International Food Research Journal 26(5): 1515-1523 (October 2019) Journal homepage: http://www.ifrj.upm.edu.my Feasibility of Leum Pua glutinous rice substrate for sugar syrup and vinegar production by raw starch degrading enzyme hydrolysis 1*Lomthong, T. and 2Saithong, P. 1Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathumthani 12110, Thailand 2Department of Applied Microbiology, Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand Article history Abstract Received: 11 February, 2019 Raw starch degrading enzyme (RSDE) from Laceyella sacchari LP175 was applied for Received in revised form: hydrolysis of Leum Pua glutinous rice powder (200 g/L) and compared with other brown and 25 April, 2019 black rice varieties in 250 mL Erlenmeyer flasks at 50°C for 12 h. Results showed that Leum Accepted: 4 July, 2019 Pua glutinous rice gave the highest liberated sugar syrup and anthocyanin contents at 11°Brix and 82.27 mg/L, respectively. Leum Pua glutinous rice powder (250 g/L) was up-scaled for hydrolysis in 5.0 L glass jar by simultaneous liquefaction and saccharification at 50°C for 12 h and obtained liberated sugar syrup at 13°Brix. Sugars were fermented by two mixed strains of Keywords Saccharomyces cerevisiae for 10 d without shaking at room temperature. During fermentation, total soluble solids decreased from 22 to 10°Brix while alcohol content increased to 15% (w/v). Thai rice Leum Pua glutinous rice wine was subjected to acetic acid surface culture fermentation (SCF) Laceyella sacchari LP175 with Acetobacter aceti TISTR 354 at room temperature. After 6 d of fermentation, acidity Wine and vinegar of Leum Pua glutinous rice vinegar increased to 5.7%. Antioxidant activity and vinegar fermentation concentration were determined during acetic acid fermentation for 6 d. Results suggested the Leum Pua glutinous rice feasibility of producing Thai rice vinegar using cold hydrolysis with raw starch degrading enzyme without the heating process. © All Rights Reserved Introduction et al., 2014), apple (Qi et al., 2017), plum (Williams et al., 2016), tomato (Koyama et al., 2017), onion Leum Pua glutinous rice (Oryza sativa L., variety (Lee et al., 2017) and purple sweet potato (Wu et Leum Pua) is a local Thai glutinous rice with purple al., 2017). Vinegar has several advantages, including seeds and high nutrition value as compared to white application as a food additive for preservation against and other coloured rice varieties (Pornputtapitak et spoilage (Budak et al., 2014). Ali et al. (2016) al., 2018). Boonsit et al. (2010) reported that Leum reported that vinegar could be used as a medicine with Pua glutinous rice contains higher antioxidants a positive effect on biomarkers for diabetes, cancer and bioactive compounds such as anthocyanins, and heart diseases. A recent report described vinegar γ-oryzanol and phenolic compounds than white rice. production by surface culture fermentation (SCF) Raw rice seeds contain high starch content which using a stainless steel deep long tray as a container at can be used as a carbon source for microbial growth room temperature which gave high contents of acetic and fermentation of various end products (Joo et al., acid in a short time period (Saithong et al., 2017). 2009). Cold hydrolysis by raw starch degrading enzyme Vinegar is obtained from microbial fermentation at low temperature (40 - 50°C) has been compared through biotechnological processes. Production of to the conventional hydrolysis process at above vinegar consists of two steps which are alcoholic gelatinization temperature (90 - 105°C) (Cinelli et and acetic fermentations of liquid juice substrates al., 2015; Lomthong et al., 2018). Hydrolysis of (Kulkarni, 2015). Currently, various substrates are starch-based materials at low temperature without used for vinegar fermentation such as mushroom (Li the heating process can reduce energy consumption *Corresponding author. Email: [email protected] 1516 Lomthong, T. and Saithong, P./IFRJ 26(5) : 1515-1523 and cost. Recently, cold hydrolysis by raw starch flask containing 100 mL of YM medium (3 g/L degrading enzyme for the production of various kinds yeast extract, 3 g/L malt extract, and 5 g/L peptone; of fermentation products was reported for bioethanol supplemented with 10 g/L glucose). Cultures were (Lomthong et al., 2016) and lactic acid (Okano et al., incubated at 30°C overnight, and 10% (v/v) was 2018). However, scant information exists regarding used as inoculum for wine fermentation. A. aceti vinegar or acetic acid fermentation by raw starch TISTR 354 at 10% (v/v, 108 CFU/mL) was grown on degrading enzyme. One advantage of hydrolysis at a Leum Pua glutinous rice inoculum liquid medium low temperature by rice starch degrading enzyme containing 7.0 g Leum Pua glutinous rice powder, 7.0 (RSDE) is the preservation of enzyme activity in the mL of 95% ethanol and 76.0 mL of distilled water, rice grains. Takahashi et al. (1971) reported that the and incubated at 30°C without shaking for 96 h for presence of α-glucosidase in rice grains increased use as the vinegar starter in acetic fermentation. glucose hydrolysis. Moreover, hydrolysis at low temperature also maintains bioactive compounds Hydrolysis of Leum Pua glutinous rice powder by which are denatured at high temperature. Therefore, raw starch degrading enzyme the production of vinegar from Thai rice was The hydrolysis of Leum Pua glutinous rice (250 investigated in the present work via low temperature g/L) was performed in a 50 mL reaction mixture of saccharification by RSDE from Laceyella sacchari 300 U/mL RSDE produced by L. sacchari LP175 LP175, and reported as the first application of raw in 0.1 M phosphate buffer (pH 6.5) in a 250 mL starch degrading enzyme to produce a healthy product Erlenmeyer flask. Other varieties of brown and through a biotechnological process. black rice including Riceberry, Black Jasmine, and Sangyod were used to compare the yield of sugar Materials and methods syrup production and anthocyanin contents. Reaction mixtures were incubated at 50°C without shaking Substrates for 12 h, and the total soluble solid and anthocyanin Four varieties of brown and black rice (Riceberry, contents of the samples were subsequently Leum Pua glutinous, Black Jasmine and Sangyod) determined. were purchased from the Or Tor Kor fresh market, Bangkok, Thailand. Each substrate was ground to Production of sugar syrup in a 5.0 L glass jar tank powder using an electric grinder and stored in dry Hydrolysis of Leum Pua glutinous rice was up- condition until further use. The total starch assay scaled in a 5.0 L glass tank containing 3.0 L reaction (Megazyme International Ireland, Wicklow, Ireland) mixture of enzyme and Leum Pua glutinous rice was performed by the Cassava and Starch Technology powder (250 g/L). The reaction was incubated at Research Unit (CSTRU) at Kasetsart University. 50°C for 12 h. Samples were taken at time intervals to determine total soluble solid and anthocyanin Microorganism and inoculum preparation contents. Scanning electron microscopy was used to Laceyella sacchari LP175, a thermophilic study the native and digested rice powder with crude filamentous bacterium, was obtained from the enzyme at optimum conditions. All samples were Department of Microbiology, Kasetsart University, washed with pure water, dried at 50°C overnight and Bangkok, Thailand. Raw starch degrading enzyme then examined under a scanning electron microscope production by the LP175 strain was carried out in a (model SU8020, Hitachi, Tokyo, Japan) at 10.0 kV. 3.0 L airlift fermenter using a 2.0 L working volume Hydrolysis products were qualitatively detected by of optimized medium: 4.93 g/L cassava starch, 2.8 g/L thin-layer chromatography (TLC) (Sassaki et al., 2008). A silica gel TLC plate (Merck, 20 × 20 cm) yeast extract, 0.5 g/L K2HPO4, 0.5 g/L MgSO4.7H2O, was coated with a solvent mixture of ethyl acetate:2- and 1.0 g/L CaCl2 (pH 6.5) (Lomthong et al., 2015). Fermentation was operated at 45°C with an aeration propanol:glacial acetic acid:water (4:2:2:1, v/v), rate of 0.5 vvm for 36 h (Lomthong et al., 2016). developed by spraying with a solution of orcinol- Two strains of yeast (Saccharomyces cerevisiae sulfuric acid and then heated at 100°C for 10 min. var. montache and S. cerevisiae var. kyokai) and Glucose and maltose were used as standards. Acetobacter aceti TISTR 354 were obtained from the Department of Applied Microbiology, Institute of Wine fermentation Food Research and Product Development (IFRPD), Sugar syrup from the Leum Pua glutinous Kasetsart University, Thailand and used for wine and rice residue was applied as substrate for wine vinegar fermentation, respectively. The yeast strains fermentation in a medium containing Leum Pua were grown separately in a 500 mL Erlenmeyer glutinous rice, with initial sugar concentration Lomthong, T. and Saithong, P./IFRJ 26(5) : 1515-1523 1517 adjusted to 22°Brix by addition of sucrose powder. Analysis For alcoholic fermentation, 5% (v/v) of both strains was inoculated into the same tank which contained Alcohol and soluble solid contents hydrolysed Leum Pua glutinous rice sugar syrup in An ebulliometer (Dujardin-Salleron, Paris, 1:1 ratio of S. cerevisiae var. kyokai and S. cerevisiae France) was used to assess the total alcohol content var. montache. The fermentation was incubated by measuring the difference in boiling points between at 30°C for 10 d. Fermentation was stopped pure water and the wine (Kocabey et al., 2016). Based when alcohol content exceeded 10% (w/v) as the on the comparison, percentage alcohol (v/v) was optimum concentration for application to the Surface determined as a percentage. Soluble solid contents Culture Fermentation (SCF) process for acetic acid were investigated at 20°C using a refractometer fermentation as described by Saithong et al.