Application of Saccharomyces cerevisiae and Yarrowia lipolytica interaction in vinegar brewing

Abstract

The application belongs to the technical field of vinegar brewing, and provides application of Saccharomyces cerevisiae and Yarrowia lipolytica in vinegar brewing. A high-yield ester and low-yield fusel excellent indigenous Saccharomyces cerevisiae CGMCC 15729 and a high-yield erythritol Yarrowia lipolytica SHMCC D25204 strain are interacted to optimize the inoculation ratio, and the two strains of yeast are mixed in high density according to the optimized ratio to prepare a composite yeast inoculum, which is applied to the alcohol fermentation stage of Shanxi old vinegar brewing, so that the content of fusel substances in the wine mash is reduced, and the content of total esters and volatile aroma substances is increased; finally, the total ester content of the new Shanxi vinegar is greater than or equal to 2.50 g / 100 mL, the erythritol content is greater than or equal to 0.85 g / 100 mL, the total acid content is greater than or equal to 5.50 g / 100 mL, and the amino acid nitrogen content is greater than or equal to 0.35 g / 100 mL. The types of volatile flavor substances are more abundant, and the contents of characteristic ester substances such as ethyl acetate, ethyl lactate, ethyl hexanoate and ethyl phenylacetate are obviously increased, so that the vinegar presents a more rich and harmonious ester aroma characteristic.

Description

Technical Field

[0001] This invention belongs to the field of vinegar brewing technology, specifically involving the application of the interaction between brewing yeast and lipophilic yeast in vinegar brewing. Background Technology

[0002] Vinegar, a traditional fermented condiment in my country, relies on the synergistic metabolism of a complex microbial community during fermentation to develop its characteristic flavor profile, characterized by sourness, mellowness, sweetness, aroma, and umami. Vinegar brewing typically involves two stages: alcoholic fermentation and acetic acid fermentation, with alcoholic fermentation being the crucial initial step. During this stage, yeast converts fermentable sugars in the raw materials into ethanol through sugar metabolism, providing substrates for subsequent acetic acid fermentation. Simultaneously, volatile substances such as esters, higher alcohols, and aldehydes produced during yeast metabolism further contribute to the formation of vinegar's characteristic aroma. Therefore, the composition and metabolic characteristics of the microorganisms during alcoholic fermentation directly influence the subsequent acetic acid fermentation process and the flavor quality of the finished vinegar; there is a consensus in the industry that "good wine is essential for good vinegar."

[0003] During alcoholic fermentation, in addition to producing ethanol, yeast also generates higher alcohols such as n-propanol, isobutanol, isoamyl alcohol, and 2-phenylethanol through amino acid metabolism and sugar metabolism. Appropriate amounts of higher alcohols can impart aroma and complex flavor to fermentation products, forming an important component of the traditional flavor system of fermented foods. However, excessive accumulation of higher alcohols can produce irritating odors and unpleasant flavors such as spiciness and bitterness, reducing the quality of the fermentation products. Furthermore, the microbial composition of the fermentation system is also a crucial factor influencing fusel oil formation. Besides yeast's own metabolism, the growth, reproduction, and metabolic activities of other microorganisms during fermentation can also promote the formation of higher alcohols. Therefore, regulating the structure of dominant functional microbial communities and enhancing the metabolic direction of target strains are important ways to reduce fusel oil formation and improve the flavor and quality of fermented foods.

[0004] The patent with publication number CN117903958A, "low fusel alcohol and high ester production Saccharomyces cerevisiae mutagenesis strain AR28 and its application", obtained a low fusel alcohol and high ester production Saccharomyces cerevisiae strain through mutagenesis screening. When applied to the fermentation process of rice wine, it achieved a reduction in fusel alcohol content and an increase in ester content. Compared with existing strains, the alcohol content was 24.80% (v / v) and the fusel alcohol content was 289.84 mg / L.

[0005] Erythritol, a functional sugar alcohol with low calories, high tolerability, and good sweetness, has high application value in the food industry. While some yeasts possess strong erythritol synthesis capabilities, currently high-yield erythritol yeasts are mainly used in sugar alcohol production. In summary, research on the application of low-fusel alcohol and high-ester yeasts mainly focuses on alcoholic beverages, and primarily revolves around the screening, improvement, and functional optimization of single strains. There are few reports on research into the vinegar brewing process, especially the alcoholic fermentation stage of Shanxi aged vinegar, on enhancing the regulation of fermentation metabolism and improving the composition of flavor compounds through functional yeasts. Summary of the Invention

[0006] This invention addresses the problems in existing technologies for Shanxi aged vinegar, such as high fusel oil content, insufficient ester aroma accumulation, and a single source of sweetness in the natural fermentation system. Furthermore, current yeast enhancement technologies are mostly limited to optimizing the function of a single strain, failing to establish a synergistic regulatory system between low-fusel oil-producing strains and functional sugar alcohol-producing strains. This makes it difficult to simultaneously achieve multi-dimensional quality improvements in "reducing fusel oils, enhancing ester aroma, increasing sweetness, and strengthening functionality." The invention provides the application of the interaction between *Saccharomyces cerevisiae* and *Yarrowia lipolytica* in vinegar brewing. By interacting low-fusel oil-producing, high-ester-producing *Saccharomyces cerevisiae* with high-erythritol-producing *Yarrowia lipolytica*, and utilizing the complementary metabolic functions of the two yeast strains, a composite yeast fermentation system is constructed and applied to the alcoholic fermentation stage of Shanxi aged vinegar. This aims to reduce the accumulation of fusel oils, promote the formation of ester aroma compounds, and simultaneously increase erythritol content, enriching the flavor composition.

[0007] This invention is achieved by the following technical solution: the application of the interaction between Saccharomyces cerevisiae and Yersinia lipolytica in vinegar brewing. Excellent indigenous Saccharomyces cerevisiae CGMCC 15729, which produces low fusel oils and high esters, and Yersinia lipolytica SSHMCC D25204, which produces high erythritol, were screened from Shanxi aged vinegar mash. The inoculation ratio was optimized through strain interaction, and a compound microbial agent was prepared by simultaneous high-density mixing and cultivation according to the optimized ratio. This agent was then applied to the alcoholic fermentation stage of vinegar brewing. Alcoholic fermentation yielded mash, which was then subjected to solid-state acetic acid fermentation, smoking, and leaching to obtain new leached vinegar.

[0008] The preparation method of the Saccharomyces cerevisiae-Yersinia lipolytica compound inoculum is as follows: Saccharomyces cerevisiae CGMCC 15729 seed culture and Yersinia lipolytica SSHMCC D25204 seed culture are mixed at a 1:1 ratio and inoculated into 75L of yeast extract liquid culture medium at a 3% inoculation rate. The mixture is cultured at 30℃ and an aeration rate of 0.6L / min for 24 hours, during which 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled, and once the cold trap temperature stabilized at -50℃, the frozen material was placed inside. Vacuum freeze drying was performed using a segmented temperature control mode to obtain a *Saccharomyces cerevisiae*-*Yersinia lipolytica* compound inoculum, with viable cell counts reaching 10-1. 9 CFU / g, vacuum packaged and stored at 4℃.

[0009] The method for preparing the *Saccharomyces cerevisiae* CGMCC 15729 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 15729 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0010] The method for preparing the seed culture of *Yarrowia lipophila* SMHCC D25204 is as follows: cultured in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Yarrowia lipolyticis* SHIMCC D25204 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium at a concentration of 3.5 CFU / mL. The fermenter was incubated at 30°C, with an aeration rate of 0.2 L / min and a rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0011] The brewing yeast ( Saccharomyces cerevisiae CGMCC 15729 was obtained from Shanxi aged vinegar mash. It is the strain described in the invention publication number CN 109666594 B, entitled "A method for enhancing the production of Shanxi aged vinegar using superior indigenous high-yield alcohol brewing yeast and Candida albicans". In this invention, after being cultured in sorghum juice medium for 72 hours, the total ester content was 6.58 g / 100 mL, the total fusel oil content was 25.45 mg / 100 mL, and the total ester compound content was 10.7535 mg / mL. It is deposited at the China General Microbiological Culture Collection Center on May 7, 2018. The lipophilic yeast ( Yarrowia lipolyticaSHIMCC D25204, with a residual reducing sugar content of 11.79 g / 100 mL and an erythritol content of 3.33 g / 100 mL in liquid fermentation medium, is preserved at Shanghai Preservation Microbial Co., Ltd. The liquid fermentation medium is prepared as follows: 200 g glucose, 8 g yeast extract, 5 g ammonium citrate, 0.25 g potassium dihydrogen phosphate, 0.25 g anhydrous magnesium sulfate, 0.01 g copper sulfate pentahydrate, 0.05 g zinc sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, and distilled water is added to 1000 mL. The mixture is then sterilized at 121°C for 20 min. The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0012] The specific brewing process includes the following steps: sorghum is crushed to 50-60 mesh, sorghum and water are mixed at a mass-to-volume ratio of 1:4.5, heated to 90-95℃, 0.1% of heat-resistant α-amylase is added by mass of sorghum, the mixture is kept warm for 80-90 minutes, the temperature is lowered to 60℃, and then 0.1% of saccharifying enzyme is added by mass of sorghum, and the mixture is kept warm for 20-30 minutes. Once the temperature drops to 28-32℃, pump the mixture into an alcohol fermentation tank, add 60% Daqu (a type of starter culture) of sorghum and 0.1% of a compound yeast of brewing yeast and Yeastra lipolytica of sorghum, and ferment for 8 days until the alcohol content reaches 8-9% vol. The fermentation process ends to obtain mash. The fermented mash is then thoroughly mixed with 100% wheat bran, 60% rice bran, and 40% rice husks of sorghum. Simultaneously, 10% of a fermented mash is added for acetic acid fermentation for 12 days until the total acid content reaches 5-6 g / 100 mL. The acetic acid fermentation is then completed, and the mash is further treated with fumigation and leaching to obtain fresh leached vinegar.

[0013] The resulting new leached acetic acid is reddish-brown with a rich color, and contains ≥2.50 g / 100 mL of total esters, ≥0.85 g / 100 mL of erythritol, ≥5.50 g / 100 mL of total acid, and ≥0.35 g / 100 mL of amino acid nitrogen.

[0014] Compared with the prior art, the present invention has the following significant advantages: This invention screened a superior indigenous brewing yeast strain, CGMCC 15729, from Shanxi aged vinegar mash. This strain produces low levels of fusel alcohols and high levels of esters. During alcoholic fermentation, it can reduce the formation of fusel alcohols such as isoamyl alcohol and isobutanol, while promoting the synthesis of characteristic ester aroma compounds such as ethyl acetate, ethyl lactate, and ethyl butyrate. This is beneficial for improving the basic flavor of the mash and enhancing the potential for flavor compound formation during subsequent acetic acid fermentation.

[0015] The brewing yeast ( Saccharomyces cerevisiae CGMCC 15729 was obtained from Shanxi aged vinegar mash. It is the strain described in the invention announcement number CN 109666594 B, entitled "A method for enhancing the production of Shanxi aged vinegar using superior indigenous high-yield alcohol brewing yeast and Candida albicans". It is deposited at the China General Microbiological Culture Collection Center on May 7, 2018.

[0016] The *Yersinia lipolytica* strain used in this invention is a commonly used strain in the industrial production of erythritol. This invention selects the *Yersinia lipolytica* strain SHIMCC D25204 and innovatively applies it to the vinegar fermentation process. It can efficiently convert glucose into erythritol, imparting a naturally rounded, refreshing, and palatable sweetness to the vinegar without the need for external sweeteners. This effectively buffers the sharp acidity of the vinegar and significantly optimizes the overall flavor harmony and palatability.

[0017] This invention optimizes the inoculation ratio of *Saccharomyces cerevisiae* strain CGMCC 15729 and *Yersinia lipolyticis* strain SMHCC D25204, and prepares a compound microbial agent by simultaneously mixing and high-density cultivation according to the optimized ratio. This agent is then applied to the alcoholic fermentation stage of vinegar brewing. During fermentation, it reduces the formation of fusel oils, promotes the formation of esters and volatile aroma compounds, and increases the content of erythritol. The newly brewed vinegar produced using this process has a reddish-brown appearance, a rich and harmonious aroma, a sweet and mellow taste, and a mild and pleasant acidity, significantly enhancing the typical flavor characteristics of the product. Compared with the comparative example, the total fusel oil content of the mash is significantly reduced, while the total ester, erythritol, total acid, and amino acid nitrogen content of the newly brewed vinegar are significantly increased, achieving a comprehensive improvement in the flavor quality and taste harmony of Shanxi aged vinegar. Attached Figure Description

[0018] Figure 1The biomass results of the interaction between *Saccharomyces cerevisiae* strain CGMCC 15729 and *Yarrowia lipolyticis* strain SGMCC D25204 are shown in the figure. The top figure shows the biomass of *Yarrowia lipolyticis* strain SGMCC D25204, and the bottom figure shows the biomass of *Saccharomyces cerevisiae* strain CGMCC 15729. A: B represents *Yarrowia lipolyticis* strain SGMCC D25204: *Saccharomyces cerevisiae* strain CGMCC 15729. Figure 2 The results of fusel alcohol determination for the interaction between Saccharomyces cerevisiae strain CGMCC 15729 and Yersinia lipolytica strain SHOMCC D25204 are shown in the figure. In the figure, A and B represent Yersinia lipolytica SHOMCC D25204 and Saccharomyces cerevisiae strain CGMCC 15729, respectively. Figure 3 The results of total ester and erythritol determination for the interaction between Saccharomyces cerevisiae strain CGMCC15729 and Yersinia lipolytica strain SHOMCC D25204 are shown in the figure. In the figure, A:B represents Yersinia lipolytica SHOMCC D25204 and Saccharomyces cerevisiae strain CGMCC 15729. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0021] Equivalent technologies of the specific embodiments described herein that are apparent to those skilled in the art through routine experimentation are included in this application.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The thermostable α-amylase and saccharifying enzyme used in the following examples were purchased from Shandong Longket Enzyme Preparation Co., Ltd. The Angel Yeast inoculum used in the following comparative examples was purchased from Angel Yeast Co., Ltd. The fungal DNA extraction kit used in the following examples was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0023] Example 1: Screening, identification and preservation of yeast strains with low fusel oil production and high ester production (1) Initial screening of yeast strains with low fusel oil production and high ester production: Ten indigenous yeast strains isolated from Shanxi aged vinegar mash were activated and inoculated into PDA liquid medium, and cultured at 30℃ to achieve a cell concentration of 10. 8 At CFU / mL, 3% inoculum was added to sorghum juice culture medium and incubated at 30 ℃ for 72 h (the incubator was sealed with six layers of gauze for the first 24 h and with plastic sheeting for the next 48 h). After 72 h of fermentation, the content of fusel oils and total esters in the fermentation broth was measured. The specific determination method is as follows: Methods for determining fusel oils: The contents of higher alcohols such as n-propanol, isobutanol, isoamyl alcohol, and phenylethanol were determined by dispersive liquid-liquid microextraction combined with gas chromatography-mass spectrometry (LLME-GC-MS). 7 mL of diluted fermentation broth sample was placed in a 15 mL centrifuge tube, and an extraction system consisting of dichloromethane, acetonitrile, and 4-methyl-2-pentanol (internal standard) at a concentration of 0.4536 g / L was added. After thorough extraction with shaking, the mixture was centrifuged at 5000 rpm to separate the layers. The lower organic phase was collected, filtered through a 0.22 μm organic filter membrane, and then analyzed by GC-MS.

[0024] Total ester determination method: The total ester content of the fermentation broth was determined by the saponification method.

[0025] Table 1 shows that different yeast strains exhibited significant differences in fusel oil production levels and ester formation capabilities during fermentation. CGMCC 15729, JY530, and JY561 strains showed lower fusel oil production levels, with total fusel oil contents of 25.45 mg / 100 mL, 28.70 mg / 100 mL, and 31.02 mg / 100 mL, respectively, while maintaining higher total ester contents of 6.58 g / 100 mL, 4.18 g / 100 mL, and 3.91 g / 100 mL, respectively. Therefore, these three strains were selected as the subjects for subsequent aroma compound analysis.

[0026] Table 1 Screening of yeasts with low fusel oil production and high ester production (2) Re-screening of aroma-producing ability of yeast strains with low fusel oil production and high ester production: The volatile flavor compounds of the three yeast strains with low fusel oil production and high ester production obtained above were determined. A DB-FFAP polar capillary column (30 m × 0.25 mm × 0.25 μm) was used for separation, with high-purity helium (≥99.999%) as the carrier gas and a flow rate controlled at 1 mL / min. The injection system used manual injection mode with an injection volume of 1 μL and a non-split flow program. The column temperature gradient was set as follows: initial temperature of 40 °C for 5 min, followed by a programmed temperature increase to 240 °C at a rate of 10 °C / min and a holding time of 2 min. The mass spectrometry detection system parameters were set as follows: conduction line temperature 280 °C, ion source temperature 230 °C, electron impactionization (EI) mode, electron energy 70 eV, and mass scan range 20–350 amu.

[0027] As shown in Table 2, five classes of volatile flavor compounds—esters, alcohols, acids, ketones, and aldehydes—were detected in the fermentation broths of the three yeast strains, with significant differences in flavor metabolism among the different strains. A comprehensive comparison of the composition of various volatile compounds indicates that CGMCC 15729 exhibits superior aroma-producing characteristics. A total of 15 ester compounds were detected in CGMCC 15729, with a total ester content of 8.6252 mg / mL, higher than that of JY530 and JY561. Among these, the contents of major aroma-contributing substances such as ethyl butyrate, ethyl acetate, ethyl lactate, ethyl hexanoate, and phenylethyl acetate were all higher than those of other tested strains, contributing a richer ester aroma to the fermentation system.

[0028] Meanwhile, the total alcohol content of CGMCC 15729 was 1.2855 mg / mL, with a low isoamyl alcohol content, indicating that CGMCC 15729 can effectively reduce the accumulation of higher alcohols while forming aroma compounds. The total aldehyde content of CGMCC 15729 was 0.0085 mg / mL, lower than the other two strains, indicating a low level of undesirable flavor compounds. Comprehensive analysis shows that CGMCC 15729 is characterized by abundant esters, low higher alcohol production, and a harmonious flavor composition.

[0029] Table 2. Determination of volatile flavor compounds in superior yeast strains. Note: "-" indicates that it was not detected.

[0030] Example 2: Screening of high-yielding erythritol-producing Yersinia lipolytica strains: Ten Yersinia lipolytica strains, including nine laboratory-preserved strains and the preserved strain SMHCC D25204, were evaluated for fermentation performance. The fermentation broth cell concentration (OD200) was measured.600 The residual sugar content and erythritol yield were measured to screen for dominant erythritol-producing strains. The 10 *Yarrowia lipolytica* strains were activated and inoculated into PDA liquid medium, then cultured at 30°C to achieve a cell concentration of 10⁻⁶. 6 When the concentration of CFU / mL is 3%, it is inoculated into the liquid fermentation medium and placed in a constant temperature incubator at 30℃ for 72 hours (the incubator is sealed with six layers of gauze for the first 24 hours and with plastic sheeting for the next 48 hours). The fermentation is completed after 72 hours.

[0031] The above-mentioned PDA liquid culture medium preparation method is as follows: 200 g of potatoes, peeled and cut into pieces, add 1000 mL of distilled water, boil for 20 min, filter with gauze, add 20.0 g of glucose, 2.0 g of peptone, 2.0 g of potassium dihydrogen phosphate, and 1.0 g of magnesium sulfate to the filtrate, add distilled water to 1000 mL, and autoclave at 121℃ for 20 min.

[0032] The above-mentioned liquid fermentation culture medium is prepared as follows: 200 g glucose, 8 g yeast extract, 5 g ammonium citrate, 0.25 g potassium dihydrogen phosphate, 0.25 g anhydrous magnesium sulfate, 0.01 g copper sulfate pentahydrate, 0.05 g zinc sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, and distilled water to make up to 1000 mL. The medium is then autoclaved at 121°C for 20 min.

[0033] The specific measurement method is as follows: (1) Fermentation broth cell concentration (OD) 600 The method for determining the OD of the fermentation broth (nm) is as follows: The OD of the fermentation broth is determined using a UV spectrophotometer. 600nm To assess cell growth parameters.

[0034] (2) The method for determining reducing sugars is as follows: the determination shall be carried out in accordance with GB / T 5009.7—2003; (3) The determination method for erythritol was as follows: Agilent 1100 high-performance liquid chromatography was used. 5.0 mL of fermentation broth was centrifuged at 8000×g for 10 min. The supernatant was diluted appropriately with ultrapure water and filtered through a 0.22 μm organic filter membrane. The erythritol content of the filtrate was determined by HPLC. Chromatographic conditions: mobile phase was 5 mmol·L⁻¹. -1 H2SO4 was used, and the chromatographic column was an Aminex HPX-87H sugar analysis column (300 mm × 7.8 mm). The column temperature was 35℃, and the flow rate was 0.6 mL·min. -1 The injection volume was 10 μL, the detector was a differential refractive index detector, and the detector temperature was 30℃.

[0035] (4) Apparent rate of erythritol formation: Where: Qp: erythritol production rate (g / L·h); P: erythritol concentration in fermentation broth after 72h (g / L); t: fermentation time (72 h).

[0036] As shown in Table 3, the erythritol production results of the 10 yeast strains are as follows: strain SHIMCC D25204 has an erythritol content of 3.33 g / 100 mL and a production rate of 0.46 g / L·h, both of which are higher than those of the other strains.

[0037] Table 3. Results of erythritol production by 10 strains of *Yersinia lipolytica*. Note: The initial reducing sugar content is 20g / 100ml.

[0038] Example 3: Interaction between low-fusel-producing, high-ester-producing *Saccharomyces cerevisiae* and high-erythritol-producing *Yersinia saccharizoides*: The selected high-erythritol-producing *Yersinia saccharizoides* SMHCC D25204 and low-fusel-producing, high-ester-producing *Saccharomyces cerevisiae* CGMCC 15729 were mixed at different volume ratios (v / v) of 0:1, 1:1, 1:0, 1:5, and 1:10, and inoculated at a total inoculum of 3%. The mixture was inoculated into PDA liquid medium and cultured at 30°C for 24 hours until the cell concentration reached 10⁻⁶. 6 At CFU / mL, the yeast was inoculated into sorghum juice medium at an inoculation ratio of 1% and an inoculation volume of 3%, respectively. After incubation at 30℃ for 72 h, the viable cell count of the two yeast strains was measured to investigate whether there was any inhibitory effect between the yeast strains. At the same time, the contents of fusel oils, total esters, esters and erythritol in the fermentation broth were measured.

[0039] The method for preparing the PDA liquid culture medium is as follows: Peel and cut 200 g of potatoes into pieces, add 1000 mL of distilled water, boil for 20 min, filter with gauze, add 20.0 g of glucose, 2.0 g of peptone, 2.0 g of potassium dihydrogen phosphate, and 1.0 g of magnesium sulfate to the filtrate, add distilled water to 1000 mL, and autoclave at 121℃ for 20 min.

[0040] The method for preparing the sorghum juice culture medium is as follows: 200g of sorghum is crushed into four or six pieces, and moistened with 4 times the amount of water for 4 hours. Then, it is gelatinized at a temperature of 85~90℃ for 1 hour with constant stirring. 0.5g of heat-resistant α-amylase (enzyme activity 70000U) is added and the mixture is cooked at 100℃ for 60 minutes until it is no longer sticky to the touch and there is no raw core. The mixture is then cooled to room temperature, filtered, and the filtrate is sterilized at 121℃ for 20 minutes for later use.

[0041] Different mixing ratios significantly affected the co-culture performance of the two yeast strains. Figure 1 , Figure 2 as well as Figure 3It was found that, compared with single-strain culture, the fermentation system of *Saccharomyces cerevisiae* CGMCC 15729 and *Yarrowia lipolytica* SMHCC D25204 in a 1:1 ratio exhibited a better synergistic effect. This combination contained 4.72 mg / 100 mL of n-propanol, 6.31 mg / 100 mL of isobutanol, 11.86 mg / 100 mL of isoamyl alcohol, and 4.86 mg / 100 mL of 2-phenylethanol, resulting in a reduced total fusel oil content of 27.75 mg / 100 mL, lower than the pure culture group. Simultaneously, the total ester content reached 1.92 g / 100 mL, higher than all other ratios. Erythritol content was 0.98 g / 100 mL, maintaining a high accumulation level. Furthermore, the viable counts of *Yarrowia lipolyticis* and *Saccharomyces cerevisiae* in this combination reached 7.78 lg CFU / mL and 7.52 lg CFU / mL, respectively, indicating that the two strains have good co-culture adaptability in the sorghum juice fermentation system. Considering the survival ability, fusel oil reduction ability, ester formation ability, and erythritol accumulation level of the two yeasts, the optimal interaction ratio of CGMCC 15729 and SMHCC D25204 was determined to be 1:1.

[0042] Example 4: Preparation of a compound inoculum from *Saccharomyces cerevisiae* (low fusel oil production, high ester production) and *Yersinia lipolytica* (high erythritol production): The preparation method of the *Saccharomyces cerevisiae*-*Yersinia lipolytica* compound inoculum was as follows: *Saccharomyces cerevisiae* seed culture CGMCC 15729 and *Yersinia lipolytica* seed culture SMHCC D25204 were mixed at a 1:1 ratio, and 3% inoculated into 75L of yeast extract liquid culture medium. The mixture was cultured at 30℃ and an aeration rate of 0.6L / min for 24 hours. During this period, 0.2% (v / v) of glucose from the yeast extract liquid culture medium was added every 8 hours until the cell concentration reached 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added to the sludge at a volume-to-weight ratio (v / w) of 3:1. After thorough mixing, the mixture was spread evenly onto the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 hours. The freeze dryer was pre-cooled beforehand, and the frozen material was placed inside after the cold trap temperature stabilized at -50℃. Vacuum freeze drying was performed using a segmented temperature control mode to obtain a compound bacterial agent with a viable count of 10-1. 9 CFU / g, vacuum packaged and stored at 4℃.

[0043] The method for preparing the *Saccharomyces cerevisiae* CGMCC 15729 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 15729 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0044] The method for preparing the seed culture of *Yarrowia lipophila* SMHCC D25204 is as follows: cultured in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Yarrowia lipolyticis* SHIMCC D25204 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium at a concentration of 3.5 CFU / mL. The fermenter was incubated at 30°C, with an aeration rate of 0.2 L / min and a rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL.

[0045] The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20 min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5 g skim milk powder, 8 g whey powder, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; Protectant 2: 5 g trehalose, 3 g sucrose, 5 g glucose, 0.5 g monosodium glutamate, and 50 mL of phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20 min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio (v / v) of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The segmented temperature-controlled vacuum freeze-drying parameters are set as follows: first segment: -60℃, 15min; second segment: -25℃, 1920min; third segment: 0℃, 420min; fourth segment: 20℃, 120min.

[0046] Example 5: Application of compound microbial agent for strengthening Shanxi aged vinegar (60% Daqu and 0.1% compound microbial agent added during the alcoholic fermentation stage): Sorghum is crushed to 50-60 mesh, and sorghum and water are mixed at a mass-volume ratio (w / v) of 1:4.5. The mixture is heated to 90-95℃, and 0.1% of heat-resistant α-amylase is added by mass of sorghum. The mixture is kept warm for 80-90 minutes, cooled to 60℃, and then 0.1% of saccharifying enzyme is added by mass of sorghum. The mixture is kept warm for 20-30 minutes. Once the temperature drops to 28-32℃, pump the mixture into an alcohol fermentation tank, add 60% Daqu (a type of starter culture) of sorghum and 0.1% of a compound yeast of brewing yeast and Yeastra lipolytica of sorghum, and ferment for 8 days until the alcohol content reaches 8-9% vol. The fermentation process ends to obtain mash. The fermented mash is then thoroughly mixed with 100% wheat bran, 60% rice bran, and 40% rice husks of sorghum. Simultaneously, 10% of a fermented mash is added for acetic acid fermentation for 12 days until the total acid content reaches 5-6 g / 100 mL. The acetic acid fermentation is then completed, and the mash is further treated with fumigation and leaching to obtain fresh leached vinegar.

[0047] Comparative example: In the brewing process of Shanxi aged vinegar, only 60% of Daqu (a type of starter culture) is added during the alcoholic fermentation stage, and Angel yeast inoculant with a sorghum mass fraction of 0.1% is added at the same time. The rest of the process is the same as in Example 5.

[0048] The specific measurement method is as follows: 1. Physicochemical indicators: The determination methods for total acid, amino acid nitrogen, non-volatile acid, total ester, total soluble solids and reducing sugar content are as follows: The determination is carried out in accordance with the method of the national standard GB / T 19777—2013 "Geographical Indication Product Shanxi Aged Vinegar".

[0049] 2. Volatile aroma compounds: Separation was performed using a DB-FFAP polar capillary column (30 m × 0.25 mm × 0.25 μm) with high-purity helium (≥99.999%) as the carrier gas at a flow rate of 1 mL / min. The injection system was manual, with an injection volume of 1 μL and a non-split flow program. The column temperature gradient was set as follows: initial temperature of 40 °C for 5 min, followed by a programmed increase to 240 °C at a rate of 10 °C / min and a hold for 2 min. The mass spectrometry detection system parameters were set as follows: conduction line temperature 280 °C, ion source temperature 230 °C, electron impact ionization (EI) mode, electron energy 70 eV, and mass scan range 20–350 amu.

[0050] As shown in Table 4, the total alcohol content of the comparative example after fermentation with conventional Angel yeast was 299.80 mg / L. In Example 5, the total alcohol content was only 225.91 mg / L after co-fermentation with a low fusel alcohol-producing Saccharomyces cerevisiae and a high erythritol-producing Yersinia spp., and the total fusel alcohol content was significantly reduced. Among them, the core fusel alcohol components with strong irritation, such as isoamyl alcohol, isobutanol, n-propanol, and phenylethanol, all showed significant reductions. Isoamyl alcohol decreased from 60.15 mg / L to 45.31 mg / L, and isobutanol decreased from 60.15 mg / L to 45.31 mg / L. The compound microbial agent inhibited the formation of higher fusel alcohols through the amino acid deamination pathway during the alcoholic fermentation stage, effectively reducing the accumulation of undesirable flavor precursors such as spiciness and bitterness. In Example 5, the total amount of various esters increased to 426.42 mg / L, and characteristic fruit aroma esters such as ethyl butyrate, ethyl acetate, and γ-nonanolide were enriched in large quantities. This achieved the reduction of fusel alcohols and weakened the irritation while enriching the aroma base of the mash, laying an excellent flavor foundation for subsequent acetic acid fermentation.

[0051] Table 4. Determination of flavor compounds at the end of alcoholic fermentation. As shown in Table 5, compared with the comparative example, Example 5 significantly improved the quality of Shanxi aged vinegar. The total acidity was 5.68 g / 100 mL, and the non-volatile acidity, amino acid nitrogen, total esters, and erythritol were 2.32 g / 100 mL, 0.41 g / 100 mL, 2.87 g / 100 mL, and 0.92 g / 100 mL, respectively, representing increases of 25.41%, 28.13%, 33.49%, and 70.37%. Erythritol, in particular, gave the product a unique refreshing sweetness and improved the taste harmony.

[0052] Table 5. Determination of Physicochemical Indicators of Freshly Rinseed Oil As shown in Table 6, the total ester content of Example 5 was 864.33 mg / L, which was significantly higher than that of the comparative example. Characteristic aroma esters such as ethyl butyrate and nonyl lactone were significantly enriched, providing the fruity and milky flavor of vinegar. The total alcohol content was 180.41 mg / L, and fusel alcohols such as isoamyl alcohol and isobutanol were effectively suppressed, avoiding the spicy and bitter taste. The total acid content was 671.59 mg / L, and pyrazines supplemented the caramel flavor, significantly improving the aroma richness and palatability of the product.

[0053] Table 6. Determination of volatile aroma compounds in fresh vinegar. Note: "-" indicates that it was not detected.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the interaction between *Saccharomyces cerevisiae* and *Yarrowia lipolytica* in vinegar brewing, characterized by: Excellent indigenous Saccharomyces cerevisiae CGMCC 15729, which produces low fusel alcohols and high esters, and Yersinia lipolytica S. SMHCC D25204, which produces high erythritol, were screened from Shanxi aged vinegar mash. The inoculation ratio was optimized through strain interaction, and a compound microbial agent was prepared by synchronous mixing and high-density culture according to the optimized ratio. This agent was then applied to the alcoholic fermentation stage of vinegar brewing. Alcoholic fermentation yielded mash, which was then subjected to solid-state acetic acid fermentation, smoking, and leaching to produce new leached vinegar.

2. The application according to claim 1, characterized in that: The preparation method of the Saccharomyces cerevisiae-Yersinia lipolytica compound inoculum is as follows: Saccharomyces cerevisiae CGMCC 15729 seed culture and Yersinia lipolytica SSHMCC D25204 seed culture are mixed at a 1:1 ratio, and then inoculated into 75L of yeast extract liquid culture medium at a 3% inoculation rate. The mixture is cultured at 30℃ and an aeration rate of 0.6L / min for 24 hours, during which 0.2% (v / v) of glucose from the yeast extract liquid culture medium is added every 8 hours until the cell concentration reaches 10... 8 The bacterial sludge was obtained by centrifuging at 8000 r / min for 10 min with CFU / mL. The sludge was then mixed thoroughly and spread evenly on the freeze dryer tray to a thickness of 1 cm. The material was then frozen at -60℃ for 6 h. The freeze dryer was started in advance for pre-cooling. After the cold trap temperature stabilized at -50℃, the frozen material was placed in and vacuum freeze-dried using a segmented temperature control mode to obtain a composite inoculum of Saccharomyces cerevisiae and Yeastra lipolytica, with a viable count of 10⁻⁶ cells / mL. 9 CFU / g, vacuum packaged and stored at 4℃; The method for preparing the *Saccharomyces cerevisiae* CGMCC 15729 seed culture is as follows: culture the culture in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Saccharomyces cerevisiae* CGMCC 15729 (CFU / mL) was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium. The culture was incubated at 30℃, aeration rate of 0.2L / min, and rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL; The method for preparing the seed culture of *Yarrowia lipophila* SMHCC D25204 is as follows: cultured in PDA liquid medium until the cell concentration reaches 10... 8 A 3.5% inoculum of *Yarrowia lipolyticis* SHIMCC D25204 was inoculated into a 5L fermenter containing 2.5L of yeast extract liquid medium at a concentration of 3.5 CFU / mL. The fermenter was incubated at 30°C, with an aeration rate of 0.2 L / min and a rotation speed of 200 r / min for 24 hours, achieving a cell concentration of 10⁻⁶. 8 CFU / mL; Yeast extract ( Yarrowia lipolytica SMHCC D25204 contained 11.79 g / 100 mL of residual reducing sugar and 3.33 g / 100 mL of erythritol in liquid fermentation medium. It was deposited at Shanghai Preservation Microbial Co., Ltd. The liquid fermentation medium is prepared as follows: 200 g glucose, 8 g yeast extract, 5 g ammonium citrate, 0.25 g potassium dihydrogen phosphate, 0.25 g anhydrous magnesium sulfate, 0.01 g copper sulfate pentahydrate, 0.05 g zinc sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, and distilled water is added to 1000 mL. The mixture is then autoclaved at 121°C for 20 min. The method for preparing the yeast extract liquid culture medium is as follows: 1g yeast extract, 2g glucose, 0.3g yeast extract, 100mL water, sterilized at 121℃ for 20min for later use. The method for preparing the vacuum freeze-drying protectant is as follows: Protectant 1: 5g skim milk powder, 8g whey powder, 50mL phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20min; Protectant 2: 5g trehalose, 3g sucrose, 5g glucose, 0.5g monosodium glutamate, 50mL phosphate buffer solution with pH 6.5, sterilized at 121℃ for 20min; After sterilization, protectant 1 and protectant 2 are cooled to room temperature and mixed evenly at a volume ratio of 1:1 to obtain the bacterial cell vacuum freeze-drying protectant; The parameters for the segmented temperature-controlled vacuum freeze-drying process are set as follows: First segment: -60℃, 15min; Second segment: -25℃, 1920min; Third segment: 0℃, 420min; Fourth segment: 20℃, 120min.

3. The application according to claim 2, characterized in that: The vinegar brewing process includes the following steps: Sorghum is crushed to 50-60 mesh; sorghum and water are mixed at a mass-to-volume ratio of 1:4.5; the mixture is heated to 90-95℃; 0.1% (by mass) of heat-resistant α-amylase is added; the mixture is kept at this temperature for 80-90 minutes; the temperature is then lowered to 60℃; 0.1% (by mass) of saccharifying enzyme is added; the mixture is kept at this temperature for 20-30 minutes; once the temperature drops to 28-32℃, the mixture is pumped into an alcohol fermentation tank, and 60% (by mass) of sorghum is added. The yeast and 0.1% (by weight) of brewing yeast-Yersinia lipolytica compound inoculant were added to sorghum and fermented for 8 days to achieve an alcohol content of 8-9% vol. After the alcohol fermentation was completed, the mash was obtained. The fermented mash was then thoroughly mixed with 100% (by weight) of wheat bran, 60% (by weight) of rice bran, and 40% (by weight) of rice husks. At the same time, 10% (by weight) of vinegar mash was added for acetic acid fermentation for 12 days to achieve a total acid content of 5-6 g / 100 mL. After the acetic acid fermentation was completed, the mash was smoked and then leached to obtain new vinegar.

4. The application according to claim 3, characterized in that: The resulting new leached vinegar is reddish-brown with a rich color, and contains ≥2.50g / 100mL of total esters, ≥0.85g / 100mL of erythritol, ≥5.50g / 100mL of total acid, and ≥0.35g / 100mL of amino acid nitrogen.

Images