Rhodotorula mucilaginosa H04 and yellow wine co-fermentation process thereof

Abstract

The invention relates to a rhodotorula mucilaginosa strain and a yellow wine co-fermentation process thereof, and belongs to the technical field of fermentation engineering and strains. The invention provides the rhodotorula mucilaginosa with the fermentation characteristics of low temperature resistance, acid resistance, high yield of beta-glucosidase and aroma production, sequential inoculation is adopted, the rhodotorula mucilaginosa H04 is firstly inoculated for fermentation for two days, then the saccharomyces cerevisiae is inoculated, and the yellow wine co-fermentation is carried out by an equal-proportion 20 DEG C low-temperature fermentation process. The yellow rice wine meeting national standard requirements of yellow rice wine can be obtained after 20 days of fermentation, and the volatile flavor type, content, taste and aroma sensory quality of the yellow rice wine after fermentation are improved.

Description

Technical Field

[0001] This invention relates to a strain of red yeast and its co-fermentation process for rice wine, belonging to the fields of fermentation engineering and biotechnology. Background Technology

[0002] Yellow rice wine is a traditional fermented alcoholic beverage made primarily from rice through the fermentation of various yeasts. It is widely popular among consumers for its rich aroma, mellow taste, and abundance of nutrients. However, the traditional brewing process is complex and relies heavily on manual labor, resulting in low production efficiency, high labor intensity, and inconsistent product quality. With technological advancements and the demands of industrialized production, mechanization and automation have been introduced into yellow rice wine brewing to improve efficiency, ensure product quality and stability, and reduce labor intensity and costs. While mechanized brewing has improved efficiency and stability, it also suffers from drawbacks such as loss of flavor compounds, high equipment costs, and limited production flexibility, impacting yellow rice wine quality, enterprise costs, and market adaptability.

[0003] Non-Saccharomyces cerevisiae can produce highly active extracellular enzymes, such as β-glucosidase, lipase, protease, and carboxysulfonase. These enzymes facilitate the production of esters, alcohols, and aldehydes. Currently, non-Saccharomyces cerevisiae are used in the production of fermented foods through co-fermentation to improve the flavor and quality of the food. Rhodotorula glutinis is a type of non-Saccharomyces cerevisiae with strong environmental adaptability and is a yeast strain with significant development potential. Rhodotorula glutinis is rich in protein, fat, carbohydrates, and ash, and also contains abundant β-carotene, astaxanthin, vitamin E, nucleotides, and other bioactive substances.

[0004] Red yeast rice has broad development and application prospects in food, medicine, bio-fermentation, agricultural biocontrol yeast, feed, and environmental protection. In patent application CN202210506023.4, the inventors applied selenium-enriched red yeast rice to feed additives. In patent application CN201911282192.9, the inventors used marine red yeast rice to produce mannan and carotenoids. In patent application CN202310164279.6, the inventors applied red yeast rice to soy sauce fermentation, achieving good results in increasing the fat, aroma, and quality of soy sauce. In patent application CN202311652888.2, the inventors applied red yeast rice to fermented bean curd for post-fermentation, increasing the coconut milk flavor in the bean curd. Furthermore, in patent applications CN201911034025.2 and CN202411202241.4, red yeast rice was used to ferment tobacco stems and tobacco shreds, improving product quality. The Rhodotorula glutinis used in this study was screened from fermented cigar tobacco leaves, aiming to explore its application potential in rice wine production. Regarding the application of Rhodotorula glutinis in alcoholic beverages, in patent application CN108315144A, the inventors used Rhodotorula glutinis in synergistic fermentation with Rhizopus oryzae to improve the flavor and quality of sweet rice wine. In patent application CN202410623108.X, the inventors used Rhodotorula glutinis to ferment fruit juice, increasing the content of the aroma compound ethyl hexanoate in the juice. In patent application CN202311835325.7, the inventors explored the application of Rhodotorula glutinis in the production, regulation of aroma compounds, fermentation, and brewing.

[0005] Rhodotorula mucilaginosa has significant application value in alcoholic beverage production. Co-fermentation with Saccharomyces cerevisiae can enhance the aroma and improve the color of dry red wine. Co-fermentation with Saccharomyces cerevisiae can also enrich the aroma of kiwi wine. However, there are currently no studies or reports on its application in the production of rice wine to enhance flavor and quality. Rice wine is a low-alcohol fermented beverage made from rice through multi-strain co-fermentation. It is rich in amino acids and organic acids, and its product characteristics and production process are well-suited to the fermentation characteristics of Rhodotorula mucilaginosa. Rice is a suitable substrate for Rhodotorula mucilaginosa fermentation. Therefore, utilizing Rhodotorula mucilaginosa in conjunction with Saccharomyces cerevisiae in the production of rice wine has promising application prospects and value. Summary of the Invention

[0006] This invention provides a strain of red yeast H04 and its co-fermentation process for rice wine, which can be applied to modern mechanized rice wine production processes to improve the flavor and quality of mechanized rice wine.

[0007] This invention provides a strain of Rhodotorula mucilaginosa, strain H04, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241401, located at Wuhan University, Wuhan, China.

[0008] In one embodiment of the present invention, the monoclonal colony morphology of the Rhodotorula glutinis H04 is characterized by being round, orange-red, opaque, and having raised features.

[0009] In one embodiment of the present invention, the aroma-producing red yeast H04 is used as a fermenting agent in the production of rice wine.

[0010] In one embodiment of the present invention, the Rhodotorula glutinis H04 simultaneously possesses fermentation characteristics such as low temperature resistance, acid resistance, high production of β-glucanase, and aroma production.

[0011] In one embodiment of the present invention, a method for preparing rice wine is provided, comprising co-fermentation of fermentation products produced by fermentation of Rhodotorula glutinis H04, Rhodotorula glutinis H04 inoculum, or Rhodotorula glutinis H04 combined with other microorganisms to prepare rice wine.

[0012] In one embodiment of the present invention, the red yeast is a solid inoculum or a liquid inoculum.

[0013] In one embodiment of the present invention, the Rhodotorula glutinis H04 inoculant comprises live cells of Rhodotorula glutinis, freeze-dried Rhodotorula glutinis obtained by freeze drying, immobilized Rhodotorula glutinis cells obtained by immobilization technology, or Rhodotorula glutinis existing in any other form.

[0014] In one embodiment of the present invention, the other microorganisms refer to the aroma-producing red yeast in any form along with other microorganisms such as yeast, lactic acid bacteria, and Bacillus.

[0015] In one embodiment of the present invention, the co-fermentation refers to the co-fermentation of Rhodotorula glutinis H04 with Saccharomyces cerevisiae or Angel yeast. The co-fermentation of Rhodotorula glutinis H04 with Saccharomyces cerevisiae or Angel yeast involves first inoculating Rhodotorula glutinis H04 for pre-fermentation for two days, and then inoculating Saccharomyces cerevisiae or Angel yeast for post-fermentation. Saccharomyces cerevisiae or Angel yeast and Rhodotorula glutinis H04 participate in the fermentation in a 1:1 ratio. The pre-fermentation temperature in the co-fermentation of Rhodotorula glutinis H04 with Saccharomyces cerevisiae or Angel yeast is a low-temperature fermentation at 20°C.

[0016] In one embodiment of the present invention, the rice wine prepared by co-fermentation of the red yeast H04 and brewer's yeast can meet the national standard for rice wine in 20 days compared with fermentation by brewer's yeast alone. After fermentation, the types and contents of volatile flavors, as well as the sensory quality of taste and aroma of the rice wine are improved.

[0017] The beneficial effects of this invention are: (1) This invention provides a strain of red yeast obtained from fermented cigar tobacco leaves, which has the fermentation characteristics of being resistant to low temperature, acid, producing high levels of β-glucanase, and producing aroma.

[0018] (2) When the red yeast provided by this invention is used for the co-fermentation of rice wine in the production of rice wine by sequential inoculation (first inoculating red yeast H04 for two days of fermentation and then inoculating brewing yeast or Angel yeast), equal proportion, and low temperature fermentation (20℃), compared with normal fermentation, the 20-day fermentation time can meet the national standard requirements for rice wine. After the fermentation is completed, the types and contents of volatile flavors of rice wine, as well as the sensory quality of taste and aroma are improved. Attached Figure Description Figure 1 Growth curves of different Rhodotorula glutinis Figure 2 Ethanol tolerance of different Rhodotorula glutinis Figure 3 Temperature tolerance of different Rhodotorula glutinis Figure 4 Acid tolerance graph of different Rhodotorula glutinis Figure 5 Volatile flavor compounds of different red yeasts Figure 6 Sensory quality evaluation of rice wine Figure 7 The effect of different inoculation ratios on the flavor of Shaoxing wine Figure 8 The effect of different pre-fermentation temperatures on the flavor of Shaoxing wine Figure 9 The effect of different inoculation times on the flavor of Shaoxing wine Figure 10 Volatile flavor compounds in co-fermented rice wine Figure 11 Organic acids, amino acids and sensory analysis of co-fermented rice wine Detailed Implementation The specific embodiments of the present invention are described below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0019] PBS buffer: Weigh 8.0 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.24 g potassium dihydrogen phosphate, dissolve in 800 mL distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to a final volume of 1 L to obtain 0.01 M PBS buffer. Sterilize at 115℃ for 20 min, cool, and use as needed.

[0020] YPD medium: 1% yeast extract, 2% tryptone, 2% glucose, 2% agar (solid additive), sterilized at 115℃ for 20 min; Rice saccharification broth culture medium: Mix raw rice and water at a mass ratio of 1:4, add 2‰ liquefying enzyme and 1‰ saccharifying enzyme by weight of rice, control the temperature at 60℃ in a water bath, saccharify and liquefy for 4 hours, filter with gauze, sterilize at 115℃ for 20 minutes, and cool for later use.

[0021] Example 1: Isolation and purification of Rhodotorula glutinis strain (1) Preliminary screening of bacterial strains Fermented cigar tobacco leaves, yeast starter, brewing environment soil, wheat koji samples, and rice wine fermentation mash were used for strain screening. Before the experiment, the samples were temporarily stored at -4℃. Using sterile scissors, 5 g of sample was taken in a clean bench, chopped, and placed in an Erlenmeyer flask pre-filled with sterile PBS or 0.9% physiological saline. The flask was then incubated on a shaker at 37℃ and 200 rpm for 30 min. 1 mL of the culture solution was diluted to a 1:10 concentration. -3 ~10 -7 Take 100 μL of each dilution and spread it evenly on YPD solid medium. Incubate at 30℃ for 36-48 h. When colonies grow on the plate, select single colonies with the characteristics of Rhodotorula glutinis colonies and streak them for purification more than 3 times until no other morphological colonies are found on the plate. Perform 2-3 purifications until only single-clone colonies with a single morphological characteristic are found on the plate. Then, take a single-clone colony and inoculate it into liquid YPD medium. Incubate it on a shaker at 37℃ and 200 rpm for 24-48 h. Then, take an equal proportion of the bacterial solution and mix it with sterilized 30% glycerol. Transfer the mixture to cryovials, label them, and store them at -80℃.

[0022] (2) Strain identification The universal primers for synthesizing fungi, ITS1 (Primer F): 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4 (Primer R): 5'-TCCTCCGCTTATTGATATGC-3', were used. The PCR reaction system (30 μL) consisted of: 15 μL Premix Taq, 0.6 μL each of Primer F and Primer R, 0.6 μL template DNA, and 13.2 μL ddH2O. The thermal cycling parameters were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for 30 cycles; and a final extension at 72℃ for 5 min. The amplified products were sent to Qingdao Weilan Technology Co., Ltd. for sequencing, and the sequences were compared with the NCBI database for homology analysis. The sequence coverage of R. mucilaginosa (KY488461.1) was 100%, and the similarity was 99.53–99.84%. Therefore, all nine selected strains were identified as R. mucilaginosa. Strain information is shown in Table 1. Strain H04 was named during preservation. To compare the differences between different strains, a unified numbering system was implemented, with JH-6 being the experimental name for strain H04.

[0023] Table 1. Results of strain sequence alignment (3) Determination of growth curve of Rhodotorula glutinis Yeast was inoculated at a 1% inoculum in YPD liquid medium and cultured at 30℃ and 150 r / min for 48 h. Absorbance was measured at 600 nm every 4 h to plot the strain growth curve. There were no significant differences in the lag phase, logarithmic phase, and stationary phase among different strains; all were in the lag phase from 0 to 4 h, and the logarithmic phase from 4 to 28 h. After 28 h, the OD value changed little, reaching the stationary phase. Figure 1 ).

[0024] (4) Tolerance testing Ethanol and acid tolerance: The isolated strains were inoculated at a 1% inoculum into YPD liquid medium with different volume fractions of ethanol (0%–12%) and different pH values ​​(2.0–6.0), and cultured at 30℃ and 150 r / min for 48 h on a shaker. The absorbance value at a wavelength of 600 nm was measured (see details). Figure 2 and Figure 4 ) Temperature tolerance: The isolated strain was inoculated into YPD liquid medium at a 1% inoculum and cultured at 150 r / min for 48 h in eight growth gradients, from 10.0℃ to 45.0℃, with a growth gradient of 5.0℃ each. The absorbance was measured at 600 nm (see details). Figure 3 ).

[0025] (5) Determination of β-glucosidase activity in Rhodotorula glutinis A standard curve was established according to the instructions of the β-glucosidase activity assay kit from Beijing Box Biotechnology Co., Ltd. The enzyme activity calculation formula is as follows: BGL(U / mL) = (x V_antitotal) / (V_sample) T) Enzyme activity unit: 1 nmol of p-nitrophenol is generated per mL of liquid sample per hour.

[0026] In the formula: Vsample, the volume of crude enzyme solution added to the reaction system, 0.1 mL; Vreaction total, the total volume of the reaction system, 1 mL; T: reaction time, 0.5 h. This is derived from the standard curve. Table 2 β -Glucosidase activity Initial screening results: Nine strains of Rhodotorula glutinis were isolated from different samples. Tolerance tests revealed that all strains exhibited good low-temperature tolerance (see details). Figure 3 ) and acid tolerance (see details) Figure 4 However, ethanol tolerance varies considerably (see details). Figure 2 When the ethanol concentration reached 6% vol, the growth of all strains except JH-1 was inhibited. When the ethanol concentration was between 8% and 12% vol, the OD values ​​of all strains did not change significantly, indicating that the ethanol concentration completely inhibited their growth. To further investigate the differences in their aroma-producing abilities, they were inoculated into rice fermentation broth to screen for the optimal Rhodotorula glutinis.

[0027] Example 2: Analysis of volatile flavor compounds in fermentation broths of different Rhodotorula glutinis yeasts (1) Pick colonies of Rhodotorula glutinis from the plate and inoculate them into 50 mL of YPD liquid medium. Incubate at 30℃ and 180 r / min for 48 h. Then, inoculate the seed culture of each isolated strain into 50 mL of rice fermentation broth at an inoculation rate of 1% (V / V) and incubate at 30℃ on a shaker for 7 days. (2) Qualitative analysis of volatile flavor compounds in the fermentation broth of Rhodotorula glutinis was performed using the NIST14 database to determine the categories of volatile flavor compounds. Semi-quantitative analysis based on internal standards was then conducted to determine the relative contents of the main flavor compounds. The results are as follows: Figure 5 .

[0028] Secondary screening results: The types of aroma components produced by different strains varied considerably. JH-6 strain produced significantly higher levels of the main volatile flavor compounds (alcohols, aldehydes, acids, and esters) than other strains, demonstrating excellent aroma-producing and fermentation performance. Therefore, Rhodotorula glutinis JH-6 was selected for co-fermentation of rice wine with Saccharomyces cerevisiae.

[0029] Example 3: Fermentation Experiment of Yellow Rice Wine Using Red Yeast JH-6 (1) Preparation of fermentation agent Saccharomyces cerevisiae and JH-6 were dispersed on YPD agar plates to obtain single colonies. These colonies were then pre-cultured in rice fermentation broth at 30°C for 48 hours (primary seed culture). The yeast starter culture was then activated in the rice fermentation broth and cultured at 30°C for 48 hours. Triple samples of each yeast starter culture were prepared, and the yeast concentration was determined using the hemocytometer method. Different strains yielded different concentrations of starter culture, which can be directly used for co-fermentation of rice wine. (2) Fermentation of rice wine Soaking rice: Weigh out the amount of glutinous rice needed for each batch of fermentation experiment according to the ratio of 500 g for each system, soak the rice in a 5L beaker, 2.5 kg of rice per container, add water until the liquid level is 6 cm above the rice. The soaking time is generally 3-5 days before steaming rice in summer, and can be extended to 7 days in winter. After soaking, the wet rice will have a sour taste, which is produced by the metabolic process of acid production by microorganisms such as lactic acid bacteria in rice, and is a normal phenomenon. Steamed rice: Use a rice steamer to steam the rice. Place two layers of gauze on the bottom and then put the wet rice on top. Spread the wet rice evenly, but not too thickly, otherwise the rice will be undercooked when it is finished steaming. Set the temperature to about 105℃ and the steaming time to about 30 minutes. Cooling the rice: Spread the steamed rice out to cool to below 60℃ before adding the ingredients; Preparation: Mix 750g of cooked rice with 600mL of purified water, add 60mL of red yeast JH-6 for fermentation, and then add 20mL of brewer's yeast for post-fermentation. This is the experimental group. The control group directly adds 80mL of brewer's yeast and performs pre-fermentation and post-fermentation respectively. Fermentation: Pre-fermentation: 28℃ constant temperature fermentation for 5 days; stir 2-3 times a day (stir well), with an interval of 8-10 hours between stirrings; Post-fermentation: 15℃ constant temperature fermentation for 15 days.

[0030] (3) Analysis of volatile flavor and sensory evaluation of rice wine co-fermented with red yeast rice and brewer's yeast The basic physicochemical properties of the fermentation broth were determined, and the results are shown in Table 3. The alcohol content of the rice wine fermented by Rhodotorula glutinis JH-6 was significantly lower than that of the control group. The total acid and reducing sugar content both met the standards for semi-dry rice wine, and the amino acid nitrogen content was greater than 0.35 g / L. All physicochemical indicators met the national standards.

[0031] Table 3 Physicochemical Indicators of Simulated Fermentation of Yellow Rice Wine Note: 1 represents the unit (%vol); a represents the total acid content as lactic acid; the value is the mean ± standard deviation of at least three independent tests; and it indicates that there is a significant difference between different systems (P<0.05). The volatile flavor compounds of co-fermented rice wine were analyzed using HS-SPME-GC-MS, and a total of 63 volatile components were detected, including 28 esters, 12 alcohols, 11 aldehydes, and 12 acids. Esters, alcohols, acids, and aldehydes were the main volatile components in the co-fermented rice wine. The content of esters, such as isoamyl acetate, ethyl hexanoate, ethyl 3-hydroxybutyrate, and ethyl 3-phenylpropionate, was significantly increased in the co-fermented rice wine (experimental group).

[0032] Table 4. Content of volatile flavor compounds in Shaoxing wine Note: Values ​​are the mean ± standard deviation of at least three independent tests; * indicates a significant difference between the experimental group and the control group (P<0.05).

[0033] Based on the sensory characteristics of Shaoxing wine ( Figure 6 The evaluation data analysis shows that the rice wine fermented with mixed cultures of *Rhodotorula glutinis* and *Saccharomyces cerevisiae* (experimental group) exhibits a significant advantage in sensory characteristics. The mixed-culture fermented rice wine (experimental group) excels in aroma, particularly in honey and fruit aromas, consistent with the experimental results. Furthermore, in terms of taste, compared to the control group, the experimental group shows enhanced sweetness and reduced astringency, bitterness, and sourness. In summary, the sensory evaluation results indicate that the mixed-culture fermented rice wine possesses a unique flavor, a smooth taste, rich aroma, and a distinct floral and fruity fragrance, demonstrating a significant improvement in flavor quality.

[0034] Example 4: Optimization of the co-fermentation process of Huangjiu (yellow rice wine) using Rhodotorula glutinis JH-6 (1) Determination of optimal fermentation conditions Due to metabolic differences in rice wine fermentation, different yeast strains significantly affect the concentration of volatile compounds, thus influencing the quality of the rice wine. Fermentation parameters affect the yeast's reaction to aromatic or harmful substances related to the quality of fermented alcoholic beverages during biosynthesis. Therefore, selecting suitable fermentation conditions is crucial for the quality of rice wine.

[0035] Table 5 Design of different fermentation schemes (2) Effect of inoculation ratio on rice wine fermentation The volatile components in fermented rice wine were analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry. Figure 7 The results showed that 63 volatile compounds were detected in different yeast starter ratios, including 43 alcohols, 13 aldehydes, 9 acids, and 9 esters. The relative contents of these volatile compounds in different treatments were analyzed using heatmaps. The total volatile compound content in the mixed fermentation ratio (Saccharomyces cerevisiae: JH-6 = 1:1) treatment group was higher than that in the control (CK) treatment. The CS1R1 treatment increased the variety of volatile compounds, especially esters. These results confirm the contribution of Rhodotorula glutinis to the diversity of volatile components in Shaoxing wine. The effect of fermentation temperature on the volatile flavor compounds of Shaoxing wine at this fermentation ratio will be investigated later.

[0036] (3) Effect of pre-fermentation temperature on rice wine fermentation The experiment determined the optimal inoculation ratio (brewing yeast: JH-6 = 1:1) and selected three pre-fermentation temperatures (20℃, 25℃ and 30℃) to ferment rice wine. Figure 8 This indicates that the volatile compound content of rice wine fermented at 20℃ is higher than that of other experimental groups. The effect of the inoculation time of JH-6 fermenting agent on the volatile flavor compounds of rice wine will be studied at this fermentation temperature.

[0037] (4) The effect of inoculation time on the fermentation of rice wine Different inoculation times of yeast starter are core parameters for quality control of fermented alcoholic beverages, regulating quality by influencing the interplay of microbial interactions, metabolic pathways, and environmental factors. After determining the optimal inoculation ratio and pre-fermentation temperature, the optimal inoculation sequence was determined by screening different inoculation times (0d, 2d, 4d). Figure 9 This indicates that inoculating with *Saccharomyces cerevisiae* 2 days after inoculation with *Rhodotorula glutinis* has a better effect on the volatile flavor compounds of rice wine.

[0038] Therefore, by optimizing the experimental conditions through single factors, the conditions for fermenting rice wine with a mixture of Saccharomyces cerevisiae and Rhodotorula glutinis JH-6 were determined to be: first inoculate with H04, then inoculate with Saccharomyces cerevisiae 2 days later, with an inoculation ratio of JH-6 to Saccharomyces cerevisiae of 1:1, and a pre-fermentation temperature of 20℃.

[0039] (5) Changes in volatile compounds during mixed fermentation of Rhodotorula glutinis JH-6 and Saccharomyces cerevisiae. such as PCA Figure 10As shown in Figure A, the fermented rice wine samples from the CK and COF groups were relatively separated. The CK group mainly contained substances in the second and third quadrants, while the COF group mainly contained substances in the first and fourth quadrants. Significant separation was observed between different fermentation stages, particularly on day Q2 and at other times. A total of 62 volatile substances were identified in the rice wine during fermentation, including 18 alcohols, 32 esters, 6 aldehydes, and 6 acids. Esters and aldehydes were the main volatile compounds in the CK group, accounting for 75.69%-83.63% and 9.20%-13.23% of the total volatile flavor compounds, respectively. In the COF group, esters and alcohols were the main volatile compounds, accounting for 63.12%-73.18% and 14.32%-21.46% of the total volatile flavor compounds, respectively. Figure 10 B, C). The COF group of rice wine samples showed relatively significant separation of volatiles at different fermentation stages, exhibiting a clear trend. With increasing fermentation time, the concentrations of most volatile compounds gradually increased (…). Figure 10 D).

[0040] Depend on Figure 11 B identified 15 amino acids, including the 7 essential amino acids found in rice wine. The addition of JH-6 significantly affected the amino acid content. Aspartic acid was found at a higher concentration in the control group than in the experimental group, reaching the highest concentration (750 mg / L). 1 This may be because the addition of JH-6 slowed down the fermentation rate, resulting in a shorter post-fermentation time than the control group (CK). Arginine is the main bitter amino acid in rice wine and has been identified as a key factor in the bitterness of tea. Table 6 shows that the TAV analysis of each amino acid indicates that glutamic acid, alanine, and arginine have TAVs greater than 1, contributing significantly to umami, sweetness, and bitterness, respectively.

[0041] Table 6. Content of amino acids with TAV>1 in rice wine samples Depend on Figure 11As shown in C, the intensity values ​​of all eight taste attributes exceeded their respective thresholds. The flavor characteristics of the rice wine in different treatment groups were significantly affected. The umami flavor of COF rice wine was higher than that of the control group, with average relative intensities of umami of 5.10 and 7.24, respectively, a difference of 2.14. The key substance for the umami flavor of rice wine is amino acids. The increase in umami flavor in COF may be due to the limited amino acid content. Adding JH-6 to rice wine can increase the rate at which proteins are broken down into amino acids by yeast, leading to an increase in umami flavor. The difference in relative intensity of acidity between the two treatment groups was 4.00 and 2.01, respectively, indicating that the addition of JH-6 affected the acidity and bitterness of rice wine. The acidity of COF rice wine was lower than that of CK, consistent with the results in Table 1. After adding JH-6, the pH value increased and the total acid content decreased. In conclusion, the addition of JH-6 significantly altered the taste attributes of rice wine, making the umami flavor more pronounced and significantly reducing the acidity and bitterness.

[0042] Depend on Figure 11 As shown in D, the contribution rate of the first principal component PC1 in the figure is 78.9%, and the total contribution rate of PC1 and PC2 is 96%, both of which are greater than 80%. Furthermore, PC1 is the most important variable. Along PC1, relative separation between different treatment groups can be observed, indicating that there are differences in the overall taste of the two groups of rice wine, suggesting that they can represent the main taste information of the rice wine samples.

[0043] Sensory evaluation data analysis showed that the COF group of Shaoxing wine exhibited a significant advantage in sensory flavor characteristics. In terms of aroma, the honey and fruit aromas were significantly better than the control, consistent with the experimental results mentioned above. Furthermore, in terms of taste, the sweetness was enhanced, attributed to incomplete fermentation, resulting in higher reducing sugar content and enhanced sweetness. Astringency, bitterness, and sourness were reduced. In summary, the sensory evaluation results indicate that the co-fermentation strategy of JH-6 with Saccharomyces cerevisiae compensated for the ethanol production rate of the aroma-producing yeast, while also increasing the ethanol content in the sample, contributing to a fuller flavor. In contrast, the Shaoxing wine samples from the mixed-culture fermentation group performed well in terms of floral and fruity aromas. This improvement may be attributed to the production of more acetate esters and higher alcohols during mixed-culture fermentation, which helps to enhance floral, fruity, and ester aromas.

Claims

1. A method for preparing rice wine through co-fermentation, characterized in that, The yeast was prepared by co-fermentation of Rhodotorula mucilaginosa H04, or Rhodotorula mucilaginosa H04 fermentation product, or Rhodotorula mucilaginosa H04 inoculum with Saccharomyces cerevisiae or Angel yeast. The Rhodotorula mucilaginosa H04 was deposited at the China Center for Type Culture Collection on July 1, 2024, with accession number CCTCCNO: M 20241401, and the deposit address is Wuhan University, Wuhan, China.

2. The method according to claim 1, characterized in that, The co-fermentation involves first inoculating with Rhodotorula glutinis H04 for pre-fermentation, followed by inoculating with Saccharomyces cerevisiae or Angel yeast for post-fermentation.

3. The method according to claim 2, characterized in that, The co-fermentation process involves first inoculating Red Yeast A (H04) for pre-fermentation for two days, followed by inoculation with Saccharomyces cerevisiae or Angel Yeast for post-fermentation.

4. The method according to claim 1, characterized in that, The brewing yeast or Angel yeast and the red yeast rice H04 are fermented in a 1:1 ratio.

5. The method according to claim 1, characterized in that, The pre-fermentation temperature is 20℃ low-temperature fermentation.

6. The method according to claim 1, characterized in that, The aforementioned Rhodotorula glutinis H04 inoculum is a solid inoculum.

7. The method according to claim 1, characterized in that, The aforementioned Rhodotorula glutinis H04 inoculant is a liquid inoculant.

8. The method according to claim 1, characterized in that, The aforementioned Rhodotorula glutinis inoculum contains live cells of Rhodotorula glutinis, freeze-dried Rhodotorula glutinis obtained by freeze-drying, immobilized Rhodotorula glutinis cells obtained by immobilization technology, or Rhodotorula glutinis existing in any other form.

9. The method according to any one of claims 1-8, characterized in that, The quality improvement of rice wine prepared through co-fermentation includes increasing the variety and content of volatile flavor compounds such as alcohols, esters, aldehydes and acids, as well as improving the taste and aroma sensory quality of rice wine.

10. Rice wine prepared by the method according to any one of claims 1-9.

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