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

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 highquality 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.

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, nonglutinous 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 nonglutinous 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 (MUL-TISKAN 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.

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 wheatbran 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 wheatbran 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.