rice wine experiment

• Open access
• Published: 18 February 2021

Fermentation profiling of rice wine produced by Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 newly isolated from Korean fermentation starter

• Minjoo Kim 1 &
• Jeong-Ah Seo   ORCID: orcid.org/0000-0002-0566-1217 1  

Applied Biological Chemistry volume  64 , Article number:  25 ( 2021 ) Cite this article

5850 Accesses

11 Citations

Metrics details

The objective of this study was to determine the fermentation characteristics of rice wine produced by koji inoculated with Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 on moisturized wheat-bran and rice grain. We also compared rice wine samples produced in this study and three commercial Makgeolli . The alcohol content was about 12% higher in the rice wine samples fermented by wheat-bran koji than in the other samples. In all of the samples, the range of pH value was 3.8–4.6 and the total acid was below 0.5. The soluble solid content was highest in the rice wine sample prepared by the wheat-bran R . oryzae KJJ39 koji (15.5°Brix) and overall relatively higher in the samples with wheat-bran koji than rice koji . The content of reducing sugar was twofold higher in rice wine prepared by koji inoculated with R . oryzae KJJ39 than A . oryzae KSS2. The volatile acid content was higher in rice wine produced by the wheat-bran A . oryzae KSS2 koji than the others. In the analyses of five organic acids, A . oryzae KSS2 was found to produce more malic acid and fumaric acid while R . oryzae KJJ39 to do more citric acid, lactic acid and acetic acid. The rice wine sample prepared with the wheat-bran A . oryzae KSS2 koji contained much higher concentration of sucrose, maltose and amino acids. Comprehensively, the results of fermentation profiling suggest that both A . oryzae KSS2 and R . oryzae KJJ39 can be applied to the production of rice wine as a valuable fungal isolate for fermentation start.

Introduction

Makgeolli is a traditional Korean rice wine that has been consumed by Koreans for centuries. Korean rice wine is traditionally brewed using rice (as a starch) and nuruk (as a fermentation starter culture), and it involves a two-step fermentation process. The taste of Makgeolli is determined by a combination of four flavor profiles: sweet, sour, bitter, and astringent. Unlike other alcoholic liqueurs, Makgeolli is rich in nutrients, including (1) vitamin B, which is involved in human metabolism, (2) acetylcholine, which boosts liver function, and (3) organic acids. Makgeolli also contains essential amino acids such as lysine, leucine, and arginine, as well as esters such as ethyl acetate, amyl acetate, and ethyl caproate, which are responsible for the sour taste of the rice wine [ 1 , 2 , 3 , 4 , 5 ]. Besides the nutritional and functional benefits of drinking Makgeolli , the presence of raw yeast gives it a unique taste when compared to other alcoholic beverages [ 4 , 6 , 7 ]. The quality of Makgeolli is usually determined by the alcohol contents, total acid contents, organic acid concentrations and flavor profile, and these factors vary depending on the production and storage conditions [ 8 , 9 ].

Traditional nuruk samples show significant variations of microbial composition that depend on the region, environment and process of production [ 10 , 11 ]. The development and expansion of refined nuruk started in the 1970s, and this has allowed the production of alcohols of uniform quality [ 2 , 6 , 12 , 13 ]. In order to support the increasing consumption and expanding market of Makgeolli , it is crucial to standardize the quality and production process through the standardization of the brewing conditions. It is also important to develop high-quality manufacturing technologies, and to maintain and improve the quality of the rice wine to meet the standards of present-day consumers.

Most of the previous studies on Makgeolli have focused on various topics including the alcohol contents, quality traits, alcohol fermentation [ 1 , 6 , 14 , 15 ], sensory properties [ 2 , 16 , 17 , 18 ] according to the processing method of starch and nuruk and the characteristics of volatile flavors of Makgeolli prepared with different types of nuruk [ 19 , 20 , 21 , 22 ]. Also, there have been a few studies about production of rice wine using a koji or modified nuruk inoculated with a single fungus [ 23 , 24 , 25 , 26 ]. In this study, to determine and compare the fermentation characteristics, we investigated the physiochemical properties of the rice wine samples produced by both wheat-bran koji and rice koji inoculated with Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 which have been selected as high amylolytic enzyme producers in our previous study [ 11 ] and the commercial Makgeolli samples.

Materials and methods

Preparation of commercial makgeolli samples.

The commercial Makgeolli samples were purchased from the shelf within 3 days on the market and stored at − 80 °C until analyzed. The Makgeolli samples were made by three representative manufacturers, all using 100% rice without any food additives. The alcohol content of all samples was 6%, indicated on the label. Analyses have been triplicated with each sample of Makgeolli .

Preparation of wheat-bran and rice koji

Koji was prepared as previously described with an exception of the amount of wheat-bran and rice used [ 13 ]. Briefly, 50 g of steamed rice was distributed in 250 ml polypropylene bottles covered with gauze (autoclaved at 121 °C for 20 min) and inoculated with fungal spore suspension (about 5 × 10 5 spores/g of rice). Wheat-bran koji was prepared with 50 g of wheat-bran (McSun, Dongawon) added to 30 ml of distilled water using the same method to make rice koji . The solid media prepared with wheat-bran and rice were incubated at 30 °C for 24–72 h under a relative humidity of over 70%. The wheat-bran and rice koji were stored at − 20 °C until used. For this study, non-glutinous rice (Gyeonggi Chucheon, Gyeonggi, Korea) was used and prepared. Wheat-bran koji and rice koji were prepared by inoculation of A . oryzae KSS2 and R . oryzae KJJ39 (Table 1 ).

Rice wine fermentation

Rice wine fermentation was performed by the same methods as described [ 13 ]. Rice was soaked in water for 3 h, drained for 30 min, steamed for 90 min and cooled down for 20 min. The water content of the steamed rice was determined to be approximately 30% (w/w). Two hundred-fifty grams of steamed non-glutinous rice, 500 mL of water, 30 g of koji and 1.0 × 10 5 cells/g mash of Saccharomyces cerevisiae (INRA7013, Fermevin, Denmark) were mixed for fermentation. After incubation at 25 ± 3 °C for 24 h, 250 g of steamed non-glutinous rice was added to the mixture and incubated under the same condition. For further fermentation, 500 g of steamed rice and 500 mL of water were added and the samples were incubated at 25 ± 3 °C for 7 days. All samples were stored at − 80 °C. All of the fermentation process were carried out in triplicates for each type of koji .

Rice wine analysis

Ph and total acid.

The pH was measured using a pH meter (Orionstar A211, Thermo scientific, US). The total acid content was measured by titrating 10 mL of sample with 0.1 N NaOH solution until the pH became 8.2. The total acid content was calculated based on the amount of NaOH (mL) and then converted to acetic acid (%) [ 27 ].

Volatile acidity and amino acids

The volatile acids were measured by taking 30 mL of the distillate used for alcohol contents analysis, titrating it with 0.01 N NaOH to pH 8.2–8.4 and then converting it to citric acid. The amino acid was determined by the titration method using phenolphthalein as the indicator. Ten mL of sample was first titrated with 0.1 NaOH to pH 8.2. Subsequently, 5 mL of neutral formalin was added, and the solution was titrated again with 0.1 N NaOH to pH 9.2. The amino acid was calculated as a volume needed for titration after the addition of neutral formalin, and the molecular weight of glycine was used as a conversion value for calculation [ 27 ].

Measurement of soluble-solid and reducing sugar content

Soluble-solid content was measured using a digital refractometer (HI 96801, Hanna Instruments Inc, USA) and recorded in brix units. Reducing sugar was measured using the dinitrosalicylic acid (DNS) method [ 28 ]. Briefly, 1 mL of DNS reagent was added to 1 mL of sample and the mixture was heated in a water bath for 10 min. The solution was then cooled down at room temperature and 3 mL of distilled water was added. The absorbance was measured at 550 nm using a spectrophotometer (MULTISKAN Go, Thermo Scientific, USA). The reducing sugar (%) was determined based on a glucose standard curve.

Measurement of alcohol, organic acid, free sugar and amino acid

Sample preparation for analyses was followed by the previous report [ 4 ]. The Makgeolli samples were prepared by centrifugation at 1000 rpm for 10 min. The supernatant was passed through a Sep-pack C18 cartridge (Waters Co., Milford, MA, USA), followed by filtration using a membrane filter (0.45 µm, Advantec MFS, Inc, Tokyo, Japan) and then analyzed by HPLC (Ultimate3000, Thermo Dinex, Japan). An Aminex HPX-87H column (300 mm × 10 mm, Bio-Rad, USA) was used to analyze the organic acids and alcohol content in the samples, using 20 mM H 2 SO 4 (pH 2.7) as the mobile phase, a flow rate of 0.5 mL/min and an injection volume of 10 μL. Analysis was performed using an RI (ERC, Refractor MAX 520, Japan) and UV detector (210 nm). Analysis of free sugars was performed with a Sugar-pak (300 mm × 6.5 mm, Waters) column under 70 °C by heating, and using water as the mobile phase, 0.5 mL/min as the flow rate and 10 μL as the injection volume. The detector used was Shodex RI-101 (Shodex, Japan). For amino acid analysis, an Inno C18 column (150 mm × 4.6 mm, Younginbiochrom, Korea) was used with the following conditions: a column temperature of 40 °C, flow rate of 1.5 mL/min, injection volume of 0.5 μL and a mobile phase of 40 mM sodium phosphate (pH 7) and distilled water/acetonitrile/methanol (10:45:45, v/v/v %) used in a gradient. Analysis was performed using an Agilent 1260 Infinity fluorescence detector (Agilent, USA).

Statistical analysis

Data processing was performed using the KoreaPlus Statistics (embedded on SPSS Statistics25) on Windows to evaluate statistical differences in the principle components of all samples.

Results and discussion

Alcohol production.

The alcohol content is a major factor that impacts the quality of Makgeolli along with the degree of fermentation. During the fermentation of Makgeolli , ethanol is produced through the degradation of starch by the microorganisms present in nuruk . This means that the alcohol content increases with the duration of fermentation, and the air bubbles caused by the formation of carbon dioxide during the process can act as visual indicators of the degree of fermentation [ 29 ]. The alcohol contents of CA, CB, and CC were 6.0, 5.3, and 4.8%, respectively, which showed a difference up to 1.8% from the product label indicated on (Table 2 ). The rice wine samples produced by koji showed higher alcohol contents than commercial ones, ranging from 12.3–12.6% for AW and RW and 9.9–10.2% for AR and RR. In previous studies, a few isolates of A. oryzae and R. oryzae originated from Korean fermentation starters had been tested for making koji and producing rice wine [ 13 , 25 ]. In those cases, alcohol contents produced by new fungal isolates ranged from 10 to 14%. In this study, the results showed that alcohol production was similar to the previous analyses, and we found that wheat-bran koji produced more alcohol than rice koji regardless of the fungal strains.

pH and total acidity

The pH value is greatly influenced by the types and concentrations of organic acids and other acid-based substances, and it is an important indicator of the progress of fermentation and the production of alcohol in Makgeolli [ 29 ]. The pH values of CA, CB, and CC were in the range of 3.9–4.6, while the ones of AW, RW, AR and RR ranged from 4.0 to 4.4 (Table 2 ). All of the rice wine samples had pH values within the range established by the Liquor Tax Act (pH 3.8–4.4). Acidity is an important factor that affects the flavor and preservation of Makgeolli [ 30 ]. Excessive acidity indicates that abnormal fermentation has occurred, which makes the product undrinkable due to the acidic taste [ 31 ]. On the other hand, the product will be tasteless if the acidity is too low [ 32 ]. In the present study, the total acidity was 0.18–0.26% for CA, CB and CC and 0.27–0.38% for the samples prepared with wheat-bran koji and rice koji (Table 2 ). The Liquor Tax Act dictates that the optimal acidity of Makgeolli is less than 0.5%, indicating that all of the samples analyzed in this study had acceptable acidity.

Soluble-solid and reducing sugar contents

The soluble-solids content of Makgeolli greatly affects its sweetness. The CC sample contained a soluble-solid content of 11.10°Brix, which was about twofold higher than those of CA and CB which were 3.73 and 3.66°Brix, respectively (Table 2 ). Rice wine prepared with wheat-bran koji (AW and RW) showed soluble-solids contents of 13.13 and 15.47°Brix respectively, while the samples prepared with rice koji (AR and RR) had soluble-solids contents of 9.27 and 12.03°Brix, respectively. Furthermore, rice wine prepared with wheat-bran koji and rice koji showed threefold higher soluble-solid content than that of CA, which was supplemented with iso-malto-oligosaccharide during its industrial production. A previous study reported that the soluble-solids contents of other commercial Makgeolli were in the range of 2.9–4.7°Brix [ 4 ] less than the ones of the commercial samples used in this work (3.7–11.1°Brix). Their study concluded that differences in soluble-solids content are probably due to differences in the types and quantity of raw material used, the fermentation starter, and the conditions of fermentation. In the present study, wheat-bran koji was more effective as a fermentation starter than rice koji for both fungal strains used.

Glucose, fructose, and maltose are reducing sugars. During the fermentation of Makgeolli , amylase digests starch into smaller carbohydrates, eventually breaking it down into glucose molecules. Glucose is an important component used as the substrate for alcohol fermentation that greatly impacts the acidity, taste, and alcohol content of Makgeolli [ 29 ]. In this study, the reducing-sugars content of CA and CB was 0.29%, while that of CC was more than double, at 0.73% (Table 2 ). The rice wine samples inoculated with A. oryzae KSS2 (AW and AR) showed reducing-sugars contents of 0.29% and 0.32%, respectively, while those inoculated with R.   oryzae KJJ39 showed significantly higher values of 0.76% (RW) and 0.67% (RR). The results suggest that R.   oryzae KJJ39 was better than A. oryzae in production of reducing sugars.

Volatile acid concentration and amino acid ratio

Acetic acid is the predominant volatile acid in Makgeolli , but in excessive amounts it imparts an unpleasant odor similar to that of vinegar. This indicates the need to control the amount of acetic acid produced during the fermentation process of Makgeolli [ 16 ]. In this study, the range of the volatile acid concentrations was 24.8–38.8 ppm for the commercial Makgeolli samples, 51.2–60.4 ppm for rice wine prepared with wheat-bran koji , and 34.4–44.0 ppm for rice wine prepared with rice koji (Table 2 ). A previous study produced similar results, with volatile acid contents of less than 40 ppm for rice wine prepared with rice koji and less than 80 ppm for those with added plant material [ 8 ].

Amino acids play an important role in moderating the savory taste of Makgeolli . However, a high concentration of amino acids will impart a greasy taste and reduce the quality of the rice wine [ 17 ]. In the present study, the amino acid content was 0.06–0.09% in commercial Makgeolli , 0.10–0.26% in rice wine prepared with wheat-bran koji and 0.08–0.25% in rice wine prepared with rice koji (Table 2 ), shown to be similar to a previous report [ 8 ].

Organic acid concentration

The organic acid concentrations of the rice wine samples are listed in Table 3 . The citric acid concentrations varied markedly among the commercial Makgeolli samples, being 0.95 mg/mL in CA, 0 mg/mL in CB, and 0.67 mg/mL in CC. Rice wine prepared with wheat-bran koji showed citric acid concentrations in the range of 0.19–0.25 mg/mL, while they were 0.22–0.26 mg/mL in rice wine prepared with rice koji . Park et al. reported that the citric acid concentration differed significantly between different commercial Makgeolli samples, which they attributed to the type of imported rice and the quantity of rice used during the production of the Makgeolli [ 4 ]. The malic acid concentrations also differed greatly among the commercial Makgeolli samples, with values ranging from 0 to 0.15 mg/mL. For rice wine prepared with wheat-bran koji and rice koji , the malic acid concentration was 0.75 mg/mL for AW, 0.43 mg/mL for RW, 0.96 mg/mL for AR, and 0.13 mg/mL for RR. The fumaric acid concentration was 0–0.02 mg/mL in commercial Makgeolli , 0.19 mg/mL in AW, 0.09 mg/mL in RW, 0.17 mg/mL in AR, and 0.03 mg/mL in RR. These results showed that the concentrations of malic acid and fumaric acid were 2–5 times higher in the samples prepared with A.   oryzae KSS2 than in those inoculated with R.   oryzae KJJ39. The lactic acid concentrations also differed markedly among the commercial Makgeolli samples, at 0.29 mg/mL in both CA and CC, but 2.01 mg/mL in CB. Rice wine prepared with wheat-bran koji showed lactic acid concentrations of 0.77 mg/mL (AW) and 1.02 mg/mL (RW), while the AR and RR samples prepared with rice koji had lactic acid concentrations of 0.51 and 0.86 mg/mL, respectively.

It has been reported previously [ 6 ], that as fermentation progresses, the concentrations of lactic acid and succinic acid increase significantly to become the most abundant organic acids in Makgeolli . Organic acids are important ingredients that give a sour taste to the rice wine and play an important role in enhancing its taste and aroma if they are present in trace amounts. However, excessive acetic acid can alter the taste of the rice wine, reducing its quality [ 5 ]. In the present study, the commercial Makgeolli samples contained 0.12–0.30 mg/mL acetic acid, those prepared with wheat-bran koji contained 0.28 mg/mL (AW) and 0.55 mg/mL (RW), and those prepared with rice koji contained 0.14 mg/mL (AR) and 0.58 mg/mL (RR). Overall, the acetic acid concentrations were 2–3 times higher for the samples inoculated with R.   oryzae KJJ39 than for those prepared with A.   oryzae KSS2.

Free sugar concentration

The free sugar concentrations of the samples are listed in Table 4 . Glucose is a major sugar in Makgeolli that plays an important role in fermentation, since the degradation of starch into glucose allows the production of alcohol. The present study did not detect fructose, and only detected maltose in AW (2.78 mg/mL) and RR (0.61 mg/mL). The glucose concentrations were high in CC (65.51 mg/mL), RW (42.39 mg/mL) and RR (44.99 mg/mL) and low in CA (0.69 mg/mL), AW (19.64 mg/mL) and AR (1.18 mg/mL). During the fermentation of Makgeolli , starch is broken down into monosaccharides such as glucose through the action of amylase, which is produced by molds. In general, the concentration of glucose becomes high during early stages of fermentation, and then reduces as the growth of yeast and lactic acid bacteria progresses [ 8 ].

Amino acids

The concentrations of total amino acids and essential amino acids are presented in Table 5 . In the commercial Makgeolli samples, the total amino acid concentrations were 0.74–2.23 mg/mL and those of essential amino acids ranged from 0.04 to 0.80 mg/mL. Rice wine prepared with wheat-bran koji had total amino acid concentrations in the range of 1.82–9.62 mg/mL and essential amino acid concentrations of 0.64–2.90 mg/mL; the corresponding concentrations in rice wine prepared with rice koji were 0.66–6.50 and 0.22–1.77 mg/mL, respectively.

The essential amino acids (threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine) represented 27.3–35.8% of the total amino acids in all of the analyzed samples except for CA. Essential amino acids play important roles in different biological functions, such as lysine being essential for the synthesis of tissue, and methionine playing an important role in preventing fatty liver and by promoting phospholipids in the liver. Among the essential amino acids, lysine was measured much higher in AW (0.47 mg/mL) and AR (0.35 mg/mL) than the commercial products CC (0.11 mg/mL) and CA (0.01 mg/mL). Methionine was also included about hundred times higher in AW (0.13 mg/mL) and AR (0.10 mg/mL) than CA. This result suggests that A. oryzae KSS2 may better function in production of lysine and methionine rather than R.   oryzae KJJ39.

Amino acids are not only important as nutrients in Makgeolli , but they are also precursors for aroma: threonine, glycine, alanine, and serine produce a sweet taste; glutamic acid gives a umami taste; aspartic acid gives a sour taste; and leucine, isoleucine, lysine, and tyrosine give a bitter taste to the rice wine. Threonine, glycine, alanine, and serine involved in sweetness and were measured by higher level in AW (0.24–0.29 mg/mL) rather than other samples. Proline is the only amino acid that is soluble in alcohol, and it produces a pleasant aroma when heated with sugars [ 33 ]. AW (0.73 mg/mL) and AR (0.50 mg/mL) contained large amounts of proline. The amino acid concentration was 5–10 times higher in rice wine inoculated with A.   oryzae KSS2 and R.   oryzae KJJ39 than in the commercial Makgeolli samples. The rice wine samples inoculated with A.   oryzae KSS2 showed concentrations of amino acids that were 5 times higher than those in samples inoculated with R.   oryzae KJJ39 . Rice wine prepared with wheat-bran koji had a twofold higher concentration of amino acids compared with that prepared with rice koji . The results of this work will allow production of Makgeolli with higher nutritional and sensory qualities.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Kim HR, Jo SJ, Lee SJ, Ahn BH (2008) Physicochemical and sensory characterization of a Korean traditional rice wine prepared from different ingredients. Korean J Food Sci Technol 40:551–557

CAS   Google Scholar  

Lee TS, Han EH (2001) Volatile flavor components in mash of Takju prepared by using Aspergillus oryzae Nuruk s. Korean J Food Sci Technol 33:366–372

Google Scholar  

Lee SJ, Kim JH, Jung YW, Park S, Shin WC, Park CS, Hong S, Kim GW (2011) Composition of organic acids and physiological functionality of commercial Makgeolli . Korean J Food Sci Technol 43:206–212

Article   Google Scholar  

Park CW, Jang SY, Park EJ, Yeo SH, Kim OM, Jeong YJ (2011) Comparison of quality characteristics of commercial Makgeolli type in south Korea. Korean J Food Preserv 18:884–890

Woo SM, Shin JS, Seong JH, Yeo SH, Choi JH, Kim TY, Jeong YJ (2010) Quality characteristics of brown rice Takju by different Nuruk s. J Korean Soc Food Sci Nutr 39:301–307

Article   CAS   Google Scholar  

Park CS, Lee TS (2002) Quality characteristic of Takju prepared by wheat flour Nuruks . Korean J Food Sci Technol 34:296–302

Song JC, Park HJ (2003) Takju brewing using the uncooked germed brown rice at second stage mash. J Korean Soc Food Sci Nutr 32:847–854

Ha GJ, Kim NK, Je HJ, Choi SY, Seol HK, Hong GP, Lee SD (2015) Quality characteristics of Makgeolli produced in Gyeongnam province. J Agric Life Sci 49:247–257

Huh CK, Lee JW, Kim YD (2012) Quality characteristics of rice wine according to the rice wine seed mash with lactic acid concentration. Korean J Food Preserv 19:933–938

Lee WK, Kim JR, Lee MW (1987) Studies on the changes in free amino acids and organic acids of Takju prepared with different Koji strains. J Korean Agric Chem Soc 30:323–327

Carroll E, Trinh TN, Son H, Lee YW, Seo JA (2017) Comprehensive analysis of fungal diversity and enzyme activity in nuruk , a Korean fermenting starter, for acquiring useful fungi. J Microbiol 55:357–365

Kim HS, Yu TS (2000) Volatile flavor components of traditional Korean Nuruk Fungi. Korean J Appl Microbiol Biotechnol 28:303–308

Yang S, Choi SJ, Kwak J, Kim K, Seo M, Moon TW, Lee YW (2013) Aspergillus oryzae strains isolated from traditional Korean Nuruk : Fermentation properties and influence on rice wine quality. Food Sci Biotechnol 22:425–432

Han EH, Lee TS, Noh BS, Lee DS (1997) Quality characteristics in mash of Takju prepared by using different Nuruk during fermentation. Korean J Food Sci Technol 29:555–562

Han EH, Lee TS, Noh BS, Lee DS (1997) Volatile flavor components in mash of Takju prepared by using different Nuruk s. Korean J Food Sci Technol 29:563–570

Jeong ST, Kwak HJ, Kim SM (2013) Quality characteristics and biogenic amine production of Makgeolli brewed with commercial Nuruks . Korean J Food Sci Technol 45:727–734

Joung EJ, Park NS, Kim YM (2004) Studies Korean Takju using the by-product of rice milling. Korean J Food Nutri 17:199–205

Kim IH, Park WS, Koo YJ (1996) Comparison of fermentation characteristics of Korean traditional alcoholic beverage with different input step and treatment of rice and Nuruk . Korean J Diet Cult 11:339–348

Kim SH, Mun JY, Kim SY, Yeo SH (2018) Quality characteristics of glutinous rice- Makgeolli fermented with Korean yeast (SC Y204 and Y283) isolated from Nuruk . Korean J Food Preserv 25:874–884

Kwon YH, Lee AR, Kim JH, Kim HR, Ahn BH (2012) Changes of physicochemical properties and microbial during storage of commercial Makgeolli . Korean J Mycol 40:210–214

Lee Y, Yi H, Hwang KT, Kim DH, Kim HJ, Jung CM, Choi YH (2012) The qualities of makgeolli (Korean rice wine) made with different rice cultivars, milling degrees of rice, and Nuruks. J Korean Soc Food Sci Nutr 41:1785–1791

Lee JE, Lee AR, Kim HR, Lee EJ, Kim TW, Shin WC, Kim JH (2017) Restoration of traditional Korean Nuruk and analysis of the brewing characteristics. J Microbiol Biotechnol 27:896–908

So MH, Lee YS, Han SH, Noh WS (1999) Analysis of major flavor compounds in Takju mash brewed with a modified Nuruk. Korean J Food Nutr 12:421–426

So MH, Lee YS, Noh WS (1999) Improvement in the quality of Takju by a modified Nuruk. Korean J Food Nutr 12:427–432

Seo WT, Cho HK, Lee JY, Kim B, Cho KM (2012) Quality characteristics of wheat-rice Makgeolli by making of rice Nuruk prepared by Rhizopus oryzae CCS01. Korean J Microbiol 48:147–155

Son EY, Lee SM, Kim M, Seo JA, Kim YS (2018) Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae . Food Res Int 109:596–605

Korea National Tax Service Liquor Analysis of liquor regulatory (2010) National Tax Service Technical Service Institute, Korea: 35-53

Luchsinger WW, Cornesky RA (1962) Reducing power by the dinitrosalicylic acid method. Anal Biochem 4:346–347

Kim JY, Sung KW, Bae HW, Yi YH (2007) pH, acidity, color, reducing sugar, total sugar, alcohol and organoleptic characteristics of puffed rice powder added Takju during fermentation. Korean J Food Sci Technol 39:266–271

Lee SM, Lee TS (2000) Effect of roasted rice and defatted soybean on the quality characteristics of Takju during fermentation. J Nat Sci 12:71–79

Lee JS, Lee TS, Noh BS, Park SO (1996) Quality characteristics of mash of Takju prepared by different raw materials. Korean J Food Sci Technol 28:330–336

Lee WY, Rhee CH, Woo CJ (2004) Changes of quality characteristics in brewing of chungju ( sambaekju ) supplemented with dried persimmon and cordyceps sinensi s. Korean J Food Preserv 11:240–245

Lee MO, Youn JB, Park SA, Lee YS, Bin JH, Lee SH (2002) Investigation on the quality characteristics of Sansung Takju compared with commercial Takju. Rep Busan Inst Health Environ 12:48–62

Download references

Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agricultural Microbiome R&D Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA-918010-4). The authors thank S. Y. Park for English correction and editing.

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agricultural Microbiome R&D Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA-918010-4).

Author information

Authors and affiliations.

School of Systems Biomedical Science, Soongsil University, Seoul, 06978, Republic of Korea

Minjoo Kim & Jeong-Ah Seo

You can also search for this author in PubMed   Google Scholar

Contributions

MK organized the experiment, collect data and summarize a main finding for the manuscript. JAS corrected and wrote some part of the first draft manuscript and manage a final full manuscript as a corresponding author. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jeong-Ah Seo .

Ethics declarations

Competing interests.

The authors declare that they have no competing interests.

Additional information

Publisher's note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Kim, M., Seo, JA. Fermentation profiling of rice wine produced by Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 newly isolated from Korean fermentation starter. Appl Biol Chem 64 , 25 (2021). https://doi.org/10.1186/s13765-020-00582-2

Download citation

Received : 27 August 2020

Accepted : 15 December 2020

Published : 18 February 2021

DOI : https://doi.org/10.1186/s13765-020-00582-2

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Aspergillus oryzae KSS2
• Fermentation starter
• Inoculation
• Profiling wheat-bran koji
• Rhizopus oryzae KJJ39

ORIGINAL RESEARCH article

Comparison of the fermentation activities and volatile flavor profiles of chinese rice wine fermented using an artificial starter, a traditional jiuyao and a commercial starter.

• Department of Food Science and Technology, Shanghai Institute of Technology, Shanghai, China

In this study, an artificial starter culture was prepared using the core microbial species of JIUYAO to produce Chinese rice wine (CRW). The fermentation activity and flavor profiles of CRW samples fermented with traditional JIUYAO, a commercial starter culture, and our artificial starter culture were compared. The optimal protectant combination for lyophilization of the artificial starter was established as 15.09% skim milk, 4.45% polyethylene glycol, 1.96% sodium glutamate, and 11.81% maltodextrin. A comparative analysis revealed that the ethanol content of the three CRW samples was similar. The total acid content of the CRW sample fermented with the artificial starter (7.10 g/L) was close to that of the sample fermented with JIUYAO (7.35 g/L), but higher than that of the sample fermented with the commercial starter (5.40 g/L). An electronic nose analysis revealed that the olfactory fingerprints of the CRW samples fermented with JIUYAO and the artificial starter resembled each other. For both above mentioned samples, the flavor profiles determined by gas chromatography–mass spectrometry indicated some differences in the variety and content of the aroma compounds, but the key odorants (odor activity values ≥1), such as isoamyl acetate, ethyl acetate, phenyl alcohol, and isoamyl alcohol, were similar.

Introduction

Chinese rice wine (CRW), which has a high nutritional value and distinctive flavor, has been consumed for centuries ( Xu et al., 2015 ; Huang Z. R. et al., 2018 ). CRW includes many renowned types, such as Shaoxing, Jimo, and Fujian rice wine ( Jiao et al., 2017 ). The best-known CRW, Shaoxing rice wine, is generally produced using glutinous rice, traditional JIUYAO, and wheat qu ( Chen et al., 2013 ). JIUYAO is a mixed starter culture that mainly includes bacteria, molds, and yeast and is responsible for the starch saccharification and fermentation in Shaoxing rice wine brewing ( Liu et al., 2018 ). The microorganisms in JIUYAO are believed to play a crucial role in the fermentation activity and unique flavor formation of Shaoxing rice wine ( Xie et al., 2007 ). Zhao et al. (2020) revealed that Proteobacteria and Firmicutes are the dominant bacterial phyla in JIUYAO. Our previous study ( Chen et al., 2020 ) found that only five core species, two from the Weissella genus and one each from the Pediococcus , Saccharomycopsis , and Rhizopus genera, play a key role in the flavor formation and fermentation activity of CRW.

Currently, commercial starters that mainly include yeast and Rhizopus are widely used in the industrial brewing of CRW ( Ferrer-Gallego et al., 2014 ). Although the fermentation period of CRW brewed with commercial starters is comparable to that of CRW brewed with traditional JIUYAO ( Huang et al., 2019 ), the flavor of the former is inferior due to the limited microbial variety in the commercial starters ( Liu et al., 2019 ). However, as traditional JIUYAO is usually handcrafted, its limitation is the instability of the resulting CRW’s quality and flavor profiles across batches ( Zhao and Zhang, 2010 ). Therefore, it would be of great significance to develop an artificial starter culture as a replacement for traditional JIUYAO that could yield CRW batches of high quality and uniform flavor.

A microbial consortium selected as a starter culture should be able to withstand subsequent fermentation. Therefore, lyophilization techniques and low temperatures (refrigeration or freezing) are usually used to stabilize the starter culture ( Abadias et al., 2001 ; Pradelles et al., 2009 ). Lyophilization is a simple technique to maintain a high number of viable microorganisms in a starter culture inoculum in powder form, as it involves few procedural steps. However, this preservation process can cause cell damage or lead to cell death ( Ale et al., 2015 ). Cell mortality during lyophilization can be minimized by optimizing freeze-drying conditions and using lyoprotectants ( Cerrutti et al., 2000 ; Hubálek, 2003 ), such as proteins, skim milk, sugars, and other biopolymers ( Coulibaly et al., 2016 ). Although freeze-dried bacterial powder with protectants has been used as a starter culture ( Chen H. et al., 2015 ), its use in place of traditional JIUYAO for CRW brewing has not been documented.

In this study, an artificial starter culture was prepared by combining the core microorganisms from traditional JIUYAO that contribute to the unique flavor of CRW, and then freeze-drying them to powder form using a lyoprotectant combination optimized through a response surface optimization experiment. The fermentation activity and flavor profile of the CRW samples fermented with this artificial starter were compared with those of samples fermented with traditional JIUYAO and a commercial starter culture. The use of such an artificial starter can improve and standardize the fermentation activity and aroma quality of CRW, and further promote the industrial production of traditional CRW.

Materials and Methods

Jiuyao, starter culture and lyoprotectants.

JIUYAO samples were obtained from Zhejiang Tapai Shaoxing Rice Wine Co., Ltd., Shaoxing city, Zhejiang province, China. The samples were refrigerated at 4°C before being transported to the laboratory. A commercial starter culture composed of Saccharomyces cerevisiae and Rhizopus oryzae was purchased from a commercial yeast and yeast extract manufacturer in China. Five microbial species, namely Pediococcus pentosaceus (CCTCC M 2019323), Weissella cibaria (BNCC206838), W. confusa (CICC23465), Saccharomycopsis fibuligera (CCTCC M 2019324), and R. arrhizus (CCTCC M 2019325), were selected and isolated as the core functional microbes from the JIUYAO samples, due to their key role in the fermentation activity and flavor formation of CRW, as determined in our previous study ( Chen et al., 2020 ). To ensure that the samples had the ability to produce ethanol, S. cerevisiae (NKCCMR NK3. 00156) was also added to the artificial starter ( Chen et al., 2020 ). The isolated strains were stored at −80°C and used after reactivation by successive subcultures in steamed rice. Briefly, for activation, W. cibaria , W. confusa , and P. pentosaceus (2% vol/vol) were inoculated into MRS broth (Merck, Darmstadt, Germany) and incubated at 37°C for 18 h, whereas S. fibuligera , S. cerevisiae , and R. arrhizus (2% vol/vol) were inoculated in MEB liquid medium (Merck, Darmstadt, Germany) and incubated at 30°C for 24 h. The inoculum concentrations of the individual core bacterial species were determined according to the amount of JIUYAO added to the CRW, the total number of bacterial and fungal colonies in the JIUYAO, and the abundance of core microbial species in JIUYAO as described in our previous study ( Chen et al., 2020 ). The final concentrations of the core microbes that were combined to form the artificial starter culture were P. pentosaceus (8.6 × 10 3 CFU/g), S. fibuligera (9.6 × 10 3 CFU/g), R. arrhizus (10.6 × 10 2 CFU/g), W. cibaria (2.2 × 10 3 CFU/g), W. confuse (2.2 × 10 3 CFU/g), and S. cerevisiae (2.0 × 10 4 CFU/g).

For use as lyoprotectants, skim milk was purchased from Fonterra Ltd. (New Zealand), and polyethylene glycol, sodium glutamate, and maltodextrin of food grade were purchased from WanBang Co. Ltd. (Zhengzhou, Henan Province, China).

Freeze-Drying of the Starter

All of the protectants used in the experiment were dissolved in distilled water to obtain various concentrations. They were sterilized at 115°C for 15 min and stored at 4°C until use.

Different concentrations of the protectant solutions were added to the culture pellets of the core microbes in the ratio 1:1 (vol/vol). After mixing, the samples were pre-frozen at −80°C for 2 h, placed in a vacuum freeze-dryer at −80°C and 0.162 mbar vacuum for 48 h, and finally stored at −20°C until use ( Qi et al., 2017 ).

The lyophilization survival factor ( SF L ) was calculated following the formula given by Hubálek (2003) :

where CFU/mL initial is the number of viable cells before lyophilization, and CFU/mL final is the number of viable cells after lyophilization.

Experimental Design for Optimization of the Lyoprotectant Composition

A lyoprotectant is usually added to the target solution of cells before the freeze-drying process. It adds a matrix around the cells that protects them from drying and freezing and increases their survival ability ( Abadias et al., 2001 ). Ten lyoprotectants were selected and mixed thoroughly with the core microbes, and the survival factors of the microbes were determined as an indicator of their survival in our preliminary study (data not shown). Finally, four lyoprotectants with the best protective effects were selected for our experiment: skim milk, polyethylene glycol, sodium glutamate, and maltodextrin. These selected lyoprotectants were diluted to different concentration gradients (skim milk and maltodextrin: 5, 10, 15, 20 and 25%; polyethylene glycol: 1, 3, 5, 7 and 9%; sodium gluconate: 0.5, 1, 1.5, 2 and 2.5%), and their optimal concentrations were determined based on the cell survival factor.

The lyoprotectant composition was further optimized using a four-factor, three-level Box–Behnken design and three levels of the N = 27 test with Y (lyophilization survival rate) as the response value. The factor levels are shown in Table 1 . The following polynomial equation was used:

Table 1. Experimental factor levels of the Box–Behnken design.

where Y is the predicted response; A, B, C, and D are independent variables representing the concentration of the four protectants skim milk, polyethylene glycol, sodium glutamate, and maltodextrin, respectively; a 0 is the second-order reaction constant; a 1 , a 2 , a 3 , and a 4 are the linear coefficients; a 11 , a 22 , a 33 , and a 44 are the quadratic coefficients; and a 12 , a 13 , a 14 , a 23 , a 24 , and a 34 are the interaction coefficients ( Crowell and Ough, 1979 ).

CRW Brewing

The main steps in CRW brewing are rice soaking, steaming, cooling, starter addition, and fermentation. Briefly, glutinous rice (100 g) was soaked in 100 mL water for 12 h at 25°C. After steaming the soaked rice for 20 min and then cooling it to 25–30°C, rice fermentation was initiated by adding JIUYAO, the artificial starter, or the commercial starter at a final concentration of 0.002 g/g steamed rice. The fermentation process was performed at 29°C for 30 h. The acid content, ethanol content, and saccharification power were determined as described in our previous study ( Chen et al., 2020 ). All chemical determination experiments were performed in triplicate.

Flash GC Electronic Nose Detection

A HERACLES flash GC electronic nose (Alpha M.O.S., Toulouse, France) equipped with an MXT-5 column and an MXT-1701 column was used for the aroma analysis of the CRW samples. This instrument can perform a complete data analysis owing to its integration with classical gas chromatography (GC) functionalities and electronic nose (e-nose) olfactory fingerprint software.

Briefly, 5 mL of each CRW sample was added to a separate 20-mL vial and incubated at 25°C for 30 min. Hydrogen was circulated at a constant flow rate of 1 mL/min, and 5 mL of headspace gas was injected into the GC port at 200°C. The temperature changes in the GC column were as follows: 50°C for 2 s, a 1°C/s ramp to 80°C, and then a 3°C/s ramp to 250°C with a 15 s hold. The temperature of the detector was 260°C, and each sample was analyzed five times.

Volatile Compound Analysis

The volatile compound profiles of the CRW samples were analyzed using the headspace solid-phase microextraction (HS-SPME)/gas chromatography–mass spectrometry (GC-MS) approach ( Yu et al., 2019b ). Briefly, 5 mL of each CRW sample was added to 20 μL of internal standard (2-octanol, 410 mg/L) in a 15-mL headspace glass vial. A fiber (50 μm DVB/CAR/PDMS, Supelco Inc., Bellefonte, Pennsylvania, United States) was exposed to the headspace of the glass vial for 50 min at 50°C.

An Agilent 7890 GC instrument was coupled to a 5973C MS detector (Santa Clara, CA, United States). A capillary HP-Innowax column from Agilent Technologies (60 m × 0.25 mm × 0.25 μm) was used to perform the chromatographic separation. After extraction, the fiber was immediately introduced into the GC instrument, and desorption was performed at 250°C for 5 min. The temperature changes were as follows: the initial temperature was maintained at 40°C, increased to 120°C at a rate of 3°C/min, held for 5 min, and then increased to 200°C at a rate of 3°C/min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The transfer line temperature was 250°C. The mass spectrometers were operated in the electron ionization mode at 70 eV, with a scan range of m/z 30–450.

The compounds were identified by comparing their retention indices (RIs) with those reported in the literature and matching their MS spectra with those in the NIST 11 database. The RIs were determined in relation to those of the C 5 –C 30 alkane standards (Sigma-Aldrich, St. Louis, MO, United States). To evaluate the sensory contributions of the compounds to the flavor of the CRW samples, their odor activity values (OAVs) were obtained, given by the ratio of the compound concentration in the sample to the threshold concentration in water ( Van Gemert, 2003 ).

Statistical Analysis

XLSTAT version 7.5 (Addinsoft, New York, NY, United States) was used to analyze the GC-MS data. Design Expert software (version 9, Stat-Ease Inc., Minneapolis, MN, United States) was used for the regression and graphical analysis of the experimental data. The optimal values of the four protectants were calculated using response surface methodology. The regression equations of the models were evaluated using the F -test for the analysis of variance. A principal component analysis was performed using WinMuster version 1.6.2 (Alpha M.O.S., Toulouse, France). Origin version 9.0 (Origin Lab Inc., Hampton, MS, United States) and SPSS version 19.0 (SPSS Inc., Chicago, IL, United States) were used for further data analysis.

Results and Discussion

Effects of lyoprotectants on the survival factor of the artificial starter after freeze-drying.

Lyophilization has several limitations, such as the formation of ice crystals, altered permeability of the cell membrane, and denaturation and inactivation of sensitive proteins ( Yang et al., 2012 ; Lbg et al., 2020 ). The role of a lyoprotectant is to prevent these adverse effects. Skim milk, polyethylene glycol, sodium glutamate, and maltodextrin were selected as the protective agents for the artificial starter culture prepared in this study. The results of single-factor experiments indicated that with 15% skim milk, 5% polyethylene glycol, 2% sodium glutamate, or 10% maltodextrin as individual protective agents during freeze-drying, the maximum survival rate of the artificial starter culture was 85.55, 80.94, 83.83, or 85.01%, respectively ( Figure 1 ). The survival factor of the artificial starter gradually improved with increasing concentrations of the protective agents, but after a certain concentration, the survival rate remained unchanged or slowly decreased. This phenomenon was in line with the results reported by Chen et al. (2019) , who found that extremely high concentrations of protective agents accelerated the repolymerization of proteins in the cells, resulting in poor survival of the artificial starter.

Figure 1. Effects of different concentrations of four lyoprotectants on the survival factor of the artificial starter culture. (A) Sodium glutamate, (B) polyethylene glycol, (C) skim milk, and (D) maltodextrin.

Predictive Modeling for the Concentration of Protective Agents

To further improve the survival factor of the microbes in the artificial starter, a predictive model was established for the optimal protectant combination comprising skim milk, polyethylene glycol, sodium glutamate, and maltodextrin. Based on the combination of the Box–Behnken design and the response surface method, an empirical quadratic model was selected to establish the correlation between the independent variables and the response of the survival factor, as follows:

The F -value of the model was 35.22, indicating that this model was significant. The p -values of the lack of fit ( p = 0.3293) and the model ( p < 0.0001) ( Table 2 ) indicated that the actual corresponding survival factor values exhibited a good fit with this model.

Table 2. Regression model analysis of variance.

The effects of the tested factors on the survival factor were visualized in the response surfaces ( Figure 2 ). The interaction terms of the concentrations of skim milk, polyethylene glycol, sodium glutamate, and maltodextrin demonstrated statistical significance ( p < 0.0001). The survival factor peaked at 0.942 when the concentrations of skim milk, polyethylene glycol, sodium glutamate, and maltodextrin were 15.09, 4.45, 1.96, and 11.81%, respectively. Using this combination, the survival factor increased significantly by approximately 10% relative to the use of single protective agents before optimization. This suggests that the established predictive model could effectively predict the survival factor. Hence, an artificial starter was established with the optimal protectant combination of 15.09% skim milk, 4.45% polyethylene glycol, 1.96% sodium glutamate, and 11.81% maltodextrin. To further verify the predictive value of the response surface optimization, three repeat experiments were performed using the optimal protectant combination. The average value of the survival factor of the artificial starter was 0.931, and the fitting rate of the predicted value was 98.83%, indicating that the predicted value and the actual value had a good fit. Together, these data suggest that the determined optimal protectant composition can significantly improve the survival factor of the artificial starter.

Figure 2. Response surface and contour plots describing the interactive effects of (A) skim milk + polyethylene glycol, (B) skim milk + sodium glutamate, (C) skim milk + maltodextrin, (D) polyethylene glycol + sodium glutamate, (E) polyethylene glycol + maltodextrin, and (F) sodium glutamate + maltodextrin on the survival factor of the artificial starter culture.

Fermentation Activity Analysis of the CRW Samples Brewed With Different Starters

We compared the fermentation activities of the three CRW samples, each brewed with either traditional JIUYAO, the commercial starter, or the artificial starter prepared with the optimal lyoprotectant composition obtained by the response surface method. Saccharification capacity is a key factor that significantly influences wine fermentation ( Yao et al., 2018 ). JIUYAO showed the highest saccharification capacity at 290 ± 3.2 mg/g h, followed by the artificial starter (275 ± 5.3 mg/g h), while the commercial starter showed the lowest capacity (200 ± 4.6 mg/g h). The fermentation activity (ethanol and acid content) of the three CRW samples during fermentation is shown in Figure 3 . As CRW is mostly fermented in an open environment, rapid growth of the yeast strains in the starter is required to produce ethanol at the initial stage to inhibit bacterial overgrowth and avoid spoilage ( Wu et al., 2015 ; Yang et al., 2018 ). The changes in ethanol content during fermentation were similar across the three samples, suggesting comparable ethanol production capacities of all three starter cultures. The total acidity of the wines brewed with JIUYAO and the artificial starter were similar, and were higher than that of the wine brewed with the commercial starter throughout the whole fermentation process. These results suggest that the fermentation activity of the CRW sample fermented with the artificial starter was comparable to that of the sample fermented with JIUYAO, but more vigorous than that of the sample fermented with the commercial starter.

Figure 3. Fermentation performance results of the three Chinese rice wine samples fermented with three different starter cultures. (A) Changes in the total acid contents during the fermentation process. (B) Changes in the ethanol contents during the fermentation process.

Electronic Nose Measurements

Combining the high-efficiency separation ability of GC and the biological simulation of the sense of smell, the electronic nose can provide a comprehensive aroma profile of volatile flavor compounds ( Wu et al., 2012 ). As shown in Figure 4 , the flavor profile radar chart of the CRW samples brewed with the three different starters displays the data intuitively. The overall aroma peak appearance of the samples brewed with JIUYAO and the artificial starter is relatively similar, whereas the peak areas are reduced at multiple positions for the sample fermented with the commercial starter, indicating an overall reduction in aroma intensity. To enable better data discrimination between the three samples, a principal component analysis was performed to identify patterns associated with their individual components ( Figure 5 ). The principal components of the CRW samples fermented with the artificial starter and JIUYAO were closer and overlapped in the same quadrant, suggesting similar aroma profiles, whereas those of the CRW sample brewed with the commercial starter appeared in a different quadrant. These results indicate similarities in the aroma profiles between the CRW samples brewed with the artificial starter and JIUYAO, but some differences in the CRW sample brewed with the commercial starter. These differences may be ascribed to the microbial species composition. Five core species isolated from JIUYAO formed the functional microbial species of the artificial starter culture, whereas the commercial starter contained only two functional microbial species, namely S. cerevisiae and R. oryzae .

Figure 4. Flavor profile radar chart of three Chinese rice wine samples brewed with three different starter cultures. (A) MXT-5-FID, (B) MXT-1701-FID.

Figure 5. Principal component analysis of the three Chinese rice wine samples brewed with three different starters.

Volatile Flavor Compounds of the CRW Samples Fermented With Different Starters

In total, 51 flavor compounds, including 27 esters, 8 alcohols, 1 aldehyde, 4 phenols, 2 ketones, and 5 acids, were detected in the three CRW samples by HS-SPME/GC-MS ( Table 3 ). A total of 50, 39, and 20 aroma compounds were identified in the CRW samples fermented with JIUYAO, the artificial starter, and the commercial starter, respectively.

Table 3. Relative contents of volatile compounds in the Chinese rice wine samples fermented by different starter cultures (μg/kg, n = 3), as identified by HS-SPME/GC-MS.

Esters are the most important and common volatile aroma compounds that impart floral and fruity sensory properties to wine ( Huang L. et al., 2018 ), and can be synthesized by yeast and other microorganisms during fermentation ( Comuzzo et al., 2006 ). In the present study, esters formed the largest group of flavor compounds, with 26 and 13 ester compounds detected in the CRW samples fermented with JIUYAO and the artificial starter, respectively. The ester content of the wine fermented with the artificial starter (6685.06 μg/kg) was comparable to that of the wine fermented with JIUYAO (7076.73 μg/kg), but significantly ( p < 0.05) higher than that of the wine fermented with the commercial starter (4794.15 μg/kg). Notably, isobutyl acetate, n-caprylic acid isobutyl ester, ethyl heptanoate, ethyl nonanoate, phenethyl acetate, and ethyl caprylate were the only esters identified in the CRW samples brewed with JIUYAO and the artificial starter. Thus, these ester compounds may be significantly correlated with the core microbial species used in the artificial starter.

Alcohols formed the second largest category of flavor compounds in the CRW samples, and are known to be the key aroma components of wine, especially brewed rice wine ( Wang et al., 2014 ). Alcohols are produced through the metabolism of sugars and the decarboxylation and dehydrogenation of amino acids ( Hernandez-Orte et al., 2008 ). Nine, eight, and two alcohol compounds were detected in the CRW samples fermented with JIUYAO, the artificial starter, and the commercial starter, respectively. Only two alcohols, namely isobutanol and phenylethyl alcohol, were identified in the wine fermented with the commercial starter. The alcohol content of the CRW samples fermented with JIUYAO and the artificial starter was 15,212.26 and 14,341.84 μg/kg, respectively, but that of the sample fermented with the commercial starter was only 1683.33 μg/kg. These results indicate that the core microorganisms used in the artificial starter could produce considerably more alcohol compounds during CRW fermentation.

To assess the complex olfactory effects of the different aroma compounds, individual OAVs were calculated ( Bavcar et al., 2011 ). As shown in Table 4 , 14 volatile compounds with OAVs ≥1 were identified in the three CRW samples, including 8 esters, 4 alcohols, 1 acid, and 1 aldehyde. Twelve aroma compounds with OAVs ≥1 were found in the CRW samples fermented with JIUYAO and the artificial starter, but only five were identified in the sample fermented with the commercial starter. Among these compounds, ethyl caprylate, 1-octen-3-ol, ethyl decanoate, ethyl butyrate, furfural, isoamyl alcohol, ethyl propionate, isobutanol, and n-decanoic acid were detected only in the samples brewed with JIUYAO and the artificial starter. These compounds contribute to the pleasant aroma profile of CRW due to their desirable aroma and low odor threshold ( Yu et al., 2019a ). For example, ethyl butyrate imparts an apple-like aroma to rice wine. Isoamyl alcohol is a powerful aroma agent with a banana flavor, which can improve the taste of wine by reducing the bitter-tasting amino acids (leucine) ( Chen Z. et al., 2015 ). These results, together with those of the electronic nose analysis, indicate that the flavor profile—especially the key aroma compounds—of the CRW fermented with the artificial starter is similar to that of the CRW fermented with JIUYAO. Thus, the artificial starter is a potential substitute to JIUYAO to aid the industrial production of high-quality CRW with a stable flavor profile.

Table 4. OAVs of volatile compounds detected in the three Chinese rice wine samples fermented with three different starter cultures.

An artificial starter was prepared for high-efficiency industrial CRW production, and the fermentation activities and flavor profiles of CRW samples fermented with JIUYAO, a commercial starter culture, and our artificial starter culture were compared. The optimal lyoprotectant combination was determined as 15.09% skim milk, 4.45% polyethylene glycol, 1.96% sodium glutamate, and 11.81% maltodextrin, using the response surface optimization method. These three different starters had equivalent fermentation activity in terms of the alcohol content of the fermented CRW samples. The sample brewed with the artificial starter showed similar acid content and volatile compound profiles to those of the sample brewed with JIUYAO. Although the aroma compound content of the CRW sample fermented with the artificial starter was lower than that of the sample fermented with JIUYAO, the same main aroma compounds, such as isoamyl acetate, ethyl acetate, phenyl alcohol, and isoamyl alcohol (OAV ≥ 1), were found in both and contributed highly to the flavor of the CRW. Our artificial starter culture is a promising substitute to traditional JIUYAO to aid the industrial production of high-quality CRW with a stable flavor profile. Further studies should be devoted to in-depth analysis of the associations between the core microbes and flavor substances to enhance the activity and stability of the artificial starter during batch production of CRW.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Author Contributions

CC wrote the manuscript and performed the statistical analyses. ZL performed the statistical analyses and flash GC electronic nose detection. WZ compared the fermentation activities between different samples. HT determined the flavor profiles of samples by gas chromatography–mass spectrometry. JH and HYua established the optimal protectant combination for lyophilization of the artificial starter. HYu designed the research. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (32172336) and Capacity Project of Local Colleges and Universities of the Science and Technology Commission of Shanghai, China (21010504100).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

The authors are grateful to the Zhejiang Tapai Shaoxing Rice Wine Co., Ltd. for their generous supply of materials for this study.

Abadias, M., Benabarre, A., Teixidó, N., Usall, J., and ViñAs, I. (2001). Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake. Int. J. Food Microbiol. 65, 173–182. doi: 10.1016/S0168-1605(00)00513-4

CrossRef Full Text | Google Scholar

Ale, C. E., Otero, M. C., and Pasteris, S. E. (2015). Freeze-drying of wine yeasts and oenococcus oeni and selection of the inoculation conditions after storage. J. Bioprocess. Biotech. 5:248.

Google Scholar

Bavcar, D., Cesnik, H. B., Cus, F., and Kosmerl, T. (2011). The influence of skin contact during alcoholic fermentation on the aroma composition of Ribolla Gialla and Malvasia Istriana Vitis vinifera (L.) grape wines. Int. J. Food Sci. Tech. 46, 1801–1808. doi: 10.1111/j.1365-2621.2011.02679.x

Cerrutti, P., Huergo, M., Galvagno, M., Schebor, C., and Buera, M. (2000). Commercial baker’s yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices. Appl. Microbiol. Biot. 54, 575–580. doi: 10.1007/s002530000428

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, C., Liu, Y., Tian, H., Ai, L., and Yu, H. (2020). Metagenomic analysis reveals the impact of JIUYAO microbial diversity on fermentation and the volatile profile of Shaoxing-jiu. Food Microbiol. 86, 103321–103326. doi: 10.1016/j.fm.2019.103326

Chen, H., Chen, S., Li, C., and Shu, G. (2015). Response surface optimization of lyoprotectant for lactobacillus bulgaricus during vacuum freeze-drying. Prep. Biochem. Biotech. 45, 463–475. doi: 10.1080/10826068.2014.923451

Chen, H., Tian, M., Chen, L., Cui, X., Meng, J., and Shu, G. (2019). Optimization of composite cryoprotectant for freeze-drying Bifidobacterium bifidum BB01 by response surface methodology. Artif. Cells Nanomed. Biotechnol. 47, 1559–1569. doi: 10.1080/21691401.2019.1603157

Chen, S., Xu, Y., and Qian, M. C. (2013). Aroma characterization of Chinese rice wine by gas chromatography-olfactometry, chemical quantitative analysis, and aroma reconstitution. J. Agric. Food Chem. 61, 11295–11302. doi: 10.1021/jf4030536

Chen, Z., Jia, R., Liu, Z., Zhang, W., Chen, S., Rao, P., et al. (2015). Microbial community structure and dynamics during the traditional brewing of Fuzhou Hong Qu glutinous rice wine as determined by culture-dependent and culture-independent techniques. Food Control 57, 216–224. doi: 10.1016/j.foodcont.2015.03.054

Comuzzo, P., Tat, L., Tonizzo, A., and Battistutta, F. (2006). Yeast derivatives (extracts and autolysates) in winemaking: release of volatile compounds and effects on wine aroma volatility. Food Chem. 99, 217–230. doi: 10.1016/j.foodchem.2005.06.049

Coulibaly, W. H., N’guessan, K. F., Coulibaly, I., Cot, M., Rigou, P., and Djè, K. M. (2016). Influence of freeze-dried yeast starter cultures on volatile compounds of tchapalo, a traditional sorghum beer from cte d’ivoire. Beverages 2:35. doi: 10.3390/beverages2040035

Crowell, E. A., and Ough, C. S. (1979). A modified procedure for alcohol determination by dichromate oxidation. Am. J. Enol. Vitic. 30, 61–63.

Ferrer-Gallego, R., Hernández-Hierro, J., Rivas-Gonzalo, J. C., and Escribano-Bailón, M. (2014). Sensory evaluation of bitterness and astringency sub-qualities of wine phenolic compounds: synergistic effect and modulation by aromas. Food Res. Int. 62, 1100–1107. doi: 10.1016/j.foodres.2014.05.049

Hernandez-Orte, P., Cersosimo, M., Loscos, N., Cacho, J., Garcia-Moruno, E., and Ferreira, V. (2008). The development of varietal aroma from non-floral grapes by yeasts of different genera. Food Chem. 107, 1064–1077. doi: 10.1016/j.foodchem.2007.09.032

Huang, L., Ma, Y., Tian, X., Li, J. M., Li, L. X., Tang, K., et al. (2018). Chemosensory characteristics of regional Vidal icewines from China and Canada. Food Chem. 261, 66–74. doi: 10.1016/j.foodchem.2018.04.021

Huang, Z. R., Guo, W. L., Zhou, W. B., Li, L., Xu, J. X., Hong, J. L., et al. (2019). Microbial communities and volatile metabolites in different traditional fermentation starters used for Hong Qu glutinous rice wine. Food Res. Int. 121, 593–603. doi: 10.1016/j.foodres.2018.12.024

Huang, Z. R., Hong, J. L., Xu, J. X., Li, L., Guo, W. L., Pan, Y. Y., et al. (2018). Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine. Food Microbiol. 76, 487–496. doi: 10.1016/j.fm.2018.07.014

Hubálek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46, 205–229. doi: 10.1016/S0011-2240(03)00046-4

Jiao, A., Xu, X., and Jin, Z. (2017). Research progress on the brewing techniques of new-type rice wine. Food Chem. 215, 508–515. doi: 10.1016/j.foodchem.2016.08.014

Lbg, A., Ahc, A., and Og, B. (2020). Storage stability and sourdough acidification kinetic of freeze-dried Lactobacillus curvatus N19 under optimized cryoprotectant formulation. Cryobiology 96, 122–129. doi: 10.1016/j.cryobiol.2020.07.007

Liu, S., Chen, Q., Zou, H., Yu, Y., and Zhang, S. (2019). A metagenomic analysis of the relationship between microorganisms and flavor development in Shaoxing mechanized huangjiu fermentation mashes. Int. J. Food Microbiol. 303, 9–18. doi: 10.1016/j.ijfoodmicro.2019.05.001

Liu, Z., Wang, Z., Lv, X., Zhu, X., and Li, N. (2018). Comparison study of the volatile profiles and microbial communities of Wuyi Qu and Gutian Qu, two major types of traditional fermentation starters of Hong Qu glutinous rice wine. Food Microbiol. 69, 105–115. doi: 10.1016/j.fm.2017.07.019

Pradelles, R., Vichi, S., Alexandre, H., and Chassagne, D. (2009). Influence of the drying processes of yeasts on their volatile phenol sorption capacity in model wine. Int. J. Food Microbiol. 135, 152–157. doi: 10.1016/j.ijfoodmicro.2009.07.019

Qi, K., He, C., Wan, H., Man, H., and Wu, Y. (2017). Response surface optimization of lyoprotectant from amino acids and salts for bifidobacterium bifidum during vacuum freeze-drying. Acta Univ. Cibiniensis Ser. E Food Technol. 21, 3–10. doi: 10.1515/aucft-2017-0009

Van Gemert, L. (2003). Compilations of Odour Threshold Values in Air, Water and Other Media. Zeist: Oliemans, Punter & Partners B.V.

Wang, P., Mao, J., Meng, X., Li, X., Liu, Y., and Feng, H. (2014). Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region. Food Control 44, 58–63. doi: 10.1016/j.foodcont.2014.03.018

Wu, K. N., Tan, B. K., Howard, J. D., Conley, D. B., and Gottfried, J. A. (2012). Olfactory input is critical for sustaining odor quality codes in human orbitofrontal cortex. Nat. Neurosci. 15, 1313–1319. doi: 10.1038/nn.3186

Wu, Z., Xu, E., Jie, L., Zhang, Y., Fang, W., Xu, X., et al. (2015). Monitoring of fermentation process parameters of Chinese rice wine using attenuated total reflectance mid-infrared spectroscopy. Food Control 50, 405–412. doi: 10.1016/j.foodcont.2014.09.028

Xie, G. F., Li, W. J., Jian, L., Yu, C., Hua, F., Zou, H. J., et al. (2007). Isolation and identification of representative fungi from Shaoxing rice wine Wheat Qu using a polyphasic approach of culture-based and molecular-based methods. J. Inst. Brew. 113, 272–279. doi: 10.1002/j.2050-0416.2007.tb00287.x

Xu, E., Jie, L., Wu, Z., Li, H., and Jiao, A. (2015). Characterization of volatile flavor compounds in Chinese rice wine fermented from enzymatic extruded rice. J. Food Sci. 80, 1476–1489. doi: 10.1111/1750-3841.12935

Yang, C., Zhu, X., Fan, D., Mi, Y., Luo, Y., Hui, J., et al. (2012). Optimizing the chemical compositions of protective agents for freeze-drying bifidobacterium longum BIOMA 5920. Chin. J. Chem. Eng. 20, 930–936. doi: 10.1016/S1004-9541(12)60420-0

Yang, Y. J., Xia, Y. J., Lin, X. N., Wang, G. Q., Zhang, H., Xiong, Z. Q., et al. (2018). Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity. Food Res. Int. 108, 83–92. doi: 10.1016/j.foodres.2018.03.036

Yao, L. D., Ju, X., James, T. Y., Qiu, J. Z., and Liu, X. Y. (2018). Relationship between saccharifying capacity and isolation sources for strains of the Rhizopus arrhizus complex. Mycoscience 59, 409–414. doi: 10.1016/j.myc.2018.02.011

Yu, H., Xie, T., Xie, J., Ai, L., and Tian, H. (2019b). Characterization of key aroma compounds in Chinese rice wine using gas chromatography-mass spectrometry and gas chromatography-olfactometry. Food Chem. 293, 8–14. doi: 10.1016/j.foodchem.2019.03.071

Yu, H., Xie, T., Qian, X., Ai, L., and Tian, H. (2019a). Characterization of the volatile profile of Chinese rice wine by comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry. J. Sci. Food Agr. 99, 5444–5456. doi: 10.1002/jsfa.9806

Zhao, G., and Zhang, G. (2010). Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. J. Appl. Microbiol. 99, 333–338. doi: 10.1111/j.1365-2672.2005.02587.x

Zhao, X., Wang, Y., Cai, W., Yang, M., and Shan, C. (2020). High-throughput sequencing-based analysis of microbial diversity in rice wine koji from different areas. Curr. Microbiol. 77, 1–8. doi: 10.1007/s00284-020-01877-9

Keywords : Chinese rice wine, artificial starter, JIUYAO, flavor profiles, response surface methodology

Citation: Chen C, Liu Z, Zhou W, Tian H, Huang J, Yuan H and Yu H (2021) Comparison of the Fermentation Activities and Volatile Flavor Profiles of Chinese Rice Wine Fermented Using an Artificial Starter, a Traditional JIUYAO and a Commercial Starter. Front. Microbiol. 12:716281. doi: 10.3389/fmicb.2021.716281

Received: 28 May 2021; Accepted: 24 August 2021; Published: 20 September 2021.

Reviewed by:

Copyright © 2021 Chen, Liu, Zhou, Tian, Huang, Yuan and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Haiyan Yu, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• Biomed Res Int

Effect of Temperature on Chinese Rice Wine Brewing with High Concentration Presteamed Whole Sticky Rice

Dengfeng liu.

1 Key Laboratory of Industrial Advanced Process Control for Light Industry of Ministry of Education, Jiangnan University, Wuxi 214122, China

Hong-Tao Zhang

2 Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China

Weili Xiong

3 Shaoxing Nverhong Wine Company Limited, Shangyu, Zhejiang 312000, China

Chi-Chung Lin

Lihua jiang.

Production of high quality Chinese rice wine largely depends on fermentation temperature. However, there is no report on the ethanol, sugars, and acids kinetics in the fermentation mash of Chinese rice wine treated at various temperatures. The effects of fermentation temperatures on Chinese rice wine quality were investigated. The compositions and concentrations of ethanol, sugars, glycerol, and organic acids in the mash of Chinese rice wine samples were determined by HPLC method. The highest ethanol concentration and the highest glycerol concentration both were attained at the fermentation mash treated at 23°C. The highest peak value of maltose (90 g/L) was obtained at 18°C. Lactic acid and acetic acid both achieved maximum values at 33°C. The experimental results indicated that temperature contributed significantly to the ethanol production, acid flavor contents, and sugar contents in the fermentation broth of the Chinese rice wines.

1. Introduction

Chinese rice wine, a natural and nondistilled wine, is very popular in China and its market is speedily increasing [ 1 ]. The annual consumption is about 1.4 million tons. Hitherto, the Chinese rice wine brewing process is mainly controlled by experienced technician rather than by scientific instruments. This technician control method causes each batch of Chinese rice wine with different flavors. Currently, how to standardize all batches of Chinese rice wine with the same flavor is still an unresolved issue. Good taste becomes more important than ever for the Chinese rice wine. Young drinkers have more choices for drinks. Consequently, the wine should be with good and consistent taste to attract more customers. It is thus very important to study the effects of temperature on Chinese rice wine brewing.

Similar to sake and other rice wine varieties, the fermentation process of Chinese rice wine brewing can be divided into two stages: the main stage (also called primary fermentation) and the second stage (also called postfermentation). In the main stage, pre-steamed rice, Saccharomyces cerevisiae yeast culture, and wheat qu are mixed and fermented for 96 h [ 2 ]. During the entire process of Chinese rice wine brewing, the main stage is the core of Chinese rice wine brewing and determine the Chinese rice wine quality.

The main stage of the fermentation process is a typical simultaneous saccharification and fermentation (SSF) process as well as a semisolid state and semiliquor state fermentation (SSSLF) process. As the concentration of pre-steamed rice and wheat in mash is very high (can be as high as 45%), the SSF and SSSLF process may decrease yeast cell growth inhibition with high sugar concentration and facilitate ethanol production in Chinese rice wine brewing. The concentration of ethanol can thus be high and even more than 20% (v/v) in the final mash at the end of the main stage fermentation [ 3 ].

Temperature effects on wine fermentation have been widely investigated in beer [ 4 ], grape wine, and other ethanol fermentations [ 5 ]. Research results suggested that temperature can affect glycerol and ethanol production [ 6 ]. The effects of temperature, pH, and sugar concentration on the growth rates and cell biomass of wine yeasts were studied in grape juice wine [ 7 ]. Fermentation temperature can affect the microbial population during grape-must fermentation [ 8 ] and then affect the ethanol production of grape wine. Both yeast strain and temperature can affect the grape-wine fermentation rate and wine quality [ 9 ]. Redón et al. [ 10 ] found that temperature can affect membrane lipid composition of Saccharomyces cerevisiae yeast species and then affect ethanol production. In addition, appropriate pH value also is necessary for yeast growth and ethanol production [ 11 ].

The proportions of sugars, glycerol, ethanol, and organic acids are primarily responsible for the delicate taste and flavors of Chinese rice wine [ 12 , 13 ]. Especially, organic acid (i.e., lactic acid) and ethanol can produce esterification in the long time storage stage and form the wine's good taste and smell. In addition, sugar contents in Chinese rice wine determine the wine types. In the National Standard of China GB 13662–2000, Chinese rice wine is divided into four types according to the concentrations of the total sugar: dry type (total sugar ≤ 15 g/L), semidry type (15 g/L < total sugar ≤ 40 g/L), semisweet type (40 g/L < total sugar ≤ 100 g/L), and sweet type (total sugar > 100 g/L).

In the past, pursuit of high ethanol concentration is the main goal for wine fermentation. At present, volatile compounds in wine have become the new important parameters to evaluate the wine quality [ 4 , 6 , 14 – 16 ]. It is clear that sugar and volatile acids can influence the taste of drink and juice [ 17 – 19 ]. Volatile organic acids are important to the flavor and taste characteristics of the Chinese rice wine [ 20 ]. Especially, lactic acid was the most important volatile acid [ 21 ] and constituted over 90% of the total volatile acids.

Due to the increasingly recognized importance of sugars and acids and their relationship to wine quality, it is important to investigate the effect of temperature on the yeast fermentation, organic acid, and glycerol compound during Chinese rice wine brewing. The experiment which simulated Chinese rice wine fermentation process was implemented at various temperatures (18°C, 23°C, 28°C, and 33°C) in a scale-down level. Based on previous research, 33°C is the highest temperature designed in plant fermentation process, 28°C is the desired temperature for this yeast cell growth [ 22 ], and 25°C–28°C is the desired temperature for the start of the fermentation, as 5°C was a temperature gradient and 23°C and 18°C were picked for comparison purposes [ 23 ]. As a result, these four temperatures were chosen. Natural fermentation (the surrounding temperature is 16°C and labeled as RT) was added as the control.

The results of the study contributed significantly to the understanding of the role of temperature in ethanol, organic acids, glycerol, and sugars kinetics during Chinese rice wine brewing and also provided useful information to improve the quality of Chinese rice wine.

2. Materials and Methods

2.1. microorganisms for fermentation.

Saccharomyces cerevisiae Su -25 (Shaoxing Nverhong Rice Wine Plants, Zhejiang, China) was used and stored at 4°C. Chinese wheat qu (Shaoxing Nverhong Rice Wine Plants, Zhejiang, China) was used to hydrolyze rice starch which was stored at room temperature. Sticky rice bought from a local market (Vanguard Market, Wuxi, China) was used.

2.2. Small-Scale Chinese Rice Wine Brewing with Designed Experiments

2.2.1. yeast medium and yeast culture.

Yeast extract peptone dextrose medium (YPD) includes glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, and agar 20 g/L.

Liquid yeast extract peptone dextrose medium (LYPD) includes glucose 20 g/L, peptone 20 g/L, and yeast extract 10 g/L.

The yeast strain was stored at 4°C on slants of yeast peptone dextrose agar medium (YPD). The yeast inoculum was transferred to a new slant of YPD and cultured for 24 h at 28°C. A sloop of yeast culture was added to 50 mL LYPD medium (250 mL flask) and cultured at 28°C for 18 h as yeast seed. The prepared yeast seed was diluted at a ratio of 1 : 10 with new LYPD (100 mL in 500 mL bottle) medium and cultured at 28°C for another 18 h.

2.2.2. Batch Fermentation

Fermentation experiments were conducted in a 7 L tank fermenter (BioFlo IV, NBS Edison, NJ, USA). Sticky rice was steamed for 45 min and cooled at room temperature to 26°C. 1200 g steamed rice (dry weight), 204 g wheat qu , 120 mL yeast seed culture, and 2400 mL tap water were mixed in the fermenters. During the entire fermentation process, the pH was not controlled; aeration and agitation both were set at 0 value. The temperature was maintained at preset value as RT, 18°C, 23°C, 28°C, and 33°C.

The fermentation process was maintained at constant temperature as designed for 4 days. 2 mL of samples from each experiment was taken out every 2 h and centrifuged at 10000 r/min for 3 min, filtered (0.22  μ m, PVC membrane), and quickly analyzed with high-performance liquid chromatography (HPLC). The HPLC method was used to identify each component with the elution time and quantified by using the spiking technique.

2.3. Chemicals

Submicron-filtered HPLC-grade water was used. D-glucose, D-fructose, maltose, maltotriose, and sulfuric acid purchased from Sigma-Aldrich (St Louis, MO, USA) were used. HPLC-grade lactic acid, acetic acid, succinic acid, citric acid, malic acid, tartaric acid, propionic acid, ethanol, and acetonitrile purchased from Fisher (Pittsburgh, PA, USA) were used.

2.4. Analysis of Enological Parameters

In order to compare the effect of various fermentation conditions on the Chinese rice wine brewing process, several enological parameters were determined offline right after the samples were taken out of the fermenter.

The concentrations of sugars, glycerol, ethanol, and organic acids were determined with HPLC (Agilent 1200 series). At the predetermined time, wine samples of 1 mL were taken for analyses. Durapore (PVDF, 0.45  μ m pore) membrane filters (Fisher, Pittsburgh, PA, USA) were used to filter wine samples. An Aminex HPX-87H column (300 × 7.8 mm) (Bio-Rad Labs, Richmond, CA, USA) was used to determine the concentrations of the sugars, glycerol, ethanol and organic acids. A Bio-Rad HPLC column heater was used to maintain column temperature as 55°C. A Bio-Rad 125-0131 guard cartridge (Bio-Rad Labs, Richmond, CA, USA) was used to protect column. The eluted compounds (sugars, glycerol, ethanol and organic acids) were detected with an G1314B VWD detector (Agilent 1200 Series, Santa Clara, CA, USA) and an G1362A RID detector (Agilent 1200 Series, Santa Clara, CA, USA) simultaneously. The solvent delivery system was driven by a G1311A quaternary pump (Agilent 1200 Series, Santa Clara, CA, USA).

Every 1000 mL mobile phase consisted of 1270  μ L sulfuric acid and 60 mL acetonitrile. The samples were eluted with the mobile phase at a flow rate of 0.5 mL/min. Injection volume for each sample was 20  μ L per fixed loop with run time as 30 minutes.

HPLC system (Agilent 1200 Series, Santa Clara, CA, USA) was used. Separate calibration standard curves were constructed.

The statistical analysis of the final lactic acids and ethanol concentrations was performed with SAS software (version 9.3; SAS Institute, Cary, NC).

3. Results and Discussion

3.1. effect of temperatures on ethanol production in fermentation mash.

Temperature is an important controlling parameter controlling the Chinese rice wine quality. However, it is still not clear why it affects the quality of Chinese rice wine. As ethanol is the main product of Chinese rice wine, exploring the effect of temperature on ethanol production is necessary for optimizing rice wine fermentation.

In this work, ethanol production under various temperatures (RT, 18, 23, 28, and 33°C) was investigated and presented in Table 1 . It is clear that with the temperature increasing, the ethanol yield increased from 9.8% (v/v) to 12.2% (v/v) and then decreased from 12.2% (v/v) to 10.4% (v/v) and 3.6% (v/v) with the temperature increasing from 18°C to 23°C, 28°C, and 33°C, respectively. It is to be noted that the ethanol production at 11.2% (v/v) was achieved at RT condition. Ethanol production at 23°C was the highest compared to other temperatures in this work. However, Lee found that the optimal temperature for growth was 34°C, while the specific ethanol production rate was maximal at 37–43°C with Saccharomyces uvarum [ 24 – 26 ]. It is conceivable that a suitable temperature would facilitate Saccharomyces cerevisiae Su -25 to use sugars to produce ethanol during the Chinese rice wine brewing process. Low temperature slows down reaction rate, but excessive temperature accelerates cellular aging, and aged cells reduce ethanol production.

Ethanol concentration (%, v/v ± SD) under various temperatures.

Detailed evaluation of the effect of various temperatures on ethanol production is an efficient way to analyze the kinetics of ethanol fermentation during Chinese rice wine production which are shown in Figure 1 .

The profiles of ethanol concentration under various temperatures.

For the first 20 hours, the ethanol concentration increased with temperature. The highest concentration was achieved at 33°C and the lowest concentration was detected at 18°C. From 20 h to 70 h, the profiles of ethanol concentration under 23°C, 28°C, and RT were all similar. After 20 h, ethanol concentration under 33°C only has just a little fluctuation. At 18°C the ethanol concentration kept growing and was much higher than that of 33°C, but slightly lower than that at other temperatures at the 70 h. However, from 70 h to 100 h, the profile of ethanol concentration at RT and 23°C increased quickly, which only slightly increased at 28°C. From 100 h to 140 h, the ethanol concentration at 18°C increased quickly. However, under the other temperature, there were only slight variations of the ethanol concentrations. At 33°C, the ethanol concentration only slightly increased from 20 h to 140 h. All these data indicate that temperatures have different effect on ethanol production kinetics at different fermentation stages.

Ethanol production kinetics at various stages of Chinese rice wine brewing under designed temperatures was further analyzed ( Table 2 ). At the stages of 0–47 h, highest ethanol production was achieved at 28°C. After 47 h, highest ethanol production was reached at 23°C. Consequently, a two-stage temperature controlling strategy is better for the enhancement of ethanol production in the Chinese rice wine brewing process.

Effect of various temperatures on the ethanol concentration (%, v/v ± SD) at different stages.

3.2. Effect of Temperatures on Sugars and Glycerol Concentrations in Fermentation Mash during the Main Stage

Sugars in the fermentation mash of Chinese rice wine not only are important nutrient components for rice wine production but also contribute to its taste and flavor. Chinese rice wine (commonly known as Shaoxing huangjiu) is divided into four types according to its total sugar contents as shown in the Introduction. Among it, semidry rice wine is the most popular [ 27 , 28 ].

The production of sugars and glycerol in the fermentation mash of Chinese rice wine fermentation with designed experiments, including glucose, maltose, maltotriose, and glycerol, was analyzed. The fermentation profiles of sugars and glycerol are shown in Figure 2 . The glycerol concentration is shown in Figure 2(a) in a similar way to the changes of ethanol level, the highest concentration of glycerol was achieved at 23°C at 3.5 g/L. For the batch tested under RT condition, the concentration of glycerol was 3.2 g/L, and for the batch at 18°C and 33°C the concentration of glycerol was 2.5 g/L and 3.5 g/L, respectively. The fermentation kinetics for fructose is shown in Figure 2(b) . The highest concentration of fructose production is 0.42 g/L at 23°C followed by conditions at RT, 18°C, 28°C, and 33°C.

Time course of changes in productive concentration of sugars and glycerol under various temperatures: (a) glycerol, (b) fructose, (c) maltotriose, (d) maltose, and (e) glucose.

However, the concentrations for maltotriose at various temperatures are different from that for glycerol and fructose. The maximum concentrations of maltotriose at various temperatures were in the descending order of 33°C 18°C, 28°C, RT, and 23°C ( Figure 2(c) ). It is clear that all the concentrations of fructose at various temperatures have similar pattern. Fructose concentrations began to accumulate and reached maximum at 40 h, and then all the concentrations of fructose at various temperature exhibited little variations. Considering the facts that saccharification process was completed at the end of 40 h, the fructose cannot be used by Saccharomyces cerevisiae Su -25 and other microbial cells. The profiles of maltose concentration are shown in Figure 2(d) . Similar profiles for maltose concentration to maltotriose concentration are observed under all temperature conditions tested. The highest concentration of maltose of 54.5 g/L in final fermentation mash was attained at 33°C at 140 h, and 12.4 g/L at 18°C. For other conditions, the concentrations of maltose were all low. The highest concentrations of total sugars were below 105 g/L at all temperatures during the entire process. A concentration lower than that can conceivably inhibit yeast cell growth and fermentation [ 29 ]. This experimental result agrees with previous research [ 2 ]. The glucose concentrations at different temperatures are all low and under 4 g/L during the fermentation process. The results suggest that glucose should not be the main sugar used by Saccharomyces cerevisiae Su -25 and other microbial cells to produce ethanol and acids during Chinese rice wine fermentation.

The fermentation kinetics of maltotriose and maltose are quite similar. This phenomenon can be explained below. Under low and high temperature, the fermentation rate is low. As maltose and maltotriose were utilized slowly by the microbial cells, both the observed residual maltose and maltotriose were higher. However, the low fermentation rate is different between low and high temperatures. The cellular metabolic activity at low temperature is generally low which conceivably caused retarded ethanol biosynthesis. On the contrary, at higher temperature, cellular aging process was accelerated which reduced ethanol formation at most of the fermentation processes. In addition, Saccharomyces cerevisiae Su -25 conceivably uses maltose but not glucose from which ethanol and favors were produced. This is different from that previously reported in the literature [ 3 , 30 ].

3.3. Effect of Temperature on Organic Acid Composition in Fermentation Mash

A certain amount of acids played an important role in the flavor of rice wine and gradually converted into aromatic esters during storage. Therefore, the total acid content in the fermentation mash of Chinese rice wine is a key parameter to evaluate and control the fermentation process during industrial Chinese rice wine brewing [ 27 ]. The production of acid metabolites, including acetic acid, lactic acid, and succinic acid, was analyzed.

The fermentation profiles of organic acids are shown in Figure 3 . The kinetics of succinic acid fermentation at various temperatures is similar to that of ethanol production. The highest concentration of succinic acid was achieved at 23°C, and the lowest concentration was at 33°C ( Figure 3(a) ). The concentration of lactic acid increased quickly during the fermentation process at 33°C ( Table 3 ). The final concentration of lactic acid is about 6 times higher compared to that under other conditions ( Figure 3(b) ). Similar phenomena were observed in the concentration of acetic acid, which was 2 to 5 times higher compared with the concentration of acetic acid at other temperatures ( Figure 3(c) ). In addition, the concentration of tartaric acid at 33°C was higher than that at other temperatures ( Figure 3(f) ). Propionic acid was not observed in the fermentation mash.

Time course of changes in the concentration of organic acids under various temperatures: (a) succinic acid, (b) lactic acid, (c) acetic acid, (d) pyruvate acid, (e) malic acid, and (f) tartaric acid.

Effect of changing temperature on lactic acid concentration (g/L ± SD) at different stages.

Only the concentrations of lactic acid and acetic acid at 33°C are statistically significantly higher than that at other temperatures which agrees with that of Mao [ 31 ]. Lactic acid was mainly produced by Lactobacillus during Chinese rice wine brewing [ 32 ]. At high temperature (33°C), yeast cell ( Saccharomyces cerevisiae Su -25) growth (generally optimal at 28°C), was inhibited from producing ethanol with the substrate of glucose. Nevertheless, Lactobacillus can grow well to produce lactic acid from glucose at high temperature (33°C). The fermentation kinetics for succinic acid, pyruvic acid, and malic acid are similar to that of ethanol production. All the three acids are the by-products of Saccharomyces cerevisiae Su -25 during ethanol fermentation. This essentially agrees with previous research [ 23 , 33 ].

4. Conclusion

Results from this study have shown that different fermentation temperatures affect the levels of sugars, glycerol concentration, and organic acid in the fermentation medium for Chinese rice wine production. The highest concentration of ethanol was achieved at 23°C. The lowest concentration of ethanol was at 33°C.

Higher temperatures can enhance organic acid production through stimulation of the growth of Lactobacillus . The concentrations of acetic acid, tartaric acid, and lactic acid were statistically significantly higher at 33°C than those at other temperatures (Tukey's test was used for analysis of variance to find significant differences among various treatments at P = 0.05 level). Lactic acid was mainly produced by Lactobacillus . High temperature can speed up the growth of Lactobacillus and production and accumulation of lactic acid.

Although it is proven that temperatures can affect the production of ethanol, glycerol, and organic acid, their optimal level in Chinese rice wine fermentation and how to accurately control their ratio through controlling temperatures are still not clear. Consequently, developing a kinetic model to describe the effect of temperatures on ethanol, glycerol, and organic acid production during Chinese rice wine fermentation is needed and is currently in progress.

Acknowledgments

The authors thank Drs. Feng Ding, Ya Guo, Ru Dai, and Ti Zhang and Ms. Connie Liu and Mr. Lakdas Fernando for their technical assistance. Shaoxing Nverhong Rice Wine Company is acknowledged for the material support. This work was supported in part by research grants from the National Science Foundation of China (21276111, and 21206053), Zhejiang Science and Technology Project (2011C12033), China Scholarship Council (no. 2010679023), and Jiangnan University P.h.D. Research Fund (no. 20110149).

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Information

• Author Services

Initiatives

You are accessing a machine-readable page. In order to be human-readable, please install an RSS reader.

All articles published by MDPI are made immediately available worldwide under an open access license. No special permission is required to reuse all or part of the article published by MDPI, including figures and tables. For articles published under an open access Creative Common CC BY license, any part of the article may be reused without permission provided that the original article is clearly cited. For more information, please refer to https://www.mdpi.com/openaccess .

Feature papers represent the most advanced research with significant potential for high impact in the field. A Feature Paper should be a substantial original Article that involves several techniques or approaches, provides an outlook for future research directions and describes possible research applications.

Feature papers are submitted upon individual invitation or recommendation by the scientific editors and must receive positive feedback from the reviewers.

Editor’s Choice articles are based on recommendations by the scientific editors of MDPI journals from around the world. Editors select a small number of articles recently published in the journal that they believe will be particularly interesting to readers, or important in the respective research area. The aim is to provide a snapshot of some of the most exciting work published in the various research areas of the journal.

Original Submission Date Received: .

• Active Journals
• Find a Journal
• Proceedings Series
• For Authors
• For Reviewers
• For Editors
• For Librarians
• For Publishers
• For Societies
• For Conference Organizers
• Open Access Policy
• Institutional Open Access Program
• Special Issues Guidelines
• Editorial Process
• Research and Publication Ethics
• Article Processing Charges
• Testimonials
• Preprints.org
• SciProfiles
• Encyclopedia

Article Menu

• Subscribe SciFeed
• Recommended Articles
• PubMed/Medline
• Google Scholar
• on Google Scholar
• Table of Contents

Find support for a specific problem in the support section of our website.

Please let us know what you think of our products and services.

Visit our dedicated information section to learn more about MDPI.

JSmol Viewer

Optimization of the brewing conditions of shanlan rice wine and sterilization by thermal and intense pulse light, 1. introduction, 2.1. optimization of fermentation conditions of srw, 2.1.1. single-factor experiment results, 2.1.2. response surface model analysis of sensory evaluation, 2.1.3. response surface optimization of the interaction between various factors, 2.2. effect of sterilization treatment on the physicochemical properties of srw, 2.3. effects of different sterilization treatments on the free amino acids in srw, 2.4. difference of flavor metabolites before and after sterilization by pca, 2.4.2. screening and analysis of metabolites of flavor difference in srw before and after sterilization treatment, 3. materials and methods, 3.1. chemicals and reagents, 3.2. qiuqu preparation and srw brewing, 3.3. single-factor and response surface experiments, 3.4. sensory evaluation method of srw, 3.5. sterilization of srw, 3.6. determination of srw’s physicochemical properties, 3.7. determination of free amino acids in srw, 3.7.1. sample preparation and extraction, 3.7.2. uplc conditions, 3.7.3. lc-ms/ms analysis, 3.8. determination of flavor metabolites, 3.8.1. sample preparation and treatment, 3.8.2. gc-ms analysis, 3.9. statistical analysis, 4. conclusions, author contributions, institutional review board statement, data availability statement, conflicts of interest, sample availability.

• Yang, Y.; Xia, Y.; Wang, G.; Tao, L.; Yu, J.; Ai, L. Effects of boiling, ultra-high temperature and high hydrostatic pressure on free amino acids, flavor characteristics and sensory profiles in Chinese rice wine. Food Chem. 2019 , 275 , 407–416. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Chen, D.; Mosher, W.; Wiertzema, J.; Peng, P.; Min, M.; Cheng, Y.; An, J.; Ma, Y.; Fan, X.; Niemira, B.A.; et al. Effects of intense pulsed light and gamma irradiation on Bacillus cereus spores in mesquite pod flour. Food Chem. 2020 , 344 , 128675. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ojha, K.S.; Tiwari, B.K.; O’Donnell, C.P. Chapter Six-Effect of Ultrasound Technology on Food and Nutritional Quality. Adv. Food Nutr. Res. 2018 , 84 , 207–240. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Timmermans, R.A.H.; Mastwijk, H.C.; Berendsen, L.B.J.M.; Nederhoff, A.L.; Matser, A.M.; Van Boekel, M.A.J.S.; Groot, M.N.N. Moderate intensity Pulsed Electric Fields (PEF) as alternative mild preservation technology for fruit juice. Int. J. Food Microbiol. 2019 , 298 , 63–73. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Jung, K.; Song, B.-S.; Kim, M.J.; Moon, B.-G.; Go, S.-M.; Kim, J.-K.; Lee, Y.-J.; Park, J.-H. Effect of X-ray, gamma ray, and electron beam irradiation on the hygienic and physicochemical qualities of red pepper powder. LWT 2015 , 63 , 846–851. [ Google Scholar ] [ CrossRef ]
• Yin, H.; Deng, Y.; He, Y.; Dong, J.; Lu, J.; Chang, Z. A preliminary study of the quality attributes of a cloudy wheat beer treated by flash pasteurization. J. Inst. Brew. 2017 , 123 , 366–372. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Mahendran, R.; Ramanan, K.R.; Barba, F.J.; Lorenzo, J.M.; López-Fernández, O.; Munekata, P.E.S.; Tiwari, B.K. Recent advances in the application of pulsed light processing for improving food safety and increasing shelf life. Trends Food Sci. Technol. 2019 , 88 , 67–79. [ Google Scholar ] [ CrossRef ]
• John, D.; Ramaswamy, H.S. Pulsed light technology to enhance food safety and quality: A mini-review. Curr. Opin. Food Sci. 2018 , 23 , 70–79. [ Google Scholar ] [ CrossRef ]
• Basak, S.; Mahale, S.; Chakraborty, S. Changes in quality attributes of pulsed light and thermally treated mixed fruit beverages during refrigerated storage (4 °C) condition. Innov. Food Sci. Emerg. Technol. 2022 , 78 , 103025. [ Google Scholar ] [ CrossRef ]
• Shen, D.; Labreche, F.; Wu, C.; Fan, G.; Li, T.; Shi, H.; Ye, C. Preparation and aroma analysis of flavonoid-rich ginkgo seeds fermented using rice wine starter. Food Biosci. 2021 , 44 , 101459. [ Google Scholar ] [ CrossRef ]
• Tian, T.; Yang, H.; Yang, F.; Li, B.; Sun, J.; Wu, D.; Lu, J. Optimization of fermentation conditions and comparison of flavor compounds for three fermented greengage wines. LWT 2018 , 89 , 542–550. [ Google Scholar ] [ CrossRef ]
• Singh, A.; Kocher, G.S. Standardization of seed and peel infused Syzygium cumini -wine fermentation using response surface methodology. LWT 2020 , 134 , 109994. [ Google Scholar ] [ CrossRef ]
• Jin, T.-Y.; Saravanakumar, K.; Wang, M.-H. Effect of different sterilization methods on physicochemical and microbiological properties of rice wine. Beni-Suef University. J. Basic Appl. Sci. 2018 , 7 , 487–491. [ Google Scholar ] [ CrossRef ]
• Ha, S.-J.; Kim, Y.-J.; Oh, S.-W. Effect of high hydrostatic pressure (HHP) treatment on chemical and microbiological properties of Makgeolli. J. Korean Soc. Appl. Biol. Chem. 2013 , 56 , 325–329. [ Google Scholar ] [ CrossRef ]
• Wang, P.; Mao, J.; Meng, X.; Li, X.; Liu, Y.; Feng, H. Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region. Food Control 2014 , 44 , 58–63. [ Google Scholar ] [ CrossRef ]
• Xie, G.-F.; Yang, D.-D.; Liu, X.-Q.; Cheng, X.-X.; Rui, H.-F.; Zhou, H.-J. Correlation between the amino acid content in rice wine and protein content in glutinous rice. J. Inst. Brew. 2016 , 122 , 162–167. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Zhang, C.L.; Hao, S.; Xiao, Z.L.; Zhi, G.H.; Wei, X.L.; Dong, L.D. Microbial communities and amino acids during the fermentation of Wuyi Hong Qu Huangjiu. LWT 2020 , 130 , 109743. [ Google Scholar ] [ CrossRef ]
• Gao, H.; Wang, W.; Xu, D.; Wang, P.; Zhao, Y.; Mazza, G.; Zhang, X. Taste-active indicators and their correlation with antioxidant ability during the Monascus rice vinegar solid-state fermentation process. J. Food Compos. Anal. 2021 , 104 , 104133. [ Google Scholar ] [ CrossRef ]
• Bhagat, B.; Chakraborty, S. Potential of pulsed light treatment to pasteurize pomegranate juice: Microbial safety, enzyme inactivation, and phytochemical retention. LWT 2022 , 159 , 113215. [ Google Scholar ] [ CrossRef ]
• Wang, J.; Yuan, C.; Gao, X.; Kang, Y.; Huang, M.; Wu, J.; Liu, Y.; Zhang, J.; Li, H.; Zhang, Y. Characterization of key aroma compounds in Huangjiu from northern China by sensory-directed flavor analysis. Food Res. Int. 2020 , 134 , 109238. [ Google Scholar ] [ CrossRef ]
• Jin, Z.; Cai, G.; Wu, C.; Hu, Z.; Xu, X.; Xie, G.; Wu, D.; Lu, J. Profiling the key metabolites produced during the modern brewing process of Chinese rice wine. Food Res. Int. 2021 , 139 , 109955. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Yang, Y.J.; Xia, Y.J.; Hu, W.Y.; Tao, L.; Liu, H.D.; Xie, C.L.; Bai, W.D.; Ai, L.Z. Soaking induced discrepancies in oenological properties, flavor profiles, microbial community and sensory characteristic of Huangjiu (Chinese rice wine). LWT 2020 , 139 , 110575, Prepublish. [ Google Scholar ] [ CrossRef ]
• Yang, Y.; Ai, L.; Mu, Z.; Liu, H.; Yan, X.; Ni, L.; Zhang, H.; Xia, Y. Flavor compounds with high odor activity values (OAV > 1) dominate the aroma of aged Chinese rice wine (Huangjiu) by molecular association. Food Chem. 2022 , 383 , 132370. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lu, Y.; Sun, F.; Wang, W.; Liu, Y.; Wang, J.; Sun, J.; Mu, J.; Gao, Z. Effects of spontaneous fermentation on the microorganisms diversity and volatile compounds during ‘Marselan’ from grape to wine. LWT 2020 , 134 , 110193. [ Google Scholar ] [ CrossRef ]
• Chen, S.; Xu, Y. The Influence of Yeast Strains on the Volatile Flavour Compounds of Chinese Rice Wine. J. Inst. Brew. 2010 , 116 , 190–196. [ Google Scholar ] [ CrossRef ]
• Zhao, C.; Su, W.; Mu, Y.C.; Jiang, L.; Mu, Y. Correlations between microbiota with physicochemical properties and volatile flavor components in black glutinous rice wine fermentation. Food Res. Int. 2020 , 138 , 109800. [ Google Scholar ] [ CrossRef ]
• Wu, Q.; Chen, B.; Xu, Y. Regulating yeast flavor metabolism by controlling saccharification reaction rate in simultaneous saccharification and fermentation of Chinese Maotai-flavor liquor. Int. J. Food Microbiol. 2015 , 200 , 39–46. [ Google Scholar ] [ CrossRef ]
• Xu, S.; Ma, Z.; Chen, Y.; Li, J.; Jiang, H.; Qu, T.; Zhang, W.; Li, C.; Liu, S. Characterization of the flavor and nutritional value of coconut water vinegar based on metabolomics. Food Chem. 2022 , 369 , 130872. [ Google Scholar ] [ CrossRef ]
• Li, L.; Ma, X.W.; Zhan, R.L. Profiling of volatile fragrant components in a mini-core collection of mango germplasms from seven countries. PLoS ONE 2017 , 12 , e0187487. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Wang, Y.; Yang, C.; Li, S. Volatile characteristics of 50 peaches and nectarines evaluated by HP–SPME with GC–MS. Food Chem. 2009 , 116 , 356–364. [ Google Scholar ] [ CrossRef ]

Click here to enlarge figure

Share and Cite

Wu, X.; Zhang, Y.; Zhong, Q. Optimization of the Brewing Conditions of Shanlan Rice Wine and Sterilization by Thermal and Intense Pulse Light. Molecules 2023 , 28 , 3183. https://doi.org/10.3390/molecules28073183

Wu X, Zhang Y, Zhong Q. Optimization of the Brewing Conditions of Shanlan Rice Wine and Sterilization by Thermal and Intense Pulse Light. Molecules . 2023; 28(7):3183. https://doi.org/10.3390/molecules28073183

Wu, Xiaoqian, Yunzhu Zhang, and Qiuping Zhong. 2023. "Optimization of the Brewing Conditions of Shanlan Rice Wine and Sterilization by Thermal and Intense Pulse Light" Molecules 28, no. 7: 3183. https://doi.org/10.3390/molecules28073183

Article Metrics

Article access statistics, further information, mdpi initiatives, follow mdpi.

Subscribe to receive issue release notifications and newsletters from MDPI journals

Winemakers Corner

Your resource for home winemaking, wines, and brewing.

How To Brew Rice Wine

As someone who is passionate about wine, I have always been captivated by the craft of making rice wine. It is a captivating process that requires patience, accuracy, and a bit of ingenuity. Today, I …

Written by: John Ward

Published on: August 15, 2024

As someone who is passionate about wine, I have always been captivated by the craft of making rice wine. It is a captivating process that requires patience, accuracy, and a bit of ingenuity. Today, I am thrilled to share my own observations and detailed instructions on how to brew rice wine.

Understanding Rice Wine

Rice wine, also known as sake, is a traditional Japanese alcoholic beverage that has gained popularity worldwide. It is made from fermented rice grains and has a complex flavor profile. The brewing process involves converting starches in rice into sugars, and then fermenting those sugars into alcohol.

Gathering Your Ingredients

To begin your rice wine brewing journey, you’ll need the following ingredients:

• High-quality rice: Look for short-grain rice varieties such as sushi rice or glutinous rice.
• Koji rice: This is steamed rice that has been inoculated with koji mold spores. Koji rice plays a crucial role in converting starches into sugars.
• Yeast : Choose a sake yeast strain specifically designed for rice wine brewing. It provides the necessary fermentation to convert sugars into alcohol.
• Water: Use filtered water to ensure the purity of your rice wine.

The Brewing Process

1. Preparing the Rice:

Begin by washing the rice thoroughly under cold water until the water runs clear. Soak the rice in water for about 1 hour to soften the grains.

2. Steaming the Rice:

Transfer the soaked rice to a steamer and steam it until it becomes tender and fully cooked. This usually takes around 30 minutes.

3. Cooling the Rice:

Spread the steamed rice onto a large tray or flat surface, and allow it to cool to room temperature. This step is crucial to prevent the yeast from dying due to high heat.

4. Preparing the Koji Rice:

In a separate bowl, mix the koji rice with a small amount of water to form a paste-like consistency. This will activate the koji mold spores and initiate the fermentation process.

5. Mixing the Ingredients:

In a sterilized container, combine the cooked rice, koji rice paste, and yeast. Gently mix them together, ensuring that all the rice grains are coated with the koji rice paste.

6. Fermentation:

Transfer the mixture to a fermentation vessel , such as a glass jar or ceramic crock. Make sure to seal it tightly to create an anaerobic environment. Store the vessel in a cool, dark place with a consistent temperature around 15-20°C (59-68°F).

7. Patience is Key:

Allow the rice mixture to ferment for about 2-4 weeks. During this time, the yeast will convert the sugars in the rice into alcohol, resulting in the distinct flavor and aroma of rice wine.

Final Steps

After the fermentation period, strain the liquid through a cheesecloth or fine mesh strainer to remove any solids. The resulting liquid is your homemade rice wine.

Transfer the rice wine to sterilized bottles or containers, and refrigerate it to slow down further fermentation. The flavors of rice wine will continue to develop over time, so it’s advisable to let it age for a few months before enjoying.

Brewing rice wine at home is a rewarding and enjoyable process, allowing you to create your own unique flavors and experiment with different techniques. Remember, practice makes perfect, so don’t be discouraged if your first batch doesn’t turn out exactly as expected. With time and experience, you’ll be able to craft delicious rice wine that will impress your friends and family.

So, grab your ingredients, roll up your sleeves, and embark on this exciting journey of brewing rice wine. Cheers!

About John Ward

Hey beer lovers! Get ready for an experience as we explore the world of cloning beer recipes. Imagine being able Read more

Are you a wine enthusiast looking to explore the world of winemaking in ways? Get ready for a journey into Read more

Welcome to the realm of winemaking, where we unlock the hidden potential of humble grapes and transform them into something Read more

Immerse yourself in the realm of sangria. A delightful blend that harmonizes fruits and wine creating a symphony of flavors. Read more

How To Create A Wine Brand

How to bring wine on royal caribbean.

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• My Bibliography
• Collections
• Citation manager

Save citation to file

Email citation, add to collections.

• Create a new collection
• Add to an existing collection

Add to My Bibliography

Your saved search, create a file for external citation management software, your rss feed.

• Search in PubMed
• Search in NLM Catalog
• Add to Search

Optimization of the Brewing Conditions of Shanlan Rice Wine and Sterilization by Thermal and Intense Pulse Light

Affiliations.

• 1 School of Food Science and Engineering, Hainan University, Haikou 570228, China.
• 2 Key Laboratory of Food Nutrition and Functional Food of Hainan Province, Haikou 570228, China.
• 3 School of Biomedical Engineering, Hainan University, Haikou 570228, China.
• PMID: 37049943
• PMCID: PMC10096255
• DOI: 10.3390/molecules28073183

This study aimed to optimize the brewing conditions of Shanlan rice wine (SRW) and select a suitable sterilization method. The response surface method experiment was used to optimize the brewing process of SRW. LC-MS/MS (liquid chromatography-tandem mass spectrometry) and GC-MS (gas chromatography-mass spectrometry) were used to analyze the physicochemical components, free amino acids, and flavor metabolites of the thermal-sterilized SRW and the SRW sterilized by intense pulsed light (IPL), respectively. Results showed that the optimum fermentation conditions of SRW were as follows: fermentation temperature, 24.5 °C; Qiuqu amount (the traditional yeast used to produce SRW), 0.78%; water content, 119%. Compared with the physicochemical properties of the control, those of the SRWs separately treated with two sterilization methods were slightly affected. The 60 s pulse treatment reduced the content of bitter amino acids, maintained sweet amino acids and umami amino acids in SRW, and balanced the taste of SRW. After pasteurization, the ester content in wine decreased by 90%, and the alcohol content decreased to different degrees. IPL sterilization slightly affected the ester content and increased the alcohol content. Further analysis of the main flavor metabolites showed that 60 s pulse enhanced the important flavor-producing substances of SRW. In conclusion, 60 s pulse is suitable for sterilizing this wine.

Keywords: Shanlan rice wine; free amino acids; intense pulse light sterilization; pasteurization; volatile compound.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Effect of a single factor…

Effect of a single factor on the quality of SRW. ( a )…

Response surface plot for sensory…

Response surface plot for sensory evaluation as a function of, ( a )…

The difference of 21 free…

The difference of 21 free amino acids between unsterilized and other sterilized SRW.

Principal component analysis (PCA) of…

Principal component analysis (PCA) of flavor metabolites in SRW before and after sterilization.…

The proportion of the identified…

The proportion of the identified flavor metabolites in the composition classification.

Volcanic map of flavorful metabolites…

Volcanic map of flavorful metabolites of SRW before and after sterilization. ( a…

Similar articles

• Flavor characteristics of Shanlan rice wines fermented for different time based on HS-SPME-GC-MS-O, HS-GC-IMS, and electronic sensory analyses. Hao Y, Li J, Zhao Z, Xu W, Wang L, Lin X, Hu X, Li C. Hao Y, et al. Food Chem. 2024 Jan 30;432:137150. doi: 10.1016/j.foodchem.2023.137150. Epub 2023 Aug 21. Food Chem. 2024. PMID: 37634344
• Characteristics of traditional Chinese shanlan wine fermentation. Yang D, Luo X, Wang X. Yang D, et al. J Biosci Bioeng. 2014 Feb;117(2):203-207. doi: 10.1016/j.jbiosc.2013.07.010. Epub 2013 Sep 5. J Biosci Bioeng. 2014. PMID: 24012384
• The dynamics of physicochemical properties, microbial community, and flavor metabolites during the fermentation of semi-dry Hakka rice wine and traditional sweet rice wine. Qian M, Ruan F, Zhao W, Dong H, Bai W, Li X, Huang X, Li Y. Qian M, et al. Food Chem. 2023 Aug 1;416:135844. doi: 10.1016/j.foodchem.2023.135844. Epub 2023 Mar 4. Food Chem. 2023. PMID: 36893639
• Research progress on the brewing techniques of new-type rice wine. Jiao A, Xu X, Jin Z. Jiao A, et al. Food Chem. 2017 Jan 15;215:508-15. doi: 10.1016/j.foodchem.2016.08.014. Epub 2016 Aug 6. Food Chem. 2017. PMID: 27542505 Review.
• Research Progress on Flavor and Quality of Chinese Rice Wine in the Brewing Process. Mao X, Yue SJ, Xu DQ, Fu RJ, Han JZ, Zhou HM, Tang YP. Mao X, et al. ACS Omega. 2023 Aug 30;8(36):32311-32330. doi: 10.1021/acsomega.3c04732. eCollection 2023 Sep 12. ACS Omega. 2023. PMID: 37720734 Free PMC article. Review.
• Correlation between changes in flavor compounds and microbial community ecological succession in the liquid fermentation of rice wine. Xu JZ, Zhang YY, Zhang WG. Xu JZ, et al. World J Microbiol Biotechnol. 2023 Nov 20;40(1):17. doi: 10.1007/s11274-023-03844-5. World J Microbiol Biotechnol. 2023. PMID: 37981595
• Yang Y., Xia Y., Wang G., Tao L., Yu J., Ai L. Effects of boiling, ultra-high temperature and high hydrostatic pressure on free amino acids, flavor characteristics and sensory profiles in Chinese rice wine. Food Chem. 2019;275:407–416. doi: 10.1016/j.foodchem.2018.09.128. - DOI - PubMed
• Chen D., Mosher W., Wiertzema J., Peng P., Min M., Cheng Y., An J., Ma Y., Fan X., Niemira B.A., et al. Effects of intense pulsed light and gamma irradiation on Bacillus cereus spores in mesquite pod flour. Food Chem. 2020;344:128675. doi: 10.1016/j.foodchem.2020.128675. - DOI - PubMed
• Ojha K.S., Tiwari B.K., O’Donnell C.P. Chapter Six-Effect of Ultrasound Technology on Food and Nutritional Quality. Adv. Food Nutr. Res. 2018;84:207–240. doi: 10.1016/bs.afnr.2018.01.001. - DOI - PubMed
• Timmermans R.A.H., Mastwijk H.C., Berendsen L.B.J.M., Nederhoff A.L., Matser A.M., Van Boekel M.A.J.S., Groot M.N.N. Moderate intensity Pulsed Electric Fields (PEF) as alternative mild preservation technology for fruit juice. Int. J. Food Microbiol. 2019;298:63–73. doi: 10.1016/j.ijfoodmicro.2019.02.015. - DOI - PubMed
• Jung K., Song B.-S., Kim M.J., Moon B.-G., Go S.-M., Kim J.-K., Lee Y.-J., Park J.-H. Effect of X-ray, gamma ray, and electron beam irradiation on the hygienic and physicochemical qualities of red pepper powder. LWT. 2015;63:846–851. doi: 10.1016/j.lwt.2015.04.030. - DOI
• Search in MeSH

Grants and funding

Linkout - more resources, full text sources.

• Europe PubMed Central
• PubMed Central

Miscellaneous

• NCI CPTAC Assay Portal

• Citation Manager

NCBI Literature Resources

MeSH PMC Bookshelf Disclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

SENSORY ACCEPTABILITY OF PINILISA RICE WINE A PRODUCT DEVELOPMENT

• Jones Rural School, Jones Isabela
• This person is not on ResearchGate, or hasn't claimed this research yet.

Discover the world's research

• 25+ million members
• 160+ million publication pages
• 2.3+ billion citations
• Recruit researchers
• Join for free
• Login Email Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google Welcome back! Please log in. Email · Hint Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google No account? Sign up

How to make rice wine?

Rice wine is a traditional alcoholic beverage enjoyed by many cultures around the world. Made primarily from fermented rice, this drink has a unique flavor and aroma that adds depth to various dishes and can also be savored on its own. If you’ve ever wondered how to make rice wine, you’re in luck! In this article, we will explore the step-by-step process of brewing rice wine and provide answers to frequently asked questions related to this delightful beverage.

Do you like this article? Yes No

Thank you! Please keep reading.

In this article:

To make rice wine, also known as sake, you will need the following ingredients and equipment: – High-quality rice – Koji rice or koji starter – Water – Yeast – A large pot or fermentation vessel – Airtight containers for storage

1. Rinse the rice thoroughly to remove any impurities and excess starch. 2. Cook the rice according to the package instructions until it is soft and tender. 3. Allow the rice to cool to room temperature. 4. Sprinkle koji rice or koji starter over the cooled rice. Koji is a mold that helps break down the rice starches into sugars, which the yeast will then convert into alcohol. 5. Mix the koji rice with the cooked rice, ensuring it is evenly distributed. 6. Dissolve the yeast in warm water and add it to the rice mixture. Stir well. 7. Transfer the mixture to a large pot or fermentation vessel, making sure there is some headspace for the fermentation process. 8. Cover the vessel tightly and store it in a warm, dark place for fermentation. 9. Allow the mixture to ferment for approximately 1-2 weeks, stirring it daily to prevent the growth of harmful bacteria. 10. After fermentation, strain the liquid from the rice solids using a cheesecloth or fine mesh strainer. 11. Transfer the strained liquid, which is now rice wine, to airtight containers for storage. 12. Store the rice wine in a cool, dark place for a minimum of 1-2 months to allow it to mature and develop complex flavors. 13. Once matured, your homemade rice wine is ready to be enjoyed!

Frequently Asked Questions about making rice wine:

1. can i use any type of rice to make rice wine.

While some varieties of rice are better suited for making rice wine, such as short-grain japonica rice, you can experiment with different types to find the flavor profile that suits your taste.

2. Where can I find koji rice or koji starter?

Koji rice or koji starter can usually be found at specialty Asian grocery stores, or you can even make your own by cultivating the mold spores on steamed rice.

3. Do I need specialized equipment for making rice wine?

While specialized equipment can help streamline the process, you can start with basic kitchen tools like a large pot, cheesecloth, and airtight containers.

4. How important is temperature during fermentation?

Maintaining a constant temperature between 20-30°C (68-86°F) is crucial for the fermentation process of rice wine. Drastic temperature fluctuations could negatively affect the outcome.

5. How can I ensure a successful fermentation?

Proper sanitation and hygiene are key to a successful fermentation. Clean all equipment thoroughly and ensure airtight seals to avoid contamination.

6. Can I adjust the alcohol content in my homemade rice wine?

Yes, the alcohol content can be adjusted by controlling the fermentation time and the amount of sugar in the initial mixture.

7. How long does the fermentation process take?

The fermentation process typically takes 1-2 weeks, but this can vary depending on factors such as temperature and desired flavor.

8. Can I drink rice wine immediately after fermentation?

While you can consume rice wine immediately after fermentation, it is recommended to let it mature for at least 1-2 months to enhance its flavor.

9. How should I store rice wine?

Store rice wine in a cool, dark place, away from direct sunlight. Proper storage will extend the shelf life and maintain the quality of your homemade rice wine.

10. Can I use homemade rice wine for cooking?

Absolutely! Homemade rice wine adds depth and flavor to various savory dishes, particularly in marinades and sauces.

11. What should I do if my rice wine smells foul or has visible mold?

If your rice wine smells bad or you notice visible mold, it is best to discard it. Foul odor or mold growth indicates bacterial contamination.

12. Can rice wine go bad?

If stored properly, rice wine has a relatively long shelf life and can be enjoyed for up to several years. However, once opened, it is best consumed within a few months to maintain its quality.

By following these steps and experimenting with different techniques and ingredients, you can perfect the art of making rice wine. Enjoy the sense of accomplishment when savoring a glass of your homemade rice wine, and don’t forget to share it with friends and family!

Watch this awesome video to spice up your cooking!

• Why do my teeth hurt when I eat hard food?
• How many cups in a medium onion?
• Is ketchup spicy?
• How to make turmeric tea from fresh turmeric?
• How much is beer at wrigley field?
• What is salsa macha?
• Where is johnathan from hells kitchen now?
• Is liquid nitrogen safe to eat?

About Julie Howell

Julie has over 20 years experience as a writer and over 30 as a passionate home cook; this doesn't include her years at home with her mother, where she thinks she spent more time in the kitchen than out of it. She loves scouring the internet for delicious, simple, heartwarming recipes that make her look like a MasterChef winner. Her other culinary mission in life is to convince her family and friends that vegetarian dishes are much more than a basic salad. She lives with her husband, Dave, and their two sons in Alabama.

Leave a Comment Cancel reply

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here .

Loading metrics

Open Access

Peer-reviewed

Research Article

Discovery of electromagnetic polarization in Asian rice wine deterioration process and its applications

Roles Conceptualization, Supervision, Writing – review & editing

Affiliations Department of Mathematical and Information Sciences, Shaoxing University, Shaoxing, China, Institute of Artificial Intelligence, Shaoxing University, Shaoxing, China, Visiting Scholar, Department of AOP Physics, University of Oxford, Oxford, United Kingdom, National Engineering Research Center for Chinese Rice Wine (Branch Center), Shaoxing University, Shaoxing, China

Contributed equally to this work with: Xuejing Cao, Xue Cheng, Dongqin Sun

Roles Software, Writing – original draft

Affiliations Department of Mathematical and Information Sciences, Shaoxing University, Shaoxing, China, School of Life Sciences, Shaoxing University, Shaoxing, China

Roles Investigation, Validation, Visualization

Affiliation Department of Mathematical and Information Sciences, Shaoxing University, Shaoxing, China

Roles Software

Roles Formal analysis, Validation

Roles Resources, Supervision

Roles Validation, Visualization, Writing – review & editing

Roles Formal analysis, Writing – original draft

Roles Formal analysis

Affiliations Department of Mathematical and Information Sciences, Shaoxing University, Shaoxing, China, Yingfu Tech Group Co. Ltd, Hong Kong, China

Roles Resources

Affiliations Visiting Scholar, Department of AOP Physics, University of Oxford, Oxford, United Kingdom, Oxford Industrial Holding Group, Hong Kong, China

Roles Data curation, Resources

* E-mail: [email protected]

Affiliations National Engineering Research Center for Chinese Rice Wine (Branch Center), Shaoxing University, Shaoxing, China, School of Life Sciences, Shaoxing University, Shaoxing, China

• Weijia Zhang, 
• Xuejing Cao, 
• Xue Cheng, 
• Dongqin Sun, 
• Tianfang Wei, 
• Zebo Fang, 
• Jiaju Li, 
• Feiyu Chen, 
• Xinghua Liu, 

• Published: June 20, 2024
• https://doi.org/10.1371/journal.pone.0302983
• Reader Comments

Rice wine, known as yellow wine in China and Japan, possesses considerable nutritional value and holds significant global influence. This study addresses the challenge of preserving rice wine, which is prone to rancidity due to its low alcohol content. Conventional storage techniques employing pottery jars often result in substantial spoilage losses. Through rigorous investigation, this research identifies a polarization phenomenon exhibited by degraded rice wine when subjected to high-frequency microwaves(>60GHz), presenting a pioneering method for detecting spoilage, even within sealed containers. Employing a multi-channel microwave radar apparatus, the study delves into the susceptibility of rice wine to electromagnetic waves across various frequencies, uncovering pronounced polarization traits in deteriorated samples within the E-band microwave spectrum. Furthermore, lab-controlled simulations elucidate a direct correlation between physicochemical alterations and high-frequency Radar Cross Section (RCS) signals during the wine’s deterioration process. A novel six-membered Hydrated Cluster hypothesis is proposed, offering insights into the molecular mechanisms underlying this phenomenon. Additionally, dielectric property assessments conducted using vector network analyzers (VNA) reveal noteworthy enhancements in the dielectric constant of deteriorated rice wine, particularly within the high-frequency domain, thereby augmenting detectability. These findings carry implications for refining rice wine preservation techniques and contribute to the advancement of non-destructive testing technologies, enabling the detection of rice wine deterioration or indications thereof, even within sealed vessels.

Citation: Zhang W, Cao X, Cheng X, Sun D, Wei T, Fang Z, et al. (2024) Discovery of electromagnetic polarization in Asian rice wine deterioration process and its applications. PLoS ONE 19(6): e0302983. https://doi.org/10.1371/journal.pone.0302983

Editor: Trung Quang Nguyen, Center for Research and Technology Transfer, VIET NAM

Received: December 21, 2023; Accepted: April 15, 2024; Published: June 20, 2024

Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data of this study are available from USX AIlab database with the following link: www.geoscience.ac.cn/usx/microwave/ricewine.zip .

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

1.1. industrial value, storage process, and easy deterioration of rice wine.

Rice wine, also referred to as yellow wine in China and Japan, boasts a brewing legacy spanning nearly 4000 years in Asia, positioning it among the world’s oldest alcoholic beverages, alongside beer and grape wine, collectively recognized as the three ancient wines [ 1 ]. Presently, Chinese rice wine maintains an annual production stability within the range of 2.5 to 3.5 million kiloliters. In contrast, global wine production stands at approximately 25 million kiloliters, highlighting that Chinese rice wine output constitutes 1/10 of the worldwide wine production. The significance of this study lies in its potential to substantially reduce production costs for rice wine.

Rice wine is esteemed for its nutritional value. Crafted from wheat and various grains as primary ingredients, its production involves the utilization of Wheat Qu, Rice Qu, or alcoholic medicine as saccharifying agents. Through a meticulous process encompassing steps such as steaming rice, yeast fermentation, wine distillation, and storage [ 2 , 3 ], rice wine achieves its distinctive flavor profile and aromatic characteristics.

Rice wine is renowned for its opulent bouquet and distinctive flavor profile, attributes stemming from the intricate interplay of diverse compounds synthesized throughout the fermentation process, encompassing esters, alcohols, aldehydes, acids, carbonyl compounds, and phenols. Notably, adherence to traditional consumption practices persists in China, Japan and other nations, underscoring the enduring cultural significance of this libation [ 4 ].

In both China and Japan, the traditional storage vessels for hand-brewed rice wine encompass a variety of materials, including pottery jars, carbon-steel tanks, and stainless-steel tanks. As shown in Fig 1 , an example of rice wine storage at Shaoxing Wine Distillery in China. Prior to sealing the rice wine within these containers, it undergoes a rigorous high-temperature decoction process, effectively neutralizing the majority of aerobic microorganisms and enzymes. Subsequently, the hot wine is promptly transferred into aging pottery jars, featuring a clay cover surface, and tightly sealed using lotus leaves, bamboo shells, or similar materials. Notably, the utilization of pottery jars is favored for its exceptional permeability, facilitating accelerated redox and esterification reactions during the aging phase. It is essential to recognize that not all rice wines are amenable to prolonged aging, with certain varieties suitable for storage periods ranging from three to five years. Long-term storage viability is contingent upon the utilization of premium ingredients, masterful craftsmanship by the winemaker, and fortuitous contributing factors [ 5 ].

• PPT PowerPoint slide
• PNG larger image
• TIFF original image

https://doi.org/10.1371/journal.pone.0302983.g001

Nevertheless, the preservation of traditional rice wine poses a challenge due to its inherent susceptibility to souring, attributed to its relatively low alcohol content (usually <20%).

Hence, souring phenomena are frequently encountered during the storage of rice wine. Presently, rice wine enterprises predominantly derive their revenue from aged rice wine. Over a 10-year span, the average deterioration rate exceeds 20%, thus emerging as the primary cost-impacting factor within the rice wine industry as a whole.

The rancidity of rice wine primarily manifests as acetic and lactic acid rancidity. Acetic acid rancidity is prevalent in low-alcohol and aerobic conditions, often stemming from compromised or inadequately sealed wine jars [ 6 , 7 ]. While disruptive, it poses less harm to the standard storage of rice wine. In contrast, lactic acid rancidity emerges as the predominant cause of deterioration during the aging process of rice wine in a closed environment. Notably, researchers have isolated Lactobacillus fructivorans and Lactobacillus acidophilus from sour rice wine. These strains exhibit high alcohol tolerance and thrive in acidic environments, contributing significantly to the occurrence of lactic acid rancidity.

In the assessment of rice spoilage, the conventional method necessitates the opening of the storage vessel for tactile examination and taste evaluation. Consequently, rice wine aged over decades can solely undergo sampling and analysis upon unsealing the container. Nonetheless, the unveiling of the vessel marks the conclusion of the entire rice wine aging process. In the event of souring, rot, or contamination, the entirety of the rice wine batch becomes unsalvageable, thereby diminishing the overall yield of the rice wine sector and consuming significant storage capacity, thereby augmenting production expenses considerably.

1.2. Chemical changes of rice wine during fermentation process

Consequently, the examination of the physico chemical attributes of degraded rice wine has garnered attention from previous researchers. Based on extant research, it has been observed that the alcohol content, sugar concentration, acidity, and pH levels of rice wine tend to decline with prolonged aging. Conversely, the levels of amino acid nitrogen and non-sugar solids exhibit varying degrees of augmentation. Ester concentrations reach their zenith at approximately five years before experiencing a modest decline thereafter, ultimately stabilizing [ 6 – 8 ].

Typically, substandard rice wine exhibits lower total sugar and alcohol levels compared to its unspoiled counterpart, accompanied by an elevated total acidity [ 9 ].

In Table 1 , it is observed that the ethyl content experiences an increase after five years followed by a decrease after eight years, a pattern mirrored by ethyl hexanoate, octanoate, caprate, and the overall content. One plausible explanation for this phenomenon is that rice wine undergoes a sequence of chemical transformations during the initial phase of storage, predominantly characterized by reactions between acids and alcohols, culminating in the generation of ester compounds. As the storage duration progresses, it is anticipated that these ester compounds, alongside associated alcohol constituents, will gradually volatilize, thereby resulting in a downward trajectory in their respective contents.

https://doi.org/10.1371/journal.pone.0302983.t001

Previous studies regarding the degradation of rice wine have predominantly centered on the examination of its chemical composition and observable physical attributes, yielding notable advancements in this domain [ 10 , 11 ]. Nevertheless, a notable gap persists in the literature concerning the potential interaction between rice wine and electromagnetic waves. Given that rice wine is conventionally stored in pottery jars, characterized by their imperviousness to infrared radiation, this aspect remains largely unexplored.

Instead of infrared radiation, our research team utilized multi-channel microwave technology for detection, yielding notable results [ 12 , 13 ]. Additionally, we successfully employed microwave technology for sub-surface machine vision, demonstrating commendable outcomes [ 14 ]. Notably, microwave, with its longer wavelength compared to infrared, proved advantageous for penetrating several centimeters or even meters into materials [ 15 – 19 ]. Given its ability to easily penetrate pottery jars storing rice wine, our team embarked on an innovative experiment using multi-channel microwave equipment. This endeavor aimed to investigate the sensitivity of rice wine to electromagnetic waves of varying frequencies throughout its aging and deterioration process.

This research aims to delve into the aging-related deterioration of rice wine, specifically exploring the distinctive polarization characteristics and electromagnetic alterations observed in deteriorated samples when exposed to specific frequencies of microwave electromagnetic waves.

The experiments unveiled a notable trend wherein deteriorated rice wine samples exhibited markedly heightened polarization characteristics in response to a specific high-frequency electromagnetic wave (specifically, the E-band microwave electromagnetic wave within the 60–70 GHz frequency range), contrasting with the good-quality rice wine samples. The research team conducted multiple corroborative experiments across diverse groups, thereby establishing this phenomenon for the first time.

This previously unreported phenomenon holds promise as a potential marker for assessing rice wine quality. Furthermore, it could serve as a valuable tool for detecting signs of rice wine deterioration, even when stored in sealed containers, and potentially for monitoring other fermentation processes that yield acidic byproducts.

2. Methods and experiments

2.1. high frequency rcs differences found in spoiled rice wine.

The samples utilized in our investigation were sourced directly from the distilleries and meticulously assessed by professional technicians to categorize their quality status as normal, mildly deteriorated, or severely deteriorated. Each sample underwent a comprehensive evaluation process to ascertain the extent of deterioration. Severely deteriorated samples exhibited pronounced acidity levels, visible microbial proliferation, and emitted off-putting odors discernible by trained sensory panelists. Conversely, mildly deteriorated samples displayed elevated acidity levels, a sour taste lacking the typical flavor profile, yet devoid of visible microbial growth. Prior to sample provision, the distillery conducted extensive physical and chemical analyses alongside sensory assessments by trained experts, furnishing sample data to validate their quality classification.

We initially devised an experiment targeting ordinary and mildly deteriorated rice wines, both sourced from the Chinese National Yellow Wine Engineering Experimental Center. Employing identical containers with known dielectric properties, as shown in Fig 2(A) and 2(B) , (two custom-made marble containers with a measured density of 2.63 g/cm 3 and a magnetic permeability of 0.99 H/m), we utilized a microwave radar probe to vertically emit electromagnetic waves into the container and capture their echo signal. The radar equipment offered a range of frequencies, including 300 MHz, 2.2 GHz, 6.8 GHz–8 GHz, 29 GHz, and 60 GHz–70 GHz.

(a): Quality inspection machine measures two wine jars with customized parameters, observing differences in echo imaging value curves. (b): Up to 69GHz high-frequency microwave detector.

https://doi.org/10.1371/journal.pone.0302983.g002

The aforementioned microwaves demonstrated efficient penetration through the structure of the 1 cm-thick marble wine container, reflecting upon encountering the interface between the wine and the container. Both wine containers exhibited identical outward appearances. One container held 2014 GYLS rice wine of standard quality, produced and sealed in 2014, boasting a pH value of 4.4. Conversely, the other container housed samples from the same 2014 batch, yet classified as spoiled by the Chinese National Rice Wine Engineering Experimental Center due to a pH value of 3.7, and a sour taste.

The experimenters meticulously observed and meticulously documented the imaging data pertaining to the microwave radar cross-sectional area (referred to as Radar Cross Section, or RCS).

Prior to conducting measurements, the radar equipment was powered on for an extended duration to ascertain its stable operational state post-warm-up. Throughout this period, the radar temperature remained constant, mitigating any potential drift in RCS values attributed to temperature fluctuations. This standardized protocol was consistently implemented for each experimental group.

At lower frequencies, no discernible distinctions were observed. Nevertheless, as the frequency progressively surpassed 60 GHz, noteworthy variations in the measured Radar Cross Section (RCS) values between the two groups became evident. Specifically, the 69GHz RCS values for the substandard rice wine group were approximately 10% higher compared to those of the superior rice wine group.

This discrepancy persisted in all the repeated tests (>20), and even after swapping and exchanging the wine containers, effectively eliminating the possibility that variations stemmed from the specific wine containers utilized in the experiments.

As elucidated in Merrill I. Skolnik’s seminal work, "Introduction to Radar Systems" [ 15 ], the radar cross-sectional area undergoes alterations contingent upon the angle, frequency, and polarization. When maintaining consistency in the first two parameters, any variation in RCS delineates shifts in the polarization characteristics.

With the angle and frequency remaining constant, and given the observed disparities in RCS values, we postulate a potential augmentation in the polarization properties of the substandard rice wine at microwave frequencies surpassing 60 GHz.

2.2. RCS measurements validation on rice wine and different liquids with known polarity sequences

Following the revelation of this phenomenon wherein inferior rice wine exhibits a significantly elevated echo RCS value above 60GHz, our subsequent inquiry aimed to corroborate the electromagnetic polarization of such subpar rice wine. To achieve this objective, we orchestrated a secondary set of experiments encompassing a broader array of severely degraded rice wine samples, normal rice wine samples from the same production batch, alongside various liquids of established polarity. Within this experimental paradigm, a high-frequency millimeter-wave radar was meticulously positioned directly above the liquid interface within the vessel, as shown in Fig 3 , sending 69 GHz high-frequency E-band microwaves towards the liquid’s surface below, and subsequently capturing their echoes.

Tested liquids: distilled water, pure ethanol, formamide, kerosene, normal rice wine, and bad rice wine.

https://doi.org/10.1371/journal.pone.0302983.g003

Fig 4 shows the result of our second experimental measurement.

https://doi.org/10.1371/journal.pone.0302983.g004

In Fig 4 , each curve corresponds to a specific type of liquid, with the peak value indicative of its RCS or polarity. Analysis of the data graph reveals that the RCS or polarity exhibited by severely degraded rice wine is only marginally lower than that of distilled water at 69 GHz, yet notably surpasses that of conventionally produced rice wine of standard quality. It’s worth noting that measurements for each liquid were derived from a consistent set of 20 samples, ensuring the experiment’s robust repeatability.

2.3. Experiments conducted across pottery jars as real industrial conditions

In order to ascertain the practical industrial applicability of this revelation and its potential to permeate pottery vessels for quality monitoring across the pottery jars, a third set of experiments was undertaken. In this experimental series, we positioned four microwave radar devices around an pottery wine jar, each separated by 90 degrees,as shown in Fig 5 . Subsequently, four independent observations were conducted concurrently, each facilitated by a dedicated computer system and overseen by a distinct supervisory experimenter.

Full experiment video uploaded at www.geoscience.ac.cn/usx/microwave/ricewine.zip .

https://doi.org/10.1371/journal.pone.0302983.g005

In this experiment, the frequency of 69 GHz was employed. Once the radar attained a stable operational state, measurements commenced. Initially, the empty wine jar was measured to establish the baseline RCS value of the radar. Subsequently, water was added to obtain the corresponding RCS value. Following this, a siphon device was utilized to pump all the water, and after thorough drying, standard quality rice wine (specifically, Jia Fan from 2014, with a pH value of 4.4) was added to measure the radar’s RCS value. The process was repeated with mildly deteriorated rice wine (Jia Fan from 2014, spoiled, pH 3.7). Given the findings from the preceding experiments, where milder deterioration exhibited lower polarity than severe deterioration, the ability to detect mild deterioration suggests the feasibility of detecting severe deterioration.

This database is publicly accessible, enabling anyone to download and peruse our experimental procedures.

As shown in Fig 6 , the observed values of air by each radar device consistently ranked as the lowest, succeeded by those of the standard quality rice wine, water, and finally, the inferior rice wine registering the highest value. Consequently, the inferior rice wine utilized in this experiment exhibited a more pronounced polarization reaction even compared to water.

https://doi.org/10.1371/journal.pone.0302983.g006

In summary, this set of experiments confirmed the presence of the electromagnetic polarization phenomenon in rice wine following deterioration.

Given that microwaves can penetrate rice wine jars, prolonged observation at a fixed position by millimeter-wave radars could enable the monitoring of RCS changes, thus indicating rice wine deterioration without necessitating container opening. Consequently, such polarization phenomenon harbors promising industrial applications.

3. Validation

3.1. validation: 8ghz/65ghz/69ghz dielectric properties of rice wine before and after deterioration measured with vector network analyzer (vna).

To validate the discovery, we employed a vector network analyzer (KeySight M9375A PXIe VNA) to assess if there are increases in relative permittivity following deterioration. The VNA facilitates the transmission of a generated microwave signal source to the open wave guide transmitting antenna through a corresponding coaxial connecting cable, utilizing the tested liquid substance as the microwave transmission medium. Interactions between the microwave and the liquid substance occur as the electromagnetic wave traverses through it. The open wave guide receiving antenna captures the transmitted signal, which is then conveyed to the VNA, equipped with an internal computer, through the coaxial connecting cable. Subsequently, the received microwave signal undergoes analysis and processing to ascertain the dielectric constant of the liquid samples.

Through the assessment of the dielectric constant of rice wine (samples shown in Figs 7 and 8 across various frequency bands, a distinct variation in dielectric constant emerged between spoiled and non-spoiled rice wines, particularly notable in high-frequency bands.

Results annotated in Table 2 .

https://doi.org/10.1371/journal.pone.0302983.g007

Report available at www.feifan-sz.cn with report or validation number 44577035.

https://doi.org/10.1371/journal.pone.0302983.g008

As depicted in Table 2 , the dielectric constant contrast between deteriorated and non-deteriorated rice wines at 69GHz surpasses 60%, indicating a pronounced significance in polarity divergence under high-frequency microwaves beyond 69GHz. Conversely, this distinction only reaches less than 10% at 8GHz.

Report can be checked at www.chinafcta.com with report number).

https://doi.org/10.1371/journal.pone.0302983.t002

This outcome underscores that in the low-frequency microwave spectrum, disparities in dielectric constants are less pronounced, whereas they become markedly discernible at higher frequencies. These differences are likely attributable to the sensitivity of the utilized equipment, offering insights into the observed phenomena within our experiments.

A conspicuous 60% distinction precludes the possibility of other potential errors. The heightened polarity of spoiled rice wine under high microwave frequencies is unequivocal. Furthermore, this discernible enhancement holds practical significance, offering potential industrial applications for non-invasive quality measurement of rice wine without the need to open its container.

To validate these findings, additional experiments were conducted using samples sourced from KuaiJiShan, another renowned rice wine producer. The expanded sample size accounted for various factors including distinct batches, manufacturers, and vessels. In this iteration, measurements of both dielectric constant and dielectric loss were performed concurrently. The outcomes are summarized in Table 3 .

https://doi.org/10.1371/journal.pone.0302983.t003

The findings from the second batch of samples, as presented in Table 3 , corroborate those of the initial batch outlined in Table 2 . Notably, substantial discrepancies in relative permittivity, encompassing both dielectric constant and dielectric loss, are evident within the high-frequency (>60GHz) microwave spectrum.

3.2. Further validation of the results and correlation between 60GHz RCS values & acidic components

The principal chemical compositional alterations in rice wine before and after deterioration primarily revolve around changes in acidic molecules content [ 20 ]. For instance, the rapid fluctuation in total acid content during the pre-fermentation phase of rice wine, as detected through near-infrared spectroscopy [ 20 ], is notable. Additionally, lactic acid bacteria engage in esterification reactions with ethanol, leading to the decomposition of weakly polar ethanol and consequent elevation in the proportion of more polar substances, such as lactic acid.

Hence, the most immediate hypothesis to consider is that the high-frequency electromagnetic polarization effect we have uncovered may be linked to alterations in the liquid components, particularly the production of acidic substances, within yellow rice wine.

In order to substantiate the connection between the total acid content present in alcoholic beverages and the RCS values identified by radar, the research team devised an additional validation experiment. This endeavor sought to explore the correlation between the total acid content inherent in spoiled rice wine and the corresponding detection values obtained from radar equipment RCS. The objective was to scrutinize the interrelationship between the total acid molecular content of liquid components and the resultant RCS values gleaned from radar imaging.

(1) Preparations.

The requisite materials for the experiment encompassed rice wine (Shanniang Brand, produced in 2019), standard sodium hydroxide titration solution, distilled water, acrylic cubic containers, a thermometer, high-frequency radar equipment, MS-H-Pro+ magnetic stirrer, pipette, Z-axis manual lift platform (HTZ-120), acidity meter, calipers, and ruler. In order to mitigate the potential influence of temperature variations on the experimental results obtained from the high-frequency radar equipment, the solution temperature was meticulously maintained at 26°C to uphold data accuracy.

(2) Procedures.

Controlled deterioration was induced in rice wine through the addition of 1 gram of yeast to 500 milliliters of Shaoxing rice wine. Subsequently, the mixture was incubated in a constant temperature chamber set at 37°C.

Deterioration became evident, with the flavor exhibiting a sour profile, commencing on the third day of observation.

In the provided equation, X denotes the total acidity content within the sample, delineated in grams per liter (g/L). C represents the concentration of the standard sodium hydroxide titration solution, expressed in moles per liter (mol/L). The variable M signifies the numerical value of the molar mass of lactic acid, articulated in grams per mole (g/mol), with a defined value of 90. V 1 corresponds to the volume of standard sodium hydroxide titration solution utilized during the titration of the sample, quantified in milliliters (mL), while V 2 signifies the volume of standard sodium hydroxide titration solution employed during the blank test, also measured in milliliters (mL). Finally, V 3 denotes the volume of the extracted sample, measured in milliliters (mL).

(3) Data collection and analysis.

Adhering to the procedures delineated in the Chinese National Testing Standard GB/T 13662–2018 "Rice Wine," total acidity measurements were systematically executed to uphold the precision and replicability of the experiment.

During each acid measurement, 10 mL aliquot was carefully transferred into a 150 mL beaker, followed by the addition of 50 mL of degassed water. Subsequently, a magnetic stirring bar was introduced into the beaker, which was then positioned atop a magnetic stirrer and set into motion. Standard sodium hydroxide titrant was incrementally added into the beaker until the pH reached 8.20, indicating the equivalence point, at which juncture the volume of 0.1 mol/L standard sodium hydroxide titrant utilized was meticulously documented. Concomitantly, a control experiment employing an equivalent volume of degassed water was conducted, with the corresponding volume of titrant utilized being meticulously recorded.

Concurrently, the 60GHz RCS radar apparatus was employed to capture imaging data. Initially, the lifting platform’s elevation was consistently set to 4.8 cm for each measurement to ensure uniform radar height. Subsequently, the liquid level was meticulously adjusted to maintain a consistent horizontal alignment throughout the measurements. The region of interest selected for measurement was the interface between the yellow wine and air, positioned 5 cm above the base of the acrylic container (factoring in the 4 cm thickness of the acrylic plate). Data acquisition was conducted, and subsequent to recording and storage, total acidity was derived utilizing a mathematical model. Linear regression analysis, facilitated by Origin2021 software, was then employed to investigate the correlation between total acidity and radar imaging values.

All total acidity and measured RCS values, along with other data, are accessible in S2 Appendix . The average total acidity within the rice wine and deteriorated rice wine group peaked at 9.90 g/L, accompanied by a notably higher average radar-measured value.

Sample testing were did 2–3 times daily, with temperature adjustments preceding each test to maintain consistency. Each test session was spaced at least one hour apart. Testing spanned from July 9, 2023, to August 5, 2023, 4 data sets were excluded from analysis due to significant errors resulting from improper experimental procedures.

The experiment revealed a discernible positive correlation between total acidity and radar scattering characteristics. As depicted in Fig 7 , both RCS and the total acidity within the rice wine solution demonstrated a consistent upward trajectory.

The findings illustrated in Fig 9 indicate a pronounced positive correlation between total acidity and radar scattering characteristics when viewed holistically.

Original data recorded in S2 Appendix .

https://doi.org/10.1371/journal.pone.0302983.g009

Drawing from the experimental findings outlined in this section, it is rational to propose that the high-frequency electromagnetic polarization effect observed in deteriorated rice wine is linked to the emergence of newly formed acidic components. In the subsequent section, a theoretical model alongside computer simulations will be presented to further elucidate these phenomena.

4. Discussion

This section of the study endeavors to explore the molecular polarization model of deteriorated rice wine under high-frequency microwaves in detail,propose a hydrated cluster model as an explanation for all above, and entail a quantitative examination of electronic molecular transitions and liquid polarization, culminating in the development of a theoretical framework via Gaussian Software computer simulation fitting.

RCS is known to exhibit a positive correlation with polarity, primarily because RCS directly mirrors the relative permittivity (ε), which in turn is closely associated with the polarity of molecules. Polar molecules like water have high permittivity (78), while less polar ones like cyclohexane have low permittivity (2).This relationship arises because ε reflects the mitigation of electrostatic effects induced by the medium. In the case of liquids comprised of polar molecules, the application of an electric field prompts molecular polarization. The resultant counteracting electric field generated by these polarized molecules partially offsets the external electric field, leading to a more rapid attenuation of electrostatic effects compared to that observed in a vacuum.

In section 3, the research team uncovered that the positive correlation between RCS and total acidity content suggests that as the total acidity increases, the polarity of the deteriorated rice wine solution intensifies.

This phenomenon can be explained from following aspects:

Firstly, ethanol and water constitute the primary components of rice wine prior to deterioration. It is well-established that pure water exhibits high polarity. Conversely, ethanol exhibits non polarity. Consequently, the polarity of rice wine primarily dictated by the proportion of water present. During the deterioration of rice wine, ethanol undergoes decomposition and concurrent increase in various polar acid forms thereby elevating the proportion of polar molecules.Notably, L. acidophilus stands out as the primary microorganism responsible for inducing the rancidity of rice wine, followed by L. fructose [ 21 ]. Both acid molecules and water molecules exhibit greater polarity than ethanol, consequently leading to an augmented radar Radar Cross Section (RCS) response. This observation aligns with the theory posited by Yu Derun [ 22 ] regarding empirical parameters of polarity and the proportional relationship between the dielectric constant of solvents and their polarity. Moreover, research by Zhao Donghui [ 23 ] and others affirms that, at the same temperature, the order of polarity for common solvents is water > acid > alcohol.

Secondly, recent spectroscopic investigations over the past decade [ 24 ] have revealed the formation of various ethanol hydrate clusters between liquid ethanol and water molecules. Prior to deterioration, of, rice wine, typically contains an alcohol content ranging from 14% to 20%, with water volume fractions ranging from 70% to 90%. Under these conditions, the hydration clusters predominantly consist of hydrogen bonds formed between water molecules and ethanol molecule hydroxyl groups, characterized as (H2O) m′(EtOH) n′, where n′<n. As the water volume content continues to rise, the hydrophilic hydration between (H2O)m clusters and ethanol molecule hydroxyl groups gradually approaches saturation. Consequently, the relative volume occupancy of water molecules in comparison to ethanol expands. Post-deterioration, water molecules further interact with the hydrophobic CH group of ethanol molecules, fostering the formation of additional C–H…O hydrogen bonds. Notably, as water content increases, the strength of hydrogen bonding intensifies, leading to a shift in the frequency of C–H stretching vibration towards the high-frequency band [ 25 ].

The emerging constituents, such as lactic acid and acetic acid, within deteriorated rice wine likely adopt analogous structural forms, leading to the formation of novel hydration clusters. Lactic acids, characterized by their polar nature, participate in the creation of fresh polar molecular clusters with water molecules. When the vibration frequency of a specific entity falls within the high-frequency spectrum, it can precipitate the phenomenon of electromagnetic response enhancement within that frequency range.

Thirdly, and most importantly, a mechanism we consider paramount involves the generation of newly formed lactic acid and acetic acid, which may amalgamate into novel molecular clusters with water molecules. This resultant configuration is anticipated to exhibit higher polarity compared to the initial ethanol–water molecule clusters, consequently amplifying the response of high-frequency radar RCS.

In addressing this aspect, we performed quantum mechanical calculations pertaining to the second point. In a seminal study conducted in 1988, Semmler et al. [ 26 ] employed Raman spectroscopy to examine the spectra of acetic acid aqueous solutions. Their findings pointed towards the prevalence of predominantly cyclic dimers and linear dimers within the acetic acid aqueous solution [ 26 ].

Following this, scholars posited that cyclic dimers exhibited instability in polar solutions. In 1990, Yamamoto et al. [ 27 ],Substantiating this notion. In 2003, Chocholousova et al. [ 28 ], highlighted that within a micro-water environment, water molecules engage with dimers. This interaction leads to the association of water molecules with acetic acid molecules through new hydrogen bonding, resulting in the formation of water-separated dimer structures (WSD).

Since 2010, studies [ 29 , 30 ] consistently show that in diluted acetic acid/water solutions, structures primarily form from complexes between acetic acid and water molecules The configurations and energies derived from quantum chemical calculations (QCC) within this investigation align closely with those observed in the gas phase. Expanding upon this research, we delved into the impact of solvent effects. QCC calculations were extended to encompass aqueous solutions, yielding geometric configurations, energies, and thermodynamic data pertaining to acetic acid/water aggregate structures in such solutions. Our findings reveal that in diluted acetic acid solutions (<10% acid content), six-membered hydrogen-bonded ring structures with individual acetic acid molecules are emerge, akin to those in spoiled rice wine. Rings with fewer than seventeen members exceed 1%, with five-membered and nine-membered rings each around 3% of the total. Refer to Fig 10 for details on the six-membered ring configurations.

Hydrogen bonds are represented by dashed lines, with corresponding values listed nearby. Lengths (R) are in units of Å, and angles are in degrees. Data outside parentheses are values in the gas phase, while data inside parentheses are values in aqueous solution.

https://doi.org/10.1371/journal.pone.0302983.g010

In the above equation, p represents the magnitude of the dipole moment, q represents the magnitude of the charge, and d represents the distance of charge distribution.

The orientation of the dipole moment coincides with the alignment of the charge distribution, and its magnitude scales directly with both the magnitude of the charge and the distance of the charge distribution, as determined by the computation method for the dipole moment.

The pertinent computations were conducted using Gaussian simulation software, yielding a dipole moment of 3.672 Debye under the B3LYP/def2-TZVPD basis set with dispersion function. In contrast, the experimentally determined average dipole moment for water stands at 1.85 Debye. This discrepancy underscores the heightened polarity and relative permittivity of acid-water cluster structures compared to clusters formed solely by water molecules. Consequently, these structures will exhibit elevated characteristics such as dielectric constant and dielectric loss.

5. Conclusion

In China, rice wine is typically stored in pottery jars [ 6 ], with a loss rate ranging from 5% to 10%. This study employs the polarization phenomenon under microwave conditions to assess the degree of deteriorated in rice wine, even in sealed containers.

The ensuing progress encompasses:

We found that distinct polarization behavior in deteriorated rice wine samples within high-frequency electromagnetic spectrum(specifically within the E-band microwave range of 60–69 GHz). As a validation, we use VNA to evaluate the dielectric parameters of diverse varieties and degrees of deterioration of rice wine both high and low-frequencies.

Subsequently, this study delved into examining the quantitative correlation between alterations in physicochemical parameters during the degradation of rice wine and the resultant high-frequency RCS signals.Established a comprehensive data system affirming the approximate linear correlation between fluctuations in total acidity and radar RCS. Further elaboration on the experimental data is provided in S2 Appendix . To elucidate the experimental findings, we put forward a ‘Hydrated Cluster hypothesis’, in consonance with experimental observations.

The insights gleaned from this research hold practical implications for the detection and mitigation of rice wine and wine deterioration during storage, particularly in large stainless-steel tanks [ 31 ], mitigating potential economic losses without opening the tanks. This research holds significance for brewing science beyond the realm of rice wine, as analogous degradation and aging phenomena occur in diverse liquid consumables like beer [ 32 ] and soy sauce [ 33 , 34 ].

Supporting information

S1 appendix. correlation between radar imaging value y and radar cross-sections (rcs)..

https://doi.org/10.1371/journal.pone.0302983.s001

S2 Appendix. Total acidity analysis and comparative evaluation with RCS values.

https://doi.org/10.1371/journal.pone.0302983.s002

Acknowledgments

We thank National Engineering Research Center for Chinese Rice Wine, Shaoxing Wine Distillery at ShanYin, and KuaiJiShan Distillery for providing samples as well as access to the field site and laboratories.

We also would like to thank Miss Jin Xie, Mr Yulin Li, Miss Dan Qiu and other group members for helping in the experiments.

• View Article
• PubMed/NCBI
• Google Scholar
• 9. Xinsheng Wang. Study on the oxidation of Chinese rice wine with storage process (Master dissertation, Hefei: Hefei University of Technology).
• 15. Merrill I. Skolnik. Introduction to Radar Systems.2nd ed. McGraw-Hill; 1981.Chapter 2.
• 16. Zhong M. Research on the application of microwave detection in cerebral hemorrhage [D]. Guilin University of Electronic Science and Technology (in Chinese); 2020.
• 22. Zhao D. Application of Excited-State Hydrogen Bond Dynamics in the Field of Fluorescent Probes and the Influence of Solvent Polarity on ESIPT [Doctoral dissertation]. Liaoning University; 2023. https://doi.org/10.27209/d.cnki.glniu.2023.002030 (in Chinese).
• 24. Skolnik MI. Radar Handbook.2nd ed. McGraw-Hill; 1990.

A mum and daughter sold their Ye Traditions homemade red rice wine on Carousell as an experiment and it became a hit

Advertisement.

As part of their heritage, the Yap family made red rice wine in their kitchen for decades and enjoyed it at every celebration. Wondering if there were others who loved it as much, they casually offered it on Carousell, calling it Ye Traditions. The outpouring of memories it elicited surprised them.

This mother-and-daughter duo started their rice wine business, Ye Traditions, to preserve their family tradition and share their heritage with others. (Photo: Ye Traditions)

This audio is generated by an AI tool.

It all started because of a passing remark Yap Jinyen made on her birthday in 2019. The millennial was enjoying the red rice wine brewed by her mother Yap Joo Eng – a hallowed family tradition for generations – when she wondered out loud if brewing rice wine was a “ disappearing art ”.

“It’s so labour intensive! Are people going to continue to brew it? Are there people out there looking for it,” she mulled.

Her mother shared her sentiments. And off the cuff, the duo decided to try selling homemade culinary red rice wine on Carousell that year. They called the business Ye Traditions , incorporating the family surname.

The elder Yap is a 60-year-old retired accountant while her daughter is a 31-year-old tech professional. It was never their intention to start a home business selling red rice wine – in fact, they did not expect many people to buy it.

But their Carousell post had a life of its own. Orders grew to 100 bottles a month and the duo found themselves starting a pre-order system because they were not able to produce their red rice wine fast enough.

Because of the large amount of orders, Jinyen even checked if they needed a license to sell their products.

“We found out that cooking wine like ours, with an alcoholic strength of 21 per cent and below, and either a salt content of 1.5g per 100ml or a sugar content of 25g per 100ml is non-dutiable, according to Singapore Customs," she said. “We also found out that home-based food businesses do not require licensing.”

Jinyen and her mother also got themselves certified for food safety and hygiene with the Singapore Food Agency.

The business steadily grew, as did their product offerings. They added their Hakka yellow rice wine in 2021, and ginger rice wine in 2023. Online orders for these rice wines quickly grew to more than 500 bottles each month in total.

They also added other traditional products such as red rice lees (a residual product of fermenting red rice wine) and fermented glutinous rice wine (unfiltered rice wine with visible rice bits) used to make traditional dishes, as well as Hakka cooked dishes such as red rice wine chicken soup and Hakka wine chicken.

Wanting to make these traditional products more accessible to seniors, the mother-daughter pair plan to open a shop in Ang Mo Kio in the second quarter of the year.

THE TASTE OF NOSTALGIA

The organic growth of orders for red rice wine by an unknown brand on Carousell was quite remarkable. But that was not the reason that mother and daughter decided to start the business.

It’s so labour intensive! Are people going to continue to brew it? Are there people out there looking for it.

Jinyen recalled a pivotal moment when one of their first customers came to their house to thank them.

“He said he wanted to replicate a dish for his family but no one knew how to make red rice wine anymore. He also said our red rice wine reminded him of his grandmother,” said Jinyen. “My mother had goosebumps and I saw her tearing up.”

This resonated with Jinyen and her mother because red rice wine also reminds the latter of her late mother-in-law.  

The first generation Yaps came to Singapore from Fuzhou, China, where red rice wine is a staple and a celebratory food. In 1983, when her grandfather passed away, her grandmother, who was doing odd jobs then, relied on bartering her homemade rice wine for rice, oil and other essentials to support her teenage children, Jinyen told CNA Women.

Joo Eng recalled how she was first introduced to red rice wine during her first meal with her mother-in-law . “I had never had red rice wine chicken before, and I was blown away. The chicken was so fragrant and tasted fantastic,” she said.

Her mother-in-law passed on the family recipe to her, and she continued to brew red rice wine and make red rice dishes for her four children , even after her mother-in-law passed away in 2004 .

“I started consuming rice wine in cooked food when I was a year old. I am told that as a child I drank bowl after bowl of red rice wine chicken soup,” Jinyen laughed. “My siblings were all raised the same way. We always have red rice wine at every birthday and every celebration.”

Starting a business with mum? This Singaporean learned valuable life lessons

The mother-daughter team behind one of Singapore’s top skincare brands

A SUPERFOOD AND STAPLE

When you mention red rice wine, most people immediately associate it with confinement cooking. “Red rice yeast is believed to improve blood circulation, according to traditional Chinese medicine,” explained Jinyen.

However, Joo Eng noted that it is a misconception that rice wine should only be used for confinement.

“Rice wine has lactic acid which is very good for digestion. It can replace hua tiao wine, which has salt content, as well soya sauce and salt to bring out the taste of your meat and vegetables when cooking,” Joo Eng said.

Jinyen added that while most millennial women come to know about red rice wine during confinement , many continue using it in their cooking after.

In the Yap family, rice wine is a significant part of their culinary culture. “Even when we dabao (take away) fish soup , we add rice wine to it to elevate the taste,” laughed Joo Eng, who added that the family consumes five bottles of rice wine each month, on average.

“Yellow rice wine has a light, gentle fragrance that is smooth, sweet and not heavy. Red rice wine tastes stronger and lingers for longer. Both have an alcohol content of around 15 per cent,” said Jinyen.

Love at first pretzel: The women behind Auntie Anne’s family-run outlets in Singapore

How this mother-daughter duo built a successful jewellery business in Singapore together

Naomi Yeo starts built-in furniture business with her mum: ‘I wanted to help you fulfil your dream’

SHARING THEIR FAMILY HERITAGE

It is a culinary tradition that the Yaps love and want to share. However, it is no mean feat to make 500 bottles of different types of rice wine out of a home kitchen each month.

Joo Eng uses the largest home-use steamer she could find – a 40cm steamer that cooks 15kg of glutinous rice each time. Each 100kg batch of rice takes her around two days to cook.

First, she sterilises everything, including the steamer, ladle and urn. After measuring and washing the rice several times, she places it in the steamer in a way that ensures every grain is cooked evenly, and stirs and checks it periodically.

After steaming, the rice is laid on the table to cool and sprinkled with red rice yeast before it is placed in the urn with water and left to ferment for around 30 to 40 days, then bottled for consumption.

“The rice wine ferments more quickly when the weather is hotter and takes longer when it rains a lot. So each batch will taste slightly different because of weather and humidity changes,” said Jinyen.

The Yaps recommend that their wine be consumed within a year, although it can be kept for longer in a cool, dark place away from sunlight.

“The longest batch we’ve kept was for five years. But because it is a fermented product, the longer you keep it, the stronger the taste. Some people may not be used to a very strong taste,” said Jinyen.

Joo Eng takes on most of the production because she is retired, and Jinyen takes care of marketing, social media and logistics, and helps out with production at weekends while juggling a full-time job.

Working together has brought mother and daughter closer. “I’m not a very patient person. But doing this with my mum has taught me the value of patience because this is not a trade where I can take shortcuts. You have to pay attention to details and do it with love,” said Jinyen.

The business has also deepened their appreciation for their heritage and family.

“My family loves celebrating together. We make our own dumplings for dragon boat festival, or come together on mid-autumn festival to solve riddles on lanterns and enjoy mooncakes. We always have rice wine at celebrations – food is just one way for our extended family to come together,” Jinyen said.

Gathering and remembering their roots is important for the Yaps. “All our grandparents came from somewhere. And they brought with them all these traditions. Why is it that we move so quickly that we forget our roots and the culture that makes us who we are?” Jinyen reflected.

The duo hopes their rice wine helps others remember their culture and loved ones too.

“Many of our customers have told us that our rice wine reminds them of beloved grandparents and grand-aunts. We made our rice wine to replicate the experience for people who have such memories,” said Joo Eng.

What is it like growing up in a family of 7 daughters in Singapore?

Creative Tings: How 3 Singaporean sisters found success supporting each other

CNA Women is a section on CNA Lifestyle that seeks to inform, empower and inspire the modern woman. If you have women-related news, issues and ideas to share with us, email CNAWomen [at] mediacorp.com.sg .

Related Topics

Recommended, recent searches, trending topics, this browser is no longer supported.

We know it's a hassle to switch browsers but we want your experience with CNA to be fast, secure and the best it can possibly be.

To continue, upgrade to a supported browser or, for the finest experience, download the mobile app.

Upgraded but still having issues? Contact us

Get the Reddit app

Welcome brewers, mazers, vintners, and cider makers!

Rice Wine Experiment

Just rinsed 5kg (give or take) of Wang Korea sweet rice.

Going to let it set overnight and split into 2 different batches.

First batch will use yeast balls.

Second batch using nuruk.

Going to see how this goes.

Edited to add updates in post rather than in comments.

First Update: So I left the rice to soak overnight. And I am starting to cook it this morning. The only vessel I have even remotely large enough is a pressure cooker. So I am going to use that in smaller batches.

1800g soaked rice 1800g water

I have no idea how long it will take to cook it properly. Good thing I have plenty of rice to test with!

Second Update: First batch of rice is done. A bit too liquidy still. Came out in a sticky mess. Reducing water on second batch to 1000g/1kg

Third Update: The second batch of rice was still very sticky and wet. Third batch I have increased the rice content to 2kg and only added 500g of water.

Fourth Update: 500g was not enough water. Added another 200g. I will probably use 750g of water per 2kg soaked rice from now on.

By continuing, you agree to our User Agreement and acknowledge that you understand the Privacy Policy .

Enter the 6-digit code from your authenticator app

You’ve set up two-factor authentication for this account.

Enter a 6-digit backup code

Create your username and password.

Reddit is anonymous, so your username is what you’ll go by here. Choose wisely—because once you get a name, you can’t change it.

Reset your password

Enter your email address or username and we’ll send you a link to reset your password

Check your inbox

An email with a link to reset your password was sent to the email address associated with your account

Choose a Reddit account to continue