Icofab2021 the 8Th International Conference on Food, Agriculture and Biotechnology

ICoFAB2021 The 8th 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 seven years ( 2014 – 2020) 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 since ICoFAB2020 to encourage continuous sharing of research knowledge and close academic networking in spite of social physical distancing.

This project was financially supported by Mahasarakham University. We hope that the 8th ICoFAB2021 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.

(Asst. Prof. Dr. Sumonwan Chumchuere) Dean of the Faculty of Technology Mahasarakham University

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

ICoFAB2021 Organizing Committee 1. Asst. Prof. Dr. Sumonwan Chumchuere 2. Dr. Sunisa Roidoung 3. Assoc. Prof. Dr. Maratree Plainsirichai 4. Assoc. Prof. Prasit Chutichudech 5. Assoc. Prof. Dr. Anut Chantiratikul 6. Assoc. Prof. Dr. Luchai Butkhup 7. Asst. Prof. Dr. Wantana Sinsiri 8. Asst. Prof. Dr. Sirirat Deeseenthum 9. Asst. Prof. Dr. Pheeraya Chottanom 10. Asst. Prof. Dr. Kedsirin Sakwiwatkul 11. Asst. Prof. Dr. Eakapol Wangkahart 12. Asst. Prof. Dr. Ruchuon Wanna 13. Asst. Prof. Dr. Chanyut Thamwan 14. Asst. Prof. Dr. Vijitra Luang-In 15. Mrs. Busaba Tharasena 16. Dr. Wanida Chuenta 17. Dr. Apichaya Bunyatratchata 18. Dr. Thitiwut Vongkampang

ICoFAB2021 Proceedings Scientific Committee

1. Prof. Hanny Wijaya, Institute Pertanian Bogor University, Indonesia 2. Prof. Yongqi Shao, Zhejiang University, China 3. Prof. Wu Xin, Chinese Academy of Sciences, Tianjin, China 4. Honorary Prof. Colin Wrigley, University of Queensland, Australia 5. Prof. Yueqiu H.E., Yunnan Agricultural University, China 6. Prof. Antonio J. Barroga, Central Luzon State University, Philippines 7. Prof. Emer, Chiba University, Japan 8. Assoc. Prof. Dr. Yuthana Phimolsiripol, Chiang Mai University, Thailand 9. Assoc. Prof. Dr. Chalor Jarusutthirak, Kasetsart University, Thailand 10. Assoc. Prof. Dr. Nyuk Ling Ma, Universiti Malaysia Terengganu, Malaysia 11. Assoc. Prof. Dr. Khamsah Suryati Mohd, Universiti Sultan Zainal Abidin, Malaysia 12. Asst. Prof. Dr. Rossaporn Jiamjariyatam, Srinakharinwirot University, Thailand

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

13. Asst. Prof. Dr. Ekapol Limpongsa, Rangsit University, Thailand 14. Asst. Prof. Dr. Teerasak Punvichai, Prince of Songkla University, Thailand 15. Asst. Prof. Dr. Po-Tsang Lee, National Taiwan Ocean University, Taiwan 16. Asst. Prof. Dr. Jareeya Yimrattanabovorn, Suranaree University of Technology, Thailand 17. Asst. Prof. Dr. Pongsak Khunrae, King Mongkut's University of Technology Thonburi, Thailand 18. Dr. S. Krishnakumari, Kongunadu Arts and Science College, India 19. Dr. K. Surekha, Kongunadu Arts and Science College, India 20. Dr. Supaporn Chunchom, Rajamangala University of Technology Isan, Thailand 21. Dr. Siriwan Panprivech, Assumption University, Thailand 22. Dr. Chukwan Techakanon, Prince of Songkla University, Surat Thani campus, Thailand 23. Dr. Cao Thị Thanh Loan, Nong Lam University, Vietnam 24. Dr. Song Xiaoming, Hangzhou Normal University, China 25. Dr. Manoch Kongchum, Louisiana State University Agricultural Center, USA 26. Dr. Ahmed Attaya, University of Massachusetts, USA 27. Dr. Sohye Yoon, University of Queensland, Australia 28. Dr. Ottavia Benedicenti, University of New Mexico, USA

Editor-in-Chief of the Science Technology and Engineering Journal (STEJ) Prof. Dr. Sirithon Siriamornpun, Mahasarakham University, Thailand

Editor-in-Chief of the Asia-Pacific Journal of Science and Technology (APST) Prof. Dr. Alissara Reungsang, Khon Kaen University, Thailand

iii

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Contents Page

Preface: i ICoFAB2021 Organizing Committee ii ICoFAB2021 Scientific Committee ii Editor-in-Chief of the Science Technology and Engineering Journal (STEJ) iii Editor-in-Chief of the Asia-Pacific Journal of Science and Technology (APST) iii

Full papers: Riceberry broken rice and soybean meal as substrates for 1 1 exopolysaccharide production by Bacillus tequilensis PS21

Thipphiya Karirat

Antioxidant potential of rice grain processed by solid state 9 2 cultivation with Cordycep militaris

Sudarat Tasoon

Optimization of microwave-assisted extraction of mulberry 16 3 twigs (Morus alba Linn.) on antityrosinase and antioxidant potential using response surface methodology

Waranya Kanyaprasit

Identification and transcriptional analysis of the metal 29 4 tolerance protein (MTP) gene family in cassava under zinc deficiency

Natlita Payap

Comparison on physical alteration of the peeled durian 44 5 stored in “OZONE BOX” odor lock and commercial packaging

Siriwan Tungsangprateep

The pretreatment condition for chromosome count and 53 6 karyotype analysis of Dimocarpus longan from Thailand

Panurat Pipatchananan

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.1

Full Length Article

Riceberry broken rice and soybean meal as substrates for exopolysaccharide production by Bacillus tequilensis PS21

Thipphiya Karirat, Worachot Saengha, Sirirat Deeseenthum and Vijitra Luang-In *

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

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

This work aimed to study the effect of riceberry broken rice and soybean meal on exopolysaccharide (EPS) production by Bacillus tequilensis PS21. Various concentrations of riceberry broken rice (RBR) as carbon source at 4, 5, and 6%, soybean meal (SBM) as nitrogen source at 1, 2, and 3% were used whilst the fermentation conditions at 37 °C and media pH 7 were constant. The results showed that as fermentation time increased from 24, 48 to 72 h, the EPS content increased significantly along with bacterial growth. RBR (4, 5, and 6%) at 72 h resulted in no significant differences in EPS production. However, SBM at 1 , 2 , and 3% resulted in a statistically significant difference in EPS content at 72 h. Therefore, the optimized conditions for EPS production were RBR at 5% and SBM at 3% for an EPS content of 28.47 g/ L. This work demonstrated a new way to add value to RBR and SBM to produce an EPS bioproduct for future applications including foods, cosmetics, and animal feeds.

Keywords: Bacillus tequilensis, exopolysaccharide, riceberry broken rice, soybean meal

Introduction

In recent years, waste has been increased daily in large quantities, adversely impacting ecosystems and human beings. The industry has a large amount of waste emitted in the production of products exported within and outside the country. By-products arising from the production process are difficult to eliminate and have a high cost. The need for methods of utilizing or recycling industrial waste is one of the current global environmental problems. Sugarcane molasses, broken rice, rice bran, and coconut water are agro- industrial wastes needed to be value added (Razack et al., 2013; Küçükaşik et al., 2011; Han and Watson, 1992, Seesuriyachan et al. , 2011). These are agro-industrial wastes rich in sugars such as glucose, fructose, and sucrose. They also contain nitrogen and vitamins that are useful for multi- beneficial exopolysaccharide (EPS) biosynthesis. Several factors influence waste fermentation

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

by microorganisms, including media composition, the fermentation conditions ( pH, temperature, concentration of oxygen), and the sources of carbon and nitrogen. (Pereira et al., 2006). Riceberry rice well- known due to its distinct dark purple color, long-grained shape, and high antioxidant characteristics. It was developed by the Rice Science Center at Kasetsart University in Thailand and is a cross between Hom Nil rice and KhaoDok Mali 105. The shards of rice grains obtained by milling are referred to as broken rice. Broken rice is defined as rice with a length shortening of more than 20% (Soponronnarit et al., 1999). Riceberry broken rice (RBR) is a major byproduct of Thai rice processing plants. After the polishing phase, this product is separated and has the same chemical composition as white rice. The byproduct accounts for 20-30% of riceberry production (approximately 1,200-1,800 tons per harvesting season). Thus, we would like to find a new way to produce better value-added bioproducts using riceberry broken rice as a carbon substrate for EPS production. Soybean meal (SBM) is a by-product of soybean extraction. It is a yellow or light brown color. Soybean meal consists of approximately 71. 6% of soybean and approximately 7% of husks. Soybean meal and soy husks are produced approximately 1,923,000 tons/ year. The most important protein source and contains large quantities of essential amino acids used to feed farm animals (Park et al. , 2002). Soy waste can be used as a nitrogen source for the production of EPS. EPSs are high molecular polymers consisting of sugar residues generated by EPS synthesizing microbes. EPS in the environment is found in the form of capsules or slime (Razack & Thangavelu, 2013; De Vuyst & Degeest, 1999). The microbial EPS offers a broad range of business applications, including food, cosmetics, pharmaceutics, milk, and medicines, as well as other industries, including medicine, wound dressing, food additives, cosmetic ingredients, medicine supplies, etc. As a result, EPS, as a value-added bioproduct, has a wide range of applications. The previous study reported that the concentration of carbon and nitrogen sources were able to affect EPS production (Lee et al., 2004; Smiderle et al., 2012). This study aimed to identify the effects various concentrations of riceberry broken rice and soybean meal on EPS production from Bacillus tequilensis PS21.

Materials and Methods

Materials

Soybean meal ( SBM) was obtained from agricultural shop in Kantharawichai District of Mahasarakham Province as the nitrogen source of EPS production. Riceberries broken rice (RBR) was obtained from the Agrarian Network of E- San Enterprise in Roi Et Province as the carbon source. Prehandling, RBR, and SBM were ground in a fine 200 mm mesh powder and stored dry.

Cultivation of a bacterial strain

A purified culture of B. tequilensis PS21 was obtained from milk kefir in (Luang-In et al., 2016; Luang- In et al. , 2018) in the Natural Antioxidant Innovation Research Unit, Biotechnology Laboratory of Mahasarakham University. The strain was aerobically cultured in sterilized Tryptic Soy Broth (TSB), pH 7.0 (17 g/L tryptone, 2.5 g/L K2HPO4, 5 g/L NaCl, and 2. 5 g/ L glucose) for 16- 24 h shaking at 37°C at 150 rpm. The resulting bacterial suspension's optical density (OD600nm) was adjusted to 0. 1 before inoculation into the fresh TSB media.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Bacterial EPS production

RBR (4, 5, and 6%) and SBM (1, 2, and 3%) were mixed in 100 mL distilled water and adjusted to pH 7 and sterilized at 110 °C for 15 min. The resulting bacterial suspension's optical density (OD600nm) was adjusted to 0. 1 before inoculation into the fresh RBR/ SBM media and then seed inoculum (3% v/v) was transferred in culture media and incubation at 37 °C at 150 rpm agitation speed for 72 h. Bacterial counts on TSB agar using serial dilutions ( 10- 2 - 10- 8) incubated at 37°C for 24 h were detected at several intervals at 72 h. A pH meter was used to measure the pH values, and the results were recorded in triplicate.

Extraction of crude EPS

The bacterial cultures were centrifuged at 4°C at 10,000 g for 15 min. Two volumes of cold ethanol (v/ v) precipitated the supernatant and were kept at 4 ° C for 24 h. The was EPS centrifuged and washed twice with 95% (v/v) ethanol at 10,000 g at 4°C for 10 minutes. Fresh EPS was dried at 60°C in an oven and kept at -20°C.

Statistical analysis

All data were collected in triplicate, and the results were displayed as standard deviations (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Duncan's multiple range test by SPSS software (demo version).

Results and Discussion

Appearance of EPS

The EPS feature of B. tequilensis PS21 is the ropy or slimy appearance of the colony. Colonies show a slimy appearance on RBR agar media and incubated for 3 days at 37°C (Figure 1A). The formation of EPS sheets hanging on the culture surface of the supernatant appeared after 24 h of EPS ethanol precipitation (Figure 1B). The EPS was sticky and whit-ish (Figure 1C- 1E).

Effects of various concentrations of riceberry broken rice and soybean meal on EPS production

The results showed that the EPS content increased significantly with bacterial growth as the fermentation time rose from 24, 48 to 72 h (Table 1). RBR (4, 5, and 6%) at 72 h resulted in no significant differences in EPS production. However, SBM at 1, 2, and 3% resulted in a statistically significant difference in EPS content at 72 h. Therefore, the optimized conditions for EPS production were RBR at 5% and SBM at 3% for the highest EPS content of 28.47 g/L. Significant increases in EPS production have come with increased concentrations of the substrates. EPS production was stimulated by the excess of carbohydrate in the medium and the limitation of carbon sources diminishes EPS synthesis (Van Geel-Scutten et al., 1998; De Vuyst & Degeest 1999). This is the first report of EPS production from RBR and SBM by Bacillus sp. with a higher yield than the previous reports by Bacillus sp (Moghannem et al., 2018; Ergene et al., 2018; Razack et al., 2013).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Figure 1 . EPS slimes and EPS extracts. (A) EPS slime from BRB on agar. (B) EPS was precipitated with ethanol at 24 h. (C) EPS extract from RBR 4% SBM 3%. (D) EPS extract from RBR 5% SBM 3%. (E) EPS extract from RBR 6% SBM 3%.

Table 1: The effects of various concentrations of riceberry broken rice and soybean meal on EPS production by B. tequilensis PS21

EPS content (g/L) Treatment Time of fermentation (h) 24 48 72 C,f B,f A,e RBR 4% SB 1% 4.63±0.04 16.00±0.07 21.70±0.04 C,e B,bc A,bc RBR 4% SB 2% 7.96±0.01 20.83±0.04 25.00±0.00 B,de A,a A,a RBR 4% SB 3% 9.63±0.09 26.43±0.08 27.47±0.05 C,de B,e A,cd RBR 5% SB 1% 10.90±0.23 17.80±0.10 23.97±0.20 B,d AB,d A,b RBR 5% SB 2% 10.86±0.56 18.26±0.57 25.67±0.23 B,b B,cd A,a RBR 5% SB 3% 15.27±0.30 19.93±0.50 28.47±0.07 C,d B,e A,e RBR 6% SB 1% 10.90±0.31 18.29±0.32 22.90±0.13 C,c B,de A,bc RBR 6% SB 2% 13.37±0.17 19.90±0.17 25.39±0.26 C,a B,b A,a RBR 6% SB 3% 19.07±0.10 21.43±0.10 28.07±0.03 * RBR: Riceberry broken rice; SBM: Soybean meal. Data are mean ± S.D. of three replicates. Different capital letters within the same row and small letters within the same column indicate significant differences (p < 0.05) according to Duncan’s multiple range test.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Figure 2. Number of microbial cells (dark lines), pH during fermentation (dotted lines) and EPS content (hyphen lines). (A) RBR 4% and SBM 1, 2 and 3%. (B) RBR 5% and SBM 1, 2 and 3%. (C) RBR 5% and SBM 1, 2 and 3%.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

According to the previous works (Lee et al. , 2004; Smiderle et al. , 2012), EPS yields may be affected by the amount of agricultural waste and the types of agricultural waste used in production, as well as the microorganisms used. The EPS production of P. pulmonarius used peanut husks at 20 g/L, resulting in the highest EPS production at 5.60 g/L (Ogidi et al., 2020). Previous reports found that Xanthomonas campestris 1866 had xanthan gum production of 10.3 g/L using cocoa husks as a carbon source (Nery et al., 2013). The highest EPS production of 426 mg/ L was detected using 60 g/ L molasses concentration (Ergene et al. , 2018). Various factors, different carbon sources and nitrogen sources were used. The results showed the best carbon source was molasses at 4% w/ v has been EPS productivity was 4. 2 g/ L. The highest EPS productivity was 4.4 g/L from yeast extract at a concentration of 3 g/L. (Moghannem et al., 2018).

Number of microbial cells and pH of B. tequilensis PS21

The results showed that the initial pH value of the fermentation dropped from initially 7. 00 to 6. 67 at 72 h. The pH decline was caused by the production of organic acid as a result of the high consumption of carbon sources for cell growth. (Shih et al. , 2006). Two other studies showed that growth in media with pH adjustment resulted in greater polysaccharides (De Vuyst et al. , 2001). The pH of culture media was an important parameter affecting cell and EPS production (Liu et al., 2009). The difference in pH values in cultured Bacillus sp. ZBP4 affected EPS production because pH played a role in the synthesis of EPS at the gene level. Therefore, the pH value affected the enzyme in the synthesis of EPS (Ergene et al. , 2018). The growth curve of B. tequilensis PS21 began at 108 CFU/ mL at 0 h and reached 1012 CFU/ mL at 72 h. (Figure 2). The B. tequilensis PS21 metabolite production followed a primary kinetics pattern, characterized by the metabolites' nearly simultaneous biosynthesis with growth and approaching a maximum rate near the end. EPS production decreased significantly after extended incubation which may probably be due to glycohydrolase degradation (Wang et al., 2014; Li et al., 2014). It can be seen that carbon and nitrogen sources are important for growth as well as EPS production because they provide the energy required for both processes.

Conclusion

The optimum conditions for EPS production from B. tequilensis PS21 showed that the maximum EPS was 28.47 g/L using 5% RBR and 3% SBM for 72 h of fermentation. Bacillus sp. yields higher yields than those from previously reported Bacillus sp. and is a new way to add value to broken rice and produce bio-products.

Acknowledgements

The authors appreciate the financial support the National Research Council of Thailand (Year 2020) through Master students funding awarded to TK. The authors would like to thank Mahasarakham University's Department of Biotechnology in Thailand for providing laboratory space.

References

De Vuyst, L., & Degeest, B. (1999). Heteropolysaccharides from lactic acid bacteria. FEMS Microbiology Reviews, 23(2), 153-177.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

De Vuyst, L., De Vin, F., Vaningelgem, F., & Degeest, B. (2001). Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. International Dairy Journal, 11(9), 687-707. Ergene, E., & Ayşe, A. V. C. I. (2018). Effects of cultural conditions on exopolysaccharide production by Bacillus sp. ZBP4. Journal of Agricultural Sciences, 24(3), 386-393. Han, Y. W., & Watson, M. A. (1992). Production of microbial levan from sucrose, sugarcane juice and beet molasses. Journal of Industrial Microbiology, 9(3-4), 257-260. Küçükaşik, F., Kazak, H., Güney, D., Finore, I., Poli, A., Yenigün, O., & Öner, E. T. (2011). Molasses as fermentation substrate for levan production by Halomonas sp. Applied Microbiology and Biotechnology, 89(6), 1729-1740. Lee, W. J., & Lucey, J. A. (2004). Structure and physical properties of yogurt gels: Effect of inoculation rate and incubation temperature. Journal of Dairy Science, 87(10), 3153- 3164. Li, W., Ji, J., Chen, X., Jiang, M., Rui, X., & Dong, M. (2014). Structural elucidation and antioxidant activities of exopolysaccharides from Lactobacillus helveticus MB2-1. Carbohydrate Polymers, 102, 351-359. Liu, J., Luo, J., Ye, H., Sun, Y., Lu, Z., & Zeng, X. (2009). Production, characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3. Carbohydrate Polymers, 78, 275-281. Luang-In, V., & Deeseenthum, S. (2016). Exopolysaccharide-producing isolates from Thai milk kefir and their antioxidant activities. LWT, 73, 592-601. Luang-In, V., Saengha, W., & Deeseenthum, S. (2018). Characterization and bioactivities of a novel exopolysaccharide produced from lactose by Bacillus tequilensis PS21 isolated from Thai milk kefir. Microbiology and Biotechnology Letters, 46(1), 9-17. Moghannem, S. A., Farag, M. M., Shehab, A. M., & Azab, M. S. (2018). Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design. Brazilian Journal of Microbiology, 49(3), 452-462. Ogidi, C. O., Ubaru, A. M., Ladi-Lawal, T., Thonda, O. A., Aladejana, O. M., & Malomo, O. (2020). Bioactivity assessment of exopolysaccharides produced by Pleurotus pulmonarius in submerged culture with different agro-waste residues. Heliyon, 6(12), e05685. Park, Y. H., Kim, H. K., Kim, H. S., Lee, H. S., Shin, I. S., & Whang, K. Y. (2002). Effects of three different soybean meal sources on layer and broiler performance. Asian- Australasian Journal of Animal Sciences, 15(2), 254-265. Pereira Duta, F., Pessôa de França, F., & de Almeida Lopes, L. M. (2006). Optimization of culture conditions for exopolysaccharides production in Rhizobium sp. using the response surface method. Electronic Journal of Biotechnology, 9(4), 0-0. Razack, S. A., Velayutham, V., & Thangavelu, V. (2013). Medium optimization for the production of exopolysaccharide by Bacillus subtilis using synthetic sources and agro wastes. Turkish Journal of Biology, 37(3), 280-288. Seesuriyachan, P., Kuntiya, A., & Techapun, C. (2011). Exopolysaccharide production by Lactobacillus confusus TISTR 1498 using coconut water as an alternative carbon source: the effect of peptone, yeast extract and beef extract. Sonklanakarin Journal of Science and Technology, 33(4), 379. Shih, I. L., Pan, K., & Hsieh, C. (2006). Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea. Process Biochemistry, 41, 1129-1135. Smiderle, F. R., Olsen, L. M., Ruthes, A. C., Czelusniak, P. A., Santana-Filho, A. P., Sassaki, G. L., & Iacomini, M. (2012). Exopolysaccharides, proteins and lipids in Pleurotus

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

pulmonarius submerged culture using different carbon sources. Carbohydrate Polymers, 87(1), 368-376. Soponronnarit, S., Wetchacama, S., Swasdisevi, T., & Poomsa-ad, N. (1999). Managing moist paddy by drying, tempering and ambient air ventilation. Drying Technology, 17(1-2), 335-343. Van Geel-Schutten, G. H., Flesch, F., Ten Brink, B., Smith, M. R., & Dijkhuizen, L. J. A. M. (1998). Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides. Applied Microbiology and Biotechnology, 50(6), 697- 703. Wang, K., Li, W., Rui, X., Chen, X., Jiang, M., & Dong, M. (2014). Characterization of a novel exopolysaccharide with antitumor activity from Lactobacillus plantarum 70810. International Journal of Biological Macromolecules, 63, 133-139.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.2

Full Length Article

Antioxidant potential of rice grain processed by solid state cultivation with Cordycep militaris

Sudarat Tasoon 1 and Luchai Butkhup 2*

1 Master student, Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham 44150, Thailand 2 Natural Antioxidant Innovation Research Unit (NAIRU), Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham 44150, Thailand

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

Rice grain (Oryza sativa L) was used as the raw material for Cordycep militaris cultivation to investigate the effects of extract condition on the phenolic content and antioxidant activities. The 15% of ethanol concentration exhibited most effective extraction of phenolic compounds from rice grain processed by solid state cultivation with C. militaris. The ethanolic extracted showed highest phenolic content (1636. 53±67. 98 mg GAE/ 100g DW) and reducing ability (FRAP, 1553.41±33.24 mg Fe2+/100g DW) and also high scavenging activity more than 75%. The ethanolic extracts obtained from the rice grain processed by solid state cultivation with C. militaris might be a potential antioxidant supplement for application in food products.

Keywords: Cordycep militaris, antioxidant activity, ethanolic extracted, rice grain

Introduction

Cordyceps militaris is a potential herbal drug and which contains a high nutritional value (Wasser et al., 1999). It has been widely used as a folk tonic food in Asia extensively. Besides their popular applications for tonic medicine, the constituents of C. militaris are now used extensively in modern systems of medicine. Contents of major bioactive components of the medicinal properties include Cordycepin, adenosine (Cunningham et al., 1950) pentastatin, carotenoids, polysaccharides, proteoglycans, terpenoids, steroids, and phenolic compounds (Bawadekji et al.,2016; Zhang et al., 2019). Cordycepin (30-deoxyadenosine), an adenosine analogue has been reported to possess various pharmacological, including immunological regulation (Noh et al., 2009;) antifungus (Sugar and McCaffrey, 1998) antileukemia (Thomadaki, Tsiapalis, and Scorilas, 2008; anticancer (Yoshikawa et al., 2008) and antioxidant ability, could terminate chain reaction, remove free radical intermediates, scavenge reactive oxygen species (ROS) (Pirakathiswaran et al., 2020). Report studies have shown that Cordyceps possesses liver protective effects (Liu et al., 2006) reduce the increase of cholesterol and triglyceride (Kim et al., 2005) and induce the T-cell and macrophages activity

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

(Liu et al., 2001) , decrease the level of c-Myc, c-Fos, and VEGF levels in the lungs and liver (Yang et al., 2005) [18] Besides these, Cordyceps contains some uncommon cyclic dipeptides, including cyclo-(Gly-Pro), cyclo-(Leu-Pro), cyclo-(Val-Pro), cyclo-(Ala-Leu), cyclo-(Ala- Val), and cyclo-(Tr-Leu) and small amounts of polyamines, such as 1,3-diamino propane, cadaverine, spermidine, spermine, and putrescine (Liu et al., 2006). Cordyceps was able to stabilize blood sugar and increase the liver enzymes hepatic glucokinase and glucose transporter isoform-2 (GLUT2) in hyperglycaemic diabetic rats (Kiho et al., 1999), supplementation reduced inflammatory markers in the hippocampus of the brain, it also raised increased precursor to serotonin and norepinephrine levels (Tianzhu et al., 2014). Therefore, in the present study of different ethanol extraction conditions on antioxidant activity and bioactive compounds of rice grain obtained for C. militaris cultivation.

Materials and Methods

Chemicals and materials 1-diphenyl-2-picrylhydrazyl radical (DPPH), Folin-Ciocalteu phenol reagent (FCR) were purchased from Sigma-Aldrich Chemical Co., (St. Louis, MO, USA). Ethanol (HPLC grade) was purchased from BDH (Poole, UK). Trolox standard (TE) (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), Quercetin standard, Gallic acid standard, FeSO4.7H2O standard and 2,4,6-Tripyridyl-5-Triazine (TPTZ) were purchased from Fluka Chemicals (Buchs, Switzerland).

Starter culture and solid-state cultivation 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) in the dark before mycelium growth after C. militaris mycelium was cultured continuously in potato dextrose broth (PDB) at 20˚C for 21 days, so as to obtain mycelium pellets starter culture. The solid-state media culture including rice grain and potato dextrose broth for the C. militaris cultivation 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 antioxidant and bioactive compound analysis

Preparation of the Ethanol Extracts Rice grain powder obtained for C. militaris cultivation was mixed with various ethanol concentration (15, 30, 45, 60 and 75%) and then extract on ice using probe ultrasonic homogenizer with a frequency of 20 kHz. The amplitude was controlled to set the ultrasonic output power at 70% of the maximum range. After sonication was completed, the solids were separated from the mixture by centrifugation 10,000 rpm for 30 min at 4°C and collect all clear supernatant was filtered through a Whatman paper (No.1) for experimentation.

Scavenging effect on DPPH radical’s assay The DPPH radical scavenging inhibition determination using 96-well plate according to the assay (Saengha et al., 2021). DPPH reagent (0.2 mM) 200 µL in methanol mixed with clean sample (100 µL) in each 96 well plate. The reaction incubated at room temperature for 30 min and measured at 515 nm using microplate reader.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Ferric Reducing Antioxidant Power (FRAP) Assay FRAP assay was determined according the assay (Saengha et al., 2021). FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM of 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM of HCl and 20 mM of FeCl3.6H2O in the ratio (10:1:1 v/v/v) and incubated at 37 °C for 30 min. Sample extract (20 µL) was reacted with 180 µL of FRAP reagent, mixed, left for 30 min and measured at 593 nm. Standards of known Fe (II) concentrations and FRAP values were reported as mg Fe (II)/g DW.

Total Phenolic Content Determination (TPC) TPC was determined using Folin-Ciocalteau assay following the assay (Saengha et al., 2021). The extract (20 µL) mixed with 100 µL of 10% Folin-Ciocalteu reagent and 80 µL of 7.5% NaHCO3, left for 30 min in a 96 well plate. The reaction mixture was measured at 765 nm using microplate reader. Results were reported as mg gallic acid equivalent (GAE)/g DW.

Total Flavonoid Content Determination (TFC) TFC was performed according to the assay (Saengha et al., 2021). Sample extract (20 µL) added in to 96 well plate and then mixed with 60 µL of deionized water, followed by 10 µL of 5% NaNO3, 10 µL of 10% AlCl3.6H2O each well, mixed and left for a minute. Then, 100 µL of 1 M NaOH was added, mixed and kept for 30 min before measurement at 500 nm. Results were reported as mg quercetin Equivalent (QE)/g DW.

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 extract condition on phenolic content and antioxidant activity

The fruiting body of C. militaris was grown at 60 days (Figure 1). C. militaris could grow on rice grain and mycelium colour will be change from white to yellow after light exposure. Then around 60 days of cultivation, fruiting body and rice grain ready to harvest. Rice grain harvested and dried at 50 ˚C in an incubator for extract with a different concentration of ethanol using probe ultrasonic homogenizer. The results clearly demonstrated that DPPH antioxidant activity increased significantly in C. militaris from 45% ethanol extraction (80.90±2.78%) when compared to 4 groups of ethanol concentration (Figure 2A). FRAP activity (Figure 2B) and TPC (Figure 3A) increased significantly were from 15% ethanol extraction which was significantly different from those of the 4 groups of ethanol concentration. The highest FRAP activity (1553.41±33.24 mg Fe2+/100g DW) and TPC (1636.53±67.98 mg GAE/100g DW) was found in 15% ethanol extraction. Likewise, the TFC results were significantly different (P < 0.05) among all ethanol concentration. Ethanol extraction of 75% led to the highest TFC (356.24 ± 90.37 mg QE/100g DW) which was significantly higher than that of the 4 groups of ethanol concentrations (Figure 3B).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

A B

C D

Figure 1. C. militaris grown on rice grain. A: 7 days after inoculated, B: C. militaris mycelium after light exposure, C: C. militaris fruiting body (60 days of cultivation), D: fruiting body and rice grain harvesting.

Figure 2. Effect of C. militaris from different concentration of ethanol extract on antioxidant activity. A) Antioxidant activity by DPPH assay. B) Antioxidant activity by FRAP assay, each value is the mean ± sd of three experiments. P < 0.05.

Ethanol extraction efficiency was the best on the grounds of safety and efficiency (Wang at al., 2014). The solvent polarity and extraction time affected the cordycepin content of C. militaris fruiting body (Xuan et al., 2019) and phenolic compounds were more efficiently extracted by high polar solvents. The phenolic compound is obviously related to the percentage of ethanol and its polarity index (Abarca-Vargas et al., 2016). The antioxidant potential of C. militaris extracts has stronger activity in the system of scavenging ability on DPPH• radicals and the chelating ability on ferrous ions. Antioxidant components in C. militaris, including total phenolic and flavonoid compounds, are important constituents because of their

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

scavenging inhibition due to their hydroxyl groups. It was reported that the content of total phenolic and flavonoid compound was the key component accounting for the antioxidant activity in many mushrooms (Cheung et al., 2003; Elmastas et al., 2007)

Figure 3. Effect of C. militaris from different concentration of ethanol extract on TPC and TFC, each value is the mean ± sd of three experiments P < 0.05.

Conclusion

Based on the results obtained, the higher antioxidant properties that the ethanolic extracts displayed might be somewhat beneficial to the antioxidant protection system of the human body against oxidative damage. The 15% of ethanol concentration exhibited most effective extraction of phenolic compounds from rice grain processed by solid state cultivation with C. militaris. Therefore, this extract condition might be a potential antioxidant supplement for application in food products. The study on the contribution of phenolic compounds to antioxidant properties of ethanolic extracts is still in progress. Due to the fact that most of the nature antioxidant compounds are relatively unstable, different solvent concentration could be taken as different antioxidant alternatives.

Acknowledgements

We thank the Department of Biotechnology, MSU and the MSU Central Laboratory for use of laboratory facilities. This transdisciplinary research is part of a dissertation which was submitted as partial fulfilment to meet requirements for the degree of Masters of Biotechnology at Mahasarakham University.

Author’s contribution

ST designed, conducted the experiments, analyzed data, and wrote the manuscript. LB anchored the review, revisions and approved the article submission. All authors listed have read and approved the manuscript for publication.

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Conflict of Interest Statement

The authors agree that this research was conducted in the absence of any self- benefits, commercial or financial conflicts and declare absence of conflicting interests with the funders.

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Tianzhu, Z., Shihai, Y., & Juan, D. (2014). Antidepressant-like effects of cordycepin in a mice model of chronic unpredictable mild stress. Evidence-Based Complementary and Alternative Medicine, 2014. Thomadaki, H., Tsiapalis, C. M., & Scorilas, A. (2008). The effect of the polyadenylation inhibitor cordycepin on human Molt-4 and Daudi leukaemia and lymphoma cell lines. Cancer Chemotherapy and Pharmacology, 61(4), 703-711. Wang, H. J., Pan, M. C., Chang, C. K., Chang, S. W., & Hsieh, C. W. (2014). Optimization of ultrasonic-assisted extraction of cordycepin from Cordyceps militaris using orthogonal experimental design. Molecules, 19(12), 20808-20820. Wasser, S. P., & Weis, A. L. (1999). Medicinal properties of substances occurring in higher basidiomycetes mushrooms: current perspectives. International Journal of Medicinal Mushrooms, 1(1). Yoshikawa, N., Yamada, S., Takeuchi, C., Kagota, S., Shinozuka, K., Kunitomo, M., & Nakamura, K. (2008). Cordycepin (3′-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A 3 receptor followed by glycogen synthase kinase-3β activation and cyclin D 1 suppression. Naunyn- Schmiedeberg's Archives of Pharmacology, 377(4-6), 591-595. Yang, J., Zhang, W., Shi, P., Chen, J., Han, X., & Wang, Y. (2005). Effects of exopolysaccharide fraction (EPSF) from a cultivated Cordyceps sinensis fungus on c- Myc, c-Fos, and VEGF expression in B16 melanoma-bearing mice. Pathology- Research and Practice, 201(11), 745-750. Zhang, J., Wen, C., Duan, Y., Zhang, H., & Ma, H. (2019). Advance in Cordyceps militaris (Linn) Link polysaccharides: Isolation, structure, and bioactivities: A review. International Journal of Biological Macromolecules, 132, 906-914. Zhou, X., Gong, Z., Su, Y., Lin, J., & Tang, K. (2009). Cordyceps fungi: natural products, pharmacological functions and developmental products. Journal of Pharmacy and Pharmacology, 61(3), 279-291.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.3

Full Length Article

Optimization of microwave-assisted extraction of mulberry twigs (Morus alba Linn.) on antityrosinase and antioxidant potential using response surface methodology

Waranya Kanyaprasit 1 and Luchai Butkhup 2*

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

Response surface methodology (RSM) has been used to optimize the extraction conditions of bioactive components with relatively high antityrosinase and antioxidant activity from mulberry twigs by using microwave-assisted extraction (MAE). The results showed that the highest antityrosinase (2. 51 mg VE/ g dw) and antioxidant activity (79. 03 %scavenging and 250.03 mg VEAC/100 g dw for DPPH assay, and 1342.75 mg Fe(II)/100g dw for FRAP assay) were obtained with an extraction time of 5 min, 45% ethanol, and 70 ml/g liquid to solid ratio. In this study, MAE can be used as an alternative to conventional immersion extraction with respect to the recovery of bioactive compounds from mulberry twigs, with the advantages of shorter extraction time and reduced solvent consumption.

Keywords: mulberry twigs, microwave-assisted extraction, antityrosinase activity, antioxidant activity

Introduction

Mulberry (Morus alba L.) is a fast-growing deciduous plant that grows under different climatic conditions. Mulberry has been used in traditional Chinese medicine as an anti-diabetic, anti- hypertensive, anti-headache, and diuretic agent (Choi et al., 2013). In particular, mulberry twigs have been widely used for the healing of aching and numbness of joints in oriental medicine (Zhu, 1998). Mulberry contains bioactive compounds including phenolic compounds especially stilbene groups which can inhibit enzyme tyrosinase activity and as an antioxidant, so its potential in cosmetic (Batubara et al., 2010). Oxyresveratrol was an aglycone of mulberroside A and showed strong inhibit enzyme tyrosinase activity (Kim et al., 2010). Bioactive substances especially phenolic groups could be extracted from mulberry twigs by several conventional methods, such as maceration, percolation, soxhlet extraction and reflux extraction. However, these traditional methods are often inefficient, as well as solvent-

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

and time-consuming. Therefore, some new extraction methods came into being to overcome these problems, including Ultrasonic-assisted extraction (UAE), pressurized liquid extraction, enzyme-assisted extraction and microwave-assisted extraction (MAE). MAE is a novel method used to improve production yield. Microwave radiation has a destructive effect on cell structure, which makes the active substance dissolve into the solvent quickly, thus obtaining higher extraction efficiency in a shorter time (Vinatoru et al., 2017). At the same time, the microwave-assisted extraction also has strong advantages in stability and reproducibility (Chan et al., 2011). To our knowledge, no report could be found in the literature on the extraction of antioxidants from mulberry twigs by microwave-assisted extraction. The objective of this study was to investigate the effect of microwave on the extraction efficiency of mulberry twigs on antityrosinase and antioxidant capacities, as well as to optimize the parameters of this process by response surface methodology.

Materials and Methods

Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid), and FeCl3 were purchased from Sigma-Aldrich (USA); Folin-Ciocalteus’s phenol reagent and TPTZ (2,4,6-tripyridyl-S-triazine) were purchased from Fluka (Switzerland); acetonitrile and milli-Q water were of HPLC grade and other chemicals were of analytical grade.

Microwave-Assisted Extraction (MAE)

Mulberry twigs (Morus alba L.) of the cultivars, Nakhonratchasima 60 was obtained from Silk Innovation Center, Mahasarakham University, Thailand. The MAE process was performed by microwave equipment with controlled microwave power (2000 W). Dried mulberry twigs sample (1.000 g) was placed in a centrifuge tube and then mixed with ethanol aqueous solutions of different concentrations and volumes. After extraction, the mixture was cooled with running water, centrifuged at 4200xg for 10 min and the supernatant was kept at 4oC for subsequent experiments.

Experimental design and statistical analysis

Microwave -assisted extraction optimized the experimental design using RSM. A Central Composite Design (CCD) consisting of twenty experimental runs was employed including six- star points (α = 1.682) points, eight factorial points and six central points. The independent variables were the ethanol concentration (X1, 30-60%), liquid to solid ratio (X2, 50-90 ml/g), and extraction time (X3, 1-10 min) (Table 1) while dependent variables (response) were antityrosinase (Y1) and antioxidant activity (Y2). The range values of the three independent variables were determined by preliminary study. Experiments were performed in replicate and the average values were used as the response, Y.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Table 1. Independent variables and code levels of central composite design (CCD).

Level Independent Variable Units -∞ -1 0 +1 +∞ Ethanol concentration (X1) % v/v 15 30 45 60 75 Liquid-to-Solid ratio (X2) ml/g 30 50 70 90 110 Extraction time (X3) min 1 3 5 7 9

The CCD matrix contains 20 experiments with 6 replicates of center points. The response values of the model were analyzed by Design-Expert 7.0 and the data were subjected to multiple regression analysis to fit the quadratic equation:

(1)

where Y refers to the predicted response values of antityrosinase and antioxidant activity; β0 is the constant coefficient; βi, βij and βii represent the coefficients of linear, quadratic and interaction terms, respectively; Xi and Xj are independent variables; ε is the residual error. Analysis of variance (ANOVA) was used to analyze the statistical significance of the fitting model and each term of the fitted model. The interaction effect of each variable on the response value was shown on the 3D surface plot.

Determination of antioxidant activity

DPPH scavenging activity

The radical scavenging activity of medicinal plant extracts were evaluated according to the method of Brand-Williams et al. (1995) with some modification. One hundred microliters of plant extracts were mixed with 100 μl of the 0.2 mM DPPH solution. The mixture was thoroughly mixed and left to stand for 1 h at room temperature in the dark. The absorbance was read at 520 nm using a microplate reader spectrophotometer (Synergy HT, BiotTek instruments, USA). The antioxidant activity of medicinal plant extracts was expressed as EC50 (mg/ml), the extract dose required to scavenge 50% of DPPH free radicals. A smaller EC50 value corresponds to a higher antioxidant activity.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was carried out according to the method of Benzie and Strain (1996) with some modifications. Briefly, 270 μl of freshly FRAP reagent containing 300 mM acetate buffer (pH 3.6), 20 mM FeCL3.6H2O, 10 mM TPTZ in 40 mM HCl in the proportion of 10:1:1 (v/v), respectively, was mixed with 30 μl medicinal plant extracts. After incubated in the dark at room temperature for 30 min, the absorbance at 595 nm was measured using a microplate reader spectrophotometer (Synergy HT, BiotTek instruments, USA). A standard curve of ferrous sulfate solution (FeSO4.7H2O) was used for calculation of FRAP and expressed as mg Fe (II)/100g dw.

Tyrosinase inhibition

Tyrosinase inhibition activity was determined using the modified dopachrome method with L-DOPA as the substrate (Masuda et al., 2005). A 96-well microtiter plate was used to measure

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

absorbance at 490 nm. Each well contained 40 μL of sample with 80 μL of phosphate buffer (0.1M, pH 6.8), 40 μL of tyrosinase (100 units/mL). After incubated at room temperature for 10 min, 40 μL of L-DOPA (2.5 mM) was added. The mixture was incubated for 10 min at room temperature and absorbance was measured at 490 nm using a microplate reader spectrophotometer (Synergy HT, BiotTek instruments, USA). Each sample was accompanied by a blank containing all components except L-DOPA. L-ascorbic acid was used as positive controls. The results were compared with a control consisting of phosphate buffer in place of the sample. The percentage of tyrosinase inhibition was calculated as follows: [(Acontrol − Asample)/Acontrol] × 100%

Statistical Analysis

All experiments were performed in triplicate and the results were displayed as mean. The data were analyzed by Design Expert 7.0 software and SPSS 16.0 statistics software (IBM Corp., Armonk, NY, USA). The statistical significance was investigated by one-way ANOVA and the significance level was p < 0.05.

Results and Discussion

Fitting the Models

An optimization of extraction conditions for the extraction of mulberry twigs with relatively high antityrosinase and antioxidant capacities was conducted using RSM. The extraction efficiency of bioactive compounds was affected by extraction conditions including extraction solvent concentration, extraction temperature and extraction time (Tan et al. , 2011). RSM is accepted as a powerful tool in optimizing experimental conditions to maximize various responses (Zhao et al., 2011). For RSM, the levels of independent variables for the extraction of mulberry twigs with relatively high antityrosinase and antioxidant activities was selected based on the results obtained from our preliminary experiments. The experimental design and corresponding response data are shown in Table 2. Twenty experiments were designated and six were zero-point tests performed to estimate the errors. The ranges of ethanol concentration (15%– 75%), liquid to solid ratio (30– 110 ml/ g), and extraction time (1– 9 min) were used. Antityrosinase and antioxidant activities (DPPH, FRAP) were used as responses in the RSM experimental design. Predicted response Y for extraction of mulberry twigs could be obtained by applying multiple regression analysis on the experimental data. The predicted quadratic polynomial models are shown in Table 3. The models were checked using a numerical method including the coefficient of determination (R2). R2 provided a measure of how well future outcomes are likely to be predicted by the model. In the models, X2 and X2X3 were associated with synergistic effects on the antityrosinase activity whereas X1 and X3 were associated with antagonistic effects. In addition, X1, X2 and X2X3 were associated with synergistic effects on the antioxidant activity whereas X3 and X1X3, were associated with antagonistic effects. The R2 of the models for antityrosinase and antioxidant activity was 0. 9545 and 0. 8647-0. 9787, respectively, which showed suitable fitting of the model in the designed experiments (Table 4).

Moreover, the coefficient of variation (CV) was 12.14 and 4.40-7.93, respectively, which indicates that a relatively lower value of CV showed a better reliability of the response model.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Response surface optimization of MAE

To visualize the relationship between the response and experimental levels of the independent variables for the antityrosinase and antioxidant capacities, three-dimensional (3D) surface and contour plots were constructed according to the quadratic polynomial model equations of Table 3.

The influence of the variables and their interaction on the responses can be seen Figures 1-4. As shown in Figure 1A, shows the effect of the interaction of ethanol concentration and liquid to solid ratio on antityrosinase activity. The inhibition activity of tyrosinase was lower when the extraction conditions had higher ethanol concentrations and the inhibition of tyrosinase enzyme was higher when the ratio between ethanol to mulberry twigs as higher. The optimum extraction ethanol concentration was in the range of 30-45% and the ratio of ethanol per mulberry twigs was in the range of 70-90 ml/gram which has an inhibitory effect on tyrosinase greater than 2.74 mg VE/g dw. Figure 1B shows the effect of the interaction of ethanol concentration and extraction time on antityrosinase activity. Tyrosinase inhibitory activity was higher with lower ethanol concentration and shorter extraction time. At the highest point of the graph, the concentration of ethanol used for extraction was suitable in the range of 30-45% and the extraction time was in the range of 3-5 minutes, which would inhibit the tyrosinase enzyme activity greater than 2.43 mg VE/g dw. Figure 1C shows the effect of the interaction of liquid to solid ratio and extraction time on antityrosinase activity. The inhibition of tyrosinase was higher with higher liquid to solid ratio and shorter extraction time. At the highest point of the curve, the optimum liquid to solid ratio was in the range of 70-90 ml/gram and the extraction time was 3-5 minutes, which would have an inhibitory effect on the tyrosinase enzyme greater than 2.70 mg VE/g dw. Figure 2A, 3A and 4A shows the effect of the interaction of ethanol concentration and liquid to solid ratio on the antioxidant activity (DPPH and FRAP). The antioxidant activity of DPPH and FRAP was higher when the ethanol concentration decreased and the liquid to solid ratio increased. The optimum point was in the region where the concentration of ethanol extracted was in the range of 30-45% and the liquid to solid ratio was in the range of 70-90 ml/g. The antioxidant DPPH was greater than 271.23 mg VEAC/100 g dw and the reducing ability was greater than 1332.05 mg Fe(II)/100g dw. Figure 2B and 3B shows the effect of the interaction of ethanol concentration and extraction time on the antioxidant activity (DPPH). From the graph, it was found that when the ethanol concentration and extraction time were increased, the DPPH scavenging activity was increased, indicating that the optimum point was in the concentration region. The ethanol concentration was in the range of 45-60% and the extraction time was 3-5 min. The DPPH scavenging activity was higher than 79.34% and 244.08 mg VEAC/100 g dw. Figure 4B shows the effect of the interaction of ethanol concentration and extraction time on the antioxidant activity (FRAP). The results in the same way as DPPH, from the curves of FRAP, it was found that the reducing ability was increased when the ethanol concentration and extraction time increased. The optimum point is in the region where the ethanol concentration and extraction time were in the range of 30-45% and 3-5 min, respectively, resulting in the reducing ability greater than 1349.06 mg Fe(II)/100g dw. Figure 2C and 3C shows the effect of the interaction of extraction time and liquid to solid ratio on the antioxidant activity (DPPH). The DPPH scavenging activity was lower when the liquid to solid ratio increased whereas the extraction time increased resulting in the DPPH scavenging activity was higher. This indicates that the optimum point is in the region where the liquid to solid ratio and extraction time were in the range of 50-70 ml/g and 3-5 min,

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Table 2. The design and the corresponding actual values of CCD.

Antityrosinase DPPH assay FRAP X1 X2 X3 activity (mg VE/g %Scavenging mM VEAC/100 g (mg Fe(II)/100g dw) Run dw) dw Ethanol Liquid-to- Extraction Observe predicte Observe predicte Observe predicte Observed predicte concentration Solid ratio time (min) d d d d d d d (%) (ml/g) 1 30 (-1) 50 (-1) 3 (-1) 1.74 1.88 80.48 76.62 211.97 186.24 1134.9 1029.27 2 60 (1) 50 (-1) 3 (-1) 1.24 1.46 85.58 81.98 187.83 181.51 1300.16 1245.89 3 30 (-1) 90 (1) 3 (-1) 3.39 3.17 68.31 63.27 374.63 365.11 1344.84 1329.55 4 60 (1) 90 (1) 3 (-1) 2.72 2.93 83.71 83.03 383.31 364.07 1509.74 1456.53 5 30 (-1) 50 (-1) 7 (1) 1.11 1.30 80.34 77.71 123.12 138.74 693.97 740.51 6 60 (1) 50 (-1) 7 (1) 0.63 0.71 78.98 80.72 65.48 71.38 654.23 662.85 7 30 (-1) 90(1) 7 (1) 2.97 3.15 68.02 68.32 300.67 303.37 1407.21 1454.81 8 60 (1) 90 (1) 7 (1) 1.94 2.20 85.16 85.72 217.58 239.69 1188.52 1287.50 9 15 (-2) 70 (0) 5(0) 2.77 2.56 60.44 64.40 235.74 242.40 1069.64 1079.65 10 75 (2) 70 (0) 5 (0) 1.37 1.19 87.83 87.17 177.03 174.00 1132.43 1128.97 11 45 (0) 30 (-2) 5 (0) 0.63 0.51 79.87 82.39 63.35 66.81 604.80 653.81 12 45 (0) 110 (2) 5 (0) 4.11 3.83 73.27 74.09 412.83 413.99 1621.20 1578.73 13 45 (0) 70 (0) 1 (-2) 3.01 2.77 68.82 73.76 283.77 312.37 1158.24 1269.05 14 45 (0) 70 (0) 9 (2) 1.62 1.46 79.17 77.54 165.47 140.49 915.52 811.26 15 45 (0) 70 (0) 5(0) 2.29 2.51 79.03 79.03 247.12 250.03 1304.09 1342.72 16 45 (0) 70 (0) 5(0) 2.68 2.51 78.70 79.03 253.11 250.03 1339.3 1342.72 17 45 (0) 70 (0) 5(0) 2.60 2.51 78.27 79.03 248.32 250.03 1343.25 1342.72 18 45 (0) 70 (0) 5(0) 2.62 2.51 78.33 79.03 251.14 250.03 1367.51 1342.72 19 45 (0) 70 (0) 5(0) 2.64 2.51 78.30 79.03 249.52 250.03 1332.26 1342.72 20 45 (0) 70 (0) 5(0) 2.62 2.51 78.23 79.03 247.37 250.03 1327.46 1342.72

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Table 3. Coded and processed variables levels used in experimental design for RSM.

2 Responses Model equations R CV (%)

Antityrosinase Y = – 2.85168 + 0.069023X1 + 0.083864X2 + 0.9545 12.14 activity 0.13733X3 -4 -3 – (3x10 )X1X2– (1.41667x10 )X1X3 + -4 (1.25x10 )X2X3 -4 2 -4 2 – (7.08586x10 )X1 – (2.1108x10 )X2 – 2 0.024545X3 DPPH Y = + 93.86884 – 0.038205X1 – 0.69727X2 + 0.8647 4.40 (%Scavenging) 1.74216X3 + 0.012000X1X2 – 0.019667X1X3 + 0.024688X2X3 -3 2 -4 2 – (3.60051x10 )X1 – (5.03409x10 )X2 – 2 0.21128X3 DPPH (VEAC) Y = –194.48276 + 5.43866X1 + 5.48976X2 + 0.9787 7.93 22.98852X3 -3 + (3.07083x10 )X1X2 – 0.52196X1X3 – 0.089031X2X3 2 -3 2 – 0.046487X1 – (6.02102x10 )X2 – 2 1.47523X3 FRAP Y = – 801.57622 + 42.15406X1 + 21.79947X2 + 0.9596 6.51 61.10364X3 – 0.074704X1X2 – 2.45238X1X3 + 2.58759X2X3 2 2 2 – 0.26490X1 – 0.14153X2 – 18.91011X3 CV, coefficient of variation; X1, ethanol concentration; X2, liquid to solid ratio; X3, extraction time.

Table 4. Regression coefficients for different antioxidant potential as responses.

Term Antityrosinase DPPH assay FRAP activity %Scavenging mM VEAC/100 g (mg Fe(II)/100g (mg VE/g dw) dw dw) X1 -0.34*** 5.69*** -17.10*** 12.33 X2 0.83*** -2.09* 86.80*** 231.24*** X3 -0.33*** 0.94 -42.97*** -114.45*** X1X2 -0.090 3.60** 0.92 -22.41 X1X3 -0.042 -0.59 -15.66* -73.57* X2X3 0.005 0.99 -3.56 103.50*** 2 X1 -0.16** -0.81 -10.46** -59.60*** 2 X2 -0.084 -0.20 -2.41 -56.61*** 2 X3 -0.098 -0.85 -5.90 -75.64*** C.V. % 12.14 4.40 7.93 6.51 R2 of model 0.9545 0.8647 0.9787 0.9596 *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

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respectively, resulting in the DPPH scavenging activity was higher than 7859% and 245.28 mg VEAC/100 g dw.

Solid :

Liquid : :

A: Ethanol concentration

Extraction time : C

A: Ethanol concentration

Extractiontime

: C

B: Liquid : Solid

Figure 1. Response surface plot and contour plot of the interactions between different factors. Interaction effect of ethanal concentration and liquid to solid (A), ethanal concentration and extraction time (B), extraction time and liquid to solid (C) on antityrosinase activity.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Solid :

Liquid : :

A: Ethanol concentration B

Extraction time

: C

A: Ethanol concentration

Extraction time : :

B: Liquid : Solid Figure 2. Response surface plot and contour plot of the interactions between different factors. Interaction effect of ethanal concentration and liquid to solid (A), ethanal concentration and extraction time ( B) , extraction time and liquid to solid ( C) on antioxidant activity (%scavenging).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Solid :

Liquid : B

A: Ethanol concentration

Extraction time

: C

A: Ethanol concentration

Extraction time : C

B: Liquid : Solid

Figure 3. Response surface plot and contour plot of the interactions between different factors. Interaction effect of ethanal concentration and liquid to solid (A), ethanal concentration and extraction time ( B) , extraction time and liquid to solid ( C) on antioxidant activity ( mM VEAC/100 g dw).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Solid :

Liquid : :

A: Ethanol concentration

Extraction time

: C

A Ethanol concentration :

Extraction time

: C

B: Liquid : Solid

Figure 4. Response surface plot and contour plot of the interactions between different factors. Interaction effect of ethanal concentration and liquid to solid (A), ethanal concentration and extraction time (B), extraction time and liquid to solid (C) on antioxidant activity (FRAP, mg Fe(II)/100g dw).

Figure 4C shows the effect of the interaction of extraction time and liquid to solid ratio on the antioxidant activity (FRAP). It was found that the reducing ability was increased when the liquid to solid ratio increased whereas the extraction time increased resulting in the reducing ability decreased. The optimum point is in the region where the liquid to solid ratio

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

and extraction time were in the range of 50-75 ml/g and 5-7 min, respectively, resulting in the reducing ability greater than 1265.62 mg Fe(II)/100g dw.

Conclusion

In the present study, response surface methodology was used to optimize the microwave- assisted extraction (MAE) of bioactive compounds with relatively high antityrosinase and antioxidant activities from mulberry twigs. A central composite design was used to determine the optimum process parameters and the second order polynomial models for predicting responses were obtained. Ethanol concentration, liquid to solid ratio and extraction time were the most significant factor affecting antityrosinase and antioxidant capacities and the optimal extraction conditions were 45% ethanol, 70 ml/g and 5 min. Under optimized conditions the experimental values were very close to the predicted values. As such, it may be said that MAE is an effective and practical method for obtaining bioactive compounds with relatively high antityrosinase and antioxidant activities.

Acknowledgements

The authors would like to thank Department of Biotechnology, Mahasarakham University for providing laboratory facilities to complete this study.

Conflicts of Interest

The authors declare no conflict of interest.

References

Batubara, I., Darusman, L.K., Mitsunaga, T., Rahminiwati, M., Djauhari, E. 2010. Potency of Indonesia medicinal plant astyrosinase inhibitor and antioxidant agent. J Biol Sci.10: 138-144. Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology, 28: 25–30. Chan, C.H., Yusoff, R., Ngoh, G.C., Kung, F.W.L. 2011. Microwave-assisted extractions of active ingredients from plants. J. Chromatogr. A. 1218, 6213–6225. Choi, S.W., Jang, Y.J., Lee, Y.J., Leem, H.H., and Kim, E.O. 2013. Analysis of functional constituents in mulberry (Morus alba L.) twigs by different cultivars, producing areas, and heat processings. Prev. Nutr. Food Sci.18(4): 256-262. Esclapez, M.D., Garcia-Perez, J.V., Mulet, A., Carcel, J.A. 2011. Ultrasound-assisted extraction of natural products. Food Eng. Rev. 3, 108–120. Hsieh, C.W., Cheng, J.Y., Wang, T.H., Wang, H.J., Ho, W.J. 2014. Hypoglycaemic effects of Ajuga extract in vitro and in vivo. J. Funct. Food. 6, 224–230. Kim, K.J., Kim, M., Cho, G.S., Kim, K.M., Kim, W.S., Lim, H.Y. 2010. Biotransformation of Mulberroside a from Morus alba results in enhancement of tyrosinase inhibition. Ind Microbiol J. 37: 631-637. Masuda T, Yamashita D, Takeda Y, Yonemori S. 2005. Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Biosci Biotechnol Biochem. 69:197-201.

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Tan, P.W., Tan, C.P., Ho, C.W. 2011. Antioxidant properties: Effects of solid-to-solvent ratio on antioxidant compounds and capacities of Pegaga (Centella asiatica). Int. Food Res. 18: 557–562. Vinatoru, M., Mason, T.J., Calinescu, I. 2017. Ultrasonically assisted extraction (UAE) and microwave assisted extraction (MAE) of functional compounds from plant materials. Trac-Trend. Anal. Chem. 97: 159–178. Zhao, L.C., Liang, J., Li, W., Cheng, K.M., Xia, X., Deng, X., Yang, G.L. 2011. The use of response surface methodology to optimize the ultrasound-assisted extraction of five anthraquinones from Rheum palmatum L. Molecules, 16: 5928–5937. Zhu YP. 1998. Chinese materia medica: Chemistry, pharmacology and applications. Harwood Academic Publishers, Newark, NJ, USA. p 273-274.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.4

Full Length Article

Identification and transcriptional analysis of the metal tolerance protein (MTP) gene family in cassava under zinc deficiency

Natlita Payap, Triwarat Rujikiadtichok and Nimnara Yookongkaew *

Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

Available of zinc in cassava plants is essential for plant developmental stages as well as crop production. This paper was explored the molecular mechanism of zinc transport in cassava plants. Zinc deficient symptom was found in upper cassava leaves under zinc deficiency and identified the metal tolerance proteins (MTPs) family in cassava genome. MTPs are a metal cation efflux transporter that participated in zinc homeostasis. Computational analysis showed 12 MeMTP members in cassava genome with exons ranging from 1-12. Most of the MTP proteins were predicted to localize in plasma membrane and tonoplast except MeMTP4 and MeMTP8, which localized in ER membrane. Functional annotation verified that MeMTPs were cation efflux proteins/ zinc transporters belonging to cation diffusion facilitator superfamily (CDF). Most of the cis-acting elements in MeMTP promoters were phytohormone responsive. TGA-element was identified in MeMTP2, MeMTP5-7 and MeMTP10 promoters, indicating its role in auxin regulation. MeMTP proteins were divided into three groups according to phylogenetic relationship. Moreover, RNA expression of 6 candidate MeMTP genes including MeMTP1-5 and MeMTP12 was evaluated under zinc deficiency. MeMTP1 was up-regulated in roots and leaves under zinc deficiency whereas the expression of MeMTP2, MeMTP4 and MeMTP12 were tissue-specific. These findings will provide an important foundation of the MTPs in zinc homeostasis mechanism of cassava plants.

Keywords: zinc, zinc deficiency, cassava, metal tolerance proteins, gene expression

Introduction

Cassava (Manihot esculenta) is the second-most significant economic crops contributing to Thailand economy. Recently, it has been reported that production and harvesting area of cassava decreased continuously compared to planting area. Reduction of cassava yield results from viral diseases and inappropriate fertilizer management. Moreover, in Northeastern regions of the country, zinc deficiency also results in poor quality of cassava production. The main causes of zinc deficiency in the crops are an alkaline soil, calcareous soil and excess

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macronutrient application (Alloway, 2009; Howeler, 2002; Noulas et al., 2018; Takrattanasaran et al., 2013). Zinc is a micronutrient necessary for cell metabolisms such as cofactors in several enzymes, protein synthesis and precursors for auxin production (Broadley et al., 2012). Cassava plants appealing early symptoms of zinc deficiency may develop more fibrous roots rather than storage roots, which causes a reduction in yield. Normally, cassava plants accumulate zinc 50-100 µg/g in mature leaves but less than 20 µg/g in zinc-deficient leaves (Howeler, 2002). In cassava, zinc deficiency is characterized by chlorosis in interveinal regions of upper leaves. Growing of cassava plants in zinc-deficient soils for a long period will stop plant growth (Howeler, 2002). To understand the zinc homeostasis in cassava plants whether mobilization, uptake, translocation or storage, the study of zinc uptake mechanisms in molecular level is not negligible. The knowledge also provides the baseline data for precision agriculture and farming management to improve crop production and quality. Zinc in a form of Zn2+ in soils is taken up across the membrane of root cells before transported to the upper part of the plants (Palmgren et al., 2008; Ricachenevsky et al., 2015; Scott Aleksander Sinclair & Krämer, 2012). These mechanisms require transporter proteins to sustain zinc homeostasis throughout plant organs. Among major protein families responsible for zinc transport, the CDF (Cation Diffusion Facilitator) families, called MTP families in plants, function as a metal efflux transporter from cytoplasm and involve in sequestration of zinc into intracellular compartments such as vacuole and endoplasmic reticulum (ER) (Gupta et al., 2016). The MTPs transport divalent cations, mainly Zn2+ but also Mn2+, Fe2+, Cd2+, Co2+ and Ni2+ (Ricachenevsky et al., 2013). CDF members from several species, including bacteria, fungi, mammals and plants have been classified into three subgroups due to protein sequence analysis: Zn-CDF, Fe/Zn-CDF and Mn-CDF (Montanini et al., 2007). The members of MTP family are different upon plant species. For instant, rice, Arabidopsis, wheat, poplar and tobacco have 12, 12, 20, 22 and 26 MTP members, respectively (Gao et al., 2020; Kawachi et al., 2008; Liu et al., 2019; Vatansever et al., 2017). Although MTPs are a crucial metal transporter linked to zinc homeostasis in many plants, the knowledge of MTPs in cassava is still limited. Here, we identified cassava MTP gene family (MeMTPs) and studied the transcription levels of 6 MeMTP genes under zinc deficiency. The coding regions and protein sequences of these MTP orthologs were analyzed with bioinformatics and online-based platforms.

Materials and Methods

Plant Materials

Cassava (Manihot esculenta) cultivar Kasetsart 50 (KU50) cuttings was kindly provided from the Thai Tapioca Development Institute in Nakhon Ratchasima Province, Thailand.

Plant Growth and Sample Collection

Cassava stems were cut into small sections with 2- 3 nodes and nursed under hydroponics system for 3 weeks until leaves and roots were fully developed. The healthy plants were transferred to 3- liter pots containing Hoagland Solution ( Hoagland & Arnon, 1950) with

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2. 5 µM ZnSO4 (control) or without ZnSO4 (Zn deficient condition) for 3 weeks. The daily average temperature in a greenhouse ranged from 28˚C in the morning to 38˚C in the afternoon. Youngest fully expanded leaves and fibrous roots were collected from 3 biological replicates at 3 weeks then frozen immediately in liquid nitrogen and kept at -80˚C until use for RNA extraction.

Identification of MeMTPs

To identify cassava MTP proteins. MTP protein sequences from Arabidopsis and rice databases were used as queries to search for homologous sequences in Manihot esculenta Phytozome database by BLASTP. We predicted putative transmembrane regions using TMHMM and predicted subcellular localizations of MeMTP proteins using PSORT (Peabody et al. , 2016), CELLO (Yu et al., 2006) and Plant-mPLoc (Chou & Shen, 2010) servers.

Phylogenetic Analysis and Gene Structure

MeMTP protein sequences were aligned with MTP sequences from Arabidopsis (Arabidopsis thaliana) and rice ( Oryza sativa) using ClustalW. Phylogenetic tree was constructed by MEGAX ( Kuma et al. , 2018) via the neighbor- joining ( NJ) method with 1000 bootstrap replicates. MeMTP gene structures were determined using the Gene Structure Display Server (GSDS) program (Hu et al., 2015).

Prediction of Cis-acting Regulatory Elements

DNA sequences located 2. 0 kb upstream of MeMTP genes were acquired from Phytozome database for promoter analysis and the cis-acting regulatory elements on the promoter region were predicted using PlantCare (Lescot et al., 2002).

RNA Extraction and RT-PCR Analysis

Total RNA was extracted from leaves and roots using the Plant RNA Purification Reagent, then treated with TURBO DNase™ Treatment (Thermo Fisher Scientific) to remove genomic DNA. Synthesis of cDNA was performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Expression of 6 MeMTP genes was analyzed by RT-PCR using GoTaq® Green Master Mix (Promega) and normalized to Me18S and MeGAPDH genes. The transcript level of MeZIP1 was used as a positive control under zinc deficiency. The primers used for RT-PCR are listed in Table 1.

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Table 1. Primer characteristics

Gene name Primer sequences Nucleotide Tm Amplicon size size (bp) (˚C) (bp) gDNA cDNA MeMTP1 Forward 5'-CCGTTGGGAGAAGTAGGATATG-3' 22 60 120 120 Reverse 5'-GTGCTACTGCAATCAAGAGC-3' 20 MeMTP2 Forward 5'-TGCTGCCCATTTGCTTTC-3' 18 58 124 124 Reverse 5'-TGCACCTAGAATCTCAACCC-3' 20 MeMTP3 Forward 5'-GCCATTGCTCGACTGATCTA-3' 20 60 118 118 Reverse 5'-CATGCTGATGACCCAACAAG-3' 20 MeMTP4 Forward 5'-CAGAACCTCGGGAAACAAG-3' 19 60 209 123 Reverse 5'-GGGTCTATCCACCAGTAGAA-3' 20 MeMTP5 Forward 5'-TGGGAATTGGGCTCTTCA-3' 18 60 282 131 Reverse 5'-GTACACACGATCAGGCTTTC-3' 20 MeMTP12 Forward 5'-ACCCAGTCTCACCATTCA-3' 18 58 181 124 Reverse 5'-CAACTCCAACACTTCCCATC-3' 20 MeZIP1 Forward 5'-TCACTTACGCAGGATTTGG-3' 19 58 209 124 Reverse 5'-GACTCTGAGAAGCACCTAAAG-3' 21 Me18S Forward 5'-CGGAGAGGGAGCCTGAGAAA-3' 20 60 120 120 Reverse 5'-CAGACTCGAAGAGCCCGGTATTA-3' 23 MeGAPDH Forward 5'-CGACTGTCCATGCAACTAC-3' 19 60 212 111 Reverse 5'-CACCAGTGGAACTAGGAATG-3' 20

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Results and Discussion

Structural and functional annotation of cassava MTP gene family

Twelve putative MTP genes were identified in cassava genome on phytozome database as shown in Table 2. All their protein sequences were similar to the sequences in rice and Arabidopsis, which were used as queries. The MTP genes were classified into 3 groups (Figure 1). The exon-intron organization analysis of MeMTP genes showed number of exons ranging from 1-12. MeMTP1-4 contained no intron in their gene structures whereas MeMTP6 had highest number of introns and exons ( Table 2, Figure 1) . We found that MeMTP9 and MeMTP10 shared 90% homology according to the sequence alignment and contained the same number of exons. The notion that these genes was originated as a single gene before the divergent would be considered. The predicted proteins coded from MeMTP genes consisted of 351-876 amino acids. Most of them were likely to localize in plasma membrane and tonoplast except MeMTP4 and MeMTP8, which had high potential to localize in ER membrane. Moreover, our hypothesis that MeMTPs are transmembrane proteins was supported by the presence of transmembrane helices (TMHs) in their structures (Table 2). Six TMHs are common in plant MTPs (Arrivault et al., 2006; Fujiwara et al., 2015; Gustin et al., 2011; Kawachi et al., 2008; Liu et al., 2019). In cassava, most of the MeMTPs contained 4-6 TMH domains, thus verifying the character of the MTP family. However, MeMTP6 did not hold any TMHs. In addition, the MeMTP6 was likely to be a mitochondrial protein, which was unique among other MeMTPs. Further in vivo experiments would be performed to unveil the cellular localization of MeMTP6 and its roles in plant metal homeostasis. Interestingly, MeMTP12 was a largest protein with highest TMH domains, indicating the distinctive biological function. These characters have also been reported in MTP12 from other plants (Fujiwara et al., 2015; Gao et al., 2020; Liu et al., 2019; Vatansever et al., 2017). Functional annotation of the translated sequences from PANTHER, PFAM, KOG and KEGGORTH databases verified that cassava MTPs were cation efflux proteins/zinc transporter belonging to cation diffusion facilitator superfamily ( CDF) . Gene Ontology ( GO) also classified MeMTPs as transmembrane proteins functioning in delivery of cations within or across a cell. However, the possibility to be an iron transporter would be considered in MeMTP6, MeMTP8, MeMTP9, MeMTP10 and MeMTP11 according to KOG annotation (Table 3).

Figure 1. MeMTP gene structures. Blue boxes indicate untranslated 5՛ and 3՛ regions (UTR); green boxes indicate exons; black lines indicate introns.

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Table 2. MeMTP genes encoding MTP proteins along with their general details in cassava

Gene name Locus name No. of Exons Protein length No. of TMHs Subcellular localization

MeMTP1 Manes.05G016000.1 1 407 6 plasma membrane/tonoplast MeMTP2 Manes.01G239300.3 1 390 6 plasma membrane/tonoplast MeMTP3 Manes.02G000600.1 1 415 6 plasma membrane/tonoplast MeMTP4 Manes.15G105000.1 1 404 5 ER membrane/tonoplast MeMTP5 Manes.09G035800.1 10 404 6 plasma membrane/tonoplast MeMTP6 Manes.05G065800.1 12 499 0 plasma membrane/mitochondria/tonoplast MeMTP7 Manes.03G080500.1 9 351 4 plasma membrane/tonoplast MeMTP8 Manes.08G042500.1 7 407 5 ER membrane/tonoplast MeMTP9 Manes.S031300.1 6 413 4 plasma membrane/tonoplast MeMTP10 Manes.16G046900.1 6 402 5 plasma membrane/tonoplast MeMTP11 Manes.10G037600.1 6 394 4 plasma membrane/tonoplast MeMTP12 Manes.S020800.1 3 876 13 plasma membrane/tonoplast

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Analysis of cis-acting elements in the MeMTP promoters

Promoter regions are crucial for regulation of gene expression at the transcriptional level. To underly mechanisms in transcriptional regulation of MeMTP genes, cis- acting elements in MeMTP promoter regions were determined 2. 0 kb upstream of a transcription start site. The results showed that most of the elements in MeMTP promoters were phytohormone responsive. Among them, the ethylene responsive element (ERE) was the most abundant one existed in all MeMTP genes, especially MeMTP4 that contained up to 13 replicates. Therefore, the expression of MeMTP4 may be regulated by ethylene signaling. The ERE was also found in TaMTP1A and TaMTP4D promoters of common wheat (Vatansever et al. , 2017). ERE is a binding site of ethylene responsive factors (ERFs), which are transcription factors responding to ethylene signaling (Binder, 2020). Under metal stress, the production rates of ethylene are increased in plants (Keunen et al. , 2016). Ethylene may stimulate the downstream signaling cascades including ERFs to trigger the expression of ERE responsive genes, including MTPs, resulting in the production of MTP efflux transporters to balance the concentration of metals in a cell. TGA- element was identified in MeMTP2, MeMTP5- 7 and MeMTP10 promoters, indicating its role in auxin regulation. Zinc is a precursor in a tryptophan synthesis for auxin production (Begum et al. , 2016). Thus, auxin signaling may be promoted by zinc uptake via MTP transporters. Additional elements related to stress response, TC-rich repeats, were also found in MeMTP1, MeMTP2 and MeMTP3 promoters (Table 4). Therefore, MeMTP genes could be regulated by multiple stimuli.

Phylogenetic relationship of the MTP proteins

To further analyze the phylogenetic relationship of MeMTP proteins to their orthologs from Arabidopsis and rice, the tree was constructed using a total of 36 MTP proteins, comprising 12 from cassava, 12 from Arabidopsis and 12 from rice. As a result, the MeMTPs were divided into three major groups. Mn-CDF Group contained 6 MeMTP proteins (MeMTP4, 6, 8, 9, 10 and 11), while MeMTP7 was a single gene in Zn/Fe-CDF group. Zn-CDF Group contained 5 MeMTPs including MeMTP1, 2, 3, 5 and 12. Almost all of cassava MTP nomenclatures were categorized in the same manner of Arabidopsis MTPs, except MeMTP4 that shift to the same clade as OsMTP4 in rice. The result suggests that cassava MTP genes might be in the evolutionary history of the MTP gene family in Arabidopsis and rice (Figure 2). Although the member of cassava MTP family is comparable to those reported in Arabidopsis and rice ( Gustin et al. , 2011; Kawachi et al. , 2008; Montanini et al. , 2007) , different protein numbers are presented in various plants. The highest number has been reported in tobacco with 26 MTP proteins ( Liu et al. , 2019) . It is possible that species containing high number of MTPs may be the result of gene duplication during evolution and each gene may start to develop its distinct functions. In plants, metal transporters in the CDF family have been called the Metal Tolerance Proteins (MTPs) according to their functions in sequestration of metal (Me2+) into organelles such as vacuole as well as the extracellular space to reduce cellular damage from high concentration of metals (Ricachenevsky et al., 2013). The plant MTPs have been clustered into 3 groups; the Zn-CDFs, Fe/Zn-CDFs, and Mn-CDFs, based on phylogenetic analysis and metal selectability (Ricachenevsky et al., 2013).

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Table 3. Functional annotation of MeMTP proteins based on PANTHER, PFAM, KOG, GO and KEGGORTH databases

Gene name

Database Database ID Functional annotation

MeMTP1 MeMTP2 MeMTP3 MeMTP4 MeMTP5 MeMTP6 MeMTP7 MeMTP8 MeMTP9

MeMTP10 MeMTP11 MeMTP12 PANTHER PTHR11562 CATION EFFLUX PROTEIN/ ZINC TRANSPORTER           

PANTHER PTHR11562 METAL TOLERANCE PROTEIN      

PANTHER PTHR11562:SF18 MITOCHONDRIAL METAL TRANSPORTER 1-RELATED  PANTHER PTHR11562:SF13 ZINC TRANSPORTER 7 

PFAM PF01545 Cation efflux family             PFAM PF16916 Dimerization domain of Zinc Transporter     KOG KOG1482 Zn2+ transporter    KOG KOG1484 Putative Zn2+ transporter MSC2 (cation diffusion facilitator    superfamily)

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Gene name

Database Database ID Functional annotation

MeMTP1 MeMTP2 MeMTP3 MeMTP4 MeMTP5 MeMTP6 MeMTP7 MeMTP8 MeMTP9

MeMTP10 MeMTP11 MeMTP12

KOG KOG1485 Mitochondrial Fe2+ transporter MMT1 and related transporters      (cation diffusion facilitator superfamily) GO GO:0006812 The directed movement of cations, atoms or small molecules with a net positive charge, into, out of or within           a cell, or between cell, by means of some agent such as a transporter or pore. GO GO:0016021 The component of a membrane consisting of the gene products and protein complexes having at least          some part of their peptide sequence embedded in the hydrophobic region of the membrane. KEGGORTH K14689 Solute carrier family 30 (zinc   transporter), member 2 KEGGORTH K14692 Solute carrier family 30 (zinc   transporter), member 5/7 KEGGORTH K14696 Solute carrier family 30 (zinc  transporter), member 9

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Table 4. Predicted cis-elements in the promoter regions of MeMTP genes

Gene Stress Phytohormone responsive name responsive TC- rich ABRE ERE CGTCA- TGACG- TGA- GARE- TCA- repeats motif motif element motif element MeMTP1 1 3 9 MeMTP2 2 2 2 1 1 2 MeMTP3 1 2 3 MeMTP4 13 MeMTP5 2 3 2 2 1 1 MeMTP6 1 2 2 2 1 MeMTP7 1 2 2 1 1 MeMTP8 7 3 3 MeMTP9 1 2 2 2 1 MeMTP10 3 3 2 2 2 1 2 MeMTP11 5 8 1 1 MeMTP12 1 8

Figure 2. Phylogenetic analysis of MTP proteins in cassava, rice and Arabidopsis. The phylogenetic tree was constructed using neighbor-joining (NJ) method with 1000 bootstrap replicates.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

In our case, the phylogenetic relationship also showed 3 main groups of MTPs similar to previous reports especially the Arabidopsis MTPs, except MeMTP4 that did not lie in the Zn-CDF groups (Figure 2) even the MeMTP4 was annotated as a zinc transporter (Table 3). MeMTP4 showed sequence similarity to rice MTP. In monocot such as rice and wheat, MTP4 ( also called MTP8. 1 in rice) has been characterized into Mn- CDFs ( Chen et al. , 2013; Vatansever et al., 2017). However, a metal sensitivity assay in Saccharomyces cerevisiae has shown that a poplar MTP4 could not transport either zinc, iron or manganese (Gao et al. , 2020). Therefore, roles of MTP4 might be different among plant species.

Morphological changes of KU50 cassava shoot under zinc deficiency

During the early growth stages, cassava plants were observed for 3 weeks for zinc deficiency symptoms. Initially, the plants exhibited interveinal chlorosis on young leaves then the leaves began developing small white, or light-yellow chlorotic spots between veins. The longer zinc deficient period, the smaller and more chlorotic appeared on the new emerging leaves. Moreover, foliar lobes became narrow, and curl upward compared to the control. These results provided evidence that zinc- deficient conditions were crucial for cassava development especially in the early growth stages (Figure 3). It has been reported that zinc deficiency is the most common symptom in cassava plants grown in high pH and calcareous soils due to the low availability of zinc to plant roots. Under severe conditions, plants may reduce growth and die at young age (Howeler, 1995; Watananonta et al., 2004).

Figure 3. Morphological changes of 3-week-old KU50 cassava shoot under zinc deficiency

Expression analysis of MeMTP genes in KU50 cassava leaf and root tissues under zinc deficiency

Gene identification using bioinformatic tools facilitates us to predict the existence of MTP gene family in cassava. However, verification of gene expression in vivo will provide further meaningful information to support the computational annotations. Here, we studied the expression of 6 candidate MeMTP genes including MeMTP1- 5 and MeMTP12, in cassava leaves and roots after transferring to zinc deficiency for 3 weeks (Figure 4). RT-PCR data showed that the expression level of MeMTPs was expressed differently. Under zinc deficient conditions, the transcript level of MeMTP1 was slightly increased in both leaves and roots, while MeMTP5 was up-regulated in roots, suggesting that these genes play roles in cassava plants when zinc is scarce.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Previous studies have shown that overexpression of zinc transporters AtZIP1 (zinc- regulated transporter- like protein) and AtMTP1 in cassava plants triggers high zinc concentration in storage roots but reduces the tuber size. The overall yield also decreases due to zinc deficiency symptom in the aerial parts of the plants (Gaitán-Solís et al., 2015). These data imply that MTP1 may correlate with zinc transportation in plants. Recent study has shown that Arabidopsis AtMTP2 transcript level in roots elevates under zinc deficiency condition. Analysis of mtp2 mutant demonstrates the role of MTP2 in zinc translocation from roots to shoots when the zinc status in shoots is low (Scott A Sinclair et al. , 2018). From our results, cassava MeMTP2 expressed specifically in roots but did not response to zinc concentration. The MTP2 orthologs in different plants may have diverse function. The expression levels of MeMTP4 and MeMTP12 were higher in leaves rather than roots under both + zinc and - zinc conditions. These results suggest that the expression of MeMTP2, MeMTP4 and MeMTP12 are tissue- specific. However, MeMTP3 was rarely detectable in all conditions so that MeMTP3 may not be a major gene for zinc transport compared to others in the MTP family. The plant MTP protein family are well known for metal tolerance via sequestration of zinc into intracellular compartments. Some of MTP transcripts are up-regulated under zinc toxicity (Gupta et al. , 2016). It is possible that the cassava MTP family may involve in both zinc deficiency and toxicity conditions, depending on the function of each individual protein. Study of MeMTP gene expression under zinc toxicity would provide further information of MTPs in cellular and molecular levels. Moreover, additional transcriptomic experiments in cassava under zinc deficiency would be developed for genome-wide identification of other zinc responsive genes.

Figure 4. Expression of MeMTP genes in cassava leaves and roots under zinc deficiency

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Conclusion

The MeMTP proteins have an influence on zinc transport in several plants. In this study, twelve MeMTP genes were discovered in cassava genome. MeMTP1 was up-regulated in roots and leaves under zinc- deficient condition whereas the expression of MeMTP2, MeMTP4 and MeMTP12 were tissue-specific. These genes may be responsible for zinc balance in cassava plants.

Acknowledgements

The authors would like to thank the Thai Tapioca Development Institute for supporting cassava cutting, National Research Council of Thailand (1232329) and Faculty of Science, Silpakorn University (SRIF-JRG-2563-12) for funding support.

References

Alloway, B. ( 2009) . Soil factors associated with zinc deficiency in crops and humans. Environmental Geochemistry and Health, 31: 537–548. Arrivault, S. , Senger, T. , & Krämer, U. (2006). The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply. The Plant Journal, 46(5), 861-879. Begum, M. C., Islam, M., Sarkar, M. R., Azad, M. A. S., Huda, A. N., & Kabir, A. H. (2016). Auxin signaling is closely associated with Zn-efficiency in rice (Oryza sativa L.). Journal of Plant Interactions, 11(1), 124-129. Binder, B. M. (2020). Ethylene signaling in plants. Journal of Biological Chemistry, 295(22), 7710-7725. Broadley, M., Brown, P., Cakmak, I., Rengel, Z., & Zhao, F. (2012). Function of nutrients: micronutrients. In Marschner's mineral nutrition of higher plants ( pp. 191- 248) : Elsevier. Chen, Z., Fujii, Y., Yamaji, N., Masuda, S., Takemoto, Y., Kamiya, T., Yusuyin, Y., Iwasaki, K. , Kato, S. , Maeshima, M. , Ma, J. F. , & Ueno, D. (2013). Mn tolerance in rice is mediated by MTP8. 1, a member of the cation diffusion facilitator family. Journal of Experimental Botany, 64(14), 4375-4387. Chou, K., & Shen, H. (2010). Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLOS ONE, 2010, 5: e11335. Fujiwara, T. , Kawachi, M. , Sato, Y. , Mori, H. , Kutsuna, N. , Hasezawa, S. , & Maeshima, M. (2015). A high molecular mass zinc transporter MTP12 forms a functional heteromeric complex with MTP5 in the Golgi in Arabidopsis thaliana. The FEBS Journal, 282(10), 1965-1979. Gaitán-Solís, E. , Taylor, N. J. , Siritunga, D. , Stevens, W. , & Schachtman, D. P. (2015). Overexpression of the transporters AtZIP1 and AtMTP1 in cassava changes zinc accumulation and partitioning. Frontiers in Plant Science, 6, 1-11. Gao, Y., Yang, F., Liu, J., Xie, W., Zhang, L., Chen, Z., Peng, Z., Ou, Y., & Yao, Y. (2020). Genome-wide identification of metal tolerance protein genes in Populus trichocarpa and their roles in response to various heavy metal stresses. International Journal of Molecular Sciences, 21(5), 1-25. Gupta, N. , Ram, H. , & Kumar, B. (2016). Mechanism of Zinc absorption in plants: uptake, transport, translocation and accumulation. Reviews in Environmental Science and Bio/Technology, 15(1), 89-109.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Gustin, J. L., Zanis, M. J., & Salt, D. E. (2011). Structure and evolution of the plant cation diffusion facilitator family of ion transporters. BMC Evolutionary Biology, 11(1), 1-13. Hoagland, D. R., & Arnon, D. I. (1950). The water-culture method for growing plants without soil. Circular. California agricultural experiment station, 347(2nd edit). Howeler, R. H. (1995). Mineral nutrition of cassava. Paper presented at the Mineral Nutrient Disorders of Root Crops in the Pacific. Proc. Workshop held in Nuku’alofa, Kingdom of Tonga. Howeler, R. H. ( 2002) . Cassava mineral nutrition and fertilization. Cassava: biology, production and utilization, 115-147. Hu, B., Jin, J., Guo, A., Zhang, H., Luo, J & Gao, G. (2015). GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics, 31(8):1296-1297. Kawachi, M., Kobae, Y., Mimura, T., & Maeshima, M. (2008). Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn2+/H+ antiporter of Arabidopsis thaliana, stimulates the transport activity. Journal of Biological Chemistry, 283(13), 8374-8383. Keunen, E. , Schellingen, K. , Vangronsveld, J. , & Cuypers, A. (2016). Ethylene and metal stress: small molecule, big impact. Frontiers in Plant Science, 7, 1-18. Kumar, S., Stecher, G. , Li, M. , Knyaz, C. , & Tamura, K. ( 2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35:1547-1549. Lescot, M. , Déhais, P. , Thijs, G. , Marchal, K. , Moreau, Y. , Van de Peer, Y. , Rouzé, P. , & Rombauts, S. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic acids Research, 30(1), 325–327. Liu, J., Gao, Y., Tang, Y., Wang, D., Chen, X., Yao, Y., & Guo, Y. (2019). Genome-wide identification, comprehensive gene feature, evolution, and expression analysis of plant metal tolerance proteins in tobacco under heavy metal toxicity. Frontiers in Genetics, 10, 1-20. Montanini, B., Blaudez, D., Jeandroz, S., Sanders, D., & Chalot, M. (2007). Phylogenetic and functional analysis of the Cation Diffusion Facilitator ( CDF) family: improved signature and prediction of substrate specificity. BMC genomics, 8(1), 1-16. Noulas, C. , Tziouvalekas, M. , & Karyotis, T. (2018). Zinc in soils, water and food crops. Journal of Trace Elements in Medicine and Biology, 49, 252-260. Palmgren, M. G. , Clemens, S. , Williams, L. E. , Krämer, U., Borg, S. , Schjørring, J. K. , & Sanders, D. (2008). Zinc biofortification of cereals: problems and solutions. Trends in Plant Science, 13(9), 464-473. Peabody, M. A., Laird, M. R., Vlasschaert, C., Lo, R. & Brinkman, F. S. (2016). PSORTdb: expanding the bacteria and archaea protein subcellular localization database to better reflect diversity in cell envelope structures. Nucleic Acids Research, 44(D1):D663-8. Ricachenevsky, F. K., Menguer, P. K., Sperotto, R. A., & Fett, J. P. (2015). Got to hide your Zn away: molecular control of Zn accumulation and biotechnological applications. Plant Science, 236, 1-17. Ricachenevsky, F. K., Menguer, P. K., Sperotto, R. A., Williams, L. E., & Fett, J. P. (2013). Roles of plant metal tolerance proteins (MTP) in metal storage and potential use in biofortification strategies. Frontiers in Plant Science, 4, 1-16. Sinclair, S. A., & Krämer, U. (2012). The zinc homeostasis network of land plants. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1823(9), 1553-1567. Sinclair, S. A., Senger, T., Talke, I. N., Cobbett, C. S., Haydon, M. J., & Krämer, U. (2018). Systemic upregulation of MTP2- and HMA2- mediated Zn partitioning to the shoot supplements local Zn deficiency responses. The Plant Cell, 30(10), 2463-2479.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Takrattanasaran, N., Chanchareonsook, J., Johnson, P. G., Thongpae, S., & Sarobol, E. (2013). Amelioration of zinc deficiency of corn in calcareous soils of Thailand: zinc sources and application methods. Journal of Plant Nutrition, 36(8), 1275-1286. Vatansever, R., Filiz, E., & Eroglu, S. (2017). Genome-wide exploration of metal tolerance protein ( MTP) genes in common wheat ( Triticum aestivum) : insights into metal homeostasis and biofortification. Biometals, 30(2), 217-235. Watananonta, W. , Tungsakul, S. , Phetprapi, P. , & Howeler, R. H. (2004). The response of micronutrients on root yield of 2 cassava varieties. Paper presented at the Proceedings of the 42nd Kasetsart University Annual Conference, Kasetsart, Thailand, 3-6 February 2004. Yu, C. S., Chen, Y. C., Lu, C. H., & Hwang, J. K. (2006). Prediction of protein subcellular localization. Proteins: Structure, Function and Bioinformatics, 64:643-651.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.5

Full Length Article

Comparison on physical alteration of the peeled durian stored in “OZONE BOX” odor lock and commercial packaging

Siriwan Tungsangprateep 1*, Waree Jaruwattanayon 1, Kasinee Saowakon 2, Papitchaya Kongchinda 2 and Rujira Deewatthanawong 2

1 Thailand Institute of Scientific and Technological Research 196 Phahonyothin Rd., Ladyao, Chatuchak, Bangkok 10900 2 Thailand Institute of Scientific and Technological Research 35 Mu 3, Khlong Luang, Pathum Thani 12120, Thailand

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

This study investigated the physical changes including oxygen (O2) and carbon dioxide (CO2) gas content, weight loss, color change and firmness of peeled durian stored at 4 ° C for 15 days in “Ozone Box” odor locking package compared to commercial package. Testing plan was a Complete Randomized Design ( CRD) experiment. Statistical analysis was conducted by comparison of the mean t-test at 95% confidence level. It showed that the oxygen gas in Ozone Box decreased significantly when compared to commercial package. On the other hand, carbon dioxide increased dramatically in the first 5 days of storage. It has been indicated that Ozone Box has complete barrier properties against ingress of oxygen and carbon dioxide through the development of the air tight seal structure. The durian in Ozone Box has less weight loss than commercial package in 5-7 days of storage. The Ozone Box absolutely retained moisture inside the air tight seal structure comparing to the commercial package which had a seal opening. There was no significant difference of the color and firmness of the durian in both packages throughout the storage period.

Keywords: odor lock packaging, durian, OZONE BOX, stored or peeled

Introduction

Durian (Durio zibethinus Murr.) is considered an economic fruit with high value and high potential for export. Thailand is the number one producer and exporter of durian and durian- based products in the world. For domestic distribution, there are both whole durian and peeled durian available. Because the durian is a fruit that has a peel or parts that are not eaten up to 70% on average, the production of fresh peeled durian, which is ready to eat, is a good choice to expand the fresh durian market. It also saves space and transportation costs compared to selling whole durian, however, durian is a family of Bombacaceae and responds well to ethylene gas (Tropical Climacteric Fruit).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

When the durian is ripe, it produces a sulfur compound with a unique strong odor and can cause allergic reactions (Belgis et al., 2017 and Cannon and Ho, 2018). Therefore, the “Ozone Box” durian odor locking package, which can contain the durian odor 100%, was developed by Thai Packaging Center, the Thailand Institute of Scientific and Technological Research (TISTR) with Safer Pack Thailand Co., Ltd. It is well received in commercial use as well as being strong enough for real application. However, information on the changes in properties of peeled durian stored in the package was still needed for determining the release date and helping to ensure the use and sale. The objective of this research study was to study changes in physical properties during storage including weight loss color change and firmness in durian odor locking package, "Ozone Box" compared to the commercial packaging.

Materials and Methods

Peeled durian meat preparation

The durian was selected from Monthong variety with similar maturity. The durian skin was washed and sterilized before peeling at 25 ° C, then lowered to -20 ° C for 20 minutes, then the peeled durian was packed in a package. The study plan consisted of 2 treatments, 3 repetitions per treatment, 3 packages per repetition. Box including The first treatment: commercial boxes were not completely sealed. Therefore, no complete gas retention condition occurs, resulting in a slight durian odor after closing the package. Place about 500 grams of durian in the package as shown in Figure 1.

Figure 1. Dimensions of commercial box package and peeled durian in commercial box package

Figure 2. Dimensions of commercial box package and peeled durian in commercial box package

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

The second treatment, Ozone box package, which has been developed to have a tight sealing structure like an air tight seal to keep the durian odor inside so that it does not escape into the environment outside. Therefore, there was no odor after closing the package. Place about 500 grams of durian in the package as shown in Figure 2. Store at 4 ° C for 15 days. Sampling for recording before storage and after storage on day 5, 7, 9, 12 and 15.

Data recording

The amounts of gases in the package were evaluated. These were reported in percentage (%) by measuring the amount of oxygen (O2) and carbon dioxide (CO2) in the package with a gas meter. The Weight loss was recorded. The recorded weights of the durian before storage and after storage were calculated according to the following formula:

Percentage of weight loss = (Weight before storage - weight after storage)  100 Weight before storage

The color change was determined. The durian color was measured at 3 points (head, middle, tail) with a color meter and reported in the CIE system L * a * b * where L * is used to set the brightness (Lightness). L = 0, the resulting color is darkened to black. L = 100, the resulting color will light up in white. a * determines red or green color. a is +, a reddish-colored object a is -, a greenish-colored object b * determines the yellow color or blue b is +, yellowish object b is -, a bluish color object

The hardness was measured. Peeled durian was measured at 3 points (head, middle, tail) with a food texture analyzer and reported the values in Newton (N).

Statistical analysis of data

Testing plan was a Complete Randomized Design (CRD) experiment. Statistical analysis was conducted by comparison of the mean t-test at 95% confidence level.

Results and Discussion

The amount of gas inside the package

Ozone packaging offers complete barrier properties against ingress of oxygen and carbon dioxide through the development of an air tight seal, as evidenced by the oxygen content of the package (Figure 3 and Table 1 and 2) which fell sharply to 0.25% for the first five days and to the highest of only 1.06% later throughout the storage period. This is because peeled durian uses oxygen that exists only in the head space for breathing and oxygen from the outside cannot penetrate into the package. This corresponds to a rapid increase of carbon dioxide to 31.73% in the first five days caused by respiration of the durian and accumulation of carbon dioxide gas inside the package resulting in the high content in the range of 31.73% - 49.62% throughout the storage period. On the other hand, the peeled durian in the commercial package which still 46

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

allows oxygen and carbon dioxide through the package has oxygen content in the package similar to that of the outside air. It was in the range of 18.37-18.85 percent throughout the storage period. Comparably, the amount of carbon dioxide is similar to that of the outside air. The range is 0.70-1.16% throughout the storage period.

Commercial box 25 Commercial box Ozone box 60 Ozone box 20 50

(%)

(%) 2 2 30

O 10

CO 20 5 10

0 0 0 5 10 15 20 0 5 10 15 20 Storage time (days) Storage time (days)

Figure 3. The amount of oxygen (A) and carbon dioxide (B) in the commercial package and Ozone package containing peeled durian.

Table 1. Oxygen gas content (percentage) in commercial package and Ozone package containing peeled durian.

Storage time (days) Treatments 0 5 7 9 12 15 Commercial 21 18.37±0.33 18.68±0.59 18.59±0.99 18.79±0.01 18.85±0.02 box Ozone box 21 0.25±0.17 1.06±0.51 0.55±0.13 0.71±0.16 0.23±0.09 F-test - * * * * *

Note: * means that there was a statistically significant difference at a 95 percent confidence level. ns means that there was not a statistically significant difference at a 95 percent confidence level.

Table 2. Carbon dioxide gas content (percentage) in commercial package and Ozone package containing peeled durian.

Storage time (days) Treatments 0 5 7 9 12 15 Commercial 0.03 1.16±0.13 1.53±0.10 1.44±0.28 0.99±0.11 0.70±0.06 box Ozone box 0.03 31.73±1.91 35.60±0.73 36.70±1.91 43.72±2.48 49.62±2.54 F-test - * * * * *

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Weight loss

Weight loss of peeled durian in ozone package and the commercial package were shown in Figure 4 and Table 3. Both have weight loss increased over the storage period. During days 5- 7 of storage, it was found that peeled durian in ozone package had significantly less weight loss than commercial package. This was because ozone package retained all of the moisture inside the air tight seal structure, while the commercial package had a seal opening, so moisture does not accumulate. However, after the seventh day of storage, the weight loss of peeled durian in both packages was not significantly different. This is due to the very high respiration rate of peeled durian in Ozone package, resulting in weight loss that is no different from the peeled durian in commercial package. This was consistent with the results of Kwanhong (2017) and Boonthanakorn (2020).

Commercial box Ozone box 1 0.8 0.6 0.4

Weight loss (%) loss Weight 0.2 0 0 5 7 9 12 15 Storage time (days)

Figure 4. Weight loss of peeled durian in commercial package and Ozone package

Table 3. Weight loss (percentage) of peeled durian in commercial package and Ozone package

Storage time (days) Treatments 0 5 7 9 12 15 Commercial box 0 0.33±0.02 0.44±0.04 044±0.01 0.61±0.05 086±0.09 Ozone box 0 0.16±0.06 021±0.01 0.35±0.13 0.55±0.07 0.62±0.06 F-test - * * ns ns ns

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Firmness

The firmness of the peeled durian in the commercial package and Ozone package prior to storage was 7.86 N. After that, the firmness of the peeled durian in both packages decreased similarly and no significant difference was observed over the storage period. At the end of the storage, it was found that the firmness of the peeled durian in commercial package and Ozone package were 5.56 N and 5.08 N, respectively (Figure 5 and Table 4). Obviously, there was only a slight decrease in the firmness due to the low metabolism of peeled durian, especially compared to durian skin (Booncherm and Siriphanich, 1991 and Wongs-Aree, C. and Noichinda, S. 2014).

Commercial box Ozone box 10 8 6

4 Hardness (N) Hardness 2 0 0 5 7 9 12 15 Storage time (days)

Figure 5. Firmness of peeled durian in commercial package and Ozone package

Table 4. Firmness (Newton) of peeled durian in commercial package and Ozone packaging

Storage time (days) Treatments 0 5 7 9 12 15 Commercial box 7.86±0.92 5.74±0.40 5.60±0.49 6.15±0.30 6.02±0.23 5.56±1.62 Ozone box 7.86±0.92 7.40±1.65 3.95±0.73 6.48±1.16 6.16±1.02 5.08±1.01 F-test ns ns ns ns ns ns

Color change

The changes in L * (brightness) of the peeled durian in commercial package and the Ozone package is relatively constant over the storage period and there was no significant difference in both packages with L* between 87. 39-87. 90 in commercial package and 87. 39-88. 52 in Ozone package, respectively (Figure 6 (A), and Table 5).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Commercial box Ozone box A 100

80 60

40 value L* 20 0 0 5 7 9 12 15

0 0 5 7 9 12 15 -1

-2

value a* -3

-4

b* value b* 10

0 0 5 7 9 12 15

Storage time (days)

Figure 6. Color change: L * (A), a * (B) and b * (C) values of peeled durian in commercial package and Ozone package

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Table 5. Color change, L * value of peeled durian in commercial package and Ozone package

Storage time (days) Treatments 0 5 7 9 12 15 Commercial 87.39±0.18 87.43±0.22 87.83±0.13 87.80±0.33 87.90±0.10 87.83±0.28 box Ozone box 87.39±0.18 88.38±0.22 87.82±0.25 87.86±0.20 88.27±0.30 88.52±0.21 F-test ns * ns ns ns ns

Note: *means that there was a statistically significant difference at a 95 percent confidence level. ns means that there was not a statistically significant difference at a 95 percent confidence level.

Table 6. Color change, a * value of peeled durian in commercial package and Ozone package

Note: *means that there was a statistically significant difference at a 95 percent confidence level. ns means that there was not a statistically significant difference at a 95 percent confidence level.

Table 7. Color change, b * value of peeled durian in commercial package and Ozone package

Storage time (days) Treatments 0 5 7 9 12 15 Commercial 36.81±0.58 36.30±1.06 37.91±0.92 37.10±0.65 37.66±0.47 37.97±0.80 box Ozone box 36.81±0.57 35.77±1.43 37.95±0.65 37.92±1.19 35.98±2.34 36.24±1.44 F-test ns ns ns ns ns ns

Note: *means that there was a statistically significant difference at a 95 percent confidence level. ns means that there was not a statistically significant difference at a 95 percent confidence level.

The change in a * of the peeled durian was negative (-a *) because of the yellowish color of the fresh durian. The a* of the peeled durian in both packages was relatively constant and not significantly different over the storage period with a* between (-) 2.66 - (-) 3.09 in commercial package and (-) 2.73 - (-) 3.21 in Ozone package, respectively (Figure 6 (B), Table 6). The change in b* of the peeled durian in both packages, like the a* , was somewhat constant and no statistical difference was observed at the 95% confidence level over the retention period. The b* values were between 36.30-37.97 for commercial package and 35.77- 37.95 for Ozone package, respectively (Figure 6 (C) and Table 7).

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

In summary, the color change in peeled durian in commercial packaging and ozone packaging was virtually unchanged over the storage period, and there was no significant difference between the peeled durian in either package. This is due to the same main cause with firmness, which was a low metabolism in the peeled durian, especially when compared to the durian skin (Booncherm and Siriphanich, 1991 and Wongs-Aree, C. and Noichinda, S., 2014)

Conclusion

Ozone packaging has completely barrier properties for oxygen and carbondioxide gases. With the development of a tight structure (Air tight seal), it can be used to preserve peeled durian without sinificantly physical differences i.e weight loss, firmness and color with 95% confidence level.

References

Belgis, M. , Wijaya, C. H. , Apriyantono, A. , Kusbiantoro, B. , & Yuliana, N. D. (2017). Volatiles and aroma characterization o fseveralla i(Duriokutejensis) and durian (Durio zibethinus) cultivars grown in Indonesia. Scientia Horticulturae, 220, 291–298. Booncherm, P. and Siriphanich, T. (1991). Postharvest Physiology of Durian Pulp and Husk. Kasetsart J. (Nat.Sci.Suppl.) Vol.25: 119-125. Cannon, R. J., & Ho, C.-T. (2018). Volatile sulfur compounds in tropical fruits. Journal of Food and Drug Analysis, 26(2), 445–468. Wongs-Aree, C. and Noichinda, S. (2014). Postharvest Physiology and Quality Maintenance of Tropical Fruits in Postharvest Handling: A System Approach. Third Edition. Edited by Wojciech J. Florkowski, Robert L. Shewfelt, and Stanley E. Prussia. Academic Press. 275-312.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

DOI: 10.14457/MSU.res.2021.6

Full Length Article

The pretreatment condition for chromosome count and karyotype analysis of Dimocarpus longan from Thailand

Panurat Pipatchananan 1,2, Pathrapol Lithanatudom 2,3, Isara Patawang 2 and Suparat Kunkeaw Lithanatudom 4*

1Graduate Master’s Degree Program in Biology, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand 2Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand 3Research Centre in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand. 4Program in Genetics, Faculty of Science, Maejo University, Chiang Mai, 50290, Thailand

*Corresponding author’s email: [email protected]

Received: 21 July 2021 Revised: 21 August 2021 Accepted: 23 August 2021

Abstract

Chromosome number and karyotype analysis reveal an important information for plant evolutionary study and plant breeding program. However, very few studies were conducted on cytogenetics of Dimocarpus longan ( Longan) , an economic important subtropical fruit in Thailand. This research aims to develop the practical procedure for longan chromosome preparation to investigate the chromosome number and karyotype analysis in 8 longan cultivars from Thailand. The chromosome preparation was optimized using longan shoot tip and root tip cells. Two pretreatment chemicals which are p-dichlorobenzene and 8-hydroxyquinoline were selected and treated at 4°C in different time points consisted of 1, 3, 6 and 24 hours. All treated samples were then hydrolysed in 1N HCl at 60°C for 7 minutes and stained with carbol fuchsin at room temperature for 15 minutes. The result showed that root sample pretreated with p- dichlorobenzene at 4°C for 1 hour was the best condition for longan chromosome analysis. This condition was further used for chromosome investigation in 8 Thai’s longan cultivars which are Baiyoke, Plueakkhao, Phetsakorn, Krob- Ka- Ti, Haewkrae, E- daw, BiewKhiew Chiangmai and Pingpong. The chromosome number of all studied cultivars was determined as 2n = 30. The variation of the total chromosome length from 0. 499 to 1. 293 µm while the relative length (RL) and size of chromosomes group L, M and S were observed among 8 longan cultivars. From this study, the pretreatment method can be used to investigate chromosomes of all longan cultivars. The data obtained from this study will be important information for plant breeders to develop longan varieties in Thailand. However, further chromosome investigation using approaches such as Fluorescence in situ hybridization (FISH) would be more informative for study evolution of longan in the future.

Keywords: chromosome preparation, chromosome number, karyotype, Dimocarpus longan

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Introduction

Chromosome count and karyotyping provide an important information for evolutionary study in plants. Chromosome number analysis attracts many cytotaxonomists for investigation because it is the quickest, cheapest, and easiest way to obtain the substantial data about the genome of a given species (Guerra, 2008). Squashing method is one of the most common method for the preparation of plant chromosome. The method includes pretreatment, fixation, hydrolysis and staining. The first step for chromosome analysis of any plant sample is to obtain meristematic tissue. Currently, young root tip meristem is the most used tissue for analysis because of its high rate of cell proliferation (de Paula & Pinto-Maglio, 2015). Pretreatment is another important step in the process of preparations of plant tissue to investigate the chromosome number and to establish the karyotype of a species (Rachma & Salamah, 2018). Several pretreatment agents can be used for plant chromosomes study. The most commonly used agents are colchicine, 8- hydroxyquinoline, ⍺ - bromonaphthalene, p- dichlorobenzene (PDB) and cold water, all of which inhibits the mitotic spindle (Ekong et al., 2014). Lavania (1988) used either p-dichlorobenzene, 8-hydroxyquinoline, or their mixtures with a variation of 3-5 hours of immersion at 12-14°C from the root tips of 11 species and 7 chemotypes in Cymbopogon. The results showed that 3-7 well scattered and properly contracted metaphase plates were observed in different species by their agents. In addition, Rachma et al. (2018) used cold water, p- dichlorobenzene ( P) , 8- hydroxyquinoline ( O) and PO ( their mixture) for pretreatment of the shoot tips. Each pretreatment was carried out for 3, 6, 12 and 14 hours and the results showed that different pretreatments have their own optimum condition for accumulate cells in late prophase and metaphase. Dimocarpus longan is an important subtropical evergreen fruit tree that is grown commercially in many countries. It originated from South China or Southeast Asia and is commonly called longan (dragon eye) in Asia (Lin et al., 2017). Longan is a major source of national income for Thailand since it is the biggest exporter of longan worldwide. Major longan planted area is in the upper northern provinces of Thailand, include Lamphun, Chiang Mai, Chiang Rai, Nan, Phra Yao, Lampang, Phrae and Chanthaburi (Choo, 2003). Longan is one of the “Product Champion” of Thailand regarded by Ministry of Agriculture and Cooperative and Ministry of Commerce (Jealviriyapan et al., 2000; Ramingwong et al., 2005). However, there are more than 30 longan cultivars found in Thailand and some cultivars have similar morphological characteristics. Perhaps theirs are the same cultivar but different in name because of planted location. It is difficult to distinguish the cultivar through only morphological data. Therefore, chromosome number and karyotype analysis are important genetic information for identifying cultivars more clearly. In a previous study, chromosome investigation and karyotype analysis of longan were carried out in China. The karyotype formulas of longan are 2n = 30 = 16m(2sat) + 8sm + 6sat and satellite chromosome of longan was located in No. 12 chromosome (Liuxin, 1994). In Thailand, Ramingwong et al. 2005 collected the root tips at 1 hour intervals from 7:00 a.m. – 12: 00 p. m. to determine the appropriate time for investigating longan chromosome number. The results showed that the suitable time for chromosome counting was between 9: 00 a. m. – 10: 00 a. m. However, the duration of immersion and details of cultivar analyzed were not mentioned in their study. This study attempted to establish the appropriate time and tissues processing for investigation of longan chromosome number and karyotypes of 8 longan cultivars in Thailand. The data obtained from this research provided a well reproducible method for longan’s karyotype analysis which will be useful in breeding programs, evolution, systematic and conservation of longan in the future.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

The limitation of pretreatment condition was pretreatment solution and immersion time. Accumulating evidence has suggested that different tissues may require different pretreatments and none seems to be of general applicability (Battaglia, 1957). Pretreatment solutions used in this research are p- dichlorobenzene and 8- hydroxyquinoline. Root tips and shoot tips are immerse in a pretreatment solution for 1, 3, 6 and 24 hours. The immersion period influences the size of the chromosome. The longer the immersion in the pretreatment solution the shorter the size of the chromosome (Rachma & Salamah, 2018). Therefore, the suitable condition is important to properly observe the number of chromosomes.

Materials and Methods

Plant materials

Root and shoot tips samples of 8 longan cultivars were collected from mature trees (10-15 years) planted at Maejo University, Chiang Mai, Thailand. The air layering branches from individual cultivars were used for root tips sample collection. Actively growing root and shoot tips were cut into 0.2 - 0.5 cm long and used for chromosome preparation.

Chromosome preparation

For chromosome investigation, a Feulgen squash method of Dyer (1979) was used with a slight modification. Two pretreatment chemicals which are p- dichlorobenzene and 8- hydroxyquinoline were selected to compare the efficiency of metaphases accumulation. The effect of pretreatment was observed in different time points consisted of 1, 3, 6 and 24 hours by keeping the tissue sample at 4°C. Each pretreatment group was fixed in newly prepared Carnoy’s fixative (3: 1 absolute ethanol: glacial acetic acid) for at least 24 hours and then stored in 70% ethanol at 4°C until used. To prepare a microscope slide sample, the tissues were rinsed in distilled water to remove the fixative solution and treated with 1N HCl for 7 minutes at 60°C. The tissues were removed from 1N HCl by washing in distilled water then transferred to carbol fuchsin solution for 15 minutes. The tissues were squashed by dissecting needle on a slide glass, then covered with a cover glass and observed under a microscope (Olympus CX23). The best pretreatment condition was selected for chromosome preparation for all 8 longan cultivars by observing 10 well-spread metaphases and used for karyotype analysis.

Karyotype analysis

Chromosome number was counted and the parameters of the chromosomes including total chromosome length (LT), the relative length (RL) and standard deviation of RL were analyzed from 10 metaphase cells of each individual longan cultivar. The parameters of the chromosomes derived from measurements of the metaphase chromosomes in photomicrographs according to Chaiyasut’s protocol (1989). The idiograms were generated by total chromosome length ( LT) . The size of chromosomes of all 8 longan cultivars were classified into 3 groups consisting of Group L ( Chromosome with large size) , Group M (Chromosome with medium size) and Group S (Chromosome with small size).

Results and Discussion

A simple, rapid, and reliable chromosome staining procedure for determining chromosome number is needed for plant-breeding program (Owen & Miller, 1993). A main purpose of breeding program is to transfer the genetic variability from related species into crops.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

So far, ‘E-Daw’ is the only commercially grown longan variety, about 98 percent of the total longan production (Jaroenkit et al., 2014). For this reason, bringing concern if there are some effects on ‘E-Daw’ may impact longan production in the future. Plant breeders have used interspecific genetic crosses and alien introgression lines to overcome this problem such as transfer resistance genes against pathogens (Prieto, 2020). Thus, chromosome number and karyotype of longan are one of the genetic information for plant breeder to select and combine the greatest parental plants to obtain the next generation with the best characteristics. However, investigating chromosome number of longans is hindered due to their small chromosome size. Furthermore, it is difficult to prepare the metaphase cell of longan where well- spread chromosomes are all visible in a single focal plane. At present, there is no universal agent for pretreatment and perhaps individual organisms exhibit a differential sensitivity ( Battaglia, 1957). But as the prefixing agent, p-dichlorobenzene has been commonly recommended for chromosome analysis due to its highly effectiveness (Sarbhoy, 1980). In this research, the pretreatment of tissues with 8- hydroxyquinoline failed to accumulate mitotic cells in metaphase, and prophases were relatively more abundant in any duration of immersion. In addition, pretreatment of shoot tips with p-dichlorobenzene also gave poor results in every immersion period. This may be due to some fatty suspension in the shoot tip cells (Ramingwong et al., 2005) which are difficult to squash. Nevertheless, the pretreatment of root tips with p- dichlorobenzene produced preparations superior to pretreatment with 8- hydroxyquinoline (Sharma & Mookerjea, 1955). However, the variation of immersion period with p-dichlorobenzene showed significant difference on chromosome size and arrangement. Root tip treated with p-dichlorobenzene for 3, 6 and 24 hours revealed that the chromosomes were stacked and crossed with each other, causing chromosome count to be biased. Moreover, the p-dichlorobenzene treated root tip at 24 hours resulted in the shortest chromosome size compared with other immersion periods. Based on observations made in this study, the pretreatment with p- dichlorobenzene for 1 hour is the best condition for counting longan chromosome preparation. While pretreatment conditions are important for chromosome preparation, it is imperative that hydrolysis time is also taken into consideration as this affects the chromosome as well. Inappropriate time of hydrolysis can induce differential chromosome staining with carbol fuchsin solution ( Chiamamonrat, 1990) . Fortunately, this study’ s chromosome preparation by hydrolysis using 1N HCl for 7 minutes at 60°C resulted in the most optimal condition for longan chromosome investigation. The example of metaphase cells from 8 longan cultivars pretreated with p-dichlorobenzene for 1 hour at 4°C and hydrolysed in 1N HCl for 7 minutes at 60°C was shown in Figure 1. Although, this is the best condition for counting the chromosomes, it failed to provide centromeric constrictions. The chromosomes clumped excessively that even other cell cycle stages could be obtained, thus the primary constriction was observed only in some chromosome arms as shown from the arrow in Figure 2. Therefore, to figure out the evolution of longan in Thailand further studies are required to gain more information on the karyotype using Fluorescence in situ hybridization (FISH) or other chromosome banding chromosome preparations.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Figure 1. Metaphase chromosomes of 8 longan cultivars. A: Baiyoke, B: Plueakkhao, C: Phetsakorn, D: Krob-Ka-Ti, E: Haewkrae, F: E-daw, G: BiewKhiew Chiangmai, H: Pingpong. Scale bars = 3 µm.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Figure 2. Karyotypes of 8 longan cultivars. A: Baiyoke, B: Plueakkhao, C: Phetsakorn, D: Krob-Ka-Ti, E: Haewkrae, F: E-daw, G: BiewKhiew Chiangmai, H: Pingpong. Scale bars = 3 µm.

All karyotypic parameters of 8 longan cultivars were measured as presented in Table 1. The somatic chromosome number of all cultivars was found to be 2n = 30. The total chromosome length (LT) was a determined minimum of 0.499 µm in Phetsakorn and maximum 1.293 µm in Baiyoke, whereas the lowest relative length of 4.6% was found in Baiyoke and Plueakkhao, and the highest relative length of 9.7% was observed in BiewKhiew Chiangmai. Based on the chromosome size, the idiograms were made and 5 groups of longans can be classified. Finally, the idiograms of all 8 longan cultivars showed the gradually decreasing length of the chromosome without the centromere position and different size of the chromosome pair as shown in Figure 3. The chromosome morphology of D. longan is an interesting point and should be explored further in the future.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Table 1. Mean of total chromosomes length (LT), relative length (RL) and standard deviation (SD) of RL from metaphase chromosomes in 10 cells of Dimocarpus longan ssp. longan var. longan, 2n (diploid) = 30.

LT (µm) RL±SD Chromosome pair Group Cultivar name 2n Min Max Min Max Large Medium Small

Baiyoke 30 0.640 1.293 4.6±0.005 9.3±0.005 6 8 1 1

Plueakkhao 30 0.528 1.068 4.6±0.004 9.2±0.005 6 8 1

Phetsakorn 30 0.499 0.956 4.8±0.003 9.2±0.007 5 10 0

2 Krob-Ka-Ti 30 0.664 1.280 4.8±0.003 9.3±0.006 5 10 0

Haewkrae 30 0.634 1.212 4.9±0.003 9.4±0.006 5 10 0

3 E-daw 30 0.636 1.289 4.7±0.006 9.6±0.006 5 9 1

4 BiewKhiew Chiangmai 30 0.605 1.234 4.8±0.002 9.7±0.012 4 10 1 5 Pingpong 30 0.675 1.282 5.1±0.004 9.6±0.008 3 12 0

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Figure 3. Idiogram showing lengths and sizes of chromosomes of 8 longan cultivars. A: Baiyoke, B: Plueakkhao, C: Phetsakorn, D: Krob- Ka- Ti, E: Haewkrae, F: E- daw, G: BiewKhiew Chiangmai, H: Pingpong.

ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

Conclusion

It can be concluded that the use of the root tip for chromosome counting is better than the shoot tip. The pretreatment of the longan root tip with p-dichlorobenzene for 1 hour at 4°C is effective in arresting the chromosome at metaphase and hydrolysed in 1N HCl at 60°C for 7 minutes gave the best staining results while 8- hydroxyquinoline gave unsatisfied result. This established protocol can be used with all longan cultivars in Thailand but centromeric constrictions were not readily observable.

Acknowledgement

This research was funded by Thailand Science Research and Innovation (TSRI) (PP), Thailand Science Research and Innovation (TSRI) DBG6180017 (SKL) and Chiang Mai University (PL) due to the financial support and laboratory facilities for our research.

Author contribution

PP and SKL performed research. SKL and PL designed the research study. PP and SKL collected and provided samples. PP, SKL, PL and IP analyzed the data. PP, SKL and PL wrote the manuscript. All authors approved the final version of this manuscript.

References

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ICoFAB2021 The 8th International Conference on Food, Agriculture and Biotechnology

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