, Pleurotus osttreatus and Lentinus squarrosulas Mont. were reduced production of IL-1β and Pleurotus osttreatus, Isaria tennipes and Lentinus squarrosulas Mont. were significantly suppressed IL-6 secretion. From this result showed that mushroom extracts had anti-inflammatory effects on LPS-stimulated PBMC.
Conclusion
These results showed that mushroom extracts have significant anti-inflammatory effects in vitro through reduction of LPS-induced pro-inflammatory mediators. All the conducted experiments in the present study have demonstrated that ethanolic extract of mushroom reduce the level of pro-inflammatory cytokines that participated in the prolonging of chronic inflammation. Moreover, further investigation about different model of in vitro and in vivo are important to investigate the potential application of these extracts and need to be assessed prior to clinical treatment of inflammation in variety of diseases caused by a serve immune response.
Acknowledgements
The authors gratefully acknowledges the financial support provided by TISTR for providing Research scholarship.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.14 International Conference on Food, Agriculture and Biotechnology
QUALITY CHARACTERISTICS OF REDUCED-FAT VIENNA SAUSAGE USING RICE FLOUR AND SKIM MILK POWDER MIXTURE AS A FAT REPLACER
SAKUNWIWAT, W, SRIPUI, J, ROJANAKORN, T*
Department of Food Technology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
*Corresponding author: [email protected]
Abstract:
The present work investigated the quality characteristics of Vienna sausage containing a mixture of rice flour and skim milk powder (RFSKM) as a fat replacer. Six formulations of Vienna sausage with different amounts of RFSKM (0, 20, 40, 60, 80 and 100%) were produced and evaluated for physicochemical properties and sensory quality. The addition of RFSKM to the sausage significantly increased emulsion stability, viscosity, moisture, and protein content and lowered fat content, calorific value, and cooking loss (p≤0.05). A rise in RFSKM level led to a significant increase in hardness and chewiness and a reduction of lightness of the sausages. Most sensory likability scores of sausage samples with up to 40% RFSKM were comparable to those of the full-fat control sample. The reduced-fat Vienna sausage with acceptable sensory attributes and lower calories can, therefore, be successfully developed with the incorporation of 40% RFSKM.
Keywords: Vienna sausage; Rice flour; Skim milk powder; Fat replacer
Introduction
Vienna is one of the emulsion-type sausages, which is very popular among Thai people due to its unique flavor and taste. Vienna sausage is a good source of protein. However, it contains high fat and low fiber content. Therefore, frequent consumption of Vienna sausage may thus be associated with obesity, cardiovascular diseases, and arterial hypertension (Olanwanit & Rojanakorn, 2019). Fat, however, provides many gratifying characteristics to Vienna sausages, including upgrading tenderness, juiciness and palatability, reducing cooking loss and stabilizing meat emulsions (Olanwanit & Rojanakorn, 2019). Consequently, a reduction of fat in Vienna sausage formulations may lead to lowered consumer satisfaction. The production of reduced-fat emulsion type sausages with good texture and high sensory acceptability can be performed by using fat replacers (Olanwanit & Rojanakorn, 2019). Fat replacers are compounds that use to give some or all of the functional properties of fat while providing fewer calories than the fat being replaced. They are classified in to 2 groups including fat substitutes and fat mimetics. Fat substitutes are lipid-like substances intended to replace fats on a one-to- one basis whereas fat mimetics are protein or carbohydrate ingredients which function by imitating the physical, textural mouth feel such as juiciness and tenderness, and organoleptic properties of real fats (Shaltout & Youssef, 2007). Fat replacers such as carbohydrate-based materials (Aktaş & Gençcelep, 2006) and protein-based materials (Osburn et al., 1997). In addition, a combination of carbohydrate and protein - i.e., a combination of wheat fiber and pig skin (Choe et al., 2013), a mixture of green banana flour and pork skin (Alves et al., 2016) or a mixture of Man sao powder and hydrolyzed collagen (Olanwanit & Rojanakorn, 2019) was
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology reported to improve the physicochemical and sensory properties of reduced-fat emulsion-type sausages. Rice flour is one of the most suitable ingredients for many food products due to its hypoallergenic properties, low fat and sodium content, mild flavor and pale appearance (Mancebo et al., 2015). The major components of rice flour are protein and starch, with approximately 8 and 80%, respectively (Hur et al., 2011). It has been reported that starch and protein in rice flour showed high water binding ability (Torbica et al., 2012). Rice flour has the potential to be used in meat industry due to its unique gelatinization properties and water binding capacity. This can retain water during the cooking process and subsequent improve the juiciness, tenderness and mouthfeel of reduced-fat food products (Chun Yoo, 2004). Therefore, rice flour is one of the potential materials that can be used as a fat replacer. Skim Milk Powder (SMP) is the product resulting from the partial removal of fat and water from pasteurized milk. It is classified as a high protein-low fat content product (Marafon et al., 2011). The major protein in skim milk powder is casein which possesses high water holding capacity and solubility and provides juiciness and tenderness to the food products that it is added (Holt et al., 2013). Therefore, skim milk powder could be used as a fat replacer. However, to our knowledge, studies focusing on the use of rice flour, skim milk powder, and water mixture as a fat replacer in emulsion-type sausages are very limited despite its potential. Therefore, the objective of this study was to determine the effect of incorporating a mixture of rice flour and skim milk powder (RFSKM) as a fat replacer on quality characteristics of Vienna sausage.
Materials and Methods
Preparation of The Rice Flour, Water, And Skim Milk Powder Mixture (RFSKM)
RFSKM was prepared by mixing rice flour, skim milk powder and distilled water in a 2:0.5: 1.35 ratio using a blender. This ratio was based on the findings of a preliminary study. Based on proximate analysis, RFSKM showed 38.56% moisture, 22.84% protein, 0.52% fat and 1.95% ash.
Production of Vienna Sausage
Six formulations of Vienna sausage with different amounts of RFSKM as a fat replacer (0, 20, 40, 60, 80 and 100% by weight) were prepared as per Olanwanit Rojanakorn (2019) with some modifications. Fresh pork shoulder and pork back-fat used in this study were purchased from the local market in Khon Kaen province. After removing visible fat and connective tissue, the lean meat was ground twice through a 5-mm and 2.5-mm plate, respectively, using a meat grinder (BIRO®, USA). The partially frozen pork back-fat was also ground using the same conditions. The ground pork plus ¼ parts of ice cubes were chopped in a silent cutter (cutter M11N Maschinen, Germany) at high speed for 1 min. Some additives including 1.5% sodium chloride, 0.3% sodium tripolyphosphate, 80 ppm sodium nitrite and ¼ parts of ice cubes were added to the ground pork and chopped at high speed for 2 min. After that 0.1% sodium ascorbate, 1% spices and seasoning, 0.25% monosodium glutamate, and ¼ parts of ice cubes were added to the comminuted meat and further chopped at high speed for 1 min. RFSKM and ground pork fat at test levels, as well as the remaining ice were added to the meat mixture and additionally chopped for 3 min at high speed. The temperature of the raw emulsion or meat batter was maintained below 10±1°C and monitored with a digital temperature probe (Kane- May, KM330, Harlow, Germany) during sausage preparation. One part of raw batter from each sausage formulation was randomly taken out and stored in the dark at 4±1◦C for emulsion
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology stability and viscosity analyses, which were done within 18 h. The other part of the raw batter was stuffed into a 14-mm-diameter plastic casing (LEM products, Canada) using a TWF-12 stuffer (DICK, Germany) and hand-linked at 10-cm intervals. All raw sausage samples were cooked in 80±1°C water bath until the internal temperature reached 72°C. The cooked samples were immediately cooled in an ice bath, vacuum packed, and stored at 4±1°C for one night prior to analyses.
Quality Analyses of The Sausage Samples
The stability of raw emulsion or raw batter was determined as per Colmenero, Ayo, and Carballo (2005). In brief, 20 g of raw batter were filled in pre-weighted plastic tubes (50 mL), centrifuged (5,000 g, 30 min at 5°C) and heated at 70±1°C for 30 min in a water bath. The tubes were cooled to 4±1°C. The total amount of fluid separated was collected and expressed as a percentage of sample weight. The WHC of all raw batter was determined as per Yang et al. (2007) with some modifications. Briefly, 15 g of raw batter samples was placed in a 250 mL plastic tube and centrifuged (Sorvall® RC6 Plus, Themo Electron Corporation USA) at 4°C for 20 min at 6,000 g. Then the samples were heated in a water bath at 85°C for 20 min, cooled to room temper- ature. After cooling, the samples were centrifuged again at 4°C for 20 min at 6,000 g. The water released from the samples was separated from the plastic tubes and weighed. The weight of the sample after water separation was also recorded. The WHC was calculated as follows:
WHC (%) = [1–(BW–AW)/WC] × 100
BW = weight of sample before heating and centrifugation (g)
AW = weight of sample after heating and centrifugation (g)
WC = weight of water content in the sample (g)
A Brookfield viscometer (Model RVT, Brookfield Engineering, USA) with spindle number 7 was used to measure the viscosity of 100 g of the raw batter from each sausage formulation. After 30 s of spindle rotation in the batter sample, a reading was taken (Shand, 2000). The viscosity of the raw batter was expressed as centipoise (cP). The temperature during viscosity measurement for each sample was also recorded (28±1°C). The cooking loss was determined by weight difference before and after cooking of 5 links of sausages from each sausage formulation in a water bath (80±1°C) until 72°C of core temperature was reached (Andrés, Garcıa,́ Zaritzky, and Califano, 2006). The proximate composition (moisture, fat, protein, and ash) of all cooked sausage samples was determined as per AOAC procedures (AOAC, 2000). Carbohydrate was calculated by difference method. Total calories (kcal) were calculated for 100 g samples using the Atwater values corresponding to fat (9 kcal g-1), protein (4.02 kcal g-1), and carbohydrates (3.87 kcal g-1) (Luisa García, Cáceres, and Dolores Selgas, 2006). The color of fresh cut cross-sections from all cooked sausage samples (9 per treatment) was measured using a Hunter colorimeter (Hunter Lab Ultrascan XE, USA) calibrated according to the guideline provided by the instrument supplier and the results were expressed as L* (lightness), a* (redness), and b* (yellowness). Texture profile analysis (TPA) of the cooked sausages were evaluated using texture analyzer (TA-XT2i, Stable Micro Systems, Surrey, UK) according to the method of Wimontham and Rojanakorn (2016) with slight modifications. Nine cylindrical slices (each 14
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology mm diameter, 15 mm long) from each sausage formulation were subjected to a two-cycle compression test using a 25 kg load cell. The samples were compressed to 40% of their original length using a 35 mm diameter probe with a cross-head speed of 2.0 mm/s. Sensory analyses of cooked sausage samples in the laboratory scale were performed using a 9-point hedonic scale test. Thirty untrained panelists who regularly consumed emulsion-type sausages evaluated their likability regarding color, odor, taste, texture, and overall liking (Stone Sidel, 1993). Before the test, sausage samples were steeped in hot water (95±2°C) in individual pans for 2 min. Two slices (2.5 cm long at 35±1°C) of each sausage formulation coded with 3-digit random numbers were served in random order to the taste panels in individual booths. The taste panelists were instructed to cleanse their palates between samples (Deda et al., 2007).
Design of Experiment and Statistical Analysis
Completely randomized design (CRD) and randomized complete block design (RCBD) were used in this study; the former for chemical and physicochemical attributes, the latter for sensory evaluation. The SPSS for Windows version 23 was used to perform the analysis of variance of the experimental data (ANOVA). The difference among each pair of treatment means at p < 0.05 was achieved using Duncan’s new Multiple Range Test (DMRT). All experiments were carried out in duplicate (n = 2), and the results were expressed as means (± SD).
Results and Discussion
Physicochemical characteristics of Vienna sausages
The effect of pork back fat substitution by a mixture of rice flour, water, and skim milk powder (RFSKM) on the physicochemical attributes of Vienna sausages is shown in Table 1-2.
Table 1: Effect of a mixture of rice flour, water and skimmed milk powder on physicochemical properties of the sausage samples
%Total Cooking Viscosity Treatment L* a* b* fluid loss (%) (x105 cP) released Control (T0) 64.27±0.49a 5.73±0.20a 11.36±0.14a 4.69±0.21a 1.55±0.11a 1.56±0.35e T20 63.39±1.23a 6.15±0.32a 12.00±0.38a 3.49±0.03b 1.32±0.47b 1.65±0.22de T40 62.96±1.30a 5.88±0.25a 11.49±0.36a 2.74±0.38c 1.06±0.57c 1.72±0.37d T60 61.55±1.79b 5.74±0.68a 12.11±0.74a 1.44±0.23d 0.69±0.61d 1.89±0.19c T80 57.86±1.81c 5.91±0.41a 11.78±0.58a 0.67±0.80e 0.38±0.10e 2.23±0.10b T100 56.37±1.74d 5.84±0.54a 11.74±0.60a 0.40±0.66e 0.35±0.15e 2.54±0.25a Means within the same column having different letters were significantly different (p≤0.05)
The addition of RFSKM significantly influenced the emulsion stability and cooking loss of the sausages (p<0.05). The values of total fluid release ranged from 0.35% to 1.55%. An increment of RFSKM led to a reduction of total fluid release, indicating higher emulsion stability. The full-fat control sample (T0) showed the highest values of fluid exudation and thus the lowest emulsion stability (p<0.05). This result might be due to the high starch and protein contents of RFSKM, which are able to form a three-dimensional gel network at high temperatures, resulting in higher capture of fat and water within the food matrix (Feiner, 2006).
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In meat/starch systems, gelatinized starch in RFSKM absorbs more water, but its presence does not cause a chemical interaction between meat proteins and starch because meat protein begins to denature before starches gelatinize (Li Yeh, 2002; Li Yeh, 2003). Alamanou et al. (1996) explained that the stabilizing effect of starches in emulsions is related to their high electrical charge and having more hydrophilic–lipophilic groups within structure, which increase the lipid and water interactions. These groups form a charged layer round fat droplets, causing mutual repulsion, reducing interfacial tension and preventing coalescence. In addition, the relatively high water absorbing ability of milk protein and its ability to form a network with the muscle protein may improve the stability of raw batter (Youssef Barbut, 2011). Olanwanit Rojanakorn (2019) demonstrated that higher emulsion stability was observed when a mixture of Man Sao powder and hydrolyzed collagen was used as a fat replacer in reduced-fat Vienna sausage. Alves et al. (2016) reported that an emulsion stability of reduced- fat bologna-type sausage increased with increasing a concentration of pork skin and green banana flour mixture. The cooking loss of Vienna samples containing various levels of RFSKM ranged from 0.40% to 4.69% (Table 1). An increase in RFSKM level resulted in a reduction of cooking loss (p<0.05). The T100 sample (100% RFSKM) showed the lowest cooking loss (p<0.05), which could be attributed to having the lowest fluid separation during cooking. Similarly, Olanwanit Rojanakorn (2019) reported a lower cooking loss when a mixture of Man Sao powder and hydrolyzed collagen was added to Vienna sausage samples as a fat mimetic. Choe et al. (2013) also reported that the addition of wheat fiber and pig skin mixture in frankfurter formulations caused a significant reduction of cooking loss. The viscosity of the raw batter with different levels of RFSKM ranged from 1.56 × 105 cP to 2.54 × 105 cP (Table 1). The incorporation of RFSKM caused a significantly greater batter viscosity (p<0.05) as compared to the full fat control sample (no added RFSKM). This result may be due to the high water-holding ability of starch and protein in RFSKM. The highest viscosity was found in the T100 sample, whereas the control (T0) exhibited the lowest viscosity (p<0.05). Similarly, Olanwanit Rojanakorn (2019) reported that the viscosity of raw batter of Vienna sausage increased with increasing concentration of a mixture of Man Sao powder and hydrolyzed collagen as a fat substitute. Choe et al. (2013) also reported that the inclusion of a mixture of wheat fiber and pig skin in frankfurter formulations led to a significant increase in the viscosity of meat emulsion. Shand (2000) included an instant or pre-gelatinized starch in a starch blend as a processing aid to increase the viscosity of the raw batter. The replacement of up to 40% pork fat with RFSKM did not alter the values of L*, a* and b* of the cooked sausages (Table 1). On the contrary, the sausage samples containing 60% (T60), 80% (T80) and 100% (T100) RFSKM had significantly lower L* values than the control sample (p<0.05). Furthermore, the significant variation of b* and a* values among all six samples (p<0.05) was not observed (Table 1). Alves et al. (2016) demonstrated that the inclusion of a mixture of pork skin and green banana flour as a fat replacer for up to 60% did not modify the L*, a*, and b* values of bologna-type sausages (p<0.05). However, the L* value of the sample containing more than 60% pork skin and green banana flour mixture was lower than the full-fat control sample. The level of pork skin and green banana flour mixture did not change the b* value of bologna-type sausage (p<0.05).
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Table 2: Effect of a mixture of rice flour, water and skimmed milk powder on the water holding capacity of the sausage samples
Treatment Water holding capacity (%) Control (T0) 80.02±0.63a T20 82.79±1.01b T40 85.74±1.21c T60 86.65±0.35d T80 91.04±0.90e T100 91.70±0.99e Means within the same column having different letters were significantly different (p≤0.05)
As seen in Table 2, the WHC was affected by level of RFSKM (p<0.05). The WHC values ranged from 80.02% to 91.70%. The WHC was lowest in the control sample (T0), which showed the highest cooking loss and total fluid released (p<0.05). As expected, the WHC increased with increasing RFSK level. This result might be due to the high water retention ability of starch and protein in RFSKM. As stated earlier, in meat/starch systems, gelatinized starch in RFSKM absorbs more water into the polymer matrix, resulting in higher WHC (Li Yeh, 2002; Li Yeh, 2003; Aktas Genccelep, 2006). In addition, the negative charge of casein protein in skim milk powder, a major component of RFSKM can chemically bind with water molecules resulting in a greater water holding ability of sausage samples (Holt et al., 2013). Yang et al. (2007) reported that addition of hydrated oatmeal into low-fat sausages improve WHC by decreasing cooking loss. Kim et al. (2019) also reported that reduced-fat frankfurter samples with konjac gel showed greater WHC, compared to the full fat sample without konjac gel.
Table 3: Effect of a mixture of rice flour, water and skimmed milk powder on the textural parameters of the sausage samples
Hardness Chewiness Adhesiveness Treatment Cohesiveness Springiness (kgf) (kgf) (kgf.sec) Control (T0) 3.272±0.479c 0.747±0.148a 0.987±0.080a 2.370±0.233d -0.043±0.002a T20 3.367±0.371c 0.735±0.101a 0.956±0.259a 2.390±0.268d -0.127±0.009a T40 3.410±0.340c 0.734±0.008a 0.964±0.218a 2.402±0.245cd -0.104±0.005a T60 3.638±0.264c 0.754±0.453a 0.972±0.198a 2.628±0.205c -0.007±0.008a T80 4.456±0.246b 0.725±0.005a 0.972±0.628a 3.143±0.229b -0.006±0.006a T100 4.811±0.274a 0.774±0.268a 0.969±0.463a 3.448±0.314a -0.071±0.009a Means within the same column having different letters were significantly different (p≤0.05)
As seen in Table 3, sausage formulation significantly affected the hardness and chewiness of sausage samples (p≤0.05). The sample containing 100% RFSKM (T100) exhibited the highest hardness and chewiness whereas the control (T0) showed the lowest value of hardness and chewiness (p≤0.05). An increase in RFSKM level resulted in a rise of hardness and chewiness of the reduced-fat samples (p≤0.05). This finding may be observed because the solid content of the sausages may increase with increasing RFSKM, resulting in a harder texture. Surprisingly, an addition of RFSKM as a fat replacer did not affect cohesiveness, springiness and adhesiveness of the sausage samples (p0.05). Olanwanit Rojanakorn (2019) reported that Vienna sausages containing Man Sao powder and hydrolyzed collagen had higher
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology hardness and chewiness than the control with no Man Sao powder and hydrolyzed collagen addition. Alves et al. (2016) also reported that the incorporation of a mixture of pork skin and green banana flour as a fat replacer induced the harder texture to frankfurter-type sausage.
Chemical composition of sausage sample
The result of RFSKM concentration on the chemical composition of Vienna sausages is presented in Table 4.
Table 4: Effect of a mixture of rice flour, water and skimmed milk powder on the chemical composition of the sausage samples
Moisture Protein Fat Ash Calorific Treatment (%) (%) (%) (%) value (kcal) control (T0) 59.60±0.67e 12.58±0.09e 16.32±0.63a 2.09±0.38a 233.87±0.87a T20 61.09±1.96d 13.09±0.06d 13.39±0.61b 2.20±0.00a 212.72±1.02b T40 63.16±0.62cd 14.04±0.40c 10.18±0.54c 2.25±0.04a 188.19±1.28c T60 65.57±3.84bc 14.27±0.09bc 6.94± 0.55d 2.39±0.28a 161.62±1.19d T80 67.58±0.23b 14.50±0.30b 3.31± 0.08e 2.42±0.07a 135.92±0.34e T100 71.96±2.28a 15.23±0.91a 0.81 ± 0.06f 2.53±0.24a 105.55±0.82f Means within the same column having different letters were significantly different (p≤0.05)
An increase in RFSKM concentration resulted in an increase in moisture and protein content of sausage samples, whereas the fat content and energy value decreased (p≤0.05). Interestingly, the ash content of the samples increased insignificantly with increasing RFSKM level (p0.05). The moisture content of the control sample (T0) was 59.60%. The values reached 71.96% in the T100 sample, possibly due to the high-water absorption of the starch and protein present in the RFSKM. The fat content of the sausages was the highest (16.32%) in the control and increasing RFSKM level significantly decreased fat contents (p≤0.05). The addition of 20%, 40%, 60%, 80% and 100% RFSKM showed 17.95%, 36.62%, 57.48%, 79.72% and 95.03% fat reduction as compared to the control, respectively. According to FDA regulation, food products can be claimed as “reduced-fat” if their fat content is reduced to at least 25% of the original fat content (US FDA, 2016). Hence, the Vienna sausage samples with 40% RFSKM or more can be classified as reduced-fat Vienna sausages. As the replacement of pork fat by RFSKM increased, the protein content of the sausage samples increased (p≤0.05). This may due to the higher protein content of the RFSKM (22.84%) when compared with the pork fat (8.35%). The calorific value of sausage samples greatly depended on the RFSKM level (p≤0.05) (Table 4). The highest calorific value was found in the control sample (T0) as 233.87 kcal/100 g, whereas the value for sausage samples containing RFSKM ranged from 212.72 to 105.55 kcal/100 g. The calorific value was 9.04% to 54.87% lower in the Vienna samples containing RFSKM as compared to that in the full-fat control sample. Namhong Rojanakorn (2016) reported that the calorific value of Vienna sausages decreased with increasing concentration of a mixture of sweet potato powder, gelatin powder, and water (in a ratio of 1:0.25:9) as a fat replacer (p≤0.05). Similarly, Olanwanit Rojanakorn (2019) indicated that reduced-fat Vienna sausage with a mixture of Man Sao powder and hydrolyzed collagen as a fat replacer possessed lower calorific value as compared to the full fat control.
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Sensory evaluation
Sensory likability scores of the sausages were affected by the level of RFSKM (Table 5).
Table 5: Effect of a mixture of rice flour, water and skimmed milk powder on sensory likability scores of the sausage samples
Treatment Color Odor Texture Taste Overall liking control (T0) 6.7±1.4a 6.5±1.7a 7.5±1.3a 7.6±1.2a 8.0±0.7a T20 6.4±1.4a 6.3±1.5a 7.0±1.3a 7.1±1.2a 7.5±1.0a a a a a a T40 6.5±1.7 5.8±1.7 7.1±1.5 7.1±1.3 7.4±0.8 T60 6.3+1.6a 5.9±1.7a 5.3±1.5b 5.7±1.3b 5.7±1.7b T80 6.4±1.7a 5.7±1.6a 5.3±1.6b 5.3±1.8b 5.6±1.9b T100 6.0±1.7a 6.0±1.7a 5.0±1.7b 5.0±1.7b 5.1±1.9b Means within the same column having different letters were significantly different (p≤0.05)
The pork fat replacement with RFSKM did not affect color and odor likability scores of sausage samples (p0.05). However, likability scores of texture, taste, and overall liking depended on sausage formulation (p≤0.05). The samples with up to 40% RFSKM had all likability scores comparable to the control (p0.05). The samples containing 60%, 80% and 100% RFSKM had lower likability scores for texture, taste, and overall liking than the full-fat control sample (T0) (p≤0.05), indicating that the incorporation of RFSKM as a fat replacer into the sausage samples at 60% RFSKM or more led to a reduction of some sensory attributes as compared to the control (p≤0.05). This result may be observed because the panelists could detect the flavor and odor of rice flour and skim milk powder when 60% RFSKM or more were added to the sausage samples. In addition, the texture of these samples was too hard for the panelists to accept. The overall liking scores of the control sample (T0) and the samples with 20% RFSKM (T20) and 40% RFSKM (T40) ranged between 8.0 and 7.4, indicating that the taste panels highly accepted these three samples. Therefore, replacement of up to 40% pork fat by RFSKM can be used to develop acceptable Vienna sausages.
Conclusion
The inclusion of a mixture of rice flour, skim milk powder and water (RFSKM) as a fat replacer in Vienna sausage led to an increase in emulsion stability, batter viscosity, moisture and protein contents, and hardness of the sausage samples. Increasing RFSKM concentration resulted in a reduction of fat content, calorific value, cooking loss, and lightness (L*) of the sausage samples. The results of sensory evaluation indicated that reduced-fat sausage samples containing up to 40% RFSKM had all sensory likability scores and textural parameters comparable to the full fat control. Therefore, reduced-fat Vienna sausages with high acceptability can be developed by the incorporation of 40% RFSKM as a fat replacer.
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Acknowledgement
The authors thank the Department of food technology, Khon Kaen University for the financial support received for the completion of the present work.
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Mancebo, C. M., Merino, C., Martínez, M. M., & Gómez, M. (2015). Mixture design of rice flour, maize starch and wheat starch for optimization of gluten free bread quality. Journal of Food Science and Technology, 52(10), 6323–6333. Marafon, A. P. , Sumi, A. , Alcântara, M. R. , Tamime, A. Y. , & Nogueira de Oliveira, M. (2011). Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins. LWT - Food Science and Technology, 44 (2), 511–519. Namhong, S., & Rojanakorn, T. (2016). Quality Evaluation of Vienna Sausage Incorporated with Sweet Potato Flour and Gelatin Mixture as a Fat Replacer. 9. In 3rd International Postgraduate Symposium on Food and Biotechnology (IPSFAB2016). Thailand: Maha Sarakham University. Olanwanit, W,. & Rojanakorn, T. (2019). Effect of hydrolysed collagen and Man-sao powder mixture as a fat replacer on quality of Vienna sausages. International Food Research Journal, 26(5), 1525-1523. Osburn, W. N., Mandigo, R. W., & Eskridge, K. M. (1997). Pork Skin Connective Tissue Gel Utilization in Reduced-Fat Bologna. Journal of Food Science, 62(6), 1176–1182. Shaltout, E. (2007). Fat Replacers and Their Applications in Food Products: A Review. 4, 16. Shand, P. J. (2000). Textural, Water Holding, and Sensory Properties of Low-fat Pork Bologna with Normal or Waxy Starch Hull- less Barley. Journal of Food Science, 65( 1) , 101– 107. Shand, P. J. (2000). Textural, Water Holding, and Sensory Properties of Low-fat Pork Bologna with Normal or Waxy Starch Hull- less Barley. Journal of Food Science, 65( 1) , 101– 107. Stone, H and Sidel, J. L. ( 1993) . Sensory evaluation practices. 2nd ed. Academic Press, Inc. New York. Torbica, A. , Hadnađev, M. , & Dapčević Hadnađev, T. ( 2012) . Rice and buckwheat flour characterisation and its relation to cookie quality. Food Research International, 4 (1), 277–283. Wimontham, T,. & Rojanakorn, T. (2016). Effect of incorporation of Gac (Momordica cochinchinensis) aril powder on the qualities of reduced- nitrite Vienna sausage. International Food Research Journal 23 (3): 1048-1055. Youssef, M. K., & Barbut, S. (2011). Effects of two types of soy protein isolates, native and preheated whey protein isolates on emulsified meat batters prepared at different protein levels. Meat Science, 87(1), 54–60.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.15 International Conference on Food, Agriculture and Biotechnology
RICE STRAW HYDROLYSATE AS A PROMISING CULTURE MEDIUM FOR ASTAXANTHIN PRODUCTION BY THE RED YEAST XANTHOPHYLLOMYCES DENDRORHOUS
PASINEE PHOPROEK 1, JIDAPHA TINOI 2,3*
1Interdisciplinary Program of Biotechnology, Graduate School, Chiang Mai University, Chiang Mai, Thailand 2Research Center on Chemistry for Development of Health Promoting Products from Northern Resources, Faculty of Science, Chiang Mai University 3Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
*Corresponding author: [email protected]
Abstract:
This research aimed to utilize rice straw as a low-cost substrate for astaxanthin production by Xanthophyllomyces dendrorhous TISTR 5730. Rice straw composed of high content of cellulose (35.92±0.51 %) and hemicellulose (21.13±1.93 %). After enzymatic hydrolysis with commercial cellulase, the rice straw hydrolysate contained the maximum of total reducing sugar content of 89.82±0.39 g/L. X. dendrorhous TISTR 5730 was cultivated on rice straw hydrolysate with 20 g/L of initial total reducing sugar as a culture medium. The maximum cell dry weight (5.47±1.45 g/L) with astaxanthin concentration (2.49±0.04 mg/L) was achieved. The kinetics parameters demonstrate the astaxanthin productivity of 0.50±0.01 mg/L/day with the astaxanthin coefficient of 0.13±0.00 mg/g sugar consumed. X. dendrorhous TISTR 5730 was capable to consume the reducing sugar in rice straw hydrolysate up to 97.08±0.03 %. The absorption spectra, HPLC profile and FTIR spectrum of the produced astaxanthin X. dendrorhous TISTR 5730 were characterized and compared to standard astaxanthin. This research indicated that rice straw hydrolysate could be used as a promising culture medium for astaxanthin production.
Keywords: Rice straw; Culture medium; Enzymatic hydrolysis; Astaxanthin production; Xanthophyllomyces dendrorhous yeast
Introduction
Rice straw is produced from the harvesting of rice as agricultural residues. Generally, rice straw was used as animal feed, paper making and fertilizer (Zhu et al., 2005). However, the limitation of used is reached due to the abundance of remained rice straw. The burning of rice straw is the only way to eliminate the unused rice straw. Resulting in a serious environmental problem by releasing a pollutant into the air and causing high risk for human health (Xiao et al., 2001). The alternative way to use rice straw is challenged by converting into fermentable sugars. Rice straw is a lignocellulosic material which composed of high content of carbohydrate about 60 % by weight which consisted of cellulose (32 %-72 %), hemicellulose (19 %-27 %) and lignin (5 %-24 %) (Binod et al., 2010). The cellulose in rice straw can be hydrolyzed by using enzyme into reducing sugar and further used as a fermentable sugar. The fermentable sugar is an interest carbon source for growth and produces the value-added compound of microorganisms. The utilization of rice straw as feedstock for microorganism is getting more attractive due to significantly decrease the production cost. Previously, rice straw was utilized to produce many high value-added substance such as ethanol, lipid, lactic acid, citric acid, and amino acid via fermentation processes (Zhang & Cai, 2008). Thus, rice straw might be a promising low-cost
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology substrate for producing high value bioactive compounds such as carotenoids, especially astaxanthin. Astaxanthin is a lipophilic pigment that appearing as orange-red color. It is classified as xanthophyll carotenoid in which the structure contained oxygen. Astaxanthin structure composed of polyene chain joins with the β-ionone ring with a hydroxyl group and keto group in both ends (Ambati et al., 2014). Due to its structure astaxanthin possess a powerful antioxidant activity by donating and scavenging free radical which 10 and 100 times greater than β-carotene and vitamin E, respectively (Higuera-Ciapara et al., 2006). The properties of astaxanthin is responsible for immune response, oxidative stress prevention, anti-inflammatory and anti-aging (Yamashita, 2015). Astaxanthin is a naturally synthesized in a microorganism such as the yeast Xanthophyllomyces dendrorhous and microalgae Haematococcus pluvialis. The red yeast X. dendrorhous is one of the important sources for astaxanthin production owing to its ability to utilize low-cost substrate, high growth rate along with short production time and easy cultivation condition. In recent years, many researchers have been interested in cost- effective of astaxanthin production by X. dendrorhous from low-cost culture media. Several researchers presented the utilization of low-cost materials for astaxanthin production such as molasses (Haard, 1988), peat hydrolysate (Martin et al., 1993), raw coconut milk (Domínguez- Bocanegra & Torres-Muñoz, 2004), mustard waste (Tinoi et al., 2006), Mussel processing (Amado & Vázquez, 2015) wastewater and Sweet sorghum (Stoklosa et al., 2019). This research aimed to utilize rice straw hydrolysate as a culture medium for astaxanthin production via the fermentation process. It could be reduced the cost of production and applied as a culture medium for further scale-up of astaxanthin production.
Materials and methods
Rice straw was collected from a field at San Sai, Chiang Mai, Thailand. Rice straw was dried and cut into small pieces. Then, the dried rice straw was ground and sieved through 60 mesh. The ground rice straw was stored in a plastic bottle.
Chemical Composition Determination
The proximate analysis of rice straw for moisture, crude protein, crude lipid, fiber, total ash and carbohydrate contents was determined as described by the Association of Official Analytical Chemists (AOAC, 2005). Moisture content was measured by the drying method. Crude protein content was determined by the Kjeldahl method and crude lipid content was done by using Soxhlet extraction with hexane as solvent. Total ash content was incinerated in a furnace at 550C. Fiber content was measured by weight difference methods. The carbohydrate content of rice straw was carried out by calculation. The lignocellulosic component of rice straw was also carried out by the detergent fiber analysis. Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and acid insoluble ash (AIA) were determined as described by Van Soest et al. (1991).
Rice straw hydrolysate preparation
Rice straw was pretreated by alkali (30 % NaOH) pretreatment combined with an autoclave for 20 min as described by Kobkham et al. (2018). The pretreated rice straw was then hydrolyzed by commercial cellulase (iKnowZyme®, 119 FPU/mL) hydrolysis in citrate buffer solution (pH 4.8) at ratio 1:30 (w/v). The mixture was incubated in an incubator shaker at 50 °C with 150 rpm for 120 h. The reducing sugar content of rice straw hydrolysate was carried out by the
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DNS method. The sugar composition was analyzed by High-performance liquid chromatography (HPLC).
Astaxanthin Production on Rice Straw Hydrolysate
Xanthophyllomyces dendrorhous TISTR 5730 was obtained from the Thailand Institute of Scientific and Technology Research (TISTR). The seed culture was prepared in the YM medium for 36 h. Then, 10 % of X. dendrorhous TISTR 5730 seed culture was inoculated into rice straw hydrolysate containing 20 g/L of total reducing sugar concentration. The mixture was incubated in an incubator shaker at 20 °C with 200 rpm for 10 days. The culture was taken every day for cell dry weight, astaxanthin content and total reducing sugar concentration determinations.
Analytical methods
The cell dry weight was determined using the gravimetric method and calculated in terms of concentration (g/L). The astaxanthin content was estimated via cell disruption and solvent extraction according to Urnau et al. (2018). The astaxanthin content was represented in terms of astaxanthin concentration (g/L) and yield (g/g dry cell basic). The characteristics of astaxanthin were analyzed and compared to standard astaxanthin. The adsorption spectra of astaxanthin were scanned by a UV/VIS spectrophotometer (Thermo Scientific EvolutionTM 201) with a wavelength in the range of 300 – 800 nm. The HPLC analysis of astaxanthin extract was performed by using the Hewlett Packard 1100 Series HPLC system coupled with a UV detector at 474 nm. The functional structure of astaxanthin was analyzed by the Fourier transform infrared spectroscopy (FTIR) (Bruker, USA). The sample was measured in ATR mode with the scanning wavenumber in the range of 4000 to 500 cm-1.
Results and discussion
Chemical Composition of Rice Straw
The chemical composition of rice straw was determined and shown in Table 1. The moisture content of rice straw was found to be 7.70±1.88 % which a slightly higher than the moisture content of rice straw about 5.40±0.20 % from the study of Ma et al. (2009). The low moisture content of rice straw would enhance the storage stability and extend the shelf life for further utilization. The crude protein was 4.17±0.26 % and crude lipid was found in a trace amount of 0.93±0.29 %. These results were correlating to the report of Malik et al. (2015), who exhibited the protein and crude lipid contents of rice straw as 4.5 and 1.0 %, respectively. Ash content contained about 15.72±0.26 % which the results were similar to 14.56±0.43 and 16 % of ash content (Harun & Geok, 2016; Malik et al., 2015). Moreover, 32.11±1.56 % of the carbohydrate content in rice straw was found and the results corresponded to the previous research of Candia-García et al. (2018), who obtained the carbohydrate content of rice straw in Columbia about 31.34±0.24 %. The fiber content was 39.37±2.23 % and the results in this study were higher than with the fiber content of 29.8 % in rice straw from the previous reported by Malik et al. (2015). The fiber in rice straw was considered an important component because of its contained the lignocellulosic composition. The results presented that rice straw contained cellulose, hemicellulose and lignin of 35.92±0.51, 21.12±1.93 and 5.42±0.35, respectively. Kobkham et al. (2018) revealed that the lignocellulosic content of rice straw was 34.40±0.03, 26.68±0.08 and 7.30±0.08 % of cellulose, hemicellulose and lignin, respectively. While Binod et al. (2010) also found a similar content of cellulose and hemicellulose about 38 and 19.7 %,
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Table 1: The chemical composition of rice straw
Chemical composition Content (%, w/w) Moisture 7.701.88 Crude lipid 0.930.29 Crude protein 4.170.26 Carbohydrate 32.111.56 Ash 15.720.26 Fiber 39.372.23 Cellulose 35.92±0.51 Hemicellulose 21.13±1.93 Lignin 5.42±0.35 Data expressed as mean±SD of triplicate experiments.
Enzymatic hydrolysis of rice straw
The rice straw was pretreated by using alkaline pretreatment with sodium hydroxide solution for delignification and increased surface area of cellulose. The main effect of sodium hydroxide on delignification demonstrates the breaking the ester bonds cross-linking lignin and hemicellulose, resulting in increasing the porosity of rice straw structure (Binod et al., 2010). The pretreated rice straw composed mainly of cellulose which could be hydrolyzed into glucose. This research performed the enzymatic hydrolysis of rice straw by using commercial cellulase. The rice straw cellulose was hydrolyzed by 119 FPU/ml of cellulase at 50C for 120 h of hydrolysis time. The reducing sugar concentration of 89.82±0.39 g/L and yields of 0.71±0.01 g/g rice straw was found to be in rice straw hydrolysate. Comparison to the previous studies, the results of this research were higher than the reducing sugar content from Ong et al. (2012). The results showed that the fermentable sugar content of 3.62 g/L was obtained from 10 U/g of cellulase for 96 h. Takano & Hoshino (2018) reported that the pretreated rice straw by using alkali pretreatment was hydrolyzed by enzymatic hydrolysis and revealed that fermentable sugar content of rice straw hydrolysate was achieved of 75.3 g/L. Moreover, the rice straw hydrolysate composition was investigated by using HPLC analysis and the results were shown in Table 2. The results represented that rice straw hydrolysate contained a high amount of glucose (82.35±2.23 %) which was broken down from the cellulose structure. However, the xylose (3.46±0.11 %) and arabinose (0.72±0.03 %) were also detected in a small amount. These pentose sugars was hydrolyzed from the hemicellulose which remained in during the pretreatment process. However, the unidentified saccharide of rice straw hydrolysate was also found about 13.47±1.67 % of total reducing sugar. These results were complementary with Xiao et al. (2001) who reported that the rice straw hydrolysate composed of glucose (81.55 %), xylose (15.67 %) and arabinose (2.73 %).
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Table 2: Total reducing sugar content and sugar composition of rice straw hydrolysate after enzymatic hydrolysis
Total reducing sugar content Total reducing sugar concentration (g/L) 89.82±0.39
Total reducing sugar yield (g/g rice straw) 0.71±0.01
Sugar composition (% of total reducing sugar)
Glucose 82.35±2.23
Xylose 3.46±0.11
Arabinose 0.72±0.03
Others 13.47±1.67 Data expressed as mean ± S.D of triplicate experiments.
The growth and Astaxanthin production on rice straw hydrolysate
The red yeast X. dendrorhous TISTR 5730 was cultivated on rice straw hydrolysate containing total reducing sugar of 20 g/L with the initial pH of 5.2 for 10 days of cultivation. The initial of total reducing sugar at 20 g/L was selected as the results of the prior study by Tinoi et al. (2006). Besides, Lui and Wu (2007) confirmed that glucose concentration of approximately 20 g/L was suitable for astaxanthin production by X. dendrorhous TISTR 5730. Figure 1 indicates the growth of X. dendrorhous TISTR 5730, astaxanthin production and remained reducing sugar concentration. At the early of cultivation, the results revealed that the reducing sugar was consumed up to 96.3±0.28 % within the first day and kept constant with the rest of the cultivation time. After the reducing sugar was consumed as a carbon source for yeast cell metabolism. The yeast cell was grown and increased to a maximum of 5.47±1.45 g/L in 4 days. Then, the cell dry weight of X. dendrorhous TISTR 5730 remained in the stationary state until 10 days of cultivation. Due to nutrients for growth containing in rice straw hydrolysate was exhausted and became into the stress condition. Therefore, the astaxanthin was initiated to accumulate inside the X. dendrorhous TISTR 5730 cell and the highest astaxanthin yields were 0.56±0.02 mg/g with astaxanthin concentration of 2.49±0.04 mg/L at 5 days of cultivation. Astaxanthin is considered as the secondary metabolites produced during the stationary phase of the X. dendrorhous cultivation. The production of astaxanthin by X. dendrorhous recognized as growth associated metabolite (Liu & Wu, 2007). In this research, astaxanthin was synthesized when the reducing sugar of rice straw hydrolysate was consumed and the lack of nutrients occurred after the highest of cell dry weight concentration. Liu & Wu (2007) reported that astaxanthin content also reached the maximum due to the stress condition around the X. dendrorhous cells. The initial pH of the rice straw hydrolysate medium was 5.2 which is slightly acidic condition. After the first day of cultivation, pH was increased to neutral and remained constant in neutral condition around 7.0 - 8.4. The previous studied confirmed that the suitable initial pH for X. dendrorhous TISTR 5730 cultivation was around 5.0 - 7.0 and considered as neutral pH (Martin et al., 1993).
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Table 3 presented the kinetic parameters of astaxanthin production on rice straw hydrolysate by X. dendrorhous TISTR 5730. The maximum specific growth rate was 0.39±0.00 day-1 and biomass yield was 0.23±0.01 g/g of total reducing sugar consumption. While the cellular astaxanthin reached to 551.92±22.11 µg/g with the productivity of 0.50±0.01 mg/L/day. The astaxanthin coefficient was 0.13±0.00 mg/g sugar consumed. Furthermore, the sugar consumption rate was 3.88±0.01 g/L/day and sugar consumption was up to 97.08±0.03 % within 5 days of cultivation. This research indicated that rice straw hydrolysate could be used as a promising culture medium which mainly composed of glucose with the small amount of xylose and arabinose for astaxanthin production by X. dendrorhous TISTR 5730. According to Xu et al. (2004) demonstrated that X. dendrorhous could be grown on the different type of sugar such as glucose, xylose, arabinose, cellobiose, maltose and sucrose. The medium containing glucose, galactose and cellobiose was revealed to obtain a high biomass concentration corresponding with the high astaxanthin content than others media which led to high astaxanthin productivity. However, a high glucose concentration could undergo inhibition of astaxanthin synthesis within the yeast cell which leads to overflow to increase ethanol production (Xiao et al., 2015). Parajó et al. (1998) represented that the eucalyptus hydrolysate supplement with peptone was used for X. dendrorhous cultivation and astaxanthin production. The results showed that astaxanthin concentration of 2.14 mg/L with astaxanthin content of 448 µg/g was obtained. While Hayman et al. (1995) displayed the astaxanthin concentration produced by X. dendrorhous on corn wet-milling was up to 2.6 mg/L with the astaxanthin content of 400 µg/g. Moreover, Yang et al. (2011) presented the astaxanthin concentration was 2.98 mg/L when X. dendrorhous was cultured on cassava hydrolysate.
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Table 3: Kinetic parameters of astaxanthin production on rice straw hydrolysate by X. dendrorhous TISTR 5730 for 5 days of cultivation
Parameters X. dendrorhous TISTR 5730 growth: X (g/L) 5.47±1.45 -1 µmax (day ) 0.39±0.00 RX/S (g/g) 0.23±0.01 Astaxanthin production: concentration (mg/L) 2.49±0.04 cellular (µg/g) 551.92±22.11 productivity (mg/L/day) 0.50±0.01 coefficient (mg/g sugar consumed) 0.13±0.00 Sugar concentration: Initial concentration (g/L) 20.00±0.00 Final concentration (g/L) 0.59±0.05 Sugar consumption rate (g/L/day) 3.88±0.01 % Sugar consumption 97.08±0.03 Data expressed as mean ± S.D of triplicate experiments.
The characterization of astaxanthin
The absorption spectra of produced astaxanthin by X. dendrorhous TISTR 5730 were analyzed in the wavelength between 350-800 nm and compared to the astaxanthin standard. Figure 2 shows the astaxanthin absorption spectra. The absorption spectra profile of produced astaxanthin presented to similar the astaxanthin standard. The maximum absorbance demonstrated at the wavelength of 474.90 nm for standard astaxanthin and 476.02 nm for the astaxanthin from X. dendrorhous TISTR 5730. The results indicated that the produced astaxanthin was identical to the standard astaxanthin.
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The HPLC analysis of the produced astaxanthin by X. dendrorhous TISTR 5730 was performed and revealed that the retention time was at 3.788 min as shown in Figure 3. The HPLC chromatogram presented only one peak of the astaxanthin compound as a major component. According to Schmidt et al. (2011) revealed that X. dendrorhous produced astaxanthin as a dominant compound up to 87 % of the carotenoid product.
Figure 3: HPLC chromatogram of astaxanthin profile from X. dendrorhous TISTR 5730 production from rice straw hydrolysate
The FTIR spectrum of astaxanthin was recorded as the transmittance (%) versus wavenumber in the range of 4000 – 400 cm-1 to determine the functional group contributions as shown in Figure 4. The spectrum comprised the strong absorption band at 1647.6 cm-1 corresponding to C=O stretching vibration which presented in the terminal ring of β-ionone. The absorption band at 1549.6 cm-1 corresponded to the C=C stretching vibration of the aromatic ring (Liu et al., 2019). And at 974.1 cm-1 attributed to the C-H in the C and C conjugate system. All the absorption band of astaxanthin in these results was agreed with the previous study of (Kaga et al., 2018).
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Conclusion
This research represented that rice straw composed of high content of cellulose and hemicellulose. The hydrolysate of rice straw was obtained by enzymatic hydrolysis and gave a high concentration of reducing sugar-containing with glucose as dominant sugar and also pentose sugars (xylose and arabinose). The 20 g/L of initial reducing sugar concentration of rice straw hydrolysate was applied as a culture medium for X. dendrorhous TISTR 5730. The successful growth and high astaxanthin production by X. dendrorhous TISTR 5730 was obtained. Thus, rice straw hydrolysate could be used as a promising culture medium in astaxanthin production X. dendrorhous TISTR 573 for reducing the cost of production and can be used in the further large-scale production.
Acknowledgements
This research project was supported by Chiang Mai University and The National Research Council of Thailand (NRCT). The authors thank Department of Chemistry, Faculty of Science and Interdisciplinary Program in Biotechnology, Graduate school and Chiang Mai University, Thailand.
References
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Parajó, J. C., Santos, V., & Vázquez, M. (1998). Production of carotenoids by Phaffia rhodozyma growing on media made from hemicellulosic hydrolysates of Eucalyptus globulus wood. Biotechnology and Bioengineering, 59(4), 501-506. Schmidt, I., Schewe, H., Gassel, S., Jin, C., Buckingham, J., Hümbelin, M., Sandmann, G., & Schrader, J. (2011). Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Applied Microbiology and Biotechnology, 89(3), 555-571. Stoklosa, R. J., Johnston, D. B., & Nghiem, N. P. (2019). Phaffia rhodozyma cultivation on structural and non-structural sugars from sweet sorghum for astaxanthin generation. Process Biochemistry, 83, 9-17. Takano, M., & Hoshino, K. (2018). Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Bioresources and Bioprocessing, 5(1), 16. Tinoi, J., Rakariyatham, N., & Deming, R. (2006). Utilization of mustard waste isolates for improved production of astaxanthin by Xanthophyllomyces dendrorhous. Journal of Industrial Microbiology and Biotechnology, 33(4), 309-314. Urnau, L., Colet, R., Soares, V. F., Franceschi, E., Valduga, E., & Steffens, C. (2018). Extraction of carotenoids from Xanthophyllomyces dendrorhous using ultrasound‐ assisted and chemical cell disruption methods. The Canadian Journal of Chemical Engineering, 96(6), 1377-1381. Van Soest, P. v., Robertson, J., & Lewis, B. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. Xiao, A., Jiang, X., Ni, H., Yang, Q., & Cai, H. (2015). Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation. Electronic Journal of Biotechnology, 18(3), 148-153. Xiao, B., Sun, X., & Sun, R. (2001). Chemical, structural, and thermal characterizations of alkali-soluble lignins and hemicelluloses, and cellulose from maize stems, rye straw, and rice straw. Polymer Degradation and Stability, 74(2), 307-319. Xu, B.-J., Li, C.-T., Mo, E.-K., & Sung, C.-K. (2004). Improved astaxanthin production of Xanthophyllomyces dendrorhous with carotenogenesis stimulating factors. Journal of Life Science, 14(3), 472-477. Yamashita, E. (2015). Let astaxanthin be thy medicine. PharmaNutrition, 3(4), 115-122. Yang, J., Tan, H., Yang, R., Sun, X., Zhai, H., & Li, K. (2011). Astaxanthin production by Phaffia rhodozyma fermentation of cassava residues substrate. Agricultural Engineering International: CIGR Journal, 13(2), 1847. Zhang, Q., & Cai, W. (2008). Enzymatic hydrolysis of alkali-pretreated rice straw by Trichoderma reesei ZM4-F3. Biomass and Bioenergy, 32(12), 1130-1135. Zhu, S., Wu, Y., Yu, Z., Liao, J., & Zhang, Y. (2005). Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis. Process Biochemistry, 40(9), 3082-3086.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.16 International Conference on Food, Agriculture and Biotechnology
THE EFFECT OF DRYING WITH CONTROLLING RELATIVE HUMIDITY OF DRYING AIR ON THE COLOR AND TEXTURE OF MANGO SHEET PRODUCTS
KWANCHANOK PRAPHUNCHONAKORN, WEERACHET JITTANIT*
Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, 50 Ngam Wong Wan Road, Chatuchak, Bangkok 10900, Thailand.
*Corresponding author: [email protected]
Abstract:
Mango sheet is one of the traditional mango products that is produced for preservation and market-value addition purposes. Currently, the factory encounters the problem about the long drying time requirement of this product. Therefore, this research aimed to increase the efficiency of drying mango sheets by proposing the hot air drying method that controlled the relative humidity (RH) of drying air. In this study, three drying schemes namely conventional hot air drying (60 C), single-stage drying (60 C/15% RH), and two-stage drying (80 C for 1 h 5 min in the 1st stage and then adjusted to 60 C/15% RH in the 2nd stage) were applied in the mango sheet drying until reaching the moisture content of approximately 16.8% wet basis (w.b.) which is comparable to the commercial product. The color and textural properties, consisting of firmness and adhesiveness of mango sheets dried by these methods were determined and compared with those of the commercial product. It appeared that all of the drying schemes required shorter drying time than that of the current practice in the factory, especially when the relative humidity of drying air was controlled. The color intensity of the sample dried by conventional hot air drying product was the lowest, followed by the two-stage, and single-stage drying respectively whereas all the color values of commercial product was greater than its counterparts. In addition, the firmness and adhesiveness of conventionally-dr ied samples were higher than the two-stage dried sample, commercial product, and single-stage dried mango sheets respectively. This research proved that the drying process that applied the relative humidity control which is the similar concept to the heat pump dryer could noticeably shorten the drying time compared to the general hot air drying methods while its product quality was not much different from the commercial product.
Keywords: Drying; Heat pump; Hot air drying; Mango sheet; Relative humidity control
Introduction
Mango is one of the most important economic fruit in Thailand due to its large production area of more than 300,000 hectares. It has captivating color, individual flavor, and abundant nutritional value (Matheyambath et al., 2016; Matulaprungsan et al., 2019). In addition, it can grow in many regions of Thailand because of the suitable geography and atmosphere (Matheyambath et al., 2016); however, mango is a highly perishable fruit because it has a short shelf life and thin peel which is susceptibility to physical injury that facilitate microorganism growth (Moalemiyan et al., 2012). So, the processing of fresh mango into mango sheets is a practical alternative to extend the mango shelf life, increase market value, and diminish the logistic cost (Chou & Chua, 2001). Mango sheet is a traditional snack made by drying a thin layer of mango puree. Its popularity causes by its sweetness taste, chewy texture and long shelf life under room temperature (Diamante et al., 2014). Sun drying is the traditional method for
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology drying mango sheet; however, the disadvantages of this method are its long drying period, uncontrollable drying condition, and unhygienic process. So far, the food industry has developed mechanical dryers to overcome these problems such as hot air dryer (Maskan et al., 2002; Diamante et al., 2014). Although hot air drying method requires a shorter time than sun drying and is more hygienic, most hot air dryers are still open systems. Moreover, the hot air in the drying chamber frequently contains high moisture as a result of moisture transfer from food (Wang et al., 2011). As a consequence, if this hot air was totally recycled without the mixing of the fresh air, the drying efficiency would decrease. Heat pump dryer (HPD) is a novel dryer that has the potential to be applied in the mango sheet drying. The benefit of heat pump drying is its closed-loop system; therefore, the process is more hygienic compared to those of the open system. Moreover, its capability to recycle heat back to the drying process leads to the high energy efficiency and subsequently energy cost saving (Kivevele & Huan, 2014). Duan et al. (2019) investigated the thermal characteristic of hawthorn cakes which were dried by a HPD in comparison with that of the conventional hot air dryer (HAD). They found that air humidity in HPD was lower than HAD around a half; consequently, HPD has a greater drying rate and energy saving than HAD. Pal et al. (2008) claimed that the lower relative humidity of drying air leads to the increase of driving force of moisture transfer from the surface of food material to the air. The key objective of the present work was to increase the efficiency of drying mango sheets by proposing the hot air drying method that controlled the relative humidity of drying air which imitated the occurrence in the heat pump dryer. The outcome of this research would be beneficial to the mango sheet producers who have the problem about the long drying time requirement and high energy consumption in the manufacturing process of mango sheet.
Materials and Methods
Sample Preparation
Mango puree and commercial mango sheet products were supplied by Woraporn Fruit Processing Co., Ltd., Chachoengsao, Thailand. Mango puree was spread into the plastic mold plate placed on the aluminum tray. The diameter of each hole of the plastic mold plate was 47 mm whereas its depth was 4 mm. The aluminum trays containing mango puree were put into the dryer in order to process the mango puree to be mango sheets.
Drying Experiments
The commercial mango sheet product was manufactured using two-stage drying methods. First, the tunnel dryer was applied at 65 C for 4-5 h. After that, the heat pump dryer was used for about 48 h by setting the drying air temperature at 60 C and 10% RH. However, the actual drying air condition in the heat pump drying chamber was not same as the setting values due to the incompatibility between the dryer capacity and the size of drying chamber. The temperature was much lower whereas the relative humidity was considerably higher than the set values. In the present study, mango puree samples were dried using three different drying schemes consisting of (1) conventional hot air drying at temperature of 60 C (Gujral & Brar, 2007) without RH control of drying air (average RH 48%), (2) single-stage drying at 60 C with RH control at 15%, and (3) two-stage drying applying temperature at 80 C for 1 h 5 min in the 1st stage and then adjusted to 60 C with RH control at 15% in the 2nd stage. The drying air velocity was in the range of 1.0-1.5 m/s. All the samples were dried until reaching the
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology moisture content of approximately 16.8% w.b. which is analogous to the moisture content of commercial product. The drying without RH control was carried out in the hot air oven (MEMMERT, UF 55, Germany) whereas a constant climate chamber (BINDER, KBF-240, Germany) was applied for the drying with RH control. The hot air drying with the RH control was conducted to imitate the operation of the heat pump dryer. After drying, the mango sheets were removed from the aluminum trays, filled in the aluminum foil bags, and kept at room temperature before quality determination. The drying experiments were conducted in triplicate for each drying scheme.
Color Measurement
Color of the mango sheets were measured using a Hunter Lab Colorimeter (Hunter Lab, Model UltraScan PRO, USA) using the L, a, b scale, where L represents lightness (0 ≤ L ≤ 100), while a (+), a (-), b (+) and b (-) represent redness, greenness, yellowness, and blueness, respectively. The colorimeter was calibrated with a black card device using illuminant D65 and the 10° standard observer. (Azeredo et al., 2006). The measurement was carried out in triplicate for the sample collected from each drying run.
Textural Property Measurement
The firmness and adhesiveness values of mango sheets were analyzed applying a texture analyzer (Stable Micro Systems, Model TA.XT.plus, UK). The method was modified from Suna & Karabacak (2019). The samples were stacked on two pieces. A light knife blade probe was used to compress the top mango sheet to 40% deformation at a pre-test speed of 1 mm/s, test speed of 2 mm/s, and post-test speed of 2 mm/s. The measurement was conducted in three replications for the sample collected from each drying run.
Statistical Analysis
The software package SPSS (ver. 16.0) was used for the statistical analysis. The significant differences between the mean values of all the experimental data were analyzed by one-way analysis of variance (ANOVA) with Duncan's New Multiple Range Test at a confidence level of 95% (p < 0.05).
Results and Discussion
Drying Characteristics
Drying kinetics of mango sheets during drying processes are illustrated in Figure 1. It appeared that at the beginning of the drying process, heat from drying air was transferred to mango puree, which had high initial moisture content; therefore, free moisture on the sample surface was rapidly transferred to the drying air. After that, the drying rates were lower because they relied on the moisture diffusion from inside to the surface of mango sheets (Traub, 2002; Argyropoulos et al., 2011). The required periods for drying mango puree samples to the final moisture content of about 16.8% w.b. were 14, 11, and 9 h in cases of applying the conventional hot air drying, single-stage, and two-stage schemes respectively. It is obvious that all of these drying experimental conditions spent shorter drying time than the current drying process of the commercial product applied in the factory. This should be due to the better drying condition control. The conventional hot air drying scheme that applied drying air temperature at 60 C
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology without RH control (average RH 48%) consumed apparently longer drying time than the single-stage and two-stage drying processes which combined relative humidity control because the higher relative humidity of the drying air decreased the moisture gradient between the sample surface and the drying air resulting in the decline drying rate and moisture diffusivity (Ju et al., 2016). For the two-stage drying method, the mango sheets were dried at 80 C in the first stage for 1 h 5 min leading to the moisture content reduction to approximately a half way between the initial moisture content and the required final moisture content. Although the first drying stage did not apply the relative humidity control of drying air, the drying rate was greater than its counterparts because the drying temperature of 80 C provided the higher driving force for moisture transfer than that of 60 C. Moreover, the drying air that was heated to the temperature of 80 C would commonly have low relative humidity. Pal et al. (2008), Garavand et al. (2011), and Ju et al. (2016) claimed that the higher drying air temperature results in a highly thermal gradient between the sample and the drying air; hence, it causes a higher heat and moisture transfers than the lower drying temperature. In the second drying stage, the drying air temperature was decreased to 60 C while the relative humidity control was applied in order to diminish the adverse effect of high temperature on the product quality and to accelerate the drying process. Therefore, the two-stage drying scheme spent the shortest drying time among all drying schemes applied in the present work. Azeredo et al. (2006) pointed out that high drying temperature could cause the reduction of mango sheet product quality such as darker color, harder texture and case hardening. That is the reason for the decrease of drying temperature to 60 C when the moisture content of mango sheets declined to half way after the first stage drying in this study.
70
) .
b 60
.
w %
( 50
40 30 20
Moisture Moisture content 10 0 0 5 10 15 Drying time (h)
60 C 48%RH 60 C 15%RH 80 C&60 C 15%RH
Figure 1: The drying characteristics of mango sheets in all drying conditions.
Color
The L, a, b values of mango sheets which were dried in the present work are shown in Table 1 and compared with the commercial product. Color values of the commercial product were the highest in every parameter followed by single-stage, two-stage, and conventional drying products, respectively. A lower L value implied that these samples are darker than the others.
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Maillard reaction is a non-enzymatic browning reaction that occurred during the drying process. It generated the appearance of brown color on the food products whereas the brown color would be more intense when raising the drying temperature and drying time (Argyropoulos et al., 2011). Due to the long drying time in the conventional hot air drying process, the Maillard browning reaction would occur in more extent and result in the lowest values of color lightness. It appeared that the mango sheet sample obtained from the two-stage drying scheme had significantly lower lightness than that of the single-stage drying although its drying time was shorter. It is because the higher drying temperature (80 C) was applied in the first drying stage while the single stage drying scheme used a lower temperature (60 C) of drying air throughout its drying process. Moreover, it appeared that the lightness value of the commercial product was the highest among all samples. It should be due to the fact that the commercial mango sheet product was dried at 65 C for 4-5 h in the first stage and then it was dried by a heat pump dryer for around 48 h at lower drying temperature than 60 C in the later stage. The photographs of all mango sheets samples are presented in Figure 2.
Table 1: Color values of mango sheets that were dried by different conditions.
Sample L a b
Commercial product 70.63 ± 0.74a 13.61 ± 0.57a 44.93 ± 0.51a Conventional hot air drying 59.72 ± 0.63d 12.67 ± 0.61b 38.24 ± 0.43d (60 C, 48% RH) Single-stage drying 63.52 ± 1.30b 13.10 ± 0.93b 40.80 ± 0.74b (60 C, 15% RH) Two-stage drying 62.58 ± 0.50c 12.46 ± 0.45c 40.04 ± 0.36c (80 C 60 C, 15% RH) Superscript with different letters a, b, c, and d in the same column show significantly different (p < 0.05).
(a) (b) (c) (d)
Figure 2: Photographs of mango sheet samples after drying; (a) Commercial product; (b) Conventional hot air drying; (c) Single-stage drying; (d) Two-stage drying.
Textural Property
The firmness and adhesiveness values of mango sheet samples are indicated in Table 2. It appeared that these values were significantly different among samples. The conventional hot air drying method provided the dried mango sheets that had the highest value of firmness and adhesiveness. It might be caused by the higher relative humidity of drying air during the
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology conventional hot air drying and subsequently the longest drying time compared to the single - stage and two-stage drying schemes. The longest drying time resulted in the non-uniformity of moisture distribution inside the samples. Although the mango sheet samples collected from all drying conditions had similar moisture content of around 16.8% w.b., they were likely to have dissimilar moisture distribution within the samples. For the sample dried by the conventional hot air drying method, their surface would have less moisture than the inside region of mango sheet sample due to its long drying time exposure. As a consequence, the sample surface would be denser and harder leading to the greater hardness and adhesiveness when measured. The firmness value of commercial mango sheet product was insignificantly different from that of the sample obtained from the two-stage drying while their adhesiveness were not much different because both drying methods applied two-stage drying concept which use higher drying temperature in the first stage and lower temperature in the second stage leading to the similar pattern of moisture distribution within the samples and subsequently comparable textural characteristics. It appeared that the mango sheets dried by the single-stage drying scheme at 60 C/15% RH had the lowest firmness and adhesiveness. It might be due to its mild drying condition along the whole drying process and its shorter drying time than the conventional hot air drying method.
Table 2: Textural properties of mango sheet samples.
Firmness Adhesiveness Sample (kgf) (kg.sec) Commercial product 3.82 ± 0.13b 1.23 ± 0.10c Conventional hot air drying 4.77 ± 0.19a 1.77 ± 0.13a (60 C, 48% RH) Single-stage drying 2.92 ± 0.11c 1.09 ± 0.18d (60 C, 15% RH) Two-stage drying 3.83 ± 0.16b 1.50 ± 0.16b (80 C 60 C, 15% RH) Superscript with different letters a, b, c, and d in the same column show significantly different (p < 0.05).
Conclusion
The drying characteristics of mango sheets revealed that at the beginning of the drying process, the drying rate was high whereas it decreased along the drying period. The required periods for drying mango sheets to the final moisture content of around 16.8% w.b. were 14, 11, and 9 h in cases of applying the conventional hot air drying, single-stage, and two-stage schemes respectively which were much shorter than the current drying practice of the factory. The relative humidity control obviously resulted in the higher drying rate and consequently shorter drying time. For the two-stage drying method, the first stage drying at 80 C for 1 h 5 min could reduce the moisture content of mango sheets to approximately a half way between the initial moisture content and the required final moisture content. The color values of commercial product were the highest in every parameter followed by single-stage, two-stage, and conventional drying products, respectively. Maillard reaction was expected to be a major cause of product color intensity. The long drying time in the conventional hot air drying process was speculated to result in the lowest values of color lightness. The lightness value of the commercial product was the highest among all samples. The firmness and adhesiveness values of mango sheet samples were significantly different among samples. The dissimilar moisture distribution within the mango sheets should be the reason for these textural property results.
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The present work proved that the drying process that applied the relative humidity control which is similar to the occurrence in the heat pump dryer could obviously shorten the drying time compared to the drying without relative humidity control whereas the product quality was not much different from the commercial product.
References
Argyropoulos, D. , Khan, M. T. , & Müller, J. ( 2011) . Effect of Air Temperature and Pre- treatment on Color Changes and Texture of Dried Boletus edulis Mushroom. Drying Technology. 29(16): 1890-1900. Azeredo, H.M.C., Brito, E.S., Moreira, G.E.G., Farias, V.L., & Bruno, L.M. (2006). Effect of Drying and Storage Time on the Physico- Chemical Properties of Mango Leathers. International Journal of Food Science and Technology. 41(6): 635-638. Babu, A. K. , Kumaresan, G. , Antony Aroul Raj, V. , & Velraj, R. ( 2018) . Review of Leaf Drying: Mechanism and Influencing Parameters, Drying Methods, Nutrient Preservation, and Mathematical Models. Renewable and Sustainable Energy Reviews. 90: 536-556. Chou, S.K., & Chua, K.J. (2001). New hybrid drying technologies for heat sensitive foodstuffs. Trends in Food Science & Technology. 12(10): 359–369. Diamante, L.M., Bai, X., & Busch, J. (2014). Fruit Leathers: Method of Preparation and Effect of Different Conditions on Qualities. International Journal of Food Science. 1-12. Garavand, A. T. , Rafiee, S. , & Keyhani, A. ( 2011) . Study on Effective moisture diffusivity, activation energy and mathematical modeling of thin layer drying kinetics of bell pepper. Australian Journal of Crop Science. 5(2): 128-131. Gujral, H.S., & Brar, S.S. (2007). Effect of Hydrocolloids on the Dehydration Kinetics, Color, and Texture of Mango Leather. International Journal of Food Properties. 6(2): 269– 279. Ju, H.Y., Law, C.L., Fang, X.M., Xiao, H.W., Liu, Y.H., & Gao, Z.J. (2016). Drying kinetics and evolution of the sample's core temperature and moisture distribution of yam slices ( Dioscorea alata L. ) during convective hot- air drying. Drying Technology. 34( 11) : 1297-1306. Kivevele, T. , & Huan, Z. ( 2014) . A Review on Opportunities for the Development of Heat Pump Drying Systems in South Africa. South African Journal of Science. 110(5/6): 1- 11. Maskan, A., Kaya, S., & Maskan, M. (2002). Hot air and sun drying of grape leather (pestil). Journal of Food Engineering. 54(1): 81–88. Matheyambath, AC., Subramanian, J., & Paliyath, G. (2016). Mangoes. Encyclopedia of Food and Health. 641-645. Matulaprungsan, B. , Wongs- Aree, C. , Penchaiya, P. , Boonyaritthongchai, P. , Srisurapanon, V., & Kanlayanarat, S. (2019). Analysis of critical control points of post-harvest diseases in the material flow of nam dok mai mango exported to Japan. Agriculture. 9(9): 1-12.
Moalemiyan, M., Ramaswamy, H.S., & Maftoonazad, N. (2012). Pectin-based edible coating for shelf-life extension of ataulfo mango. Journal of Food Process Engineering. 35(4): 572-600. Pal, U.S., Khan, M.K., & Mohanty, S.N. (2008). Heat Pump Drying of Green Sweet Pepper. Drying Technology. 26(12): 1584-1590. Suna, S., & Karabacak, Ö.A. (2019). Investigation of Drying Kinetics and Physicochemical
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Properties of Mulberry Leather (Pestil) Dried with Different Methods. Journal of Food Processing and Preservation. 43(8): 1-9. Traub, D. A. ( 2002, September 1) . The Drying Curve, Part 1. Retrieved from https://www.process-heating.com/articles/86586-the-drying-curve-part-1. Wang, D. C., Zhang, G., Han, Y. P., Zhang, J. P., & Tian, X.L. (2011). Feasibility analysis of heat pump dryer to dry hawthorn cake. Energy Conversion and Management. 52(8-9): 2919-2924.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.17 International Conference on Food, Agriculture and Biotechnology
THE EFFECT OF TEMPERATURE HUMIDITY INDEX (THI) ON EGG PRODUCTION IN PRADU-HANGDUM CHAING MAI HIGH EGG PRODUCTION STRAIN CHICKENS
KAMONNATE PIMRUENG1, DOUNGNAPA PROMKET1*, KHANITTA PENGMEESRI1, JENNARONG KAMMONGKUN2
1Animal Science, Department of Agricultural Technology, Faculty of Technology, Mahasarakham University, Maha Sarakham 44000, Thailand 2Chiang Mai Livestock Research and Breeding Center, San Pa Tong, Chiang Mai 50120, Thailand
*Corresponding author: [email protected]
Abstract:
The purpose of this research was to study the effects of climate changes on egg production under Chiang Mai Livestock Research and Breeding Center in Chiang Mai Province. This study was conducted from 300 Pradu-Hangdum Chiang Mai high Egg Production strain chickens. The data record collected from April 2019 until March 2020. The study indicated that, the weather was hot and humid. The temperature ranged from 22.28-31.23°C and humidity range were 46.70-81.80%. The highest temperature in May (31.12°C) and April (31.23°C) the lowest temperature in December (22.28°C). Daily temperature and humidity data from meteorological measurements in Chiang Mai were recorded daily over the duration of the trial period were used to calculate the temperature humidity index (THI). The THI group for effect on egg production composition was set to 4 groups (THI1 is THI ≤ 70, THI2 is ≤ 74 THI > 70, THI3 is ≤ 78 THI >74 and THI4 is THI > 78). The highest THI values (THI 4) effect on age at first egg (AFE), THI4 (144.59 day) was lower than THI1 (161.40 day), THI2 (158.22 d) and THI3 (156.17 d). AFE in summer season was lower than raining season. The correlation between TEP270 and average egg at 1 mouth (AEM) traits were found the highest correlation (0.90). Moreover, the correlation between weight at first egg (HWFE) and age at first egg (AFE) was 0.38, it meaning that HWFE resulting in AFE faster.
Keywords: Temperature humidity index ( THI) ; Egg production; Pradu- Hangdum Chiang Mai high Egg Production strain
Introduction
Thailand is located in area hot weather conditions are associated with animal production capability. Especially in hot summer and rainy season, high humidity will affect the body temperature of chickens (Amnuay et al., 2011). Higher temperatures and humidity are effected of the environment caused by heat stress directly affects the decline in egg production. The impact is increasing and can survive for a long time. The body temperature of hen is maintained 40.0-42.2 °C by thermoregulatory mechanism when the environmental temperature is within the thermoneutral zone poultry are homoeothermic. If the thermoregulation mechanism is insufficient to maintain homeothermy, the body temperature begins to rise and eventually cause to death from heat stress (Ilker & Simsek, 2013). The upper critical temperature for chickens were between 36 and 37oC are most comfortable. Health, productivity is maximized and stress is minimized at the thermoneutral zone. Growth rate and egg production of the native chickens under conventional rearing system in villages are very low. During the past several decades, importation of exotic breeds
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology have increased risk of extinction (Shahram et al., 2012). Haiping et al. (2011) report the age at first egg is one of the direct indicators for sexual maturation in female chickens. Moreover, Wuttigrai et al. (2014) reported mean of age at first egg (day) on Pradu Hangdum chickens was 203 day and Suchittra et al. (2015) reported mean of age at first egg (day) on Chee native chickens was 211 day. Egg production is an important economic characteristic in the poultry industrial (Wuttigrai et al., 2014). Nowadays, Thai native chickens have relatively low egg production. From the report found that native chickens was 3-5 sets of eggs each year, 13 eggs each set (Pin et al., 2004). Jennarong et al. (2014) studying Thai native chicken in Pradu- Hangdum Chiang Mai using the pure lines of Pradu-Hangdum Chiang Mai under Chiang Mai Livestock Research and Breeding Center and evaluation genetic parameter on egg production. The goal for breeding and selection provide Pradu-Hangdum Chiang Mai high Egg Production strain chickens. The temperature humidity index (THI) is an indication of the calculation form environmental temperature and humidity caused by heat stress conditions. Therefore, temperature and humidity variation may affect behavioral changes and physiology of the hens. The resultant heat stress comes from the interactions among air temperature, humidity, radiant heat and air speed, where the air temperature plays the major role. Laying hens can produce more number and more big eggs in indoor environmental temperature between 13-24 °C (Ilker & Simsek, 2013). The effect of heat stress on chickens found high mortality, decreased feed intake, low laying rate, egg weight in laying hens (Oguntunji & Alabi, 2010; Mutibvu et al., 2017; Cicero et al., 2018). The objective of this study was to determine effects of temperature humidity index (THI) on egg production in Pradu-Hangdum Chiang Mai high Egg Production strain chickens.
Materials and Methods
Animal and egg production data, the study was conducted from 300 Pradu-Hangdum Chiang Mai high Egg Production strain chickens under the open house with battery cages system in Chiang Mai Livestock Research and Breeding Center, SanPaTong District, Chiang Mai Province. The width and length of cages are width × length × high were 20×30×40 cm. All chickens were fed by raised in battery cages; solitary confinement has an artificial mating pattern. The analysis of climate changes (temperature humidity index; THI) on egg production in Pradu-Hangdum Chiang Mai high egg production Strain Chickens was focused. This study useds data recorded data of individual egg production, include hen weight at first egg (HWFE), age at first egg (AFE), egg weight at first (EWF), average egg weight at 270 day (AEW270), total egg production at 270 day (TEP270) and average egg at 1 mouth (AEM). Meteorological measurements in Chiang Mai were recorded daily over the duration of the trial period (Average temperature: degree Celsius) and Relative humidity (Average humidity: percentage). According to the method of (Mutibvu et al., 2017). The data record collected from April 2019 until March 2020. The temperature and relative humidity data (from April 2019 until March 2020) obtained from the records of the meteorological center closest to each meteorological measurements in Chiang Mai. The weather information included daily temperature and relative humidity recorded every 3 h, which were used to calculate the temperature humidity index (THI) by using the equation bellow (Mader et al., 2006).
THI =Td – [0.55x(RH/100)] x [Td – 58]
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Where Td is temperature in degrees Celsius and RH is relative humidity as a percentage. After calculating the THI value dividing the THI group into 4 groups as follows: THI1 is THI ≤ 70, THI2 is ≤ 74 THI > 70, THI3 is ≤ 78 THI >74 and THI4 is THI > 78.
Statistical analysis
The means of egg production traits were analyzed using PROC MEANS (SAS, 1998). The correlation among production traits used PROC CORR. The effect of THI on egg production traits was performed using the GLM procedure. The means between variables were considered significantly different at P < 0.05 (SAS, 1998).
Results and Discussion
Table 1 shows the means, standard deviation (SD), minimum and maximum for egg production characteristics of Pradu-Hangdum Chiang Mai high Egg Production Strain Chickens. This study showed the means of hen weight at first egg (HWFE) was 1,930.87 g. Moreover, means of age at first egg (AFE), egg weight at first (EWF), average egg weight at 270 day (AEW270), total egg production at 270 day (TEP270) and average egg at 1 mouth (AEM) were 153.81 day., 33.80 g., 44.80 g., 137.92 egg, 16.33 egg, respectively. According with Worawit et al. (1998) reported mean of weight at first egg of hen was 1,900 g. which similarly with this study. But, Wuttigrai et al. (2014) reported mean of age at first egg (day) in Pradu Hangdum chickens was 203 day. and accordance with Pin et al. (2004) reported mean of egg weight at first in Betong chickens was 38.53 g. and Natthakan (2015) reported mean of egg weight at first in black-bone chicken was 35.14 g.
Table 1: The descriptive data of egg production in Pradu- Hangdum Chiang Mai high egg production strain chickens.
Traits Means SD Minimum Maximum HWFE 1,930.87 19.35 1,075.00 2,482.00 AFE 153.81 11.75 128.00 198.00 EWF 33.80 5.94 18.00 58.00 AEW270 44.80 3.05 32.28 54.36 TEP270 137.92 28.76 70.00 211.00 Traits Means SD Minimum Maximum AEM 16.33 3.04 8.67 23.44 Note: abmeans within a row with different superscripts different significant (P<0.05) HWFE is hen weight at first egg (g), AFE is age at first egg (day), EWF is egg weight at first (g), AEW270 is average egg weight at 270 day, TEP270 is total egg production at 270 day, AEM is average egg at 1 mouth
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90 80 70 60 50 40 30 20 10 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec THI Temp (°C) RH (%) Critical Point
Figure 1: Average temperature, relative humidity and temperature humidity index (THI) variation during year of 2019.
Figure 1 shows temperature, relative humidity, temperature humidity index (THI) and critical point that can affect chickens due to heat stress. The highest temperature add THI found on April. Moreover, the temperature and THI values in this study were lower than 80 which had on effect on egg production. Habeeb et al. (2018) showed critical point that can affect productivity in chickens due to heat stress when THI over 82.
Table 2: Environmental conditions during the experimental periods.
Temperature (°C) Relative humidity (%) Month THI Means Minimum Maximum Means Minimum Maximum January 23.73 21.10 26.10 70.30 64.00 89.00 68.25 February 25.33 23.30 27.00 56.70 49.00 67.00 71.48 March 28.54 26.20 31.20 47.10 40.00 52.00 76.80 April 31.23 28.90 33.40 46.70 40.00 60.00 80.45 May 31.12 27.30 34.70 60.90 43.00 82.00 77.96 June 29.67 27.40 32.70 69.10 58.00 83.00 74.99 July 28.97 24.50 32.70 69.70 56.00 92.00 74.12 August 27.37 24.90 29.20 81.80 67.00 91.00 70.80 September 27.77 25.40 29.50 75.30 66.00 85.00 72.05 Temperature (°C) Relative humidity (%) Month THI Means Minimum Maximum Means Minimum Maximum October 27.81 24.50 29.80 74.00 66.00 90.00 72.27 November 26.15 24.60 28.20 71.00 64.00 80.00 70.84 December 22.28 17.70 25.40 67.20 49.00 78.00 66.89
Table 2 shows of temperature, relative humidity and temperature humidity index (THI) by Meteorological Department from Chiang Mai province. Mean maximum and minimum of temperature, relative humidity and calculated THI by the experimental period are shown in
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology table 2. There are 3 seasons in Thailand: winter season (October to January), summer season (February to May) and raining season (June to September). The highest temperature (31.23), THI (80.45) and lowest humidity (46.70) was found on April which in summer season. The effect of temperature humidity index (THI) of all 4 groups on egg production in Pradu-Hangdum Chiang Mai high Egg Production Strain Chickens was found in AFE. The AFE trait on THI4 (144.59 day) was lower than THI1 (161.40 day), THI2 (158.22 d) and THI3 (156.17 d). For means of HWFE, EWF, AEW270, TEP270 and AEM among THI groups were not significant difference (Table 3). Narongsak (2004) reported the upper critical temperature for chickens was between 36 and 37oc is most comfortable. Health, productivity is maximized and stress is minimized at the thermoneutral zone. Habeeb et al. (2018) showed critical point that can effect chickens due to heat stress when THI over 82. Therefore, the AFE of THI4 in maximum at 80.45 shows that THI4 is still in a comfortable range of animals.
Table 3: The effected of THI groups on egg production in Pradu-Hangdum Chiang Mai high egg production strain chickens.
THI groups Traits P-value 1 2 3 4 HWFE 1,935.00 1,991.37 1,938.55 1,868.16 0.83 AFE 161.40a 158.22a 156.17a 144.59b <0.01 EWF 32.47 34.47 34.25 32.345 0.48 AEW270 45.97 43.95 44.65 45.68 0.09 TEP270 146.80 134.28 135.89 144.67 0.39 AEM 17.14 16.42 16.16 16.59 0.79 Note: abmeans within a row with different superscripts different significant (P<0.05) HWFE is hen weight at first egg (g), AFE is age at first egg (day), EWF is egg weight at first (g), AEW270 is average egg weight at 270 day, TEP270 is total egg production at 270 day, AEM is average egg at 1 mouth. Temperature humidity index: THI1 is THI ≤ 70, THI2 is ≤ 74 THI > 70, THI3 is ≤ 78 THI >74 and THI4 is THI > 78.
Table 4: Egg production (LSM ± SD) between summer and rainning in Pradu-Hangdum chiang Mai high egg production strain chickens.
Seasons Traits P-value summer raining HWFE 1,839.70 1,934.01 0.33 AFE 138.40b 154.34a 0.01 EWF 31.98 33.86 0.10 AEW270 45.32 44.78 0.18 TEP270 148.90 137.54 0.47 AEM 16.74 16.31 0.41 Note: abmeans within a row with different superscripts different significant (P<0.05) HWFE is hen weight at first egg (g), AFE is age at first egg (day), EWF is egg weight at first (g), AEW270 is average egg weight at 270 day, TEP270 is total egg production at 270 day, AEM is average egg at 1 mouth Summer is February to May, raining is June to September, Winter is October to January
Table 4 shows egg production (LSM ± SD) of the different seasons in Pradu-Hangdum Chiang Mai high Egg Production Strain Chickens. For the effect of seasons, AFE in summer was the best is significant different. For HWFE, EWF, AEW270, TEP270 and AEM was no significant different in the seasons (P<0.05). Narongsak (2004), the genetic potentiality of poultry would not be utilized fully. The main season for this restriction is environment
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology constraints. In accordance with Behura et al. (2016) reported summer at means temperature was 31.71°C, so age at first egg is summer in chickens (138.40 day). Because of influence from the environment found that the hot weather makes the chicken stay comfortable. Housing system effect on the thermos physiological traits of hens by higher in battery cage than the deep litter system. Narongsak (2004) showed a comfortable temperature for chickens was between 36 oC and 37oC in the summer.
Table 5: The correlation of egg productions in Pradu-Hangdum Chiang Mai high egg production strain chickens.
Traits HWFE AFE WFE AEW270 TEP270 AEM HWFE 1.00 0.38 0.18 0.07 -0.17 -0.12 AFE 1.00 0.25 -0.16 -0.07 -0.02 EWF 1.00 -0.12 -0.06 -0.04 AEW270 1.00 0.07 0.01 TEP270 1.00 0.90 AEM 1.00 Note: HWFE is hen weight at first egg (g), AFE is age at first egg (day), EWF is egg weight at first (g), AEW270 is average egg weight at 270 day, TEP270 is total egg production at 270 day, AEM is average egg at 1 mouth
Table 5 shows the correlation of egg productions in Pradu-Hangdum Chiang Mai high Egg Production strain chickens. The correlation on egg productions between -0.17 to 0.90. The result showed correlation between TEP270 and AEM traits was the highest correlation (0.90). The highest correlation of this study showed that, the chickens high TEP270 and high AEM also. A positive correlation was found between HWFE and AFE (0.38) and between AFE and EWF (0.25). In addition, HWFE was negatively correlated with TEP270 (-0.17). Moreover, the correlations between AFE and AEW270 was negatively relationship (-0.16). In accordance with Oke et al. (2004) reported percentage lean was positive correlated with body weight and weight at first egg. Moreover, Hidalgo et al. (2011) reported percentage lean was positive correlated with age at first egg and egg weight. But, age at first and egg production, weight at first egg and egg production were negatively correlated.
Conclusions
Results of this study indicate the effect of temperature humidity index (THI) on egg production in Pradu-Hangdum Chiang Mai high Egg Production strain chickens. The AFE trait on THI4 (144.59 day) was lower than THI1 (161.40 day), THI2 (158.22 d) and THI3 (156.17 d). For HWFE, EWF, AEW270, TEP270 and AEM among THI groups not significant difference. AFE in summer season was lower than raining season. The correlation between TEP270 and AEM traits was found the highest correlation (0.90).
Acknowledgements
I would like to thank faculty of technology Mahasarakham University, Maha Sarakham, Thailand for financial support. Chiang Mai Livestock Research and Breeding Center, Thailand, for data of egg productions, which were the main information of this research.
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References
Amnuay, M., Suvit, B. & Montakan, K. (2011). Environmental improvement for high milk production for small scale farmers in western Thailand. 52(1), 0211-035. Behura, N. C., Kumar, F., Samal, L., Sethy, K., Behera, K. & Nayak, G.D. (2016). Use of Temperature-Humidity Index (THI) in energy modeling for broiler breeder pullets in hot and humid climatic conditions. J. Livestock Sci. 7, 75-83. Cicero, H. O., Cohen, J., Rankine, D., Taylor, M., Cambell, J. & Stephenson, T. (2018). Characterizing heat stress on livestock using the temperature humidity index (THI)— prospects for a warmer Caribbean. 18, 2329-2340. Habeeb, A. A., Gad, A. E. & Atta, M. A. (2018). Temperature-humidity indices as indicators to heat stress of climatic conditions with relation to production and reproduction of farm animals. ISSN: 2639-4529. Hidalgo, A. M., Nunes,M. E., Santos, A. L., Quadros, T., Ana, P. S. & Rafael, T. (2011). Genetic characterization of egg weight, egg production and age at first egg in Quails . (1), 95-99. Ilker, K. and Simsek, E. (2013). The effects of Heat stress on egg production and quality of laying hens. 12(1), 42-47. Jennarong, K., Chalermpon, B., Choosak, P. & Amnuay, L. (2014). Effect of layer breed, storage temperature and time on egg quality. Khon Kaen AGR. J. 42(1), 223-229. Mader, T. L., Davis, M. S. & Brown-Brandl, T. (2006). Environmental factors influencing heat stress in feedlot cattle. Anim. Sci. J. 84(3), 712-719. Mutibvu, T., Chimonyo, M. & Halimani, TE. (2017). Physiological responses of slow-growing chickens under diurnally cycling temperature in a hot environment. Poul. Sci. J. 19, 567-576. Narongsak, C. (2004). Physiological reactions of poultry to heat stress and methods to reduce its effects on poultry production. Thai J. 34(2), 17-30. Natthakan, M. (2015). Egg production and reproductive performance of royal project black- bone chicken in parent generation. 2(1): 28-38. Oke, U. K., Herbert, U. & Nwachukwu, E. N. (2004). Association between body weight and some egg production traits in the guinea fowl (Numida meleagris galeata. Pallas). 62, 1155-1159. Oguntunji, A.O. and Alabi, O.M. (2010). Influence of high environmental temperature on egg produvtion and shell quality a review. 66, 739-749. Pin, C., Worawit, W., Thumrunk, T. & Somsak, L. (2004). Village betong chicken production in three southernmost Thailand: A Study of phenotypic characteristics, growth, carcass Yield and Egg Performance of Betong chickens. Khon Kaen AGR. J. 20(3), 278-288. SAS. 1998. SAS User’s Guide. Version 6.12. SAS. Inst., Cary, NC. Worawit, W., Suk, W., Saim, Kh. & Banjub, H. (1998). A comparative study on egg production of 4 commercial hybrid layer strains. 33(8), 1-35. Shahram, N., Ardeshir N. J., Hassan, M. Y. & Seyed, A. F. (2012). Estimation of genetic parameters for body weight and egg production traits in Mazandaran native chicken. Trop. Anim. Health Prod. 44, 1437–1443. Suchittra, S., Worawit, S., Jisasak, S. & Kamonporn, Kh. (2015). Development of parent stock of Thai indigenous chicken: Chee. 112-118. Wuttigrai, B., Monchai, D., Banyat, L. & Thevin, V. (2014). Effects of heat stress on genetic parameters and egg production in Pradu Hang Dam Thai native chickens. Khon Kaen AGR. J. 42(3), 319-328.
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Xu, H., Zeng, H., Luo, Ch., Zhang, D., Wang, Q., Sun, L., Yang, L., Zhou, M., Nie, Q. & Zhang, X. (2011). Genetic effects of polymorphisms in candidate genes and the QTL region on chicken age at first egg. 1-9. Yukubu, A., Ekpo, E. & Oluremi, O.I.A. (2018). Physiological Adaptation of Sasso Laying Hens to the Hot-Dry Tropical Conditions. 83(2), 187-193.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.18 International Conference on Food, Agriculture and Biotechnology
THE NUTRITIVE VALUE AND BIOACTIVE COMPOUNDS OF ALFALFA (Medicago sativa) GROWN AT BURAPHA UNIVERSITY, SA KAEO CAMPUS
SUPREENA SRISAIKHAM, QUANJAI RUPITAK*
Faculty of Agricultural Technology, Burapha University, Sa Kaeo Campus, Sa Kaeo 27160, Thailand
*Corresponding author: [email protected]
Abstract:
Legume is a good quality protein source that provides nutritive value for livestock animals, particularly ruminants. It also contains bioactive compounds which can be work against oxidants that prevent diseases. Alfalfa (Medicago sativa) is a perennial flowering plant in the legume family Fabaceae with a high protein content and moderate contents of vitamin B, C, phosphorus, and zinc. The aim of this research was to evaluate the nutritive value and bioactive compounds of alfalfa at harvesting intervals of 120 days at Sa Kaeo province. Alfalfa was grown at the agricultural research facility of the Faculty of Agricultural Technology, Burapha University, Sa Kaeo Campus, Thailand. They experiment was done from September 2018– March 2019. Chemical composition and bioactive compounds (tannin, total phenolic, total flavonoids, and isoflavone) were measured. The results showed that dry matter, ash, crude protein, fat and crude fiber were 91.44%, 9.20%, 17.68%, 2.46%, and 26.48%, respectively. Whereas, tannin, total phenolic, total flavonoids, daidzein and genistein in dry alfalfa were 2.34%, 2.17 mg RE/g sample, 2.02 mg RE/g sample, 46.72 and 15.38 µg/g sample, respectively. The highest phenolic compounds were 203.60 µg/g sample. The results suggest that the alfalfa plant can be developed as supplement that is benefit to health, for dairy cattle to achieve alternative products that contains high-flavonoids level in goat or dairy cow’s milk that will be useful for ruminant and human health as well.
Keywords: Legumes; Alfalfa; Medicago sativa; Nutritive value; Bioactive compound
Introduction
Alfalfa (Medicago sativa) is the forage legume which is an important crop for livestock animals, particularly ruminants. It has been reported that many species of legumes have been used as the raw materials for animal feed, in the form of green feed, hay or pellets, especially in the tropics and sub-tropical regions. Legumes that have been used as fodder and roughage, such as alfalfa forage, hedge lucern (Desmanthus virgatus), hamata (Stylosanthes hamata), and Tha Phra Stylo (Stylosanthes guianensis) which are forage legumes that use parts of the stem, branches and leaves of plants as an animal feed sources, and in comparison to acacia or Leucaena foliage (Leucaena leucocephala) which is one of the fastest-growing leguminous trees. Legumes are resistant to frequent cutting and grazing with high palatability (Clement, 2019). Forage legumes are also high voluntary intake and ruminant animal production when feed supply is non-limiting compared to grasses or cereals (Phelan et al., 2015). In addition, legumes leaf contained both high nutrients and protein leaf that can be used in the diet of ruminant animals. However, Desmanthus virgatus and Leucaena leucocephala have an average CP (17.8% vs. 25.2%) which is moderately rich in condensed tannins (8.3% DM) (Ramirez et al., 2000; Ramirez et al., 2001). Therefore, if it is used at appropriate levels, it can be fed safely
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology and has the potentially useful to be used as a protein raw material to feed the ruminants without toxic symptoms. It has been reported that Leucaena should not be used as a major portion of the diet in non-ruminant animals because they are lower ability to tolerate mimosine than ruminants (Rushkin, 1984). Alfalfa contains numerous secondary metabolites that are useful as human nutrition (Stochmal et al., 2001). There are six phytonutrients in alfalfa including saponins (Oleszek, 1996; 2000), flavonoids (Herna´ndez et al., 1991; Bisby et al., 1994), condensed tannin (Cleef & Dubeux, 2019), coumestrol (Knuckles et al., 1976), carotenoids, and tocols (Livingston, et al., 1980; Hegsted & Linkswiler, 1980) (cited by Stochmal et al., 2001). Due to the fact that the biological activities of flavonoids, it has recently been recognized as an active principal (antioxidant, cancer-preventing, and antimicrobial) (Packer et al., 1999) which also have pharmalogical benefits (Mamta & Jyoti, 2012). Thus, these characteristics of the phytochemical composition of alfalfa offer potential sources for important health based compounds will improve our understanding of their nutritional value. Although parts of the alfalfa plant have most often been unspecified, a number of different flavonoids (Bisby et al., 1994), for example, 47 varieties of alfalfa from USDA stocks, showed that all varieties had similar profiles to flavonoids (Stochmal et al., 2001). Numerous research studies also suggest that a blend of antioxidants found in various phytochemical plants, vegetables and fruits of many types will provide better antioxidant quality when compared to antioxidants from a single food source. This research paper focuses on the nutritive value of alfalfa plants as a rich source of health benefits materials. We expected that they might be the potential of value-added raw materials sourced from alfalfa plants production and providing high-value food supplements in Thailand, or alternatively, as having a possible use as a feed ingredient in some ruminant diets.
Materials and Methods
Study area
The alfalfa (Medicago sativa) plantation was located at the experimental farm of the agricultural research facility of the Faculty of Agricultural Technology, Burapha University, Sa Kaeo Campus, Watthana Nakhon district, Sa Kaeo province in the eastern part of Thailand. The elevation was lower than 1,000 meters above sea level with dry and shallow sandy soil. The soil of the field crops in Sa Kaeo province was classified to be 49 series, including the Phon Phisai soil series (Pp), Sakon soil series (Sk) and Sa Kaeo soil series (Ska), and it is characterized by pH of 6.59 and low fertility.
Collection and chemical analysis of alfalfa plant
The alfalfa samples were randomly collected at 120 days after planting (Figure 1C) for chemical composition analysis of dry matter (DM), ash, crude protein (CP), ether extract (EE), crude fiber, tannin, total phenolic, total flavonoids and isoflavone. Shoot samples of about 20- 30 cm from the tip of the alfalfa branches were taken. Also, samples of the alfalfa plants were cut and immediately chopped into 3-4 cm length with a mechanical forage chopper and dried at 60oC for 48 h. Representative samples of alfalfa were later ground through a 1 mm screen and stored pending chemical analysis and being subjected to a proximate analysis. Chemical analyses of alfalfa samples were performed on each sample in three replications, and the analysis was repeated when the CV was > 0.05. Analytical DM was analyzed by drying samples at 135°C for 2 h, followed by hot weighing (AOAC, 1990; method 930.15). Ether extract was determined by using petroleum ether in a Soxtec System (procedure 948.15, AOAC, 1998). Crude fiber was determined with the standard methods of the AOAC (2010); method 962.09.
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The chemical analysis was expressed on the basis of the final DM. Determination of total tannins (as feed basis) was carried out by following the methods of Burns (1971) by submitting samples to the Feed analysis-2 Laboratory, Department of Animal Science, Faculty of Agriculture at Khamphaeng Saen, Kasetsart University, Khamphaeng Saen. Total phenolic content and total flavonoid compound were extracted and determined using the Folin-Cioca lteu method following the protocol of Kubola & Siriamornpun, 2011). Isoflavone extraction was determined by using the methods of Devi et al. (2009), and analyzed by high performance liquid chromatography (HPLC). Analysis was performed using Shimadzu LC-20AC pumps, a SPD-M20A diode array detector, and a LUNA C-18 column (4.6 x250 mm i.d., 5 µm). Analyses were made using three replications and the results were reported as averages with calculated standard deviations.
Morphological observation
Alfalfa plants were observed for morphological characterization on germination, seedling, stem and leaf shape, flowering period, inflorescence form and pod shape (Figure 1). All characteristics were compared to report of Undersander et al. (2011) that described the botany, vegetative stage and flowering stage of alfalfa. This observation showed that alfalfa characteristics were normally when it was grown at Sa Kaeo province.
Extraction and determination of total phenolics content
The extracts prepared from the freeze-dried alfalfa samples were approximately 5 g and were extracted with 20 ml of 80% methanol (V/V) on a shaking incubator set at 37°C for 12 h. After being filtered through a Whatman No.1 filter, the supernatant was mixed with other previously extracted supernatants of the sample under identical conditions and combined before being decanted into a vial and then stored at -20°C until the total phenolic content and the total flavonoid compounds were measured.
Determination of total phenolics content
The total phenolic content was determined using the Folin–Ciocalteu reagent as described by Kubola & Siriamornpun (2011) and gallic acid was used as standard. Three hundred microliters of alfalfa extract were mixed with 10% of the Folin–Ciocalteu reagent and 2 ml of 7.5% sodium carbonate (Na2CO3) solution. After 90 min incubation time at room temperature, the absorbance was measured at 725 nm using a spectrophotometer. The total phenolic content of the alfalfa extracts was calculated and expressed as gallic acid equivalents at a concentration of 1 to 1000 mg/L which was used as a standard. The quantitative results were expressed in grams of dry weight (mg GAE/gDW (sample)) based on the gallic acid standard curve.
Determination of total flavonoid compounds
Briefly, 500 µl of the alfalfa extract was mixed with 2.25 ml of distilled water following by the addition of 150 µl of 5% NaNO2 solution in test tube. After 6 min, 10% AlCl3.6H2O solution was added and allowed to stand for another 5 min before 1.0 ml of 1 M NaOH was added. The mixture was mixed well using a vortex. The absorbance was measured immediately at 510 nm using a UV-vis spectrophotometer. The quantitative results were expressed in mg rutin equivalents in 1 g of dried sample (mg RE/g).
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Figure 1: Characteristics of alfalfa plant: germination (A), seedling (B), samples collection at 120 days (C), flowering stage (D), compound leaf (E), inflorescence (F) and pod (G).
Results and Discussion
The results of a proximate analysis of the alfalfa plants showed that they contained DM about 91.44%. The CP was between 17.67 to 17.69%, ash 9.18 to 9.22%, crude fat 2.44 to 2.47% and CF 26.40 to 26.53% at the cutting interval of 120 days, respectively (Table 1). The CP was similar to the approximate value of alfalfa legume at cutting intervals of 30, 45, 60 and 75 days from between 18.1 down to 16.7%CP at Burapha University Sa Kaeo Campus, Watthana Nakhon District, Sa Kaeo province during the period from October 2018 to July 2019 [Study Topic: Effects of feeding Leguminosae on productive performance in goat meat]. (Unpublished raw data). A similar result was also recently published by Vintu et al. (2012), with values ranging from 24.3 (early buds), 21.3 (late buds) and down to 16.7%CP in full bloom in the harvest period of whole alfalfa plants with non-fertilizer. Although the forage value of alfalfa was high protein, there was low yield at 325.6, 619.1, 533.0, 597.3 kg/rai with cutting intervals
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology of 30, 45, 60 and 75 days respectively, this demonstrates that it was alfalfa should be combined or mixed with other feedstuffs to make it suitable for the production of animal feed in Sa Kaeo province. It is therefore suggested that alfalfa should be harvested at the beginning of the bud formation period, so that the yield of CF will also decrease markedly. The decreases in %CF, or %NDF and %ADL content with decreasing cutting intervals can possibly be attributed to the cattle eating more because of its low fiber resulting in the feed flowing too rapidly through the intestine. This increases the digestibility and consume ability of the forage for ruminants which corresponds to Srisaikham & Lounglawan (2018) who reported that cutting a stand of sunnhemp (Crotalaria juncea), which was in the same Leguminaceae family, at 55 days of harvesting time would achieve a greater yield of CF than cutting at 30 days.
Table 1: Chemical composition of the branches of alfalfa (Medicago sativa) at harvesting intervals of 120 days.
Item Alfalfa1 Alfalfa2 Alfalfa3 Alfalfa (x̄ ) DM (%) 91.46±0.03 91.44±0.03 91.43±0.05 91.44 Ash 9.22±0.12 9.18±0.15 9.19±0.14 9.20 CP (%) 17.68±0.10 17.69±0.08 17.67±0.12 17.68 EE (%) 2.44±0.01 2.46±0.02 2.47±0.02 2.46 CF (%) 26.50±0.11 26.40±0.13 26.53±0.10 26.48 Tannin (%) 2.75±0.07 2.81±0.13 2.79±0.10 2.78 1 the representation of the first replicated of alfalfa results are an of the 3 values obtained for each set with a standard deviation, 2 and 3 the representation of the second and third replicated of alfalfa results are an average of the 3 values obtained for each set with a standard deviation respectively, DM = Dry matter, CP = Crude protein, EE = Ether extract
We investigated the composition of the phytochemicals (total phenolic content, total flavonoid compounds and isoflavone (daidzein and genisten) and the type and content of flavonoid compounds of all of the alfalfa. Details of the samples are described in Table 2 and 3. The results show that the alfalfa was detected in TPC (2.17 mg GAE/g), TFC compounds (2.02 mg RE/g), daidzen (46.72 µg/g) and genisten (15.38 µg/g) and flavonoid content including catechin (434.55 µg/g), rutin (46.67 µg/g), myricetin (1354.78 µg/g), luteolin (19.41 µg/g), quercetin (183.31 µg/g) and apigenin (2267.75 µg/g). Alfalfa is a known source of phytoestrogens that exhibits estrogenic activity in plants (Seguin et al., 2004) and many groups of this substance can be identified as isoflavones and they also have high genistein, daidzein, and coumestrol (Romm et al., 2010). Phytoestrogens can be found in 3 groups of plants, namely, legumes, cereals and grasses, all of which plants are used in the farming of ruminants . Hloucalová et al. (2016) reported that the phytoestrogen content (daidzein and genistein) in fresh cut of Medicago sativa L. harvesting at flowering stage was similar to the present study at 0.057 and 0.014 mg/g of DM respectively. Due to the current health-conscious consumer trends, there is a need for a variety of alternative foods in response to the demands of modern lifestyles and consumers have begun to choose foods or products which are beneficial for their health and which are derived from natural sources. Therefore, farmers who manage to feed cows with legumes (especially alfalfa or beans) should be able to produce high phytoestrogen milk which is useful as it composed of antioxidants. They will prevent the symptoms of premature ageing of humans. Phenolic and flavonoids compounds are both known as strong antioxidants which can also reduce the risk of cardiovascular disease (Rodrigues et al., 2012). (Steinshamn et al., 2008) showed that the supplementation of forage legumes increased the phytoestrogen in milk which has both health benefits for consumers as well as adding value to the milk produced by dairy goat farmers.
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Table 2: Total phenolic content, total flavonoid compounds and isoflavone (daidzein and genisten) of alfalfa (Medicago sativa) at harvesting intervals of 120 days.
TPC TFC Daidzein Genisten Sample (mg GAE/g sample) (mg RE/g sample) (µg/g sample) (µg/g sample) Alfalfa 2.17±0.02 2.03±0.01 46.72±0.01 15.36±0.01 2.18±0.02 2.05±0.02 46.71±0.01 15.39±0.02 2.15±0.02 2.01±0.01 46.74±0.02 15.37±0.01 Mean 2.17 2.02 46.72 15.38 TPC = Total phenolic content, (TPC was determined in comparison with standard gallic acid and the results expressed in terms of mg GAE/g), TFC = Total flavonoid compounds (TFC was determined in comparison with standard rutin and the results expressed in terms of mg RE/g), (x̄ )±SD) (n = 3).
Table 3: Type and content of flavonoid compounds of alfalfa (Medicago sativa) harvested at intervals of 120 days.
Flavonoid content (µg/g sample) (Mean±SD) Sample Catechin Rutin Myricetin Luteolin Q uercetin Apigenin Kaempferol Alfalfa1 432.76 ± 5.90 47.07 ± 3.62 1354.76 ± 82.94 19.46 ± 0.61 182.55 ± 10.23 2265.41 ± 90.32 nd Alfalfa2 434.44 ± 5.93 46.49 ± 3.41 1356.81 ± 83.75 19.40 ± 0.58 184.67± 10.40 2270.33± 90.43 nd Alfalfa3 436.45 ± 5.97 46.44 ± 3.44 1352.78 ± 82.39 19.36 ± 0.56 182.71 ± 10.36 2267.52 ± 90.37 nd Total 434.55 46.67 1354.78 19.41 183.31 2267.75 - mean Values were expressed as mean ± SD of triplicate measurements. 1 = the present of the first replicated of alfalfa results are an average of the 3 values obtained for each set with a standard deviation. 2 and 3 = the present of the second and third replicated alfalfa results are an average of the 3 values obtained for each set with a standard deviation respectively.
Conclusion
This study has shown that alfalfa can be a beneficial natural nutrient composition as contains relatively high in protein with the potential bioactive compounds. These characteristics suggest that alfalfa could be a promising source of nutritional value for health. However, research on the impacts of bioactive compounds from alfalfa legumes when it is used in the diets of dairy cattle or goats in Thailand is limited. Thus, it is recommended that the use of alfalfa legumes combined with the nutrients available from other roughage sources should be used for ruminants. These can be fed to ruminants to increased phytochemical activity in milk in order to benefit the health of consumers.
Acknowledgements
The author would like to thank Mr. Theeraphan Chumroenphat in the Laboratory Equipment Center, Mahasarakham University, Thailand, for the assistance.
References
AOAC. (1990). Official Methods of Analysis (Washington, DC: Association of Official Analytical Chemists). AOAC. (1998). Official Methods of Analysis of AOAC international (16th ed.), Gaithersburg, MD., USA.
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AOAC. (2010). Official Methods of Analysis of AOAC international (18th ed., Revision 3). AOAC International, Washington DC., USA. Bisby, F.A., Buckingham, J., & Harborne, J.B. (1994). Phytochemical Dictionary of the Leguminosae: Volume 1 Plants and their constituents. Chapman and Hall, London. Burns, R.E. (1971). Method for Estimation of Tannin in Grain Sorghum. Agronomy journal, 63(3), 511-512. Cleef, F.V., & Dubeux, J. (2019). Condensed tannins in forage legumes. SS-AGR-440, Agronomy Department, UF/IFAS Extension. Retrieved from https://edis.ifas.ufl.edu/ag440 Clements, R.J. (2019). Medicago sativa (PROSEA). Plant Resources of South-East Asia. Retrieved from https://uses.plantnet-project.org/en/Medicago_sativa_(PROSEA) Devi, M.K.A., Gondi, M., Sakthivelu, G., Giridhar, P., Rajasekaran, T., & Ravishankar, G.A. (2009). Functional attributes of soybean seeds and products, with reference to isoflavone content and antioxidant activity. Food Chemistry, 114(3), 771-776. Hegsted, M., & Linkswiler, H.M. (1980). Protein quality of high and low saponin alfalfa protein concentrate. Journal of the Science of Food and Agriculture, 31, 777-781. Hernández, T., Hernández, A., & Martinez, C. (1991). Polyphenols in alfalfa leaf concentrates. Journal of Agricultural and Food Chemistry, 39, 1120-1122. Hloucalová, P., Skládanka, J., Horký, P., Klejdus, B., Pelikán, J., & Knotová, D. (2016). Determination of phytoestrogen content in fresh-cut legume forage. Animals, 6(43), 1- 15. Knuckles, B.E., deFremery, D., & Kohler, G.O. (1976). Coumestrol content of fractions obtained during wet processing of alfalfa. Journal of Agricultural and Food Chemistry, 24, 1177-1180. Kubola, J., & Siriamornpun, S. (2011). Phytochemicals and antioxidant activity of different fruit fractions (peel, pulp, aril and seed) of Thai gac (Momordica cochinchinensis Spreng). Food Chemistry, 127(3), 1138-1145. Livingston, A.L., Kohler, G.O., & Kuzmicky, D.D. (1980). Comparison of carotenoid storage stability in alfalfa leaf protein (Pro-Xan) and dehydrated meals. Journal of Agricultural and Food Chemistry, 28(3), 652-656. Mamta, S., & Jyoti, S. (2012). Phytochemical screening of Acorus calamus and Lantana camara. International Research Journal of Pharmacy, 3(5), 324-326. Oleszek, W. (1996). Alfalfa saponins: structure, biological activity, and chemotaxonomy. Advances in Experimental Medicine and Biology book series, (AEMB, volume 405), 155-170. Oleszek, W. (2000). Alfalfa saponins: Chemistry and application. In W.R., Bidlack, S.T., Omaye, M.S., Meskin, & D.K., Topham, (Eds.). Phytochemicals as Bioactive Agents. (pp 167-188). Technomic Publishing Co., Inc., Lancaster, PA and Basel, Switzerland. Packer, L., Hiramatsu, M., & Yoshikawa, T. (1999). Antioxidant food supplements in human health. Academic Press: San Diego, CA. Phelan, P., Moloney, A.P., McGeough, E.J., Humphreys, J., Bertilsson, J., O’Riordan, E.G., & O’Kiely, P. (2015). Forage legumes for grazing and conserving in ruminant production systems. Critical Reviews in Plant Sciences, 34(1-3), 281-326. Ramirez, R.G., Haenlein, G.F.W., & Núñez-González, M.A. (2001). Seasonal variation of macro and trace mineral contents in 14 browse species that grow in northeastern Mexico. Small Ruminant Research, 39(2), 153-159. Ramırez,́ R.G., Neira-Morales, R.R., Ledezma-Torres, R.A., & Garibaldi-González, C.A. (2000). Ruminal digestion characteristics and effective degradability of cell wall of browse species from northeastern Mexico. Small Ruminant Research, 36(1), 49-55.
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Rodrigues, M.R.A., Kanazawa, L.K.S., Neves, T.L.M.d., Silva, C.F.d., Horst, H., Pizzolatti, M.G., Werner, M.F.d.P. (2012). Antinociceptive and anti-inflammatory potential of extract and isolated compounds from the leaves of Salvia officinalis in mice. Journal of Ethnopharmacology, 139(2), 519-526. Romm, A., Weed, S.S., Gardiner, P., Bhattacharya, B., Lennox, C.A., Lee, R., & Winston, D. (2010). CHAPTER 19 - Menopausal Health. In A. Romm, M.L. Hardy, & S. Mills (Eds.). Botanical Medicine for Women's Health. (pp. 455-520). Saint Louis: Churchill Livingstone. Rushkin, F.R. (1984). Leucaena: promising forage and tree crops for the tropics. 2nd ed. National Research Council. Washington, DC: National Academy Press. Seguin, P., Zheng, W., & Souleimanov, A. (2004). Alfalfa Phytoestrogen Content: Impact of Plant Maturity and Herbage Components. Journal of Agronomy and Crop Science, 190(3), 211-217. Srisaikham, S., & Lounglawan, P. (2018). Effect of cutting age and cutting height on production and nutritive value of sunnhemp (Crotalaria juncea) harvest in Nakhon Ratchasima, Thailand. Acta horticulturae, 1210, 29-34. Steinshamn, H., Purup, S., Thuen, E., & Hansen-Møller, J. (2008). Effects of Clover-Grass Silages and Concentrate Supplementation on the Content of Phytoestrogens in Dairy Cow Milk. Journal of Dairy Science, 91(7), 2715-2725. Stochmal, A., Piacente, S., Pizza, C., De Riccardis, F., Leitz, R., & Oleszek, W. (2001). Alfalfa (Medicago sativa L.) flavonoids. 1. Apigenin and luteolin glycosides from aerial parts. Journal of Agricultural and Food Chemistry, 49(2), 753-758. Undersander, D., Hall, M.H., Vassalotti, P., & Cosgrove, D. 2011. Alfalfa germination & growth. Cooperative Extension Publishing. Vintu V., Stavarache M., Samuil C., & Munteanu, I. (2012). Chemical composition dynamics of alfalfa (Medicago sativa L.) at different plant growth stages. Grassland Science in Europe, 17, 394-396.
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ICoFAB2020 doi: 10.14457/MSU.res.2020.19 International Conference on Food, Agriculture and Biotechnology
NEUROBLASTOMA CELL-LINE TOXICITY DUE TO INCREASED ALZHEIMER’S-RELATED PROTEINS AND CELL DEATH SUBSEQUENT TO HIGH-DOSE PALMITIC ACID EXPOSURE
PHITTHAYAPHONG PHANSA1,2,3*, KUMFU SIRINART1,2,3, CHATTIPAKORN NIPON1,2,3, CHATTIPAKORN C. SIRIPORN1,3,4
1Neurophysiology Unit, Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand. 2Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,50200, Thailand. 3Center of Excellence in Cardiac Electrophysiology Research, Chiang Mai University, Chiang Mai, 50200, Thailand. 4Department of Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai University, Chiang Mai, 50200, Thailand.
*Corresponding author: [email protected]
Abstract:
Obesity caused by high-fat diet (HFD) consumption is one of the risk factors for the development of Alzheimer’s disease (AD). Previous studies have reported that saturated fatty acids from an HFD, particularly palmitic acid (PA), promotes Alzheimer’s pathologies, neuroinflammation and cognitive impairment. The pathological hallmarks of AD are characterized by the accumulation of amyloid-β (Aβ) peptides and phosphorylated Tau (pTau) proteins. However, the expression of Aβ peptides, and Tau proteins levels, as well as the viability of cell in neuronal cells induced by PA remain unclear. Therefore, this study aims to determine the effect of high concentrations of PA on cell viability and Alzheimer’s-related proteins of neuronal cells. In this study, SH-SY5Y was used as a neuronal cell model in order to investigate the effect of PA at a concentration of 400 and 800 µM. The results showed that neuronal cell exposed to PA at doses of 400 and 800 µM for 24 and 48 hours had decreased cell viability in a dose-dependent manner. In addition, receiving PA for 48 hours induced Aβ peptides and p-Tau proteins production. Our study suggests that high concentration of PA exposure can lead to increased risk of the development of Alzheimer’s pathologies and neuronal cell death.
Keywords: Palmitic acid; Alzheimer’s disease; Neuronal cell; Amyloid beta; Tau
Introduction
According to previous studies, long-term high-fat diet (HFD) consumption is one of the key factors in obesity around the world. Obesity enhances the impairment of learning and memory performance and the development of Alzheimer’s-like pathophysiological changes in the brains (Granholm et al., 2008). Palmitic acid (PA) is the most common form of saturated fatty acid (FA) in HFD. PA has been found to be an abundant saturated FA existing in the human body and is closely linked to causing lipotoxicity (Plötz et al., 2016). In neuronal cells, PA is known to induce endoplasmic reticulum stress, impaired cell proliferation, abnormal cell differentiation, and resulting in cellular apoptosis (Park et al., 2011; Wang et al., 2014). Alzheimer’s disease (AD), which is the most common neurodegenerative disease, is characterized by cognitive decline, memory dysfunction, and behavioral impairment. In 2015,
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46.8 million people worldwide were living with dementia and the number is expected to reach 75 million in 2030 and 131.5 million in 2050 (Martin et al., 2016). AD is caused by brain cell death, and this brain cell death happens progressively over time. An abundance of intracellular and extracellular aggregation was found in affected brain regions. Neuronal intracellular aggregates called neurofibrillary tangles consist of hyperphosphorylated Tau proteins in the form of insoluble paired helical filaments (Lee et al., 2001). The abnormal hyperphosphorylation of Tau results in the loss of Tau function in promoting microtubules stabilization, and eventually lead to cell death (Iqbal et al., 2005). Neuronal extracellular deposits called amyloid plaques consist of amyloid β (Aβ) peptides in the form of insoluble amyloid fibrils. The abnormal regulation of amyloid precursor protein (APP) causes the accumulation of Aβ resulting in the development of systemic inflammation. Excessive Aβ plaques and neurofibrillary tangles are the primary causes of AD. However, the underlying mechanism of PA-induced neuronal cell death which is associated with the upregulation of Alzheimer’s-related proteins has not been thoroughly investigated. Therefore, the purpose of this study is to investigate the effects of high concentrations of PA on cell viability and the expression of Alzheimer’s-related proteins. The concentration of PA in this study was referred to previous study that SH-SY5Y was died after exposed to 200-500 µM of PA for 24 and 48 hrs (Hsiao et al., 2014). Accordingly, we chose PA at the concentration of 400 and 800 µM to investigate the effect of high dose of PA. We hypothesized that PA is involved in the progression of AD and is related to neuronal toxicity and neuronal cell death.
Materials and Methods
Experimental Protocol
SH-SY5Y cells were divided into 3 groups according to the PA treatment protocol, each group consisted of 4 cell samples. 1) The control group: SH-SY5Y cells receiving a vehicle (0.02 mM, NaOH), 2) the PA 400 µM group: SH-SY5Y cells receiving PA at a concentration of 400 µM, 3) the PA 800 µM group: SH-SY5Y cells receiving PA at a concentration of 800 µM. To determine cell viability, SH-SY5Y cells were exposed to PA for 4, 24 and 48 hrs and viability was determined by the alamarBlue assay. To investigate the expression of Alzheimer’s-related proteins including of Aβ, APP, p-Tau and Tau, SH-SY5Y cells were exposed to PA for 48 hrs then a western blot analysis was performed. The diagrams of experiment are illustrated in Figure 1.
Cell Culture
This study used the SH-SY5Y cell line (human neuroblastoma cell line) as an in vitro model due to its wide use in neuronal function and differentiation research. SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). SHSY5Y cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) /Ham's Nutrient Mixture F12 with glutamax (1:1; v/v), sodium bicarbonate, 10% fetal bovine serum and 1% penicillin-streptomycin and incubate at 37 °C in a 5% CO2 incubator. Fresh medium was changed every 2-3 days.
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Figure 1: Diagram of experimental protocol
Preparation of Palmitic Acid Solution
First, the stock of 40 mM PA solution was prepared by dissolving 10.26 mg of PA in 0.01 M Sodium hydroxide (NaOH) in a dry bath at 80 °C for 15 minutes. The PA was purchased from Sigma-Aldrich (Dorset, UK). In addition, 1% free fatty acid (FFA)-free BSA was dissolved in DMEM at 37ºC. After that, the PA was mixed with FFA-free BSA by adding different volumes of PA stock solution to reach final concentrations of 16 and 32 mM of PA for PA200 and PA400 groups, respectively. The mixtures were incubated in water bath at 37 °C for 30 min before being treated. The vehicle control consisted of 1% of FFA-free BSA media with 0.02 mM NaOH.
Cell Viability Assessment by AlamarBlue Assay
SH-SY5Y cells were plated in 96-well plate with 1×104 cells/well then cultured in DMEM at 37 °C in a 5% CO2 incubator overnight. After that, cells were treated with vehicle and PA at concentration of 16 and 32 mM to reach the final concentration of PA in the control, PA 400 µM and PA 800 µM groups, respectively. To determine cell viability, alamarBlue reagent was then added in each well. alamarBlue reagent is composed of Resazurin dye which is used in redox based colorimetric assays to determine cellular metabolic reduction. Living cells change Resazurin from blue and non-fluorescent to pink and highly fluorescent. After 4, 24 and 48 hrs of PA exposure, the absorbance was measured at 570 nm and 590 nm. The intensity of fluorescence produced is proportional to the number of living cells. To calculate the percentage of cell viability, the absorbance of control group was used as a reference number of cells. The absorbance of PA 400 and 800 µM group was divided by the absorbance of the control group at the same time point and then multiplied by 100 as shown in this following equation.
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Alzheimer’s-Related Protein Measurement by Western Blot Analysis
SH-SY5Y cells were plated in 6-well plate with 2.5×105 cells/well overnight. Then, cells weretreated with vehicle and 16 and 32 mM of PA to reach final concentration of PA in the control, PA 400 µM and PA 800 µM group, respectively, for 48 hrs. Next, SH-SY5Y cells were lysed with radioimmunoprecipitation assay (RIPA) buffer, 1% Triton X-100 and 1% protease inhibitor and measured the concentration of total protein by Bicinchoninic acid (BCA) assay. After that, the prepared proteins were separated by electrophoresis method called SDS- PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred onto nitrocellulose membranes. Membranes were blocked for one hour with 5% nonfat dry milk or 5% BSA, according to the manufacturer recommendation, in Tris-buffer saline (pH 7.4) containing 0.1% Tween 20. Next, the membranes were probed with the primary antibodies (1:1000) against Alzheimer’s-related proteins including Aβ (Santa Cruz Biotechnology, USA), APP, p-Tau, Tau, (Cell Signaling Technology, USA), and a loading control (GAPDH) (Abcam, USA). After that, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:1000) at room temperature for 1 hour. Finally, the membranes were visualized by ClarityTM Western ECL Blotting Substrate (Bio-Rad Laboratories Ltd.) and scanned by ChemiDoc™ Touch Gel Imaging System. Image J (NIH image) analysis software was used to analyze the intensity of western blotting images.
Statistical Analysis
The data for each experiment was expressed as mean ± standard error of the mean. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test were performed on the expression of Alzheimer’s-related proteins. Two-way ANOVA analysis followed by Fisher’s LSD comparisons test was used for the cell viability assessment. GraphPad Prism software was used in this study. P-value < 0.05 was considered statistically significant.
Results
The Effects of PA on SH-SY5Y Cell Viability
The result of the cell viability assessment from alamarBlue assay is shown in Figure 2. We found that SH-SY5Y cells exposed to PA in the concentration of 400 and 800 µM had significantly decreased in the percentage of cell viability. Interestingly, the decrease of cell viability was proportional to the increase PA concentration. Cell viability of SH-SY5Y cells was significantly decreased, when exposed to 400 and 800 µM of PA for 24 and 48 hrs when compared to that condition for 4 hrs. However, the statistical analysis did not show the differences in cell viability between 24 and 48 hrs of treatment. These findings suggested that the exposure of PA at both concentrations of 400 and 800 µM for 4, 24 and 48 hrs caused neuronal toxicity and cell death to SH-SY5Y cell line. The images of SH-SY5Y cell line after exposure to 400 and 800 µM of PA for 24 and 48 hrs are shown in Figure 3.
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Figure 2: Cell viability of SH-SY5Y after exposed to 400 and 800 µM of PA for 4, 24 and 48 hrs. N = 4 per group in each time point. The values are presented as mean ± SEM. *p < 0.0001 compared with control group at the same time point, †p < 0.0001 compared with PA 400 µM at the same time point, ‡p < 0.05 compared with 4 hrs of PA exposure.
Figure 3: Image of SH-SY5Y cell line after exposure to 400 and 800 µM of PA for 24 and 48 hrs by light microscope with a magnification of 400X.
The Effects of PA on the Expression of Alzheimer’s-Related Proteins of SH-SY5Y Cells
The results demonstrated that SH-SY5Y cells exposed to 400 and 800 µM of PA for 48 hrs significantly increased Aβ/APP ratio and Tau proteins when compared to the control group (Figure 4A, B). Moreover, exposure of 800 µM of PA for 48 hrs also showed significantly increased expression of p-Tau proteins when compared to the control group (Figure 4C). However, PA at the concentration of 400 µM did not affect p-Tau proteins when compared with control (Figure 4C). The representative band was displayed in Figure 4D.
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Figure 4: The effects of PA exposure at the concentration of 400 and 800 µM for 48 hrs on the expression of Alzheimer’s-related proteins in SH-SY5Y cells. Western blot analysis and relative bar graph quantification of proteins (A) Aβ/APP, (B) Tau and (C) p-Tau that normalize with GAPDH and (D) representative bands. N = 4 per group in each time point. The values are presented as mean ± SEM. *p < 0.05 compared with control group.
Discussion
Consumption of HFD not only induces obese condition, it is also one of the major risk factors in the development of cognitive decline, dementia, and AD (Eskelinen et al., 2008). Furthermore, PA, a saturated fatty acid, has been shown to be one of the major risk factors for the development of AD (Klein et al., 2005; Marwarha et al., 2017). Regarding to the results of this study, we found that PA affected to cell viability of SH-SY5Y cells since both 400 and 800 µM of PA significantly reduced the percentage of cell viability by dose-dependent manner. Moreover, exposure of PA for 4 and 24 hrs at each dose of PA also presented the reduction of cell viability by time-dependent manner significantly. However, when SH-SY5Y cells exposed to PA for 48 hrs, the percentage of cell viability was not different from 24 hrs of exposure. To illustrate this point, if we further extend the incubation period of SH-SY5Y cells with PA, the cell viability may become the worse and show the reduction in comparison to 24 hrs of PA exposure. Furthermore, previous study showed that HFD consumption increased Aβ and Tau proteins in the brain of 3xTg-AD mice (Julien et al., 2010). However, previous studies of PA affected to the expression of Aβ together with Tau proteins in SH-SY5Y neuronal cells have not been investigated. The present study reported that PA caused the upregulation of the Alzheimer’s related proteins production such as Aβ peptides, p-Tau and Tau proteins in SHSY5Y cells. Consequently, our study provided that high concentration of PA plays a vital role in the upregulation of neuronal toxicity which is related to Alzheimer’s pathology and neuronal cell death.
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Conclusion
The results illustrated that PA was found to be a neurotoxic substance to neuronal cells as shown by upregulated levels of proteins associated with the development of Alzheimer’s pathology, including Aβ peptides, p-Tau and Tau proteins. Moreover, these Alzheimer’s related proteins were found related to the increasing of neuronal cell death. Our study suggests that long-term of HFD consumption can cause not only obesity, but also damage to neuronal cells, resulting in brain inflammation, brain cell death and further the development of Alzheimer’s disease.
Acknowledgements
This work was supported by the senior research scholar grant from the National Research Council of Thailand (SCC), a NSTDA Research Chair Grant from the National Science and Technology Development Agency Thailand (NC) and the Chiang Mai University Excellent Center Award (NC).
References
Eskelinen, M. H., Ngandu, T., Helkala, E. L., Tuomilehto, J., Nissinen, A., Soininen, H., & Kivipelto, M. (2008). Fat intake at midlife and cognitive impairment later in life: a population-based CAIDE study. International Journal of Geriatric Psychiatry, 23(7), 741-747. Granholm, A. C., Bimonte-Nelson, H. A., Moore, A. B., Nelson, M. E., Freeman, L. R., & Sambamurti, K. (2008). Effects of a saturated fat and high cholesterol diet on memory and hippocampal morphology in the middle-aged rat. Journal of Alzheimer's Disease, 14(2), 133-145. Iqbal, K., del C. Alonso, A., Chen, S., Chohan, M. O., El-Akkad, E., Gong, C.-X., Grundke- Iqbal, I. (2005). Tau pathology in Alzheimer disease and other tauopathies. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1739(2), 198-210. Julien, C., Tremblay, C., Phivilay, A., Berthiaume, L., Emond, V., Julien, P., & Calon, F. (2010). High-fat diet aggravates amyloid-beta and tau pathologies in the 3xTg-AD mouse model. Neurobiology of Aging, 31(9), 1516-1531. Klein-Platat, C., Drai, J., Oujaa, M., Schlienger, J. L., & Simon, C. (2005). Plasma fatty acid composition is associated with the metabolic syndrome and low-grade inflammation in overweight adolescents. The American Journal of Clinical Nutrition, 82(6), 1178-1184. Lee, V. M., Goedert, M., & Trojanowski, J. Q. (2001). Neurodegenerative tauopathies. Annual Review of Neuroscience, 24, 1121-1159. Martin Prince, A. C.-H., Martin Knapp, Maëlenn Guerchet, & Ms Maria Karagiannidou. (2016). World Alzheimer Report 2016. Retrieved from London: Marwarha, G., Rostad, S., Lilek, J., Kleinjan, M., Schommer, J., & Ghribi, O. (2017). Palmitate Increases beta-site AbetaPP-Cleavage Enzyme 1 Activity and Amyloid-beta Genesis by Evoking Endoplasmic Reticulum Stress and Subsequent C/EBP Homologous Protein Activation. Journal of Alzheimer's Disease, 57(3), 907-925. Park, H. R., Kim, J. Y., Park, K. Y., & Lee, J. (2011). Lipotoxicity of palmitic Acid on,neural progenitor cells and hippocampal neurogenesis. Toxicological Research, 27(2), 103- 110.
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Plötz, T., Hartmann, M., Lenzen, S., & Elsner, M. (2016). The role of lipid droplet formation in the protection of unsaturated fatty acids against palmitic acid induced lipotoxicity to rat insulin-producing cells. Nutrition & metabolism, 13, 16-16. Wang, Z., Liu, D., Zhang, Q., Wang, J., Zhan, J., Xian, X., Hao, A. (2014). Palmitic acid affects proliferation and differentiation of neural stem cells in vitro. Journal of Neuroscience Research, 92(5), 574-586. doi:10.1002/jnr.23342
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ICoFAB2020 doi: 10.14457/MSU.res.2020.20 International Conference on Food, Agriculture and Biotechnology
EFFECTS OF MASH FEED, SINKING FEED AND FLOATING FEED ON GROWTH PERFORMANCE, FEED UTILIZATION AND HEMATOLOGY OF NILE TILAPIA (Oreochromis niloticus)
KAMRUZZAMAN MUHAMMAD1,3, JINTASATAPORN ORAPIN1 CHUMKAM SRINOY2
1Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand 2Faculty of Agricultural Technology,Valaya Alongkorn Rajabhat University, Pathum Thani, Thailand 3Department of Fisheries, Ministry of Fisheries and Livestock, Bangladesh.
*Corresponding author :[email protected]
Abstract:
The present study was conducted to evaluate the effects of mash feed, sinking feed and floating feed on growth performance, feed utilization and hematology of Nile tilapia (O. niloticus). The study was assigned in Completely Randomized Design (CRD) with three treatments and four replicates. Fish with average weight 13.30± 0.85 g. (means ± SD) were fed three experimental diets mash feed, sinking feed and floating feeds that had same feed formula and proximate composition. The fish were fed experimental diets at the rate of 3.0-5.0 % of their body weight per day, twice daily for four weeks. At the end of the experiment, there were no significant (P>0.05) differences between the treatments in weight gain (WG), average daily gain (ADG), feed conversion ratio (FCR). Specific Growth rate (SGR) was significantly (P< 0.05) different among the treatments. The high SGR was found in fish fed floating feed, followed by fish fed sinking feed, whereas lower value was found in mash feed. Fish fed floating and sinking feed exhibited a significant (p<0.05) higher level of serum protein. In this case, the level of serum protein was lower in mash feed. However, no significant differences (P>0.05) were observed for red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Ht) percentage and immunoglobin (IgM) level. Results from this study demonstrated that fish fed floating and sinking feed both had better growth performance (p<0.05) than fish fed mash feed.
Keywords: Feed type; Growth performance; Hematology; Nile tilapia
Introduction
The Nile tilapia, Oreochromis niloticus is considered as one of the most important species of fish in tropical and sub-tropical aquaculture (FAO, 2012). Today, tilapia has become the shining star of aquaculture and also popularly known as ‘aquatic chicken’ and the rate of consumption has increased across the globe (Fitzsimmons, 2005). Regardless of the profits and advancement of aquaculture, this industry faces a number of problems, including high feed cost, disease and environmental pollution due to excessive uses of fish feed (FAO, 2009). The feed used in most of the commercial tilapia culture operations in Bangladesh and Thailand are floating, sinking and mash type feed. Those countries culture tilapia by purchase or production of sinking, floating and mash feeds on farms. Floating diets are usually more expensive than sinking or mash feed because of extrusion process during feed production, needs high temperature, high pressure then the extra cost add. On the other hand, sinking and mash feeds rather less expensive to produce than the floating feeds. So farmers need less investment in fish farming. There are some observations on field level that some farmers prefer floating diets for
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology feeding tilapia because it provides opportunity to observe satiation level of the fish. But sometimes due to higher turbidity of water body which hinder water visibility that makes impossible for farmers to observe satiation level of tilapia, as a result excluding the need for relying on floating feed. Previous studies in halibut, Hippoglossus hippoglossus have demonstrated that growth, nutrient utilization and production are affected by the form of the diets used (Suontama et al., 2007). Limbu (2015) found inconsistence results in Clarias gariepinus when floating and sinking feeds are compared on their effect on growth, nutrient utilization and yield, found similarity in mean weight gain and daily feed intake for fed on floating and sinking feeds. Good quality feeds help farmers to culture fishes for high growth and high profits (FAO, 2015). Good quality floating, sinking and mash feeds are crucial to the development and success of a tilapia farming industry. Floating feeds are high cost than sinking and mash feed but farmers may be convenient by feed management or by others means that floating feed more favorite for tilapia culture. But many farmers are still doubtful about the effectiveness of floating feed. Most of the research are conducted in consideration of the culture techniques of O. niloticus but few studies have been run to explore the feeding habit of tilapia in order to recommend the convenient feed for this species culture among floating, sinking and mash diets. For this reason famers do not know which feed to choose for O.niloticus culture due to scarcity of published scientific information on growth performance, nutrient utilization when this species of fish is fed on floating, sinking and mash feeds. This has been costly to Nile tilapia farmers since they tend to use the costly floating feeds. So it is mandatory to search a suitable and profitable type of feed that will meet up the Tilapia farmer’s demand which ultimately go in line with food security. In order to estimate the available nutrients derived from the feed to maximize the fish culture potential, it is essential to quantify the output from feed input to the cultured fish. Hence the present study was undertaken to study the effects of mash feed, sinking feed and floating feed of Nile Tilapia in order to recommend the appropriate feed types.
Materials and Methods
Animals
A total of 120 fingerling Nile Tilapia (Oreochromis niloticus) with average weight of 13.30 ± 0.85 g were obtained from Private Hatchery, NakornPathom, Thailand. After transportation, fishes were acclimated in 1,000 L fiber tank for 7-10 days with mash feed prior to the initiation of the experiment.
Experimental diets
In this experiment, one feed formula was used to form three types of feed, mash feed, sinking feed and floating feed. The floating feed was nursery feed, commercial fed, size 2-3 mm. from Betagro feedmill, Samutprakarn, Thailand. Sinking feed was produced by grinding the floating pellet to fine ground then re-pelleted by mincer to form sinking feed and dry by hot air oven at 80 oC for 12 hr. The mash feed was produced by grinding the floating pellet to fine ground then add water to form the dough before use. Proximate compositions of the experimental diets base on dry matter were the same that was 0% moisture, 47.48 % crude protein, 11.18 % crude lipid, 12.90 % ash, 3.09 % fiber, 25.35 % NFE as starch and sugar, 2.60 % calcium, 1.50 % phosphorus.
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Experimental design
The trial was assigned in completely randomize design (CRD) of three treatments and four replicates, T1 Mash feed, T2 Sinking feed, T3 Floating feed.
Experimental condition
A total of 120 fingerling of Nile Tilapia (Oreochromis niloticus) were randomly distributed 10 fish to each of 12 glass aquarium of 120 L. All experimental units were supplied with filtered water and continuous uniform aeration was given through the air-diffuser stones. Throughout the trial water in each aquarium (about 80%) was exchanged every two days. Water was exchanged just before feeding in morning and 1 h after feeding in evening. Each experimental feed was fed to fish at 3-5% body weight at the same amount for each treatment and each replicate. The amount of feeds required for each day was divided into two parts and feed twice per day for four weeks. After 30 minutes of feeding unconsumed feeds were siphoned out and dried to determine the exact feed consumption. Total weight of fish in each aquarium was estimated every week and the amount of feed fed to fish was adjusted accordingly.
Data collection
Growth performance At the end of the four weeks of feeding trial, fish were starved for 24 hours, after that they were then counted and weighed to determine the weight gain (WG), average daily gain (ADG), specific growth rate (SGR), feed conversion ratio (FCR) and survival rate. The following indices were calculated as the follows:
Survival rate (%) = (final no. of fish /initial no. of fish) x 100
Weight gains (WG, g / fish) = final live weight of fish – initial live weight of fish
Average Daily Growth (ADG, g/day) = net weight gain/rearing period
Specific growth rate (SGR, % body weight/day) = [ln final fish weight - ln initial fish weight] x 100/ culture period
Feed intake/feed given (FI, g/fish) = Dried feed given weight per aquarium/number of fish in aquarium
Feed intake per day(FIPD, g/fish/day) = Dried feed given weight per aquarium/(number of fish in aquariumXrearing period) Feed Conversion Ratio (FCR) = dry feed consumed / wet weight gain
At end of the experiment, three fishes were randomly chosen from each tank and were anaesthetized using clove oil. About 1.0 ml of blood was drawn from the caudal vein, using a 2.0 ml syringe with 26-G needle (Hawk et al. 1965). Three fish from each replicate were anesthetized with clove oil and blood was collected from the caudal vein using a syringe with EDTA as an anticoagulant. For serum, another three fish from each replicate were anesthetized and blood was collected without anticoagulant and left to clot. Then the clotted blood was centrifuged at 4000 × g for 10 min to separate the serum. The blood samples were used to analyzed red blood cells (RBC), hemoglobin (Hb) and hematocrit. RBC were counted
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ICoFAB2020 International Conference on Food, Agriculture and Biotechnology with a hemocytometer under the light microscope. The estimation of hemoglobin was done according to the method of Drabkin Austin (1935). Hematocrit was measured using hematocrit capillary tubes spun in Gemmy model KHT-410E Hematocrit Centrifuge. Total serum protein and immunoglobin were measured following the method of Lowry et al. (1951) using BSA as the standard protein. The feeding trial and all analysis were conducted at Nutrition and Aquafeed Laboratory, Department of Aquaculture, Kasetsart University, Bangkok, Thailand.
Statistical Analysis
All mean value data were analyzed using one-way analysis of variance (ANOVA) to compare significant differences between treatments. Duncan multiple range tests was used to compare the means of the treatment. The treatment effects were considered to be significant at p<0.05. The standard deviation of each parameter and treatment was determined and expressed as the Mean ± SD. All statistical analysis performed using statically software.
Results and Discussion
Growth performance of Nile tilapia fed different experimental diets for four weeks was shown in Table 1 and Figure 1. The different feed types had no significant (p>0.05) effects on the final weights, weight gain (WG), average daily gain (ADG) and food conversion ratio (FCR) but had significant (p<0.05) effects on the specific growth rate (SGR) where floating and sinking feed were significantly higher (p<0.05) than the group of mash feed. The present experiment clearly demonstrated the beneficial effects of floating and sinking feeds on the performance of Nile tilapia although feed contain the same nutrient concentration and fed the same amount. In this experiment, though, there were no significant differences in the growth parameters in term of WG, ADG, FCR among the type of feed but every numerical value of these parameters tended to be higher (p=0.07-0.09) than those group of fish fed mash feed. In laboratory condition of small aquarium and shallow water depth, it is easy for the fish to go to consume feed that sinking or lay on the bottom then feed intake was not big different. Fish got the same amount of feed and nutrients for promote their growth. Tilapia are able to consume floating and sinking pellets very effectively. According to a study, sinking pellets were better utilized than unpelletized feeds like mash feed (Allison et al., 1979). David et al. (2017) reported that floating feed is better for the growth performance of African catfish fingerlings. Similarly et al. (2006) demonstrated that extruded pellets are better than paste diet for the growth performance and water quality management in juvenile olive flounder aquaculture. In the present study, all growth parameters in fish fed mash feed lower than the other groups due to low water stability of mash feed and high nutrient leaching of it before fish consume the feed. This results was in accordance with the study of Kim & Sin (2006) in juvenile olive Flounder.
Table 1: Growth performance and nutrient utilization of tilapia fed mash, sinking and floating feed over the experimental period
Parameters T1: T2: T3: P-value Mash feed Sinking feed Floating feed Initial weight (g/fish) 13.68±0.87 13.05±0.68 13.17±1.07 0.584 Final weight (g/fish) 28.90±0.20 31.41±2.21 31.99±4.10 0.276 Weight gain at final (g/fish) 15.22±0.72 18.36±1.86 18.82±3.32 0.096
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Average daily gain 0.54±0.03 0.66±0.07 0.67±0.12 0.090 (g/fish/d) Specific growth rate 2.68±0.21b 3.13±0.20 a 3.16±0.29 a 0.032 (% /day) Feed intake/ feed given 21±00 21±00 21±00 1.000 (g/ fish) Feed intake per day/ feed 0.75±00 0.75±00 0.75±00 1.000 given per day (FIPD) (g/fish/day) Feed conversion ratio 1.38±0.06 1.15±0.10 1.14±0.18 0.074 Survival rate (%) 100±00 100±00 100±00 1.000 Note :Different superscript letters a, b in the same row indicate significant difference (P<0.05).
Spercific growth rate
mash sinking floating 2.500 a a b a 2.000 a b b a a b a a 1.500
1.000 SGR (%/d) SGR
0.500
0.000 1w 2w 3w 4w Time(week)
Figure 1: Specific Growth Rate of Tilapia over the experimental period.
The hematological parameters of Tilapia fed three types of feed was presented in Table 2 and Figure 2. Hematological parameters can be used as an index of health status of fish ( Blaxhall, 1972). Routine examination of blood parameters has been used in earlier studies to assess fish health status and circumstantial stress (Hesser, 1960; Blaxhall, 1972; Blaxhall Daisley, 1973; Casillas Smith, 1977). The results of this study showed that the different feed types had no significant (p>0.05) effects on Tilapia health when focusing on hematological parameters in term of the RBC count, Hb concentration, Hct percentage and IgM level but had significant (p<0.05) effect on the serum protein level where mash feed was significantly lower than those groups of sinking and floating feed. The present study has been indicated that serum protein level in the group of fish fed mash feed was significantly lower than those group of sinking and floating feed because fish might be always in stressful condition due to feed competition. Mash feed had lower water stability in water so feed mater and nutrient dissolved in water before fish consumption then caused poor water quality and induced fish stress (Kim & Sin, 2006). Lee et al. (2016) investigated on the effects of extruded pellets and dough Type
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Diets and reported that fish fed dough diet indicating that extruded diet was probably pollution free and environmentally friendly. In this instance, sinking and floating feeds could be the right choice to increase total fish production, minimize production cost including promote fish health.
Table 2: Hematology studies of tilapia fed mash, sinking and floating feed over the experimental period.
Parameters T1:Mash feed T2:Sinking T3: Floating feed p-value feed Red Blood cell count 1.41±0.43 1.90±0.92 2.04±1.14 0.593 (x105 cell/mL) Haematocrit (%) 24.00±2.58 27.00±4.69 28.00±7.07 0.540 Hemoglobin (g/dL) 23.90±4.65 26.84±6.91 27.01±2.86 0.637 Serum Protein 1.66±0.93 b 3.72±0.94 a 5.02±0.78 a 0.001 (g/dL) Immunoglobin 0.19±0.07 0.08±0.06 0.06±00 0.093 (IgM; g/L) Note :Different superscript letters a, b in the same row indicate significant difference (P<0.05).
serum protein
6 a 5
4 a
3 g/dL
2 b
1
0 mash sinking floating feed types
Figure 2: Serum protein of Tilapia over the experimental period
Conclusion
In conclusion, results from the present study, showed the beneficial effects of sinking and floating feeds over commonly used mash feed in enhancing growth performance of Nile tilapia and fish health.
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Acknowledgements
The author would like to thank Betagro Public Co., Ltd ,.Samutprakan, Thailand, for supporting the feed throughout the trial. This study was financially supported by Thailand International Cooperation Agency (TICA) and The Nutrition and Aquafeed Laboratory, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand.
References
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Seunghan Lee et al. (2016) Effects of extruded pellet and moist pellet on growth performance, body composition, and hematology of juvenile olive flounder, Paralichthys olivaceus. Fisheries and Aquatic Sciences.,19:32 Suontama et al. (2007).Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods.Aquaculture nutrition.,V.13 Issuue.4 pp 241- 255. Samwel Mchele Limbu (2015), The effect of floating and sinking diets on growth performance, feed conversion efficiency, yield and cost effectiveness of African sharptooth catfish, Clarias gariepinus reared in earthen ponds. International Journal of Fisheries and Aquatic Studies. 2(5): 253-259. Seval et al. (2009) Effect of Different Feed Types on Growth and Feed Conversation Ratio of Angel Fish (Pterophyllum scalare Lictenstein, 1823). Journal of Applied Biological Sciences 3(2): 0-1.
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