Songklanakarin Journal of Science and Technology SJST-2019-0323.R2 Srisuvor

The Properties of GABA (Gamma-Aminobutyric Acid) Drinks Supplemented with Probiotic and Oligofructose during Chilled Storage

Journal: Songklanakarin Journal of Science and Technology ManuscriptFor ID SJST-2019-0323.R2 Review Only Manuscript Type: Review Article

Date Submitted by the 19-Feb-2020 Author:

Complete List of Authors: Srisuvor, Nutthaya; Rajamangala University of Technology Krungthep, Food Technology and Nutrition

γ-aminobutyric acid (GABA), germinated , rice drink, probiotic, Keyword: oligofructose

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1 2 3 4 1 The Properties of GABA (Gamma-Aminobutyric Acid) Rice Drinks Supplemented 5 6 7 2 with Probiotic and Oligofructose during Chilled Storage 8 9 3 Nutthaya Srisuvor1 10 11 4 1Department of Food Technology and Nutrition, 12 13 14 5 Faculty of Home Economic Technology, 15 16 6 Rajamangala University of Technology Krungthep, Bangkok, 10120, Thailand 17 18 7 * Corresponding author, Email address: [email protected] 19 20 8 21 For Review Only 22 23 9 Abstract 24 25 10 The quantities of -aminobutyric acids (GABA) and total sugar of the 26 27 28 11 germinated brown rice (GBR) and germinated brown rice drinks supplemented with 29 30 12 probiotic and oligofructose (GBRD-PO) were measured by high-performance liquid 31 32 13 chromatography. The physicochemical properties (i.e., total soluble solids, total solids, 33 34 14 moisture, pH, total acidity, and apparent viscosity) and sensory properties of the GBRD- 35 36 37 15 PO were investigated during 21-days storage. The survival of probiotics was counted. 38 39 16 The results showed that the quantities of GABA, total sugar, glucose, and maltose 40 41 17 depended on the types and cultivars of the germinated rice. The quantities of GABA and 42 43 44 18 total solids increased but the sucrose and pH decreased in the GBRD-PO during chilled 45 46 19 storage. The survival of probiotics averaged 8.26 log CFU/ml throughout the storage. 47 48 20 The sensory properties of GBRD-PO were in an acceptable range. This research is a 49 50 51 21 prototype for the development of GABA quantities in the fermented rice drinks 52 53 22 supplemented with synbiotics for health. 54 55 23 56 57 58 59 60

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2 1 2 3 4 24 Keywords: -aminobutyric acid, germinated brown rice, rice drink, probiotic, 5 6 7 25 oligofructose 8 9 26 10 11 27 1. Introduction 12 13 14 28 Germinated brown rice (GBR) is an innovative food. Its major bioactive 15 16 29 compounds increase during the germination process. GBR contains levels of high 17 18 30 vitamins, minerals, fiber, antioxidants, polyphenols, inositols, ferulic acids, phytic acids, 19 20 21 31 γ-oryzanol, prolylendopeptidaseFor Review inhibitors, and  -aminobutyricOnly acids (GABA) 22 23 32 (Kayahara, Tsukahara, & Tatal, 2001; Wu, Yang, Toure, Jin, & Xu, 2013; Cho & Lim, 24 25 33 2016). GABA is a four-carbon non-protein amino acid. It is a significant component in 26 27 28 34 most microorganisms, plants, and vertebrates. The biosynthesis pathway of GABA is 29 30 35 accomplished by GABA shunt. In GABA synthesis, L-glutamate is catalyzed to form 31 32 36 GABA by glutamate decarboxylase (GAD). The quantities of GABA were dependent 33 34 35 37 on L-glutamate concentration and GAD activities (Shelp, Bown & McLean, 1999; 36 37 38 Rashmi, Zanan, John, Khandagale, & Nadaf, 2018). It has been shown that 38 39 39 environmental stresses promoted the accumulation of GABA in plant tissues (Bown & 40 41 40 Shelp, 2016). In mammals, GABA is a major neurotransmitter in the central nervous 42 43 44 41 system (Olsen, 2002; Bouche, Lacombe, & Fromm, 2003). It has multiple biological 45 46 42 functions which help to prevent Huntington's chorea, Parkinson's disease, Alzheimer's 47 48 43 disease, anxiety, depression, panic, and insomnia (Gajcy, Lochyński, & Librowski, 49 50 51 44 2010). Therefore, the germinated rice can be used as a bio-active composition in 52 53 45 functional foods, such as fresh germinated colored rice noodle, germinated 54 55 46 yoghurt, and GBR bread (Ohtsubo, Suzuki, Yasui & Kasumi, 2005; Anawachkul & 56 57 58 47 Jiamyangyuen, 2009; Cornejo, Caceres, Martínez-Villaluenga, Rosell, & Frias, 2015; 59 60

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3 1 2 3 4 48 Thao, 2018). Several researchers have studied the enhancing GABA, ferulic acid, γ- 5 6 7 49 oryzanol, tocopherols, phenolic compounds, anthocyanins, and antioxidant capacities of 8 9 50 germinated rice and GBR. (Banchuen, Thammarutwasik, Ooraikul, Wuttijumnong & 10 11 51 Sirivongpaisal, 2010; Ng, Huag, Chen & Su, 2013; Lin, Pao, Wu & Chang, 2015; 12 13 14 52 Chaiyasut et al., 2017; Singh, Simapisan, Decharatanangkoon & Utama-ang, 2017). 15 16 53 Few researchers studied GBR beverages mixed with chlorophylls and GBR beverages 17 18 54 mixed with cereals (Assumption University, 2015; Division of Rice Research and 19 20 55 Development, 2016). Recently, GBR products such as rice-balls, rice-burger, soup, 21 For Review Only 22 23 56 baked goods, wines, meat, vegetables, legumes, beverages, and dairy products are 24 25 57 widely enjoyed by consumers (Patil & Khan, 2011; Diana, Quílez, & Rafecas, 2014). 26 27 58 However, there has been no research on the development of the germinated brown rice 28 29 30 59 drinks supplemented with probiotic and oligofructose (GBRD-PO) and its properties. 31 32 60 This product does not contain the cow's milk which is suitable for the lactose intolerant. 33 34 61 This plant drink can be easily absorbed by the body. 35 36 37 62 A “probiotic” is a food supplemented with viable bacteria. It has several benefits 38 39 63 for the body, adjusting the balance of colon microorganisms, modulating 40 41 64 immunological response, and protection from diseases, such as antibiotic-associated 42 43 65 diarrhea, and colon cancer. Furthermore, probiotics prevent infections of intestinal 44 45 46 66 pathogens by creating antimicrobial agents and improving host health (Havenaar & 47 48 67 Huis in’t Veld, 1992; de Vrese & Schrezenmeir, 2008; Šuškovic et al., 2010; 49 50 51 68 Markowiak & Śliżewska, 2017). Moreover, oligofructose is a non-digestible sugar 52 53 69 which can be fermented by the intestinal bacteria. Therefore, it is considered a dietary 54 55 70 fiber for the body. It helps to improve the ecology of microorganisms in the gut (Ziemer 56 57 71 & Gibson, 1998; de Vrese & Schrezenmeir, 2008). In this research, oligofructose was 58 59 60

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4 1 2 3 4 72 used as a prebiotic together with probiotics in the GBRD considered as a synbiotic 5 6 7 73 drink. Synbiotics have been demonstrated to alter, modify, and reinstate the pre-existing 8 9 74 intestinal microflora during the passage through the upper intestinal tract and it 10 11 75 promotes increasing survival, stimulates growth, and activates probiotics metabolism 12 13 14 76 (Pandey, Naik, & Vakil, 2015). 15 16 77 The objectives of this research were to study the quantities of GABA and total 17 18 78 sugar of the germinated paddy rice (GPR), GBR, and GBRD-PO and the 19 20 79 physicochemical, biological and sensory properties of GBRD-PO during chilled storage. 21 For Review Only 22 23 80 24 25 81 2. Materials and Methods 26 27 28 82 2.1 Materials 29 30 31 83 Thai Hom Mali (HM) brown rice (white), cleaved soybean, white sesame, and 32 33 84 pure refined sugar were purchased from a supermarket in Bangkok, Thailand. Hom 34 35 36 85 Lanna (HL) paddy rice and HL brown rice (purple) were purchased from the 37 38 86 Agricultural Technology Research Institute, Rajamangala University of Technology 39 40 87 Lanna, Thailand. Oligofructose (OraftiP95, Tienen, Belgium) with a degree of 41 42 43 88 polymerization of 2-8 was received from the DPO (Thailand) Co., Ltd. Lactobacillus 44 45 89 rhamnosus TISTR 047 (TISTR 047) was purchased from the Department of Bio- 46 47 90 Science, Thailand Institute of Scientific and Technological Research. 48 49 50 91 51 52 92 2.2 Preparation of starter 53 54 55 93 TISTR 047 was inoculated in a sterilized flask of de Man, Rogosa and Sharpe 56 57 94 (MRS) broth and incubated (Memmert UN 110, Germany) at 37 C for 24 hours. The 58 59 60

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5 1 2 3 4 95 standard curve was plotted for the probiotic numbers and the optic densities at 600 nm 5 6 7 96 by using UV/VIS spectrophotometer (Biochrom Libra S22, EU). The starter probiotics 8 9 97 were prepared by incubating TISTR 047 (37 C, 18 hours); the numbers of starter 10 11 98 bacteria were assayed by comparing with the standard curve for preparing fresh culture. 12 13 14 99 They were then centrifuged (Digicen 21 R, Spain) at 3,000xg (4 C, 5 minutes), washed 15 16 100 with 0.85% sterilized saline solution, and adjusted at 8 log CFU/ml into the drinks 17 18 101 (Srisuvor, Chinprahast, Prakitchaiwattana & Subhimaros, 2013). 19 20 21 102 For Review Only 22 23 103 2.3 Preparation of GBRD-PO 24 25 26 104 HL paddy rice, and HM brown rice, HL brown rice were washed and soaked in 27 28 105 water using a rice-to-water ratio of 1:3 (w/v) (paddy rice, 24 hours; brown rice, 4 29 30 106 hours). HM and HL brown rice were incubated for 6-8 hours. They were washed again, 31 32 33 107 then incubated (paddy rice, 48 hours; brown rice, 14 hours) until 0.5-1.0 mm 34 35 108 length. The soybeans were soaked in water for 6-8 hours and washed with water. The 36 37 109 sesame seeds were roasted until brown and fragrant. All ingredients (Table 1) were 38 39 40 110 blended with hot water in a high-speed blender (5 minutes). The mixture was boiled (70 41 42 111 C, 10 minutes), filtered, and pasteurized (70 C, 5 minutes). When the mixture cooled 43 44 112 to 37 C, it was inoculated with 8 log CFU/ml of TISTR 047 starter (Srisuvor et al., 45 46 47 113 2013). The GBRD-PO was filled into 100 ml of polypropylene bottles; then refrigerated 48 49 114 (4 C, 21 days). 50 51 52 115 53 54 55 56 57 58 59 60

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6 1 2 3 4 116 2.4 Analysis of physicochemical properties 5 6 7 117 The quantities of GABA and different types of sugar, i.e., total sugar, fructose, 8 9 118 glucose, sucrose, maltose, and lactose in the GPR, GBR and GBRD-PO were analyzed 10 11 12 119 using TAS 4003 and AOAC method 982.14, respectively using high-performance liquid 13 14 120 chromatography (Shimadzu LC-6A, Japan) (AOAC, 1995; TAS, 2012a). The quantities 15 16 121 of GABA and total sugar in the GBRD-PO were analyzed at 0 and 21 days of storage. 17 18 122 The physicochemical properties of the GBRD-PO were examined on 0, 7, 14 and 21 19 20 21 123 days of storage. The totalFor soluble Review solids (TSS) were Only measured by a hand refractometer 22 23 124 (Atago 2373 WO5, Japan), the total solid (TS) and moisture were analyzed by using a 24 25 125 hot air oven (Memmert, UN 450, Germany), the pH was measured by a pH meter 26 27 28 126 (SCHOTT Lab 850, Germany), the total acidity (TA) was assayed by the titration 29 30 127 method (AOAC, 2012), and the apparent viscosity was measured using viscosity meter 31 32 128 (Brookfield LVDV-II+P, USA). 33 34 35 129 36 37 130 2.5 Analysis of probiotic survival 38 39 131 The enumerations of probiotic cells were spread on the MRS agar at 0, 7, 14 and 40 41 132 21 days of storage by plate count technique. Total plates were incubated in an anaerobic 42 43 44 133 jar (Merck) at 37 C for 72 hours. 45 46 134 47 48 135 2.6 Analysis of sensory properties 49 50 51 136 The GBRD-PO at 0 and 21 days were coded with 3 digit random numbers, 52 53 137 served at 10°C, and evaluated by 45 semi-trained panelists (students of Department of 54 55 138 Food Technology and Nutrition) for the preference test in the appearance, color, odor, 56 57 58 139 flavor, taste, texture, and overall satisfaction. The sensory evaluations used preference 59 60

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7 1 2 3 4 140 scores with 7-point hedonic scale (7- like extremely, 6- like moderately, 5- like slightly, 5 6 7 141 4- neither like nor dislike, 3- dislike slightly, 2- dislike moderately, and 1- dislike 8 9 142 extremely (Larmond, 1982). 10 11 143 12 13 14 144 2.7 Analysis of statistics 15 16 145 The Completely Randomized Design was used for the experimental design of 17 18 146 physicochemical and biological properties, and the Randomized Complete Block Design 19 20 147 was used for the evaluation of sensory properties. They were carried out over three 21 For Review Only 22 23 148 replications. These results were assayed by the analysis of variance. Means were 24 25 149 compared by Duncan' New Multiple Range Test (p  0.05). 26 27 28 150 29 30 151 3. Results and Discussion 31 32 152 3.1 Quantities of the GABA and total sugar 33 34 153 Table 2 shows the quantities of GABA and different types of sugar in the 35 36 37 154 germinated paddy rice of HL variety (GPR-HL), germinated brown rice of HM variety 38 39 155 (GBR-HM), germinated brown rice of HL variety (GBR-HL), and GBRD-PO. The 40 41 156 quantities of GABA in the GPR-HL and GBR-HL were 6.95 and 8.91 mg/100g, WB or 42 43 44 157 9.21 and 9.57 mg/100g, DB, respectively (data not shown). These results correlate with 45 46 158 Anawachkul and Jiamyangyuen’s (2009) assertion that the GABA quantities of the GPR 47 48 159 and germinated red rice were 1.5-13.1 and 2.6-21.3 mg/100g, DB, respectively during the 49 50 51 160 different germination time. The results showed that the germinated rice had a higher 52 53 161 GABA quantities than the GPR due to the soaking of the rice germ in water. It meant that 54 55 162 the germination ratio of rice was higher than the paddy rice due to water absorption and 56 57 163 hydrolytic enzyme activity. The quantities of GABA increased as the time of germination 58 59 60

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8 1 2 3 4 164 was prolonged. Karladee and Suriyong (2012) reported that the quantities of GABA 5 6 7 165 during incubation at 0 and 24 hours were 3.96-17.87 mg/100g, DB. At 24 hours, Khao 8 9 166 Dawh Mali (white) and Kum Doi Saket (purple) rice displayed the highest GABA 10 11 167 quantity (23 mg/100g, DB) of all 21 varieties. Tantakul (2009) showed that the GABA 12 13 14 168 quantities of low-amylose rice (31-37 mg/100g) were higher than high-amylose rice (21- 15 16 169 29 mg/100g), and the GABA quantities of (29-72 mg/100g) were higher 17 18 170 than low-amylose rice. The conclusion was that the quantities of GABA depended on the 19 20 171 types and cultivars of the germinated rice. It correlates with Chua, Koh, & Liu’s (2019) 21 For Review Only 22 23 172 assertion that the factors of high GABA quantities were the rice cultivars, steeping 24 25 173 conditions, provided nutrients, germination duration, and novel techniques. In this study, 26 27 174 the GABA quantity in the GBR-HM was low since the rate of germination was less than 28 29 30 175 70% and it was polished for more than 2 weeks (TAS, 2012b). Later results showed that 31 32 176 the GABA quantity in the GBRD-PO was 0.27 mg/100g, WB because this drink 33 34 177 contained GBR-HL only 1.84% of total ingredients. Besides, the pasteurization of 35 36 37 178 GBRD-PO may cause a decrease of GABA quantities from the initial GBR-HL. This 38 39 179 result was consistent with the decrease of GABA quantities in all the cooking processes 40 41 180 of germinated grains and yoghurt containing paste of germinated red rice including 42 43 181 canned GBR. (Anawachkul & Jiamyangyuen, 2009; Jongyingcharoen & Cheevitsopon, 44 45 46 182 2016; Tiansawang, Luangpituksa, Varanyanond, & Hansawasdi, 2016). Therefore, the 47 48 183 pasteurization of the GBRD-PO should use temperatures at 100 C for 10-20 minutes 49 50 51 184 because the high temperature could inactivate GAD and protease inhibiting the raise of 52 53 185 GABA (Parnsakhorn & Langkapin, 2018). Contrarily in GBR-HM, the quantity of 54 55 186 GABA increased slightly when it was produced as GBRD-PO. The result may be due to 56 57 187 the production of GABA through multiple GABA synthetic pathways. GABA may be 58 59 60

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9 1 2 3 4 188 synthesized by chemical synthesis from various bio-based or biological synthesis from 5 6 7 189 microorganisms (Rashmi et al., 2018; Koubaa, Delbecq, Roohinejad & Mallikarjunan, 8 9 190 2019). Therefore, increasing the quantities of GABA in the pasteurized drink, fermented 10 11 191 food, or commercial food could be performed by supplying probiotics with high GABA- 12 13 14 192 producing ability (Anawachkul & Jiamyangyuen, 2009). This study should be 15 16 193 investigating the steeping conditions, germination duration, and nutrients providing of 17 18 194 rice to produce high GABA before the probiotic fermentation. It has been suggested that 19 20 195 the amount of GABA used to decrease anxiety should be 300-600 mg/day (Thorne 21 For Review Only 22 23 196 Research, Inc., 2007). 24 25 197 The total sugar, glucose, and maltose of the GBR-HL were found to be in 26 27 198 reasonable quantities. The fructose, sucrose, and lactose of the GBR were undetected 28 29 30 199 since they are usually found in fruits or milk rather than rice. However, the total sugar 31 32 200 and sucrose were high in GBRD-PO because sucrose creates sweetness and is a carbon 33 34 201 source for the probiotics. Lastly, the quantities of glucose and maltose slightly dropped 35 36 37 202 in the GBRD-PO because the monosaccharide and disaccharide could be fermented by 38 39 203 probiotics. 40 41 204 42 43 205 3.2 Physicochemical properties of the GBRD-PO 44 45 46 206 Figure 1 and 2 show the physicochemical properties of the GBRD-PO during 47 48 207 chilled storage. The drinks showed no significant difference in TSS, apparent viscosity, 49 50 208 moisture, and TA. The 7-21 day samples had more TS than the production day. This 51 52 53 209 may be due to the fact that the rice starch was hydrolyzed by β-amylase on α-1, 4 54 55 210 glycosidic linkages at the non-reducing ends to produce maltose (Evans, Li & Eglinton, 56 57 211 2009). This result correlates with the increased quantity of maltose on day 21 (Table 3) 58 59 60

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10 1 2 3 4 212 and it is associated with maintaining probiotics survival (Figure 3) by using sugar as a 5 6 7 213 carbon source. The pH of the 0-day storage was higher than the 14-21 days since the 8 9 214 probiotics fermented some glucose and sucrose in the drinks to produce lactic acids, 10 11 215 decreasing the pH (Bonestroo, Kusters, de Wit & Rombouts, 1992; Costa, Fonteles, de 12 13 14 216 Jesus & Rodrigues, 2013). 15 16 217 The effects of chilled storage of the GBRD-PO on the quantities of GABA and 17 18 218 total sugar are presented in Table 3. The quantity of GABA on day 21 increased about 2 19 20 219 times from the production day. It may be due to the fact that GABA is synthesized via 21 For Review Only 22 23 220 GABA shunt, and the L-glutamate is catalyzed by GAD to form GABA (Shelp et al., 24 25 221 1999; Rashmi et al., 2018). Also, this drink was produced by supplementing TISTR 047 26 27 222 which could convert the L-glutamate to form GABA by GAD during incubation. 28 29 30 223 Enhancing GABA production of the probiotics depends on the probiotic species, co- 31 32 224 incubation strains, culture medium, L-glutamate concentration, GAD activity, pH, 33 34 225 temperature, and time (Kim, Lee, Ji, Lee & Hwang, 2009; Li, Qiu, Huang & Cao, 2010; 35 36 37 226 Shan et al., 2015; Lee, Shim, Yao, Kim & Kim, 2018). The quantities of sucrose 38 39 227 decreased slightly by day 21 since it was used for the metabolism and activities of the 40 41 228 probiotics (Costa et al., 2013). However, the maltose quantities of the GBRD-PO 42 43 229 increased slightly by day 21. It may be due to the fact that β-amylase converted the 44 45 46 230 starch in the drink into maltose (Cho & Lim, 2016). 47 48 231 49 50 232 3.3 Probiotic survival in the GBRD-PO 51 52 53 233 Figure 3 displays the survival of TISTR 047 in the GBRD-PO during 0-21 days. 54 55 234 They averaged 8.26 log CFU/ml (p > 0.05). They can survive throughout the chilled 56 57 235 storage. Probiotics live in the gut of the mammal host (de Vrese & Schrezenmeir, 2008). 58 59 60

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11 1 2 3 4 236 Their survival gradually reduced during chilled storage especially in non-dairy foods 5 6 7 237 (Costa et al., 2013; Nematollahi, Sohrabvandi, Mortazavian & Jazaeri, 2016). However, 8 9 238 combining the appropriate probiotics and prebiotics could improve the survival of 10 11 239 probiotics in fermented products (Kailasapathy & Chin, 2000). Pimentel, Madrona, 12 13 14 240 Garcia & Prudencio (2015) reported that oligofructose helped the survival of probiotics 15 16 241 by preventing injuries from the environment and acidity during juice storage. 17 18 242 Furthermore, Acevedo-Martínez, Gutiérrez-Cortés, García-Mahecha & Díaz-Moreno 19 20 243 (2018) suggested that the 5% oligofructose could be used to protect cells from acidity in 21 For Review Only 22 23 244 the mango drink at 4C by providing a substrate for metabolism, and it would help 24 25 245 probiotics to survive in the gastrointestinal tract. There are several factors involved in 26 27 28 246 enriching probiotic viability in food products, such as probiotic selection, inoculum 29 30 247 level, microbial interaction, strain adaptation, food matrix, probiotic encapsulation, 31 32 248 synbiotic combination, product formulation, processing conditions, packaging system, 33 34 249 and storage temperatures (Karimi, Mortazavian, & Da Cruz, 2011; Terpou et al., 2019). 35 36 37 250 In conclusion, these fermented conditions are suitable for the survival of TISTR 047 at 38 39 251 >8 log CFU/ml with no loss of viability during chilled storage at 4C. 40 41 252 42 43 44 253 3.4 Sensory properties of the GBRD-PO 45 46 254 Figure 4 shows the sensory properties of the GBRD-PO during chilled storage. 47 48 255 The preference scores on day 0 were all higher than on day 21, except for color. On the 49 50 st 51 256 21 day, the GBRD-PO had an increased sour odor and fermented rice taste causing a 52 53 257 decrease in overall liking scores. These results correspond with the decrease of sucrose 54 55 258 and pH (Table 3 and Figure 2). Supplementing probiotic and oligofructose did not 56 57 58 259 influence the color of the GBRD-PO during chilled storage. The overall liking scores 59 60

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12 1 2 3 4 260 were in an acceptable range. However, this GBRD-PO needs to improve the sensory 5 6 7 261 properties due to the fermented rice's sour odor and taste. The selection of probiotic 8 9 262 species to produce less acid and better flavor is a way to improve the sensory properties 10 11 263 of the product. 12 13 14 264 15 16 265 4. Conclusions 17 18 266 The quantities of GABA and total sugar in the GBR depend on the types and 19 20 267 cultivars of rice. The GBRD-PO had an increase of GABA and TS quantities but a 21 For Review Only 22 23 268 decrease of the sucrose and pH levels during chilled storage. The survival of probiotics 24 25 269 was potentially suitable to be used in functional drinks (>8 log CFU/ml). It is an 26 27 270 enriched drink from GBR which contains GABA as a synbiotic drink. Further studies are 28 29 30 271 required to improve the GABA quantities and sensory properties of the GBRD-PO to be 31 32 272 accepted as synbiotic fermented rice drink for health. 33 34 273 35 36 37 274 Acknowledgments 38 39 275 This research was funded by Rajamangala University of Technology Krungthep, 40 41 276 Bangkok. 42 43 277 44 45 46 278 References 47 48 279 Acevedo-Martínez, E., Gutiérrez-Cortés, C., García-Mahecha, M., & Díaz-Moreno, C. 49 50 280 (2018). Evaluation of viability of probiotic bacteria in mango (Mangifera indica 51 52 53 281 L. Cv. “Tommy Atkins”) beverage. Revista DYNA, 85(207), 84-92. doi: 54 55 282 10.15446/dyna.v85n207.70578 56 57 283 Anawachkul, M., & Jiamyangyuen, S. (2009). The study of GABA content and 58 59 60

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13 1 2 3 4 284 development of GABA-enriched yogurt from germinated red rice (Munpu rice). 5 6 7 285 Kasetsart Journal (Natural Science), 43, 224-231. Retrieved from 8 9 286 http://kasetsartjournal.ku.ac.th/kuj_files/2010/A1003081314396250.pdf 10 11 287 Association of Official Analytical Chemists (AOAC). (1995). Official Methods of 12 13 th 14 288 Analysis (16 ed). Washington, DC: AOAC International. 15 16 289 Association of Official Analytical Chemists (AOAC). (2012). Official Methods of 17 18 290 Analysis (19th ed). Washington, DC: AOAC International. 19 20 291 Assumption University. (2015). Petty Patent No.12346, Bangkok, Thailand: Department 21 For Review Only 22 23 292 of Intellectual Property, Ministry of Commerce. Retrieved from 24 25 293 http://patentsearch.ipthailand.go.th/DIP2013/view_public_data.php?appno=1151 26 27 294 4600005 28 29 30 295 Banchuen, J., Thammarutwasik, P., Ooraikul, B., Wuttijumnong, P., & Sirivongpaisal, 31 32 296 P. (2010). Increasing the bio-active compounds contents by optimizing the 33 34 297 germination condition of Southern Thai brown rice. Songklanakarin Journal of 35 36 37 298 Science and Technology, 32(3), 219-230. Retrieved from https://pdfs. 38 39 299 semanticscholar.org/3779/d39db0548b596449e2bf8039bc9610cbd2a6.pdf? 40 41 300 Bonestroo, M. H., Kusters, B. J. M., de Wit, J. C., & Rombouts, F. M. (1992). Glucose 42 43 301 and sucrose fermenting capacity of homofermentative lactic acid bacteria used 44 45 46 302 as starters in fermented salads. International Journal of Food Microbiology, 47 48 303 15(3-4), 365-376. doi: 10.1016/0168-1605(92)90070-J 49 50 304 Bouche, N., Lacombe, B., & Fromm, H. (2003). GABA signaling: a conserved and 51 52 53 305 ubiquitous mechanism. Trends in Cell Biology, 13(12), 607-610. 54 55 306 doi: 10.1016/j.tcb.2003.10.001 56 57 307 Bown, A. W., & Shelp, B. J. (2016). Plant GABA: Not just a metabolite. Trends in 58 59 60

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14 1 2 3 4 308 Plant Science, 21(10), 811-813. doi: 10.1016/j.tplants.2016.08.001 5 6 7 309 Chaiyasut, C., Sivamaruth, B. S., Pengkumsri, N., Saelee, M., Kesika, P., Sirilun, S., . . . 8 9 310 Peerjan, S. (2017). Optimization of conditions to achieve high content of gamma 10 11 311 amino butyric acid in germinated , and changes in bioactivities. Food 12 13 14 312 Science and Tehnology, Campinas, 37(Suppl.1), 83-93. doi: 10.1590/1678- 15 16 313 457x.33416 17 18 314 Cho, D., & Lim, S. (2016). Germinated brown rice and its bio-functional compounds. 19 20 315 Food Chemistry, 1(196), 259-271. doi: 10.1016/j.foodchem.2015.09.025 21 For Review Only 22 23 316 Chua, J-Y., Koh, M. K. P., & Liu, S-Q. (2019). 2-Gamma-aminobutyric acid: A 24 25 317 bioactive compound in foods. In H. Feng, B. Nemzer, & J. W. DeVries (Eds.), 26 27 318 Sprouted grains: Nutritional value, production, and application (pp. 25-54). 28 29 30 319 Duxford, UK: Elsevier. 31 32 320 Cornejo, F., Caceres, P. J., Martínez-Villaluenga, C., Rosell, C. M., & Frias, J. (2015). 33 34 321 Effects of germination on the nutritive value and bioactive compounds of brown 35 36 37 322 rice breads. Food Chemistry, 173(15), 298-304. doi: 10.1016/j.foodchem.2014. 38 39 323 10.037 40 41 324 Costa, M. G. M., Fonteles, T. V., de Jesus, A. L. T., & Rodrigues, S. (2013). Sonicated 42 43 325 pineapple juice as substrate for L. casei cultivation of probiotic beverage 44 45 46 326 development: Process optimization and product stability. Food Chemistry, 47 48 327 15(139), 261-266. doi: 10.1016/j.foodchem.2013.01.059 49 50 328 de Vrese, M., & Schrezenmeir, J. (2008). Probiotic, prebiotics, and synbiotics. 51 52 53 329 Advances in Biochemical Engineering/Biotechnology, 111, 1-66. 54 55 330 doi: 10.1007/10_2008_097 56 57 331 Diana, M., Quílez, J., & Rafecas, M. (2014). Gamma-aminobutyric acid as a bioactive 58 59 60

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15 1 2 3 4 332 compound in foods: A review. Journal of Functional Foods, 10, 407-420. 5 6 7 333 doi: 10.1016/j.jff.2014.07.004 8 9 334 Division of Rice Research and Development. (2016). The production of germinated 10 11 335 brown rice drink mixed with cereals. Retrieved from http://brrd. 12 13 14 336 ricethailand.go.th/index.php/2016-07-15-05-29-43/47-2016-07-15-05-53-25 15 16 337 Evans, D. E., Li, C., & Eglinton, J. K. (2009). The properties and genetics of barley 17 18 338 malt starch degrading enzyme. In G. Zhang & C. Li (Eds.), Genetics and 19 20 339 improvement of barley malt quality (pp. 143-189). New York, NY: Zhejiang 21 For Review Only 22 23 340 University. 24 25 341 Gajcy, K., Lochyński, S., & Librowski, T. (2010). A role of GABA analogues in the 26 27 342 treatment of neurological diseases. Current Medicinal Chemistry, 17(22), 2338- 28 29 30 343 2347. doi: 10.2174/092986710791698549 31 32 344 Havenaar, R., & Huis in’t Veld, J. H. J. (1992). Probiotics: A general view. In B. J. B. 33 34 345 Wood (Ed.). The Lactic Acid Bacteria, vol. 1: The lactic acid bacteria in health 35 36 37 346 and disease (pp. 151-170). London: Elsevier. 38 39 347 Jongyingcharoen, J. S., & Cheevitsopon, E. (2016). Development of UV-treated cooked 40 41 348 germinated brown rice and effect of UV-C treatment on its storability, GABA 42 43 349 content, and quality. LWT-Food Science and Technology, 71, 243-248. 44 45 46 350 doi: 10.1016/j.lwt.2016.03.044 47 48 351 Kailasapathy, K., & Chin, J. (2000). Survival and therapeutic potential of probiotic 49 50 352 organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. 51 52 53 353 Immunology & Cell Biology, 78(1), 80-88. 54 55 354 doi: 10.1046/j.1440-1711.2000.00886.x 56 57 355 Karimi, R., Mortazavian, A. M., & Da Cruz, A. G. (2011). Viability of probiotic 58 59 60

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16 1 2 3 4 356 microorganisms in cheese during production and storage: A review. Dairy 5 6 7 357 Science and Technology, 91(3), 283-308. doi: 10.1007/s13594-011-0005-x 8 9 358 Karladee, D., & Suriyong, S. (2012). -Aminobutyric acid (GABA) content in different 10 11 359 varieties of brown rice during germination. ScienceAsia, 38(1), 13-17. 12 13 14 360 doi: 10.2306/scienceasia1513-1874.2012.38.013 15 16 361 Kayahara, H., Tsukahara, K., & Tatal, T. (2001). Flavor, health and nutritional quality 17 18 362 of pre-germinated brown rice. In A. M. Spanier, F. Shahidi, T. H. Parliment, C. 19 20 21 363 Mussinan, C.-T.For Ho, & ReviewE. Tratras Contis (Eds,),Only Food Flavors and Chemistry 22 23 364 advances of the New Millennium (pp. 546-551). Cambridge, UK: RSC 24 25 365 Publishing. doi: 10.1039/9781847550859-00546 26 27 28 366 Kim, J. Y., Lee, M. Y., Ji, G. E., Lee, Y. S., & Hwang, K. T. (2009). Production of 29 30 367 gamma-aminobutyric acid in black raspberry juice during fermentation by 31 32 368 Lactobacillus brevis GABA 100. International Journal of Food Microbiology, 33 34 369 130(1), 12-16. doi: 10.1016/j.ijfoodmicro.2008.12.028 35 36 37 370 Koubaa, M., Delbecq, F., Roohinefad, S., & Mallikarjunan, K. (2019). Gamma- 38 39 371 Aminobutyric acid. In V. Peter, M. Laurence, & S. Fereidoon (Eds.), 40 41 372 Encyclopedia of Food Chemistry, Vol. 3 (pp. 528-534). Cambridge, MA: 42 43 44 373 Elsevier. doi: 10.1016/B978-0-08-100596-5.22412-8 45 46 374 Larmond, E. (1982). Laboratory methods for sensory evaluation of food. Publication 47 48 375 No. 1637. Ottawa: Food Research Institute, Canada Department of Agriculture. 49 50 51 376 Lee, K. W., Shim, J. M., Yao, Z., Kim, J. A., & Kim, J. H. (2018). Properties of kimchi 52 53 377 fermented with GABA-producing lactic acid bacteria as a starter. Journal of 54 55 378 Microbiology Biotechnology, 28(4), 534-541. doi: 10.4014/jmb.1709.09011 56 57 379 Li, H., Qiu, T., Huang, G., & Cao, Y. (2010). Production of gamma-aminobutyric acid 58 59 60

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17 1 2 3 4 380 by Lactobacillus brevis NCL912 using fed-batch fermentation. Microbial Cell 5 6 7 381 Factories, 9(1), 85-91. doi: 10.1186/1475-2859-9-85 8 9 382 Lin, Y., Pao, C., Wu, S., & Chang, C. (2015). Effect of different germination 10 11 383 conditions on antioxidative properties and bioactive compounds of germinated 12 13 14 384 brown rice. BioMed Research International, 2015(4), 1-10. 15 16 385 doi: 10.1155/2015/608761 17 18 386 Markowiak, P., & Śliżewska, K. (2017). Effects of probiotics, prebiotics, and synbiotics 19 20 387 on human health. Nutrients, 9(1021), 1-30. doi: 10.3390/nu9091021 21 For Review Only 22 23 388 Nematollahi, A., Sohrabvandi, S., Mortazavian, A. M., & Jazaeri, S. (2016). Viability of 24 25 389 probiotic bacteria and some chemical and sensory characteristics in cornelian 26 27 390 cherry juice during cold storage. Electronic Journal of Biotechnology, 21, 49-53. 28 29 30 391 doi: 10.1016/j.ejbt.2016.03.001 31 32 392 Ng, L. T., Huag, S. H., Chen, Y. T., & Su, C. H. (2013). Changes of tocopherols, 33 34 393 tocotrienols, γ-oryzanol, and γ-aminobutyric acid levels in the germinated brown 35 36 37 394 rice of pigmented and nonpigmented cultivars. Journal of Agricultural and Food 38 39 395 Chemistry, 61(51), 12604-12611. doi: 10.1021/jf403703t 40 41 396 Ohtsubo, K., Suzuki, K., Yasui, Y., & Kasumi, T. (2005). Bio-functional components in 42 43 397 the processed pre-germinated brown rice by a twin-screw extruder. Journal of 44 45 46 398 Food Composition and Analysis, 18(4), 303-316. doi: 10.1016/j.jfca.2004.10.003 47 48 399 Olsen, R. W. (2002). GABA. In K. L. Davis, D. Charney, J. T. Coyle & C. Nemeroff 49 50 400 (Eds.), Neuropsychopharmacology: The fifth generation of progress (pp. 159- 51 52 53 401 168). Philadelphia, PA: Lippincott, Williams & Wilkins. 54 55 402 Pandey, K. R., Naik, S. R., & Vakil, B. V. (2015). Probiotic, prebiotics and synbiotics- 56 57 58 59 60

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18 1 2 3 4 403 A review. Journal of Food Science and Technology, 52(12), 7577-7587. 5 6 7 404 doi:10.1007/s13197-015-1921-1 8 9 405 Parnsakhorn, S., & Langkapin, J. (2018). Effects of drying temperatures on 10 11 406 physicochemical properties of germinated brown rice. Songklanakarin Journal 12 13 14 407 of Science and Technology, 40(1), 127-134. doi: 10.14456/sjst-psu.2018.11 15 16 408 Patil, S. B., & Khan, M. K. (2011). Germinated brown rice as a value added rice 17 18 409 product: A review. Journal of Food Science Technology, 48(6), 661-667. 19 20 410 doi:10.1007/s13197-011-0232-4 21 For Review Only 22 23 411 Pimentel, T. C., Madrona, G. S., Garcia, S., & Prudencio, S. H. (2015). Probiotic 24 25 412 viability, physicochemical characteristics and acceptability during refrigerated 26 27 413 storage of clarified apple juice supplemented with Lactobacillus paracasei ssp. 28 29 30 414 paracasei and oligofructose in different package type. LWT-Food Science and 31 32 415 Technology, 63(1), 415-422. doi: 10.1016/j.lwt.2015.03.009 33 34 416 Rashmi, D., Zanan, R., John, S., Khandagale, K., & Nadaf, A. (2018). -aminobutyric 35 36 37 417 acid (GABA): Biosynthesis, role, commercial production, and applications. In 38 39 418 F.R.S. Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry, Vol. 57 40 41 419 (pp. 413-452). Cambridge, MA: Elsevier. 42 43 44 420 Shan, Y., Man, C. X., Han, X., Li, L., Guo, Y., Deng, Y., . . . Jiang, Y. J. (2015). 45 46 421 Evaluation of improved -aminobutyric acid production in yogurt using 47 48 422 Lactobacillus plantarum NDC75017. Journal of Dairy Science, 98(4), 138- 49 50 51 423 149. doi: 10.3168/jds.2014-8698 52 53 424 Shelp, B. J., Bown, A. W., & McLean, M. D. (1999). Metabolism and functions of 54 55 425 gamma-aminobutyric acid. Trends in Plant Science, 4(11), 446-451. 56 57 58 426 doi: 10.1016/s1360-1385(99)01486-7 59 60

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19 1 2 3 4 427 Singh, K., Simapisan, P., Decharatanangkoon, S., & Utama-ang, N. (2017). Effect of 5 6 7 428 soaking temperature and time on GABA and total phenolic content of 8 9 429 germinated brown rice (Phitsanulok 2). Current Applied Science Technlogy, 10 11 430 17(2), 224-232. Retrieved from 12 13 14 431 https://www.tci-thaijo.org/index.php/cast/article/view/128531/96675 15 16 432 Srisuvor, N. (2019). Preliminary properties of probiotic drinks with oligofructose. In 17 18 433 RMUTCON (Ed.), The 10th Rajamangala University of Technology International 19 20 434 Conference 2019 Proceedings (pp. 254-258). Chiang Mai, Thailand: Research 21 For Review Only 22 23 435 and Development Institute of Rajamangala University of Technology Lanna. 24 25 436 Srisuvor, N., Chinprahast, N., Prakitchaiwattana, C., & Subhimaros, S. (2013). Effect 26 27 437 of inulin and polydextrose on physicochemical and sensory properties of low-fat 28 29 30 438 set yoghurt with probiotic-cultured banana purée. LWT-Food Science and 31 32 439 Technology, 51(1), 30-36. doi: 10.1016/j.lwt.2012.10.018 33 34 440 Šuškovic, J., Kos, B., Beganovic, J., Pavunc, A. L., Habjanič, K., & Matošic, S. 35 36 37 441 (2010). Antimicrobial activity-The most important property of probiotic and 38 39 442 starter lactic acid bacteria. Food Technology and Biotechnology, 48(3), 296- 40 41 443 307. Retrieved from 42 43 44 444 https://pdfs.semanticscholar.org/a600/47ca461d122544d92a9b1f6c7089a114b2b 45 46 445 3.pdf?_ga=2.146413245.809777321 47 48 446 Tantakul, P. (2009). Pre-germinated GABA-Rice. Food Focus Thailand. Retrieved 49 50 51 447 from http://library.dip.go.th/multim6/edoc/2553/18907.pdf 52 53 448 Terpou, A., Papadaki, A., Lappa, I. K., Kachrimanidou, V., Bosnea, L. A., Kopsahelis, 54 55 449 N. (2019). Probiotics in food systems: Significance and emerging strategies 56 57 450 towards improved viability and delivery of enhanced beneficial value. Nutrients, 58 59 60

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20 1 2 3 4 451 11(7), 1591-1632. doi: 10.3390/nu11071591 5 6 7 452 Thai Agricultural Standard (TAS 4003). (2012a). Germinated Brown Rice. Bangkok: 8 9 453 National Bureau of Agricultural Commodity and Food Standards, Ministry of 10 11 454 Agriculture and Cooperatives. 12 13 14 455 Thai Agricultural Standard (TAS 4404). (2012b). Good manufacturing practices for 15 16 456 germinated brown rice. Bangkok: National Bureau of Agricultural Commodity 17 18 457 and Food Standards, Ministry of Agriculture and Cooperatives. 19 20 458 Thao, N. T. T. (2018). Effect of germinated colored rice on bioactive compounds and 21 For Review Only 22 23 459 quality of fresh germinated colored rice noodle. KMUTNB International Journal 24 25 460 of Applied Science and Technology, 11(1), 27-37. 26 27 461 doi: 10.14416/j.ijast.2017.12.008 28 29 30 462 Thorne Research, Inc. (2007). Gamma-Aminobutyric Acid (GABA). Alternative 31 32 463 Medicine Review, 12(3), 274-279. Retrieved from 33 34 464 http://www.altmedrev.com/archive/publications/12/3/274.pdf 35 36 37 465 Tiansawang, K., Luangpituksa, P., Varanyanond, W., & Hansawasdi, C. (2016). GABA 38 39 466 (γ-aminobutyric acid) production, antioxidant activity in some germinated 40 41 467 dietary seeds and the effect of cooking on their GABA content. Food Science 42 43 468 and Technology, 36(2), 313-321. doi: 10.1590/1678-457X.0080 44 45 46 469 Wu, F., Yang, N., Toure, A., Jin, Z., & Xu, X. (2013). Germinated brown rice and its 47 48 470 role in human health. Critical Reviews in Food Science and Nutrition, 53(5), 49 50 471 451-463. doi: 10.1080/10408398.2010.542259 51 52 53 472 Ziemer, C. J., & Gibson, G. R. (1998). An overview of probiotics, prebiotics and 54 55 56 57 58 59 60

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21 1 2 3 4 473 synbiotics in the functional food concept: Perspectives and future strategies. 5 6 7 474 International Dairy Journal, 8(5-6), 473-479. doi: 10.1016/S0958- 8 9 475 6946(98)00071-5 10 11 476 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 1 Table 1 Ingredients of the GBRD-PO 5 6 7 2 8 9 10 Ingredients Quantities (%) 11 12 13 GBR-HM 4.29 14 15 GBR-HL 1.84 16 17 Soybean 1.23 18 19 20 Sesame 1.23 21 For Review Only 22 Sugar 2.52 23 24 Oligofructose 3.00 25 26 27 Water 85.89 28 29 30 Total 100.00 31 32 33 3 From: Division of Rice Research and Development (2016); Srisuvor (2019) 34 35 4 36 37 38 5 39 40 6 41 42 7 43 44 45 8 46 47 9 48 49 10 50 51 11 52 53 54 12 55 56 13 57 58 14 59 60

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2 1 2 3 4 15 Table 2 Quantities of GABA and types of sugar in the GPR, GBR, and GBRD-PO 5 6 7 16 8 9 10 Quantities (mg/100g, WB) 11 Categories 12 13 GABA Total sugar Fructose Glucose Sucrose Maltose Lactose 14 15 GPR-HL 6.96 ------16 17 a a a a a a 18 GBR-HM 0.20 Nd Nd Nd Nd Nd Nd 19 20 GBR-HL 8.91 2,050 Nd b 650 Nd b 1,400 Nd b 21 For Review Only 22 GBRD-PO 0.27 4,090 Nd b Nd b 3,060 1,030 Nd b 23 24 25 17 Nd a <100 mg/100g 26 27 18 Nd b <600 mg/100g 28 29 30 19 31 32 20 33 34 21 35 36 37 22 38 39 23 40 41 24 42 43 44 25 45 46 26 47 48 49 27 50 51 28 52 53 29 54 55 30 56 57 58 31 59 60

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3 1 2 3 4 32 Table 3 Quantities of GABA and types of sugar in the GBRD-PO during chilled 5 6 7 33 storage 8 9 34 10 11 12 Storage Quantities (mg/100g, WB) 13 14 (days) ns 15 GABA Total sugar Sucrose Maltose 16 17 18 0 0.11 ± 0.00 b 4,910 ± 0.14 3,860 ± 0.12 a 1,050 ± 0.23 b 19 20 21 0.23 ± 0.00 a 4,485 ± 0.13 2,840 ± 0.13 b 1,645 ± 0.01 a 21 For Review Only 22 23 35 Fructose, Glucose, and Lactose <100 mg/100g 24 25 36 26 27 28 37 29 30 38 31 32 33 39 34 35 40 36 37 38 41 39 40 41 42 42 43 43 44 45 46 44 47 48 45 49 50 51 46 52 53 47 54 55 56 48 57 58 59 60

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