Research Article Science Technology and Engineering Journal (STEJ) Vol.6, No.1 pages 46-56

Sugar Conversion of Immature Green Rice Syrup under Solid State Fermentation

Chainarong Chuayjum1*, Wiriya Onsaard1 and Chuenjit Prakitchaiwattana2

1 Department of Agro-Industry, Faculty of Agricultrure, Ubon Ratchathani University Warinchamrap, Ubon Ratchathani, 34190, Thailand 2 Department of Food Technology, Faculty of Science, Chulalongkorn University Pathumwan, Bangkok, 10330, Thailand * Corresponding Author: [email protected]

(Received: 5th June 2020, Revised: 18th June 2020, Accepted: 23rd June 2020)

Abstract- Immature green rice contains high content of γ–oryzanol as well as γ -aminobutiric acid (GABA) and found that several bioactive active compounds such as phenolic compounds, chlorophyll, ß-carotene, tocopherol as well antioxidant activities were reported. Thus, this research is aimed to add more value of immature green rice as a substrate for Isomaltooligosaccharides (IMOs). The scope is studied a conversion from immature green rice during liquefaction using solid state fermentation (SSF) of Aspergillus oryzae TISTR 3102. The process included, gelatinization of immature green rice starter inoculation as solid state with A. oryzae and under 30 oC for 8 days. The highest syrup volume was obtained after 7 days of fermentation (p≤0.05) with increasing of total soluble solids (oBrix) (p>0.05). Moreover, found increased and the highest value was observed at the 4th day of fermentation (p≤0.05) and tended to constant through fermentation period. The highest total solid content (%) was found at the 6th day of fermentation (p≤0.05) and tended to decreasing. pH value was decreased from 5.27±0.01 to 4.73±0.01 after 7 days of fermentation (p≤0.05). Ultra-High-Performance liquid Chromatography (UHPLC) was conducted for sugar and isomaltose analysis and the highest amounts of , and isomaltose were observed in 3 and 8 days of fermentation. As monosaccharide was produced by SFF of A. oryzae, it is agreed with increasing of syrup volume, total soluble solid and reducing sugar content and dextrose equivalent. Keywords: Dough Rice Stage, Solid State Fermentation, Monosaccharide, Isomaltooligosaccharide Vol 6. No 1, January-June 2020 Sugar Conversion of Immature Green Rice 47 Syrup under Solid State Fermentation

1. Introduction product is obtained. Moreover, it has been reported that rice has been estimated to have In northeastern part of Thailand, it have a higher protein digestibility and biological been found that immature green rice is a value compared to other cereals (i.e., wheat, rice product producing from an immature corn, barley, millet and sorghum (Eggum, green paddy or dough stage rice using a 1979, Young and Pellett, 1994), Amagliani conventional method similar to parboiling et al., 2016). Thus, this research is aimed process. This rice product is normally to add more value of immature green rice produced and consumed among Asian as a substrate for Isomaltooligosaccharides countries in particular of Thailand, Lao (IMOs) which the amount of imported IMO People’s Democratic Republic (Lao could be decreased. PDR), Vietnam and Cambodia. However, IMOs, are non-digestible prebiotic different countries produce different rice oligosaccharide which pass through the varieties with different process methods, gastrointestinal tract without digestion by thus providing different characteristics of digestive enzymes, have been found to the final product. Basically, the immature increase bifidobacterial number in faces and green paddy is harvested at the 19-21 days the ratio in fecal microflora and balancing after flower blooming as it is called “dough the human gut (Kaneko et al., 1994). IMOs stage”. This rice stage was reported to have production includes 3 steps i.e. liquefaction, high sugar content than a maturity stage saccharification and transglucosidation. (Singh and Juliano, 1977) giving a special Most of liquefaction and saccharification characteristic of rice product. Unique and process were aimed to obtain high amount good attributes of immature rice product of maltose syrup by enzymatic are green color, gummy-like texture, earthy (Lee et al., 2008, Rudeekulthamrong et and green odor with a little bit sweet taste. al., 2013, Sorndech et al., 2017). Most of Moreover, (Ekasit and Jiraporn, 2013) have liquefaction and saccharification process reported that immature green rice product starting with slurry as a substrate or young flatten rice using a traditional which takes a period of time for preparing method contains high value of γ–oryzanol since the raw materials are in solid form (56-113 mg/100g) as well as γ-aminobutiric (Wang et al., 2007). Thus, an alternative acid (GABA) was observed as high as 4-8 method was proposed by using a substrate mg/100 g for this processed rice at dough as a solid form i.e. rice grain (Sawangwan stage. (Phomkong et al., 2014) have found and Saman, 2016 and Kim et al., 2017), rice that several bioactive active compounds crumbs (Pan and Lee, 2004). Moreover, such as phenolic compounds, chlorophyll, it was found that solid state fermentation ß-carotene, tocopherol as well antioxidant for IMOs production from rice has been activities have been observed in this rice successful as the glutinous rice syrup product. It could be suggested that young exhibited high prebiotic properties by flatten rice has been produced by conventional using solid-state fermentation of Aspergillus method partially of parboiling process, oryzae (Sawangwan and Saman, 2016). It thus high nutritional compound could be is well recognized that Aspergillus oryzae moved from embryo and brown layer to is an endo-enzyme producing α-amylase endosperm and thus high nutritional rice target to hydrolyze α-1,4 glycosidic bond 48 Chainarong Chuayjum, Wiriya Onsaard Science Technology and Engineering Journal (STEJ) and Chuenjit Prakitchaiwattana

of branched/linear starch during liquefaction milling. . The rice grain was then furthur step. Thereafter, β-amylase plays a role to soaked in distilled water overnight in the obtain maltose syrup in saccharification raio of water:rice gain at 1:2 and steamed stage followed by IMO synthesis is for 40 minutes until cooked by using rice ocurred during transglucosidation step by cooker. α-transgucosidase. Thus, a low degree of starch liquefaction (DE ~ 6-8) is preferred for 2.3 Preparation of fungal spore in- high maltose content during saccharification oculum process (Niu et al., 2017). Moreover, Aspergillus oryzae preparing as a solid state for Spore suspensions of A. oryzae TISTR fermentation is more practical in industrial 3102 were prepared from a fully sporulated application and it is recognized as safe and (7 days old) PDA slant culture using has been approved by WHO to apply for 10 ml of 0.85% NaCl solution (Saman food industry. Therefore, the aim of the et al., 2012) This spore suspension research is focusing of sugar conversion was used as the master suspension. Spore from immature green rice during liquefaction concentration in the inoculum was estimated using solid state of Aspergillus oryzae to by a haemacytometer (Wanichsan et al., obtain low dextrose equivalent (DE) during 2015). starch liquefaction stage.

2. Materials and Methods 2.4 Preparartion of solid state of fungal spore 2.1 Material Glutinous rice (50 g) was placed into an erlenmeyer flasks (koji) containing Aspergillus oryzae TISTR 3102 were 10% (v/v) distilled water. The contents kindly provided from Thailand Institute of the flasks were mixed thoroughly and of Scientific and Technological Research. autoclaved at 121°C for 20 minutes. Then Glutinous rice (RD6) includes dough stage spore suspension of Aspergillus oryzae (October, 2019) and maturation stage TISTR 3102 (106 spores/ml) was inoculated (November, 2019) was obtained from Pho in autoclaved rice and the mixture was Sai district, Ubon Ratchathani province, incubated at 30°C for 3 days. Thailand. All other chemicals used were analytical grade and the sugar standards were HPLC grade. 2.5 Fermentation process A rice sample was soaked in distilled water 2.2 Substrate preparation overnight in the raio of 1:2 (rice grain: water) and steamed for 40 minute by using rice Immature green rice was washed 3 times, cooker. Then the substrate was mix with and soaked in 2 % sodium chloride (NaCl) koji as prepared in 2.4 before incubated solution for 2 hours, then steamed for 20 at 30°C for 8 days using incubator. The minutes and dried until 12 % moisture syrup was collected everyday for 8 days content of paddy was obtained before duringincubation for further analysis. Vol 6. No 1, January-June 2020 Sugar Conversion of Immature Green Rice 49 Syrup under Solid State Fermentation

2.6 Analytical medthods. acetate: Acetronitrile: DI water) was used as mobile phase at constant flow rate of 1.0 2.6.1 Spore concentration ml/minute with controlling temperature at The spore concentration was 30°C. All standard compounds (glucose, estimated using a haemacytometer according maltose, ) were HPLC grade (DR to the method reported by Wanichsan et ehrenstorfer) and isomaltose (Megazyme) al., (2015). Dextrose equivalent (DE value) were calculated as described by Klanarong and 2.6.2 Syrup volume Kuakoon (2007) Volume of syrup was measure by DE value = reducing sugar x 100 (2) filtered through filter cloth before measuring total solid using a 200 milliliter measuring cylinder. Data analysis 2.6.3 pH, Total Soluble Solid (TSS) and Total Solid (TS) Analytical measurements and all data were presented by mean values with standard The pH value of syrup was determined deviation. Each parameters were analysed using a pH meter (METTLER TOLEDO by analysis of variance (ANOVA) by using AG, Schwerzenbach, Switzerland). Total completely randomized design (CRD) as Soluble Solids (TSS) was determined expermental design. Duncan’s new multiple using a hand refractometer (ATAGO, Model range test was used as determining of G-50 Brix 0-50, Japan). Total solid (TS) differences between the treatment at the values were measured using (AOAC, 2000) 95% confidentail (p≤0.05). method and calculated as follows:

(%)TS = weight of sample after drying x 100 (1) 3. Results and Discussion weight of sample before drying 2.6.4 Reducing sugar content Solid state of A. oryzae preparing as a koji was obtained with a number of spore Reducing sugar content were performed forming (2x105 spores/ml) after 3 days according to dinitrosalicylic colorimetric incubation. The koji was mixed with cooked method (DNS method) (Miller,1959) green rice before fermentation under 30 oC incubation for 8 days. Syrup volume 2.6.5 Sugar content was rapidly increased to 172 ml since day Both qualitative and quantitative of 4th (P≤0.05) and tended to constant after sugar collected from syrup were analyzed 5 days of fermentation (p>0.05), and a using Ultra-High-Performance liquid slightly decreasing was observed at day 8th of Chromatography, UHPLC (DIONEX, incubation with non-significantly difference Ultimate 3000, Sunnyvale, CA, USA) with day 7th (Fig. 1) These results agreed with Refractive Index Detector (Refrac- with the changing of total soluble solid toMax 521 ). YMC-Pack Polyamine II contents but the amount of total soluble column (4.6×250 mm) (YMC japan) was solid content was increased since day 3rd of used. A mixture of solvent (Ethanol: Ethyl fermentation and tended to constant until 50 Chainarong Chuayjum, Wiriya Onsaard Science Technology and Engineering Journal (STEJ) and Chuenjit Prakitchaiwattana

8 days of fermentation. The changing of activities of α-amylase and glucoamylase incubationsyrup volumewith non and-significantly total soluble difference solid could glucoamylaseenzyme as a result enzyme of increasing as a of resultthe total of with beday described 7th (Fig. that1) Tthehese fungal results penetrates agreed increasingsugart and of reducing the total sugar sugar (Kongt and reducinget al., with acrossthe changing the rice kernelof total before soluble hydrolysis solid sugar2018). (Kong However, et al., reducing2018). However,sugar contentstakes but place the by amountfungal amylolytic of total solubleenzymes reducingcontent tendedsugar contentto constant tended after to day constant the solid suchcontent as α-amylase was increased (Sivaramakrishnan since day 3rd et of al., after5th of day fermentation the 5th of fermentation (p>0.05). It (pcould>0.05). be It fermentation2007) and and glucoamylase tended to constant (Zambare, until 2010). 8 coulddue to be a decreasingdue to a ofdecreasing fungal synthesis of funga as l days Thisof fermentation. α-amylase catalyzethe The changing hydrolysis of syrup of synthesisthere might as bethere a decreasing might be of a substratedecreasing due of volumeinternal and α-1,4-glycosidictotal soluble solid linkages could at be the substrateto a long perioddue toof fermentationa long period process of describeinnerd thatpart ofthe the fungal amylose penetrates or amylopectin across fermentation(Yanfang et processal., 2008). (Yanfang et al., 2008). the ricein starchkernel asbefore a random hydrolys manneris takes (Nielsen, place by fungal2000) Moreover, amylolytic glucoamylase enzymes such hydrolyzes as α- amylaseα-1,4 (Sivaramakrishnan and α-1,6 glycosidic et al., bonds 2007) from and the glucoamylasenon-reducing (Zambare, ends of starch,2010). resultingThis α- in amylasethe productioncatalyzethe ofhydrolysis glucose orof disaccharideinternal α- 1,4-glycosidicsuch as maltose linkages and at isomaltosethe inner part(Uhling, of the amylose1998). Therefore, or amylopectin an increasing in starch of assyrup a randomvolume manner was (Nielsen,obtained. But2000) after Moreover, the 3rd day glucoamylaseof fermentation, hydrolyzes the amount α-1,4 andof substrate α-1,6 glycosidicmight bebonds decreased, from thusthe thenon changing-reducing of ends of starch, resulting in the production of syrup volume and total soluble content glucose or such as maltose and were constant. However, the syrup volume isomaltose (Uhling, 1998). Therefore, an and total soluble solid content were found increasing of syrup volume was obtained. coincident with reducing sugar content as But after the 3rd day of fermentation, the amoushownnt of in(sub Fig.strate 1) mightby using be DNS decreased, method. thus Itthe was changing found thatof syrup the amount volume of and reducing total solublesugar content rapidly were increased constant. (p≤0.05) However, and the the syruphighest volume amount and total was soluble observed solid at content3 days of were fermentationfound coincident due to with hydrolysis reducing by sugar fungal contentamylolytic as show enzymes.n in( Fig. In addition,1) by using preparing DNS of method.immature It was green found rice thatby starch the gelatinizationamount of reducingresulting sugar of rapidlyenzyme increasehydrolyzedd (p≤ substrate0.05) Figure 1. Changing of chemical properties of Figure 1. Changing of chemical properties and theeasily highest breaks amount the hydrogen was observed bonds withinat 3 immature green rice syrup during solid-state of immature green rice syrup during solid- days moleculesof fermentation inside of due starch to inhydroly the amorphoussis by fermentation at 30°C for 8 days. state fermentation at 30°C for 8 days. fungalpart, amylolytic causing of enzymes.unfolding ofIn theaddition, amylose preparingand amylopectin of immature polymer green chainsrice by and starch water Moreover, the highest value of total Moreover, the highest value of total gelatinizationbinding is increased, resulting causing of starchenzyme granule solids content (TS) (Fig. 2) were found on solids thcontent (TS) (Fig. 2) were found on hydrolyzedto swell. substrateThereafter, eastheily leaching breaks amylose/ the the 6 days of fermentation and slightly the 6th days of fermentationth and slightly hydrogeamylopectinn bonds within from amorphous molecules regioninside afterof decreased after day 8 of fermentation decreased after day 8th of fermentation starchgelatinization in the amorphous were degraded part, bycausing amylolytic of (p≤0.05). Generally, total solids include (p≤0.05). Generally, total solids include unfolding of the amylose and amylopectin total soluble solids and insoluble solid i.e enzymes producing from fungi to release total soluble solids and insoluble solid i.e polymer chains and water binding is carbohydrate, protein, lipid etc. Moreover, oligosaccharides by hydrolysis (Nielsen, carbohydrate, protein, lipid etc. Moreover, increased, causing starch granule to swell. enzyme containing in fungals (such as 2000). Moreover, as a long period of enzyme containing in fungals (such as Thereafter, the leaching protease) as well as amylolytic enzyme play fermentation process, inducing more digestion protease) as well as amylolytic enzyme play amylose/amylopectin from amorphous a role for hydrolysis (Saman et al 2012), region after gelatinization were degraded by thus total solid content can be increased amylolytic enzymes producing from fungi to after 3 days of fermentation. However, a release oligosaccharides by hydrolysis decreasing of total solid was observed after (Nielsen, 2000). Moreover, as a long period 5 days of fermentation. It could be of fermentation process, inducing more suggested that a substrate for fungal digestion activities of α-amylase and synthesis is decreased.

Vol 6. No 1, January-June 2020 Strategy of Energy for Commercial Building 51

a role for hydrolysis (Saman et al 2012), chromatogram of sugar analysis of immature thus total solid content can be increased rice flour fermentation (Fig. 3a) exhibited after 3 days of fermentation. However, monosaccharide compounds as peak of a decreasing of total solid was observed fructose, glucose and maltose were found after 5 days of fermentation. It could at retention time of 8, 11 and 13 minutes, be suggested that a substrate for fungal respectively. This result agreed with (Hu synthesis is decreased. et al., 2017) which soluble sugar profile of rice were found such as glucose, fructose, The changing of total solid content , raffinose and maltose. However, was agreed with the changing of pH value. glucose and maltose in immature rice syrup It wasThe observed changing that of totalpH value solid of content dough ricewas results agreed with total solid and pH value was observed after 4 day of fermentaion by agreedsyrup werewith thedecreased changing from of 5.27±0.01pH value. toIt through a fermentation process. Moreover, SSF of A. oryzae TISTR 3102 (Fig. 3b), but was4.73±0.01 observed through that pH a 7-day value period of dough of solid rice UHPLC chromatogram exhibited isomaltose a peak of fructose was no longer appear. syrupstage werefermentation decreased (P≤0.05). from 5.27 This ± 0.01 could to peak at retention time between 15-16 It is suggested that during fermentation 4.73be due ± 0.01 to fungal through metabolism a 7-day period (Yanfang of solid et minutes. process, there is metabolism of fungals by stageal., 2008). fermentation Moreover, (P≤0.05). during Thisfermentation could be using fructose as a substrate for synthesis. dueusing to solid-statefungal metabolism of A. oryzae (Yanfang produces et al., 2008). Moreover, during fermentation using These results agreed with total solid and the otherThe changi compoundsng of total such solid as organic content acidwas results agreed with total solid and pH value solid-state of A. oryzae produces the other pH value through a fermentation process. agreed with the changing of pH value. It through a fermentation process. Moreover, compounds(Konlam et such al., 2014)as organic resulting acid decreasing(Konlam et was observed that pH value of dough rice Moreover,UHPLC chromatogram UHPLC chromatogram exhibited isomaltose exhibited alof., 2014pH value.) resulting decreasing of pH value. syrup were decreased from 5.27 ± 0.01 to isomaltosepeak at retention peak at retentiontime between time between 15-16 4.73 ± 0.01 through a 7-day period of solid 15-16minutes. minutes. stage fermentation (P≤0.05). This could be due to fungal metabolism (Yanfang et al., (a) 2008). Moreover, during fermentation using

solid-state of A. oryzae produces the other compounds such as organic acid (Konlam et al., 2014) resulting decreasing of pH value.

(a)

(b) Figure 3. HPLC chromatogram of immature green rice (a) (peak 1: fructose, 2: glucose Figure 2. Changing of reducing sugar and pH of Figure 2. Changing of reducing sugar and and 3: maltose), and glutinous rice syrup pHimmature of immature green rice greensyrup duringrice fermentationsyrup during at 30°C for 8 days from SSF by A. oryzae TISTR 3102 at 30°C fermentation at 30°C for 8 days 4 day (b) (peak 1: glucose, 2: maltose, 3: isomaltose) UUHPLCHPLC analysis analysis (b) The immature green rice syrups were FigureThe peak 3. HPLCarea of chromatogram immature green of immature rice syrup daily withdrawnThe immature and analyzedgreen rice for syrups both Figuregreen 3.rice HPLC (a) chromatogram(peak 1: fructose, of immature 2: glucose green Figure 2. Changing of reducing sugar and ricewas (a) analyzed (peak 1: fructose, by comparing 2: glucose andwith 3: sugarmaltose), and quantitativewere daily and withdrawn qualitative and analysis analyzed of sugar for andisomaltose 3: maltose) standard, and curve glutinous (R2= 0.99rice ).syrup As bypH U HPLCof immature techniques. green A ricechromatogram syrup during of and glutinous rice syrup from SSF by A. oryzae both quantitative and qualitative analysis TISTRfromshown SSF 3102 in by (atTable A.30°C oryzae 14 )day, itTISTR (b)was (peak found3102 1: glucose,at that 30°C the sugarfermentation analysis at 30°Cof immaturefor 8 days rice flour 4 day (b) (peak 1: glucose, 2: maltose, 3: of sugar by UHPLC techniques. A 2:highest maltose, g lucose3: isomaltose) content was obtained in the fermentation (Fig. 3a) exhibited isoma3rd dayltose of )fermentation period which agreed monosaccharideUHPLC analysis compounds as peak of The immature green rice syrups were with reducing sugar content measured by fructose, glucose and maltose were found at The peak area of immature green rice syrup daily withdrawn and analyzed for both DNS method (Fig. 1) and tended to retention time of 8, 11 and 13 minutes, was analyzed by comparing with sugar and quantitative and qualitative analysis of sugar constant throughout fermentation2 period respectively. This result agreed with (Hu et isomaltose standard curve (R =0.99). As by UHPLC techniques. A chromatogram of shown(P>0.05). in On(Table the 1other), it hand,was found maltose that tend theed al.,sugar 2017) analysis which solubleof immature sugar profile rice offlour rice to increase with fermentation period was were found such as glucose, fructose, highest glucose content was obtained in the fermentation (Fig. 3a) exhibited 3increasedrd day of ,fermentation which the maximum period which content agreed was sucrose,monosaccharide raffinose andcompounds maltose. as However,peak of obtained on the 8th day of fermentation glucose and maltose in immature rice syrup with reducing sugar content measured by fructose, glucose and maltose were found at DNS(P≤0.05). method However, (Fig. 1the) andincreasing tended toof wretentionas observed time after of 48 ,day 11 ofand fermentaion 13 minute bys, isomaltose content was found through a SSF of A. oryzae TISTR 3102 (Fig. 3b), but constant throughout fermentation period respectively. This result agreed with (Hu et (P>0.05).period fermentation On the other process hand, .m Italtose could tend be eddue a al.,peak 2017) of fructose which solublewas no sugarlonger profile appear of. Itrice is to an activity of α-glucosidase produced suggested that during fermentation process, to increase with fermentation period was were found such as glucose, fructose, increasedfrom SSF, which of theA. maximumoryzae bycontent catalyses was there is metabolism of fungals by using sucrose, raffinose and maltose. However, obtaliberationined onof theglucose 8th day f romof fermentationnon-reducing fructose as a substrate for synthesis. These glucose and maltose in immature rice syrup (P≤0.05).ends ofHowever, oligosaccharides the increasing ofand was observed after 4 day of fermentaion by isomaltose content was found through a SSF of A. oryzae TISTR 3102 (Fig. 3b), but period fermentation process. It could be due a peak of fructose was no longer appear. It is to an activity of α-glucosidase produced suggested that during fermentation process, from SSF of A. oryzae by catalyses there is metabolism of fungals by using liberation of glucose from non-reducing fructose as a substrate for synthesis. These ends of oligosaccharides and

52 Chainarong Chuayjum, Wiriya Onsaard Science Technology and Engineering Journal (STEJ) and Chuenjit Prakitchaiwattana

The peak area of immature green equivalent (DE-value) was obtained up rice syrup was analyzed by comparing to 55.13 It could be suggested that high with sugar and isomaltose standard curve was obtained under solid-state (R2=0.99). As shown in (Table 1), it was of A. oryzae fermentation, using immature found that the highest glucose content was green rice as a substrate. In addition, there obtained in the 3rd day of fermentation period is a production of oligosaccharide such as which agreed with reducing sugar content isomaltose during fermentation by SSF of measured by DNS method (Fig. 1) and A. oryzae which the maximum content was tended to constant throughout fermentation observed on the 8th day of fermentation period (P>0.05). On the other hand, maltose (P≤0.05). tended to increase with fermentation period However, as a result of high DE-value was increased, which the maximum content was obtained in this study, it is suggested that was obtained on the 8th day of fermentation this fermentation process is not suitable for (P≤0.05). However, the increasing of IMOs synthesis. Generally, a requirement isomaltose content was found through a for IMOs production to obtain high period fermentation process. It could be maltose syrup after saccharification process due to an activity of α-glucosidase produced is required DE value between 6 to 8 (Niu from SSF of A. oryzae by catalyses et al., 2017). Therefore, high glucose syrup liberation of glucose from non-reducing ends was found under solid-state of A. oryzae of oligosaccharides and polysaccharides. fermentation, using immature green rice This enzyme is able to transfer sugar as a substrate under 30°C, is not suitable or groups of sugar residues from one for IMOs production. Therefore, the compound to another with the formation of production process may need to be modified a similar or a distinct type of linkage. Thus, by changing of fermentation conditions to an α-(1,4) link in a chain might be broken be a suitable environment for beta-amylase and the separated end could be joined to activity such as temperature and pH or can the same or different chain via either of be using both crude enzyme or SSF of A. α-(1,4) or α-(1,6) link resulting to produce niger. Since, β-amylase enzyme catalyzes molecules of maltose, isomaltose, or long the hydrolysis of the α-1,4 glycosidic bond chain of oligosaccharides. (Saman, 2012). at specific manner, cleaving off two Resulting of an increasing of glucose units (maltose) at a time (Beck and reducing sugar content, thus high dextrose Ziegler, 1989). Vol 6. No 1, January-June 2020 Sugar Conversion of Immature Green Rice 53 Syrup under Solid State Fermentation

Table 1. Sugar content and dextrose equivalent using UHPLC analysis frome solid-state by A. oryzae for immature green rice under 30°C for 8 days of fermentation.

Fructose Glucose Maltose Isomaltose Dextrose Day (mg/ml) (mg/ml) (mg/ml) (mg/ml) equivalent (DE) 0.89 2.81 2.01 0.63 0 N.D. ±0.14 ±0.0.33f ±0.50d ±0.10d 325.58 6.02 27.79 47.04 2 N.D. ±2.93d ±0.31c ±4.64c ±0.54c 353.02 12.64 32.87 55.13 3 N.D. ±5.63a ±1.54b ±1.04abc ±2.29a 340.01 13.91 35.18 51.61 4 N.D. ±3.68bc ±1.75b ±1.93abc ±2.01ba 336.83 14.14 29.35 48.46 5 N.D. ±5.84c ±1.06b ±3.47c ±0.42bc 345.00 13.84 30.05 48.30 6 N.D. ±0.91b ±0.62b ±3.79bc ±1.55bc 290.37 13.46 37.01 45.30 7 N.D. ±1.11e ±0.62b ±1.10ab ±2.16c 343.40 16.61 39.37 53.45 8 N.D. ±1.52bc ±1.00a ±1.02a ±3.09a Note: - The data shows the average and standard deviation. a-f Mean values in the same column with different letters expressed significant differences (p≤0.05) all among samples. N.D. means not detectable.

4. Conclusion immature green rice as a substrate resulting of decreasing of total solid and pH value. The production of green rice syrup by However, isomaltose was also obtained solid-state of A. oryzae TISTR 3102 at 30 under solid-state fermenation of A. oryzae, oC for 8 days fermentation was observed. but a low value was obtained which is not It was found that glucose and maltose were suitable for further IMOs synthesis. increased since 3 days of fermentation period analysed by Ultra-High-Performance 5. Acknowledgement liquid Chromatography (UHPLC). These results correspond with increasing of In this research was supported by Thailand dextrose equivalent, syrup volume, total Science Research and Innovation (TSRI) soluble solid and reducing sugar content. and Researcher for Industries (RRI) and The amylolytic enzymes plays a role Thailand Science Research and Innovation for starch hydrolysis under solid-state (TSRI) as well as MKM Premium Brands fermenation of A. oryzae under a suitable Part., Ltd., condition of fungal metabolism by using 54 Chainarong Chuayjum, Wiriya Onsaard Science Technology and Engineering Journal (STEJ) and Chuenjit Prakitchaiwattana

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