Use of strains having functions of enhancing aroma and producing exopolysaccharides and composition in fermented food

Abstract

Provided is use of Wickerhamomyces anomalus W23087 and Lactiplantibacillus plantarum L23104 derived from sourdough and a composite microbial agent thereof in fermented food, belonging to the fields of fermentation engineering and biotechnology. The Wickerhamomyces anomalus W23087 strain has a strong aroma-enhancing function and excellent fermentation performance. The Lactiplantibacillus plantarum L23104 strain can efficiently produce exopolysaccharides and enhance the stability of a fermented product. By means of compounding, the content of exopolysaccharides in the fermented food is increased, the flavor of the fermented food is enhanced, and the quality of the fermented food is improved.

Description

Application of strains and compositions with flavor-enhancing and extracellular polysaccharide-producing functions in fermented foods Technical Field

[0001] This invention relates to the application of strains and compositions with flavor-enhancing and extracellular polysaccharide-producing functions in fermented foods, belonging to the fields of fermentation engineering and biotechnology. Background Technology

[0002] Sourdough is a traditional leavening agent obtained by naturally fermenting a mixture of wheat flour and other grain flours with water using microorganisms such as yeast and lactic acid bacteria. The sourdough system is complex, composed of a rich microbial community, with yeast and lactic acid bacteria being the most important fermenting microorganisms. Aroma-producing yeasts, also known as flavor-generating yeasts, produce metabolic byproducts such as esters, higher alcohols, and lower fatty acids during fermentation, contributing to the unique fermented aroma. In recent years, aroma-producing yeasts have been widely used in fermented foods such as aged vinegar, fruit wine brewing, baijiu (Chinese liquor) flavor enhancement, and condiments, and are closely related to the formation of product flavor. The co-fermentation method of aroma-producing yeasts with brewing yeast can increase the content of ester aroma substances in fermented wines while ensuring high fermentation efficiency.

[0003] Aroma-producing yeasts play a crucial role in improving food flavor, and the discovery of novel aroma-producing yeasts holds great promise for application. Isolating and screening aroma-producing yeasts from food is a research trend. Ye Mengqi et al. isolated and screened microorganisms from apple samples and fruit storage air, purifying a strain of *V. anomala* with the strongest ester-producing ability. When artificially inoculated into apple cider for fermentation, the volatile aroma compounds in the cider changed significantly, increasing to twice their original content. Shen Guanghui et al. screened five aroma-producing yeasts with high ester production and low alcohol production from baijiu (Chinese liquor) starter cultures, including one *V. anomala* and four *Saccharomyces cerevisiae*. Inoculating *V. anomala* strain Z133 into watermelon juice for fermentation helped increase the variety and content of alcohol and ester aroma components in watermelon wine. Wang Xiaodan et al. isolated 40 yeast strains from sauce-flavored baijiu mash and screened two flavor bacteria with the strongest alcohol production ability. These two yeasts were identified as *Candida albicans* and *Pichia pastoris*, respectively, both exhibiting strong ester production capabilities.

[0004] Patent CN109456904B discloses the saccharification power of *Saccharomyces cerevisiae*, reaching 174U; patent CN109266562B discloses *Saccharomyces cerevisiae* with high ester production (especially ethyl acetate), achieving an ethyl acetate yield of 17.31 g / L; patents CN105861346B and CN114134055B, among others, disclose the flavor-producing capabilities of *Saccharomyces cerevisiae*, such as high phenylethanol production and low urea production. However, the *Saccharomyces cerevisiae* strains disclosed in existing patents suffer from relatively singular functions and insufficiently outstanding performance. Therefore, providing a strain of *Saccharomyces cerevisiae* with multiple outstanding performance characteristics has extremely high practical value and can further broaden the application fields of *Saccharomyces cerevisiae*. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention screened an abnormal Wickham yeast strain W23087 from sourdough and deposited it at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29669; and a Lactobacillus plantarum strain L23104, which was also deposited at the same center with accession number CGMCC No. 29668.

[0006] Based on this, the present invention combines *Saccharomyces cerevisiae* W23087 and *Lactobacillus plantarum* L23104 to obtain a compound microbial inoculant. The *Saccharomyces cerevisiae* W23087 and the compound microbial inoculant of the present invention have strong aroma-enhancing functions and excellent fermentation performance, which can enhance the quality and flavor characteristics of fermented foods, and have broad application prospects.

[0007] The present invention relates to Wickerhamomyces anomalus W23087, a yeast strain with excellent fermentation performance and significant aroma-enhancing or high ester production capabilities. It addresses the problems of uneven fermentation performance and unstable flavor compounds in fermented flour products, alcoholic beverages, soft drinks, condiments, and feed products. Wickerhamomyces anomalus W23087 offers the following advantages:

[0008] (1) High ester production capacity: The abnormal Wickham yeast W23087 of the present invention was fermented in YPD medium for 12 h and the total ester production reached (19.25±0.35) g / L.

[0009] (2) High saccharification ability: The saccharification power of the abnormal Wickham yeast W23087 of the present invention is 821.8U;

[0010] (3) Excellent flavor production ability: The abnormal Wickham yeast W23087 of the present invention has the ability to produce high levels of ethyl acetate and phenylethanol in the preparation of noodle products, wine, fermented fruit juice, condiments and feed, with an effect that is more than 20% higher than that of existing abnormal Wickham yeast and commercially available inoculants.

[0011] The Lactiplantibacillus plantarum L23104 used in this invention produces high levels of extracellular polysaccharides, which can act as a stabilizer to enhance the stability of fermented products, thereby improving the texture and taste of the products.

[0012] The first objective of this invention is to provide an abnormal Wickerhamomyces anomalus W23087, which was deposited at the Institute of Microbiology, Chinese Academy of Sciences on January 18, 2024, with accession number CGMCC No. 29669.

[0013] In one embodiment, the colony morphology of the *Wickham's abnormal* yeast is milky white, 1.0–2.0 mm in diameter, spherical, with protrusions, a smooth surface, neat edges, and opaque. Under an optical microscope, the cells of this strain are spherical or ellipsoidal, with obvious conidia around them.

[0014] The present invention also provides a microbial preparation containing the above-mentioned abnormal Wickham yeast W23087, its metabolites or fermentation broth.

[0015] In one embodiment, the microbial preparation is applied as a solid or liquid formulation.

[0016] In one embodiment, the number of *Wickham's abnormal* yeast per gram or milliliter of the microbial preparation is not less than 1.0 × 10⁻⁶. 8 CFU.

[0017] In one embodiment, the microbial preparation contains live cells of the aforementioned abnormal Wickham yeast W23087, or freeze-dried cells or immobilized cells, or strains containing abnormal Wickham yeast W23087 existing in any other form.

[0018] In one embodiment, the microbial agent is prepared as follows: Two loops of activated *Wickham's abnormal* yeast W23087 are inoculated into YPD medium and cultured at 28.0°C and 200.0g in a shaker for 20.0h. Afterward, enrichment culture is performed under the same conditions, followed by high-speed centrifugation for 15.0min. The precipitate is collected, washed twice with sterile water, and then washed again with sterile distilled water to prepare a yeast suspension for later use. Trehalose is added as a protectant. A 1.5–2.0 times concentration of trehalose solution is prepared and sterilized at 121°C for 20.0min. This solution is then mixed with an equal volume of the yeast suspension. The suspension is pre-frozen at -80°C for 3.0h and then freeze-dried in a freeze dryer. The freeze-dried powder is stored at -20°C.

[0019] The present invention also provides a microbial bran koji, wherein the microbial preparation contains the above-mentioned abnormal Wickham yeast W23087 or its metabolites.

[0020] In one embodiment, the microbial preparation is applied as a solid dosage form.

[0021] In one embodiment, the number of *Wickham's abnormal* yeast per gram or milliliter of the microbial preparation is not less than 1.0 × 10⁻⁶. 8 CFU.

[0022] In one embodiment, the microbial bran contains live cells of the aforementioned abnormal Wickham yeast W23087, or freeze-dried cells or immobilized cells, or strains containing abnormal Wickham yeast W23087 existing in any other form.

[0023] In one embodiment, the preparation of the microbial bran koji is as follows: Activated abnormal Wickham yeast W23087 is activated to a certain concentration of bacterial solution for later use. Weigh 20g of wheat bran, add 20mL of distilled water and stir evenly. Shake the Erlenmeyer flask to mix the material and water evenly. Sterilize at 121℃ for 40min. After cooling in a sterile room and shaking to break up the material clumps, inoculate 5‰-10% of abnormal Wickham yeast W23087 into the shake flask. After inoculation, shake each flask individually to mix evenly. Incubate at a constant temperature of 28.0℃ in an incubator. After mycelium grows in about 18h, shake the flask for the first time to loosen the clumps. After shaking evenly, spread it evenly on the bottom of the flask and continue incubation. Incubate for another 5h-6h until the material cakes obviously form, then invert the flask for 72h to ensure that the growth of the mycelium in the upper and lower parts is uniform. When inverting the flask, handle it gently to prevent the koji cake from breaking. After inverting the flask, continue culturing for about 36 hours. Then, lay the conical flask horizontally to facilitate water storage and continue culturing at a constant temperature of 25-32℃. Shake the flask regularly to ensure that the koji cake in close contact with the flask wall receives sufficient oxygen to promote the growth and reproduction of the bacterial strain. After culturing, crush the koji cake under aseptic conditions, then dispense it into sealed bags that have been sterilized at 121℃ for 30 minutes. Seal the bags, add the koji production date, and store them in a refrigerator at 4℃ for later use.

[0024] The present invention also provides the use of the above-mentioned abnormal Wickham yeast W23087 or any of the above-mentioned microbial preparations in fermented flour products.

[0025] In one embodiment, the fermented food includes, but is not limited to, bread, steamed buns, etc.

[0026] In one embodiment, the application is for bread fermentation, using the aforementioned abnormal Wickham yeast W23087 as a leavening agent.

[0027] In one embodiment, the bread fermentation involves adding the aforementioned abnormal Wickham yeast W23087 and commercially available abnormal Wickham yeast starter to the dough at amounts of 0.25 mL to 3.0 mL and 0.05 g to 0.25 g, respectively. The dough is made primarily from high-gluten flour, sucrose, sodium chloride, and drinking water, mixed in a ratio of 10:0.6:0.2:0.6, and then subjected to fermentation and baking processes.

[0028] In one embodiment, the application is for fermenting steamed buns, using the aforementioned abnormal Wickham yeast W23087 as a leavening agent.

[0029] In one embodiment, the steamed bun fermentation involves adding 1.0g of each of the aforementioned abnormal Wickham yeast W23087 and commercially available abnormal Wickham yeast starter to the dough. 100.0g of wheat flour and 50.0mL of water are added to a dough mixer, which is then slowly mixed for 3.0min followed by a fast mixing for 3.0min. The dough is divided into portions, kneaded until smooth, and then placed in a proofing box at 38.0℃ and 85% relative humidity for 50.0–80.0min. The dough is then steamed for 20.0min, and after turning off the heat, it is left to sit in the pot for 5.0min to obtain the final product.

[0030] The present invention also provides the application of the above-mentioned abnormal Wickham yeast W23087 or any of the above-mentioned microorganisms in fermented wines.

[0031] In one embodiment, the fermented food includes, but is not limited to, small-batch liquor and wine.

[0032] In one embodiment, the application is for the fermentation of small-batch liquor.

[0033] In one embodiment, the fermentation of the small-batch liquor involves using the abnormal Wickham yeast W23087 as a fermenting agent, inoculating with 1.0% w / w of abnormal Wickham yeast W23087 inoculum, and obtaining the liquor through soaking grains, initial steaming, soaking in water, re-steaming, cooling, fermentation, and distillation.

[0034] In one embodiment, the application is for wine fermentation.

[0035] In one embodiment, the fermentation of the small-batch liquor involves using the abnormal Wickham yeast W23087 and commercial yeast as fermenting agents, with an inoculation amount of 5.0% for each. Fully ripe grapes are selected, crushed, and placed into a container, with the raw materials not exceeding 3 / 4 of the container's capacity. The amount of sulfur dioxide added is 30.0 mg / L, and the amount of pectinase added is 0.02 g / L. The liquor is then cold-macerated for 48.0 hours.

[0036] The present invention also provides the use of the above-mentioned abnormal Wickham yeast W23087 or any of the above-mentioned microbial preparations in fermented beverages.

[0037] In one embodiment, the fermented beverage includes, but is not limited to, mango juice, apple cider vinegar, etc.

[0038] In one embodiment, the application is for mango juice fermentation.

[0039] In one embodiment, the mango juice fermentation involves using Abnormal Wickham Yeast W23087 as a starter culture, inoculating it with 4.0% of both the Abnormal Wickham Yeast W23087 inoculum and a commercially available Abnormal Wickham Yeast starter culture, fermenting at 25.0℃~30.0℃ for 24.0h~48.0h, pasteurizing, and then bottling.

[0040] In one embodiment, the application is for apple cider vinegar fermentation.

[0041] In one embodiment, the apple cider vinegar fermentation involves using the abnormal Wickham yeast W23087 as a starter culture. After alcoholic fermentation, acetic acid bacteria are inoculated for acetic acid fermentation. The supernatant is then collected by centrifugation and pasteurized to obtain the finished apple cider vinegar product.

[0042] The present invention also provides the use of the above-mentioned abnormal Wickham yeast W23087 or any of the above-mentioned microorganisms in fermented condiments.

[0043] In one embodiment, the fermented beverage includes, but is not limited to, sweet bean sauce and soy sauce.

[0044] In one embodiment, the application involves fermenting sweet bean sauce using the aforementioned abnormal Wickham yeast W23087 as a starter culture. 2.0%–5.0% w / w abnormal Wickham yeast W23087 and a commercially available abnormal Wickham yeast starter culture are inoculated into steamed flour. After primary and secondary fermentation in a ventilated environment, the finished sweet bean sauce is obtained.

[0045] In one embodiment, the application involves using the aforementioned abnormal Wickham yeast W23087 as a starter culture in soy sauce fermentation. Soy sauce is soaked in warm water at a 1:2 ratio for 5.0 hours, then steamed. After cooling, abnormal Wickham yeast W23087 bran koji is added at a ratio of 5.0%. Then, approximately 1.5 to 2.5 times the material weight of brine is added, resulting in a final moisture content of approximately 45% to 55% for the fermented soy sauce mash. After fermentation, the mash is further processed by rinsing, pressing, removing the mash, filtering, and sterilizing.

[0046] The present invention also provides the use of the above-mentioned abnormal Wickham yeast W23087 or any of the above-mentioned microorganisms in fermented feed.

[0047] In one embodiment, the fermented beverage includes, but is not limited to, animal feed.

[0048] In one embodiment, the application involves using Abnormal Wickham Yeast W23087 as a starter culture, inoculating it with Abnormal Wickham Yeast W23087 bran koji at a ratio of 10% w / w, stirring evenly, and then allowing it to ferment naturally.

[0049] The present invention also provides a compound microbial agent, characterized in that it contains Lactobacillus plantarum L23104 and Wickerhamomyces anomalus W23087.

[0050] The Lactobacillus plantarum L23104 was deposited with the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29668.

[0051] The aforementioned Wickerhamomyces anomalus W23087 was deposited with the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29669.

[0052] In one embodiment, the ratio of *Lactobacillus plantarum* and *Wickham's yeast* in the compound microbial agent is 1:0.8-1.2, obtained through enrichment culture, centrifugation and washing, proportional addition and freeze drying; the number of cells in the compound microbial agent per gram or milliliter is not less than 1.0 × 10⁸ CFU.

[0053] In one embodiment, the composite microbial agent includes live cells of *Lactobacillus plantarum* L23104 and *Wickham's yeast* W23087, or freeze-dried cells or immobilized cells, or strains containing *Lactobacillus plantarum* L23104 and *Wickham's yeast* W23087 existing in any other form.

[0054] The present invention also provides a product comprising any of the above-mentioned compound microbial agents; the product includes fermentation preparations, soil amendment preparations, wastewater treatment preparations, and feed additives.

[0055] The present invention also provides the application of any of the above-mentioned compound microbial agents in fermented noodle products, fermented wines, fermented fruit juices, fermented dairy products or fermented condiments.

[0056] In one embodiment, the fermented dough products include, but are not limited to, bread, steamed buns, and cakes.

[0057] In one embodiment, the fermented alcoholic beverages include, but are not limited to, rice wine and grape wine.

[0058] In one embodiment, the fermented fruit juice includes, but is not limited to, apple cider vinegar and prickly pear juice.

[0059] In one embodiment, the fermented dairy product includes, but is not limited to, stringy yogurt and cheese.

[0060] In one embodiment, the fermented condiment includes, but is not limited to, chili sauce, tomato sauce, and fermented bean curd.

[0061] In one embodiment, the bread fermentation is achieved by inoculating the compound microbial agent and flour into the flour at a mass ratio of 1 to 5:100, using high-gluten flour, sucrose, sodium chloride and drinking water as the main raw materials, mixed in a ratio of 10:0.6:0.2:0.6, and then undergoing fermentation and baking processes.

[0062] In one embodiment, the steamed bun fermentation is achieved by inoculating the compound microbial agent, commercial compound fermentation agent, and flour into the flour at a mass ratio of 1-5:1-5:200, followed by fermentation and steaming processes.

[0063] In one embodiment, the fermentation of the multigrain cake is achieved by inoculating the compound microbial agent and flour into mixed flour at a mass ratio of 1 to 5:100, followed by two proofing, fermentation, and baking processes.

[0064] In one embodiment, the rice wine fermentation is carried out by inoculating the compound microbial agent into glutinous rice at a ratio of 0.5% to 2% w / w, and then proceeding through processes such as washing, removing impurities, soaking, pulping, liquefying, saccharifying, fermenting, and filtering the glutinous rice raw materials.

[0065] In one embodiment, the wine fermentation is achieved by inoculating the compound microbial agent into grapes at a ratio of 2.5% to 5% w / w, followed by processes such as washing, crushing, cold soaking, and fermentation.

[0066] In one embodiment, the apple cider vinegar fermentation is achieved by using the compound microbial agent as a fermenting agent and then processing apple raw materials through washing, pulping, adjusting the composition, fermentation, and filtration.

[0067] In one embodiment, the prickly pear juice fermentation is achieved by inoculating the prickly pear with the compound microbial agent at a ratio of 2.0% to 5.0% w / w, followed by processes such as washing, crushing, and fermentation.

[0068] In one embodiment, the stringy yogurt fermentation is achieved by using the compound microbial agent as a starter culture, followed by pasteurization and fermentation.

[0069] In one embodiment, the application is for chili sauce fermentation.

[0070] In one embodiment, the chili sauce fermentation is carried out by inoculating the high-extracellular polysaccharide-producing Lactobacillus plantarum L23104 fermentation agent into chili peppers that have been blanched in boiling water at an inoculation rate of 1.0% w / w, adding 5.0% w / w salt, and fermenting at 30°C for 2 days.

[0071] In one embodiment, the tomato sauce fermentation is achieved by inoculating the compound microbial agent into tomatoes at a ratio of 1.0% to 5.0% w / w, followed by processes such as washing, blanching, crushing, pasteurization, and fermentation.

[0072] In one embodiment, the fermented bean curd is obtained by inoculating the compound microbial agent into soybean fodder at a ratio of 1.0% to 5.0% w / w, followed by processes such as watering, filling, vacuuming, and fermentation.

[0073] The present invention also provides a strain of Lactobacillus plantarum L23104, which was deposited with the China General Microbiological Culture Collection Center on January 18, 2024, with the accession number CGMCC No. 29668.

[0074] The fifth objective of this invention is to provide an abnormal Wickerhamomyces anomalus W23087 strain, which was deposited with the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29669. Beneficial effects

[0075] This invention screened an abnormal Wickham yeast strain W23087 from sourdough and deposited it at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29669. This strain has a strong aroma-enhancing function and excellent fermentation performance, which can enhance the quality and flavor characteristics of fermented foods and has broad application prospects.

[0076] Specifically:

[0077] (1) This invention provides an abnormal Wickham yeast W23087. The bacterial culture has a high OD value and the strain grows well within a certain temperature range, which is suitable for different temperature requirements of fermentation products.

[0078] (2) The present invention provides an abnormal Wickham yeast W23087 with flavor-enhancing function. After being cultured at 28℃ for 12.0h, the total ester content reaches the maximum of (19.25±0.35)g / L and the saccharification power is 821.8U, which can enhance the flavor characteristics of fermented products.

[0079] (3) When the high-ester-producing abnormal Wickham yeast W23087 provided by the present invention is used to ferment bread and steamed buns, the content of ethyl acetate and phenylethanol in the bread is increased by 190% compared with the control group; the total content of volatile flavor substances in the steamed buns is 82.3% higher than that in the control group, so the steamed buns have a pleasant aroma.

[0080] (4) When using the ester-producing *Anomalous Wickham yeast* W23087 provided by this invention to ferment baijiu and wine, the total ester and ethyl acetate content in the baijiu produced by *Anomalous Wickham yeast* W23087 was higher, by 36.8% and 86.9% respectively. The total amount of volatile flavor compounds in the wine produced by *Anomalous Wickham yeast* W23087 was 23% higher than that in the control group, and the fruit aroma was more intense.

[0081] (5) Using the ester-producing abnormal Wickham yeast W23087 provided by this invention to ferment mango juice and apple cider vinegar, the total sugar content of the mango juice was 21.3% lower than that of the control group, indicating that W23087 has a higher sugar utilization rate and stronger fermentation power. The total volatile aroma component content was 27.8% higher than that of the control group, which is much higher than that of mango juice from other yeasts of the same type. The content of phenylacetaldehyde and other volatile flavor substances in the apple cider vinegar was significantly higher than that of the control group by more than 35.1%, making the apple cider vinegar have a rich fruity aroma and a sweet and sour taste.

[0082] (6) The ester-producing abnormal Wickham yeast W23087 provided by the present invention was used to ferment sweet bean sauce. The total content of volatile flavor substances in the sweet bean sauce was 24.5% higher than that of the control group, making the sweet bean sauce sweet and mellow.

[0083] (7) The abnormal Wickham yeast W23087 that produces esters provided by the present invention was used to ferment the feed. The fermented feed had a special aroma and was rich in nutrients. The content of volatile ethanol was 0.31 μg / L, the content of ethanol was 0.28 μg / L, the content of acetic acid was 0.15 μg / L, and the total volatile flavor substances were 23.4% higher than those of the control group.

[0084] (8) The present invention provides a compound microbial agent containing Lactobacillus plantarum L23104, which produces high amounts of extracellular polysaccharides, and Wickham yeast W23087, which produces high amounts of esters. Under the same conditions, the extracellular polysaccharide yield of the compound microbial agent is increased by 9.33% compared with L23104, and the ester yield is increased by 8.22% compared with W23087.

[0085] (9) When using the compound microbial agent provided by the present invention to ferment flour products such as bread and steamed buns, compared with commercially available starter cultures, the polysaccharide content of the compound microbial agent after fermentation is more than 40% and 22.67% higher than that of the control group, respectively, and the volatile flavor substances are more than 34.64% and 27.71% higher than those of the control group.

[0086] (10) When using the compound microbial agent provided by the present invention to ferment alcoholic beverages such as rice wine and wine, compared with commercially available fermentation agents, the total ester content of rice wine prepared with the compound microbial agent is 0.44 g / L, which is more than 18.2% higher than that of the control group; the alcohol content of wine prepared with the compound microbial agent is 14.0% vol, while that of the control group is 12.0% vol; the content of total acid and volatile acid is 15.0% and 21.0% higher than that of the control group, respectively.

[0087] (11) When using the compound microbial agent provided by this invention to ferment beverages such as apple cider vinegar and prickly pear juice, compared with commercially available fermentation agents, the content of phenylacetaldehyde and other substances in the high-yield extracellular polysaccharide apple cider vinegar is significantly higher than that in the control group by 37.0%, making the apple cider vinegar have a rich fruity aroma and a sweet and sour taste. The total ester content in the prickly pear juice is 17.5% higher than that in the control group;

[0088] (12) When using the compound microbial agent provided by the present invention, the stringing effect is obvious during the fermentation of stringy yogurt, which improves the viscosity and adhesiveness of stringy yogurt, and the yogurt has a smooth taste. There are 40 kinds of volatile flavor substances in the experimental group, of which ethyl acetate accounts for 23.78% and acetaldehyde accounts for 19.76%, the flavor substances are richer and the milky aroma is strong.

[0089] (13) When using the compound microbial inoculant provided by this invention to ferment condiments such as chili sauce, tomato sauce, and fermented bean curd, compared with commercially available fermentation agents, the capsaicin content in chili sauce was significantly reduced to 0.28 mg / g, thus softening the spicy taste of the chili sauce. The total amount of volatile flavor compounds in tomato sauce was 7.10% higher than that in the control group, resulting in better aroma harmony, rich soy sauce flavor, and delicious taste. Fermented bean curd contained more volatile flavor compounds, giving it a mellow and wine-like aroma.

[0090] Biological Preservation Materials

[0091] The *Wickerhamomyces anomalus* provided by this invention, classified and named *Wickerhamomyces anomalus* W23087, was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29669. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0092] The *Lactiplantibacillus plantarum* provided by this invention, classified as *Lactiplantibacillus plantarum* L23104, was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29668. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0093] Figure 1 is a Gram staining diagram of the abnormal Wickham yeast W23087 of the present invention.

[0094] Figure 2 is a growth curve of the abnormal Wickham yeast W23087 of the present invention.

[0095] Figure 3 is a fermentation power curve of the abnormal Wickham yeast W23087 of the present invention.

[0096] Figure 4 is a graph showing the ester production curve of the abnormal Wickham yeast W23087 of the present invention.

[0097] Figure 5 is a flavor radar chart of the abnormal Wickham yeast W23087 of the present invention.

[0098] Figure 6 shows the Gram staining of Lactobacillus plantarum L23104 of the present invention.

[0099] Figure 7 is a phylogenetic tree diagram of Lactobacillus plantarum L23104 of the present invention. Detailed Implementation

[0100] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0101] The physicochemical indicators of fermented foods were tested as follows: pH value was determined according to the method in GB 5009.237-2016. Total bacterial count was determined according to the method in GB 4789.2-2022 National Food Safety Standard for Microbiological Examination of Food: Determination of Total Bacterial Count. Organic acid content was determined by high-performance liquid chromatography (HPLC). The texture of fermented foods was determined using a texture analyzer. The physicochemical indicators of wine, including total acid (calculated as tartaric acid), volatile acid (calculated as acetic acid), total sugar (calculated as glucose), alcohol content, total SO2, free SO2, and pH value, were determined according to the method in GB / T15038-2006 General Analytical Methods for Wines and Fruit Wines. The physicochemical indicators of apple cider vinegar were determined according to GB / T 30884-2014 Apple Cider Vinegar Beverages. Volatile flavor compounds were determined using gas chromatography-mass spectrometry (GC-MS).

[0102] Experimental materials

[0103] The sourdough dough was purchased from Dezhou City, Shandong Province.

[0104] Wickham yeast YF1503, accession number CGMCC No.1416, was purchased from the China General Microbiological Culture Collection Center.

[0105] Example 1: Isolation and identification of abnormal Wickham yeast W23087

[0106] 1. Separation and purification

[0107] Weigh 5.0 g of sourdough sample and add it to 45.0 mL of sterile physiological saline (0.85%, w / v). Vortex until the sample is uniformly suspended. Take 1.0 mL of the suspension and perform a 10-fold serial dilution to 10. -1 ~10 -7 Using a sterilized pipette tip, 100.0 μL of the diluted solution was spread onto YPD agar plates and incubated at 28.0°C for 48.0 h in a shaker. Colony morphology was carefully observed, and single colonies with good growth were selected. These single colonies were inoculated into liquid YPD medium and incubated at 28.0°C for 24.0 h. They were then streaked onto solid YPD medium to observe colony purity. This process was repeated three times until no contaminants were detected under a microscope. Strains with obvious spores were preliminarily identified as yeast by microscopic examination. The final single colonies were incubated in liquid YPD medium for 48 h, then aliquoted and stored in sterilized 25.0% v / v glycerol protectant at -80.0°C.

[0108] 2. Morphological characteristics of abnormal Wickham yeast W23087

[0109] The strain was streaked on YPD solid plates and incubated at 28.0℃ for 24.0 h. The colony morphology of the aroma-producing yeast strain was observed and photographed. When observing its cell morphology under a microscope, 1.0 mL of bacterial suspension was appropriately diluted, and one drop was placed on a glass slide and examined under a microscope stage. The strain was preliminarily classified based on its colony and cell morphology.

[0110] On YPD plates, after incubation at 28.0℃ for 24.0 h, the colonies of the aroma-producing yeast W23087 were milky white, 1.0–2.0 mm in diameter, spherical, with protrusions, smooth surface, neat edges, and opaque. Observation under an optical microscope, as shown in Figure 1, revealed that the cells of this strain were spherical or ellipsoidal, surrounded by obvious conidia, clearly conforming to the typical characteristics of yeast. Therefore, W23087 was preliminarily identified as a yeast.

[0111] 3. Strain identification

[0112] Yeast genomic DNA was extracted using a DNA extraction kit (purchased from Qingdao Haibo Biotechnology, model HBIG11), and its OD value (A260) and concentration were measured. Samples with qualified purity were diluted to about 100 ng / μL for PCR amplification.

[0113] After 1% agarose gel electrophoresis, the amplified product was purified using a product purification kit and sent to the company for sequencing. The sequencing results were compared with the NCBI database for homology analysis to find the sequence of the most homologous known species. Sequences with 99% or higher homology could be directly identified to the species level. By constructing a phylogenetic tree, the yeast was identified as *Wickham's abnormal* and named *Wickham's abnormal* W23087.

[0114] 4. Determination of total ester content in *Wickham's abnormal yeast* W23087

[0115] The activated bacterial culture was inoculated at a rate of 2.0% v / v into 10 mL of YPD liquid medium (bacterial concentration of 10). 5 The culture was incubated at 28.0℃ for 12.0 h. The total ester content was determined using the saponification reflux method. 1.0 mL of the supernatant was collected by centrifugation of the bacterial culture, diluted with water to a suitable concentration, and phenolphthalein was added. The solution was titrated with 0.1 mol / L NaOH until a faint red color appeared. An excess of 0.1 mol / L NaOH solution was then added, and the mixture was refluxed in a boiling water bath for 30 min. After cooling to room temperature, the solution was immediately titrated with 0.1 mol / L HCl until the red color just disappeared. The volume of HCl consumed was recorded, and each group was tested in triplicate. The total ester content of *Saccharomyces cerevisiae* W23087 cultured for 12.0 h was determined to be (19.25 ± 0.35) g / L.

[0116] 5. Strain preservation

[0117] The *Wickerhamomyces* strain W23087 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29669, located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The *Wickerhamomyces* ITS rDNA sequence provided by this invention is shown in SEQ ID NO. 1.

[0118] Example 2: Growth characteristics of abnormal Wickham yeast W23087

[0119] 1. Growth curve

[0120] The *Wickham's abnormal* strain W23087, preserved in glycerol as described in Example 1, was activated by inoculating it onto YPD solid medium. Single colonies were then picked and inoculated into 25.0 ml of liquid YPD medium for further culture. Finally, a 2.0% inoculum (v / v) was added to 300.0 ml of YPD broth and incubated statically at 28°C for 48.0 h. Three biological replicates were established. pH was measured every 2.0 h, and colony counts were calculated using serial dilutions. The results are shown in Figure 2. *Wickham's abnormal* strain W23087 entered the logarithmic growth phase from 20.0 h to 40.0 h, with an OD... 620 The value increases rapidly, then enters a stable phase, with the viable count reaching 10. 9 CFU / mL and maintained dynamic equilibrium over a long period of time.

[0121] 2. Temperature curve

[0122] Activated *Wickham's abnormal* yeast W23087 was inoculated into YPD liquid medium at a ratio of 2.0% (v / v) and cultured at constant temperatures of 20.0℃, 24.0℃, 28.0℃, 32.0℃, and 36.0℃ for 12.0 h. The OD620 value of the culture medium after fermentation was measured using blank YPD liquid medium as a control. The results showed that *Wickham's abnormal* yeast W23087 could grow in all temperature ranges, with high OD values ​​in the 24.0℃–32.0℃ range, indicating good growth of the strain within this temperature range. Its optimal growth temperature was 28℃.

[0123] 3. Fermentation capacity

[0124] Take 0.25g of yeast, soak it in 5.0mL of warm water for 30.0min, add 25.0g of high-gluten flour, 1.0% w / w of abnormal Wickham yeast W23087, 2.0% w / w of sugar, 1.0% w / w of sodium chloride, and 50.0% w / w of water at 40℃. Pour the mixture into a 250mL Erlenmeyer flask, cover with plastic wrap and poke a hole, weigh it, and place it in a 35℃ proofing chamber for static cultivation. Start timing and weigh it every 20.0min. The difference between the previous and next weight is the weight of CO2 lost in 20.0min. This experiment measured the CO2 loss from 0h to 5.0h. The greater the CO2 loss, the stronger the fermentation power of the baking powder. The results are shown in Figure 3. The CO2 overflow increased linearly from 0 to 4h, and then leveled off after 4h, with a maximum overflow of 0.63g.

[0125] Example 3: Determination of the saccharification power of abnormal Wickham yeast W23087

[0126] 1. Preparation of Daqu (a type of starter culture)

[0127] The activated *Saccharomyces cerevisiae* W23087 was inoculated into YPD liquid medium at a ratio of 2.0% (v / v) and cultured at 28°C for 48 hours to achieve a bacterial concentration of 10. 8 CFU / mL is prepared for use. 10g of wheat is added to 2.0mL of the prepared wheat and incubated in an incubator at 28℃ and 80% humidity for 3 days. After drying at 40℃, it becomes Daqu (a type of starter culture) for later use.

[0128] 2. Preparation of crude enzyme solution

[0129] Take 20.0 mL of the Daqu prepared above, add it to 40 mL of 0.1 mol / L (pH 4.5) acetate-sodium acetate buffer solution, and let it stand overnight at 4.0℃. Then filter the mixture with 4 layers of gauze, centrifuge the filtrate at 5000 r / min for 10.0 min, and the supernatant is the crude enzyme solution sample of abnormal Wickham yeast W23087.

[0130] 3. Determination of saccharification power

[0131] Referring to and modifying QB / T4257—2011 "General Analytical Methods for Brewing Daqu", the following steps were performed: Sodium acetate-acetic acid buffer solution, 1.0 mL of 0.5% soluble starch solution, and preheating in a 40℃ water bath for 10.0 min were added sequentially. Then, 5.0 mL of appropriately diluted crude Daqu enzyme solution was added, and the mixture was incubated in a water bath for 5.0 min. Finally, 1.0 mL of 0.1 mol / L H2SO4 was added to terminate the reaction. After the reaction was terminated, 2.0 mL of the reaction solution was mixed with 0.5 mL of 0.5% dilute iodine solution for color development, and the OD was measured. 620 Value, used to calculate enzyme activity.

[0132] The results showed that the saccharification power of the abnormal Wickham yeast W23087 was 821.8 U.

[0133] Example 4: Analysis of the aroma-producing characteristics of abnormal Wickham yeast W23087

[0134] 1. Ester production curve

[0135] Activated *Saccharomyces cerevisiae* W23087 was inoculated at a rate of 2.0% (v / v) into 10.0 mL of YPD liquid medium (inotropic concentration of 10). 5 The total ester content was measured by incubating at 28.0℃ for 18 hours (CFU / mL) and taking samples every two hours.

[0136] The total ester content was determined using the saponification reflux method. 1.0 mL of the supernatant from the fermentation broth was collected by centrifugation of the bacterial culture, diluted with water to a suitable concentration, and an appropriate amount of phenolphthalein was added. The solution was then titrated with 0.1 mol / L NaOH until a faint red color appeared. An excess of 0.1 mol / L NaOH solution was added, and the mixture was saponified and refluxed in a boiling water bath for 30 min. After cooling to room temperature, the solution was immediately titrated with 0.1 mol / L HCl solution until the red color just disappeared. The volume of HCl consumed was recorded, and each group was tested in triplicate.

[0137] As shown in Figure 4, the ester production of abnormal Wickham yeast W23087 began to level off at 6.0 h, and the maximum ester production at 12 h was (19.25±0.35) g / L, which is higher than the ester production disclosed in the prior art (CN109266562B; ester production 17.31 g / L).

[0138] 2. Analysis of volatile flavor compounds

[0139] Sample preparation

[0140] The activated abnormal Wickham yeast W23087 was inoculated at a rate of 2.0% (v / v) into 10.0 mL of YPD liquid medium and cultured at 28.0 °C for 48 h to obtain the fermentation broth.

[0141] Take 5.0 mL of the fermentation broth supernatant into a headspace vial, tighten the cap, and mix thoroughly. Use a 50 / 30 / μm DVB / CAR / PDMS extraction head to extract volatile flavor compounds from the sample. The extraction time is 40.0 min, the extraction temperature is 60.0℃, and the thermal desorption time is 5.0 min. Then perform GC-MS analysis.

[0142] The results showed that there were 46 volatile flavor compounds in the *Saccharomyces cerevisiae* W23087 culture, including 14 alcohols, 12 esters, 5 acids, 5 ketones, 4 hydrocarbons, 2 phenols, 2 pyrazines, and 2 pyrroles. The main flavor compounds were alcohols and esters, with their total peak area accounting for more than 90% of the total flavor compounds, meaning that the total relative content of alcohols and esters was as high as 90%. The contents of ethyl acetate were 230.0 μg / L, isoamyl acetate was 94.0 μg / L, phenylethanol was 50.5 μg / L, ethanol was 26.8 μg / L, and acetic acid was 13.87 μg / L.

[0143] 3. Electronic nose analysis of its fragrance characteristics

[0144] The chromatographic program was as follows: the initial temperature was 60℃, increased to 80℃ at a rate of 1.0℃ / s, then increased to 250℃ at a rate of 2.0℃ / s and held for 60s. Helium was used as the carrier gas at a flow rate of 1.0 mL / min, and the detector temperature was 260℃.

[0145] The odor radar diagram of abnormal Wickham yeast W23087 is shown in Figure 5 and Table 1.

[0146] Table 1. Types of Sensitive Substances Corresponding to Sensors

[0147] The results show that Table 1 lists the types of substances sensitive to the 12 sensors. Combined with Figure 5, it can be seen that sensors T30 / 1, P10 / 1, P10 / 2, P40 / 1, T70 / 2, and PA / 2 are sensitive to substances such as polar substances, non-polar substances (hydrocarbons, ammonia, chlorine), aromatic substances (toluene, xylene), alcohols, amines, and chlorine compounds, indicating significant differences in the samples regarding these types of substances. The remaining sensors are sensitive to nitrogen oxides, sulfides, acetone, propane, and butane, indicating no significant differences in the samples regarding these types of substances.

[0148] Example 5: Preparation of *Saccharomyces cerevisiae* W23087 inoculum

[0149] 1. Enrichment culture

[0150] Two loops of activated abnormal Wickham yeast W23087 were inoculated into YPD medium and cultured at 28℃ and 200.0 rpm for 20.0 h. 100.0 μL of the culture was then transferred into YPD fermentation medium and cultured again at 28℃ and 200 g for 48.0 h.

[0151] 2. Centrifugal washing

[0152] The fermentation broth of abnormal Wickham yeast W23087 obtained from enrichment culture was poured into a centrifuge tube and centrifuged at 3000 r / min for 15.0 min. After discarding the supernatant, the mixture was washed twice with sterile water. The resulting precipitate was washed off with sterile distilled water and a yeast suspension was prepared for later use.

[0153] 3. Add freeze-drying protectant

[0154] Trehalose was selected as a protective agent. A 10% v / v trehalose solution was first prepared and sterilized at 121℃ for 20.0 min. Then it was mixed with an equal volume of yeast suspension.

[0155] 4. Freeze-drying

[0156] After thoroughly mixing the yeast suspension with added trehalose preservative, pour it into agar plates. Seal the aliquoted plates with plastic wrap and quickly pre-freeze them in an ultra-low temperature freezer at -80°C for 3.0 hours. Immediately transfer the pre-frozen plates to a freeze dryer for freeze-drying. Once freeze-dried, quickly transfer the plates to a clean bench and seal them with plastic wrap and sealing film. Store the freeze-dried powder at -20°C. The viable count of abnormal Wickham yeast W23087 in the freeze-dried powder should not be less than 1×10⁻⁶. 8 CFU / mL.

[0157] Example 6: Preparation of Wheat Bran Koji from Abnormal Wickham Yeast W23087

[0158] Take abnormal Wickham yeast W23087 bacterial culture and adjust the concentration to 1×10⁻⁶. 6 CFU / mL, used for bran koji culture.

[0159] (1) Weigh 20g of wheat bran, add 20mL of distilled water and stir evenly. Shake the Erlenmeyer flask to mix the material and water evenly. Sterilize at 121℃ for 40min. After cooling in a sterile room and shaking to break up the material blocks, inoculate 5% v / v abnormal Wickham yeast W23087 into the shake flask.

[0160] (2) After inoculation, shake each bottle to mix evenly, and incubate at a constant temperature of 28.0℃ in an incubator. After mycelium grows in about 18 hours, shake the bottle for the first time to loosen the clumps. After shaking evenly, spread the mixture evenly on the bottom of the bottle and continue incubation.

[0161] (3) After culturing for another 5 hours until the material forms a noticeable cake, invert the bottles for 72 hours to ensure uniform growth of the bacteria in the upper and lower parts. When inverting the bottles, handle them gently to prevent the cake from breaking.

[0162] (4) After the bottle is covered, continue to incubate for about 36 hours. Place the conical flask horizontally to facilitate water storage. Continue to incubate at a constant temperature of 28℃. Shake the flask regularly to ensure that the part of the cake close to the bottle wall receives sufficient oxygen to facilitate the growth and reproduction of the strain.

[0163] (5) After the culture is completed, crush the koji cake under sterile conditions, then divide it into sealed bags that have been sterilized at 121℃ for 30 minutes, seal the bags, add the date of koji production, and store them in a refrigerator at 4℃ for later use.

[0164] The yeast spores prepared using the above method were on the order of 10. 15 Pure bran koji with CFU / g.

[0165] Example 7: Application of Abnormal Wickham Yeast W23087 in Fermented Bread

[0166] Dough fermentation and bread preparation method: High-gluten flour, sucrose, sodium chloride, and drinking water were used as the main raw materials, mixed in a ratio of 10:0.6:0.2:0.6. 0.25g of the abnormal Wickham yeast W23087 inoculum prepared in Example 5 and 0.25g of commercial leavening agent (Angel Yeast) were soaked in warm water for 30.0 min. After forming the dough, it was placed in a proofing box at 35.0℃ and incubated for 3.0 h. After fermentation, the dough was baked in an oven at 190.0℃ (top heat) and 200.0℃ (bottom heat) for 25.0 min. After baking, the bread was equilibrated at 25.0℃, sealed, and stored for further analysis.

[0167] An equal amount of abnormal Wickham yeast YF1503 (accession number CGMCC No. 1416) and a commercial starter culture (Angel Yeast) were added to the experimental group as the control group (CK). Fermentation power was determined using the CO2 loss meter method as in Example 2; bread texture properties were determined using a texture analyzer; and the determination of volatile flavor compounds in the bread was performed as in Example 4.

[0168] The physicochemical indicators of the experimental group were normal. The fermentation power of the experimental group was 2.2g, while that of the control group was 1.6g, indicating that the abnormal Wickham yeast W23087 has a strong fermentation power. The determination of volatile flavor substances showed that the main flavor substances were alcohols and esters. The contents of ethyl acetate and phenylethanol were 2.33μg / L and 2.15μg / L, respectively, while the contents of isoamyl acetate and ethanol were 0.24μg / L and 0.15μg / L, respectively. Ethyl acetate and phenylethanol contributed significantly to the overall flavor of the bread, with a relative content of over 91.9%. Compared with the ethyl acetate content of 0.76μg / L and the phenylethanol content of 0.74μg / L in the control group, the contents increased by more than 190%.

[0169] Example 8: Application of Abnormal Wickham Yeast W23087 in Fermented Steamed Buns

[0170] Take 100.0g of wheat flour, 1.0g of the abnormal Wickham yeast W23087 inoculum prepared in Example 5, 1.0g of commercially available starter culture (Angel Yeast), and 50.0mL of water in a dough mixer. Mix slowly for 3 minutes and then mix quickly for 3 minutes. Divide the dough into portions, knead until smooth, and place them in a proofing box at 38.0℃ and 85.0% relative humidity for 60.0 minutes. Steam for 20.0 minutes, and after turning off the heat, let them sit in the pot for 5.0 minutes. Remove the steamed buns and let them sit at room temperature for 1.0 hour to obtain the test samples. Use abnormal Wickham yeast YF1503 (its preservation number is CGMCCNo.1416) and commercial starter culture (Angel Yeast) as control groups.

[0171] Fermentation power was determined using the CO2 loss-in-weight method as described in Example 2; the textural properties of the steamed buns were determined using a texture analyzer; and the determination of volatile flavor compounds in the steamed buns was performed as described in Example 4. The textural results are shown in Table 2. Compared to the control group, the abnormal Wickham yeast W23087 exhibited stronger sugar utilization, producing 35.0% more gas during fermentation, causing the dough to expand. This resulted in lower hardness, higher specific volume, higher cohesion, and lower chewiness, leading to a more suitable texture.

[0172] The volatile substances in the dough were tested according to the method in Example 4. The results showed that a total of 48 volatile compounds were found, including 13 alcohols, 15 aldehydes, 7 ketones, 6 acids, 6 esters, and 1 other type of compound. The content of ethyl acetate was 1.98 μg / L, the content of isoamyl acetate was 1.43 μg / L, and the content of ethanol was 0.21 μg / L. The total content of volatile substances was 82.3% higher than that of the control group, so the steamed buns had a pleasant aroma.

[0173] Table 2 Texture characteristics of steamed buns fermented with abnormal Wickham yeast W23087

[0174] Example 9: Application of Abnormal Wickham Yeast W23087 in Small-Scale Liquor

[0175] Soak the washed rice in 80.0℃ warm water for 2.0 hours, so that the rice has a moisture content of 45.0% after soaking. Then steam it for the first time. After the steam rises, pour the soaked rice into the steamer, cover and steam for 30.0 minutes to allow the grains to expand and absorb water further. After the first steaming, submerge the rice in water at 95℃ and steam it for 2.0 hours to allow the grains to absorb water further, ensuring that the grains have no white center. Steam the rice again over high heat until steam rises above the grains, then cover and steam for 60.0 minutes. The grains should have a moisture content of 110%, be soft and tender, and feel soft to the touch. Break up the grains and cool them to about 30℃. Add 1.0% w / w of the abnormal Wickham yeast W23087 bran koji prepared in Example 6 and mix well. Incubate at 30.0℃ for saccharification for 24.0 hours until a sweet aroma is detected. First, place a layer of mixing trough at the bottom of the fermentation jar. Pour the saccharified sweet mash into the jar and press it down. Then, cover the sweet mash with the mixing trough and press it down again. Seal the fermentation jar with a plastic bag and place it in a 30℃ incubator for 7.0 days of fermentation. Add a certain amount of tap water to the distillation pot and heat it until steam rises. Then, put the fermented mash into the distillation pot, making sure the mash is evenly and loosely distributed. Adjust the heat and the cooling water speed to ensure that the distillation temperature is not too high. 1.0 kg of mash distills 150.0 mL of liquor. Use abnormal Wickham yeast YF1503 (its preservation number is CGMCC No. 1416) as a control, and follow the same procedure. Measure the alcohol content with an alcohol meter and convert it to the alcohol content at 20℃. The determination of total ester content is the same as in Example 1. The determination of volatile flavor compounds is the same as in Example 4.

[0176] The results are shown in Table 3. The physicochemical indicators of the abnormal Wickham yeast W23087 were normal. Compared with the control group, it had stronger high temperature resistance and ethanol fermentation ability, and the content of total esters and ethyl acetate was higher, by 36.8% and 86.9% respectively. The content of isoamyl acetate and phenylethanol was higher than that of the control group by 85% and 15% respectively. The higher proportion of ethyl acetate will make the aroma more harmonious and the taste better.

[0177] Table 3. Abnormal indicators of Wickham yeast W23087 fermented liquor.

[0178] Example 10: Application of Wickham's abnormal yeast W23087 in fermented wine

[0179] Fully ripe grapes were selected, crushed, and placed into a container, ensuring the container was no more than 3 / 4 full. In Example 5, *Saccharomyces cerevisiae* W23087 and a commercially available starter culture (Angel Yeast) were prepared, both inoculated at 5.0% w / w. Sulfur dioxide was added at 30.0 g / L, and pectinase at 0.02 g / L. Cold maceration was performed for 48.0 h. *Saccharomyces cerevisiae* YF1503 (CGMCC No. 1416) and the commercial starter culture served as control groups (CK). Sugar content and specific gravity were measured daily during fermentation, and the caps were pressed three times. Fermentation ended when the sugar content dropped to 4.0 g / L and the specific gravity to 0.8 g / mL. The juice was then clarified and bottled for storage. The physicochemical indicators of wine, including total acid (calculated as tartaric acid), volatile acid (calculated as acetic acid), total sugar (calculated as glucose), alcohol content, total SO2, free SO2 and pH value, were determined using the standard GB / T15038-2006 General Analytical Methods for Wine and Fruit Wine. The determination of volatile flavor substances was performed in the same manner as in Example 4.

[0180] The total sugar content of the wine made with *Aberrant Wickham yeast* W23087 was 23% lower than that of the control wine, indicating that *Aberrant Wickham yeast* W23087 has a higher sugar utilization rate and more thorough fermentation. The ethanol volume fraction in the fermentation broth was 7.2% higher than that of the control group, indicating good alcohol tolerance. The total acid and volatile acid content were 15% and 21% higher than those of the control group, respectively, giving *Aberrant Wickham yeast* W23087 a refreshing taste. Other physicochemical indicators were normal.

[0181] Among the volatile flavor compounds, there were 11 alcohols, 13 esters, and 5 other types of aroma compounds. The content of ethyl acetate was 0.23 μg / L, isoamyl acetate was 0.21 μg / L, and ethanol was 0.20 μg / L. In comparison, the content of ethyl acetate was 0.18 μg / L, isoamyl acetate was 0.16 μg / L, and ethanol was 0.18 μg / L in the control group. The total amount of volatile flavor compounds was 23% higher than that in the control group, and the fruity aroma was more intense.

[0182] Example 11: Application of Abnormal Wickham Saccharomyces W23087 in Fermented Apple Cider Vinegar

[0183] Fresh apples were washed, cut into chunks, and mixed with the same mass of pH 4.0 citric acid solution and 0.1% v / v pectinase. After soaking for 10.0 min, the apples were crushed into a puree. Enzymatic hydrolysis was performed at 40℃ for 2 h, followed by enzyme inactivation at 75℃ for 10.0 min. After cooling, the juice was extracted and filtered for later use. The initial sugar content of the puree was adjusted to 15°Brix. Alcoholic fermentation was carried out for 48.0 h using 2% w / w *Saccharomyces cerevisiae* W23087 prepared in Example 5 and a commercially available starter culture. After alcoholic fermentation, acetic acid fermentation was performed using acetic acid bacteria to obtain the acetic acid fermentation broth. The broth was centrifuged for 10 min, the supernatant was collected, and pasteurized for 20.0 min to obtain the finished apple cider vinegar. *Saccharomyces cerevisiae* YF1503 (accession number CGMCC No. 1416) and the commercial starter culture served as control groups (CK). The physicochemical properties of the abnormal Wickham yeast W23087 apple cider vinegar were determined according to GB / T 30884-2014 Apple Cider Vinegar Beverage.

[0184] The results showed that the physicochemical indicators were normal. Among them, phenylacetaldehyde accounted for 21.2% of the total volatile flavor substances (floral aroma), ethyl isovalerate accounted for 20.1% (fruity aroma), phenethyl acetate accounted for 10.9% (fruity aroma), and phenylethanol accounted for 9.6% (floral aroma). The content of these four flavor substances was significantly higher than that of the control group (35.1% to 42.4%), which made the apple cider vinegar have a rich fruity aroma and a sweet and sour taste.

[0185] Example 12: Application of Abnormal Wickham Yeast W23087 in Fermented Sweet Bean Sauce

[0186] Steamed flour was inoculated with 2.0% w / w of the abnormal Wickham yeast W23087 prepared in Example 5 and a commercially available starter culture. After 2.0 days of koji making in a ventilated environment, 10.0% salt water was added at a salt water to flour mass ratio of 1:1. After stirring evenly, the mixture was sealed and fermented for 5.0 days in an incubator at 20°C. After the fermentation, the mixture was placed in an incubator at 35.0°C for a second fermentation of 20.0 days to obtain the finished sweet bean sauce. The abnormal Wickham yeast YF1503 (its preservation number is CGMCC No. 1416) and the commercial starter culture were used as control groups (CK).

[0187] Determination of organic acids: Column: Agilent XDB-C18 (5μ, 250×4.6mm), mobile phase: 100% acetonitrile, 1.0% phosphoric acid aqueous solution (pH2.8), flow rate: 1.0mL / min, injection volume: 10.0μL, UV detection wavelength: 210.0nm, column temperature: 35.0℃.

[0188] The determination of volatile flavor compounds was the same as in Example 4. The results showed that the acetic acid content in the *Saccharomyces cerevisiae* W23087 sweet bean sauce was 27.0% higher than that in the control group. Acetic acid is derived from the oxidation of ethanol and plays a role in harmonizing the taste and flavor. A total of 47 volatile flavor compounds were detected in the *Saccharomyces cerevisiae* W23087 sweet bean sauce matrix, including 15 esters, 11 alcohols, 9 aldehydes, 6 acids, 2 phenols, 1 ketone, 1 hydrocarbon, 1 pyrazine, and 1 pyrrole. The ethyl acetate content was 1.41 μg / L, the isoamyl acetate content was 1.12 μg / L, the phenylethanol content was 0.45 μg / L, and the acetic acid content was 0.37 μg / L. The total content was 24.5% higher than that in the control group, which made the sweet bean sauce sweet and mellow.

[0189] Example 13: Application of Abnormal Wickham Yeast W23087 Bran Koji in Soy Sauce Fermentation

[0190] The abnormal Wickham yeast W23087 bran koji was prepared using the method described in Example 6. Soy sauce brewing employed a solid-state fermentation process, the flow of which is as follows: Soybeans were soaked in warm water at a 1:2 ratio for 5.0 hours, then steamed. After cooling, the abnormal Wickham yeast W23087 bran koji prepared in Example 6 was inoculated at a ratio of 5.0%, followed by approximately 1.5 times the material mass of brine, resulting in a final mash moisture content of approximately 50.0%, a salt content of approximately 5% w / v, and a pH of approximately 7.0. Fermentation of the mash: The initial fermentation temperature was controlled at approximately 40℃, lasting approximately 10 days. After the initial fermentation, an appropriate amount of concentrated brine was added to bring the salt content of the mash to approximately 15.0%, while maintaining the mash temperature at 30–32℃ for continued fermentation for approximately 20 days. After fermentation, the mash underwent subsequent processing including rinsing, pressing, removal of mash, filtration, and sterilization. The experimental group consisted of fermented wheat bran yeast of abnormal Wickham yeast W23087; the control group was a sample without inoculation.

[0191] The results showed that the ethyl acetate content in the abnormal Wickham yeast W23087 fermentation group was 0.31 μg / L, the isoamyl acetate content was 0.24 μg / L, the phenylethanol content was 0.18 μg / L, and the acetic acid content was 0.16 μg / L, with the total content being 24.5% higher than that of the control group, which made the soy sauce flavor richer.

[0192] Example 14: Improving nutrient conversion rate of abnormal Wickham yeast W23087 bran koji in feed fermentation

[0193] The fermentation process for biological feed is as follows: Rice bran, straw, and soybean meal are mixed evenly in a ratio of 1:1:2 and crushed to form a fermentable material. Water is added at a material-to-water ratio of 1:0.5, and abnormal Wickham yeast W23087 bran koji prepared in Example 5 is inoculated at a ratio of 10% w / w. The mixture is stirred evenly and allowed to ferment naturally at a temperature of 35.0℃ for 8 days. After fermentation, the mixture is dried until the moisture content is below 15.0%, thus obtaining the biologically fermented feed.

[0194] Analysis of the fermented feed quality revealed that it possessed a distinctive aroma and was rich in nutrients with a balanced amino acid profile. Compared to the control group, the content of organic acids increased by 27.87%, amino acid content by 31.04%, and crude protein content by 36.13%. The phenylethanol content was 0.31 μg / L, the ethanol content was 0.28 μg / L, the acetic acid content was 0.15 μg / L, and the total volatile flavor compounds were 23.4% higher than the control group.

[0195] Example 15: Isolation and identification of Lactobacillus plantarum L23104

[0196] 1. Separation and purification

[0197] Weigh 5.0 g of the sourdough sample and add it to 45.0 mL of sterile physiological saline (0.85%, m / v). Vortex until the sample is completely and uniformly suspended. Take 1.0 mL of the suspension and perform a 10-fold serial dilution to 10. -1 ~10 -7 Using a sterile pipette tip, 100.0 μL of the dilution solution was spread onto MRS agar plates and incubated at 37.0°C for 24.0 h in an anaerobic incubator. Colony morphology was carefully observed, and single colonies with good growth and yellowing edges were selected. These single colonies were inoculated into liquid MRS medium and incubated at 37.0°C for 24.0 h. They were then streaked onto solid MRS medium to observe colony purity. This process was repeated three times until no contamination was observed under a microscope. The purified colonies were subjected to catalase and Gram staining. Catalase-negative, Gram-positive strains were preliminarily identified as lactic acid bacteria. The final single colonies were incubated in MRS liquid medium for 48.0 h, then aliquoted and stored in sterile 25.0% glycerol protectant at –80°C.

[0198] 2. Morphological characteristics of Lactobacillus plantarum L23104

[0199] The strain was streaked on MRS agar plates and incubated at 37.0℃ for 24.0 h. Colony morphology was observed. Single colonies were picked, Gram-stained, and their cell morphology was observed under a microscope. The colonies of this strain were raised, milky white, round, smooth, translucent, and had regular edges; the cells were rod-shaped and Gram-positive. (See Figure 6.)

[0200] 3. Physiological characteristics of Lactobacillus plantarum L23104

[0201] The results of the tests using a lactic acid bacteria biochemical identification kit are shown in Table 4.

[0202] Table 4. Physiological and biochemical characteristics of Lactobacillus plantarum L23104 Note: "+" indicates that the strain reacted positively; "-" indicates that the strain reacted negatively.

[0203] 4. Strain identification

[0204] Genomic DNA of lactic acid bacteria was extracted using a DNA extraction kit, and its OD value (A260) and concentration were measured. Samples with qualified purity were diluted to about 100 ng / μL for PCR amplification.

[0205] After 1% agarose gel electrophoresis, the amplified product was purified using a product purification kit and sent to the company for sequencing. The sequencing results were compared with the NCBI database for homology analysis to find the sequence of the most homologous known species. Strains with homology of 99.0% or higher could be directly identified to the species level. By constructing a phylogenetic tree (Figure 7), the lactobacillus was identified as *Lactobacillus plantarum* and named *Lactobacillus plantarum* L23104.

[0206] 5. Fiber-drawing test of Lactobacillus plantarum L23104

[0207] Place one drop of freshly prepared 4.0% KOH aqueous solution on a clean glass slide. Using an inoculation loop, take a small amount of the freshly prepared colony from the solid culture medium and mix it in the KOH solution. Every few seconds, lift the inoculation loop and observe whether it can be drawn into threads. Repeat this process for 4-5 colonies. A positive result is indicated by the ability to draw threads from the colony with the inoculation loop; a negative result is indicated by the colony remaining in a suspension. Measure the maximum length of the drawn threads (cm). The results are expressed as averages. Initial screening yields lactic acid bacteria with thread-drawing characteristics. The results show that L23104 has the longest thread-drawing ability. The thread-drawing results are shown below.

[0208] 6. Preservation of bacterial strains

[0209] Lactobacillus plantarum strain L23104 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29668, located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The 16S rDNA sequence of Lactobacillus plantarum provided in this invention is shown in SEQ ID NO. 2.

[0210] Example 16: Extraction and determination of extracellular polysaccharides from Lactobacillus plantarum L23104

[0211] The EPS-producing strains obtained from the initial screening were activated for two generations and inoculated into 150.0 mL of 2% v / v mMRS (MRS with 5.0% sucrose added) liquid medium (bacterial concentration of 10). 5 The precipitate (CFU / mL) was incubated at 37.0℃ for 24.0 h. The tubes were then placed in a water bath and boiled for 10.0 min, then cooled to room temperature. The precipitate was removed by centrifugation at 12000g for 30.0 min at 4.0℃. 80.0% (m / v) trichloroacetic acid (TCA) solution was added to the supernatant to a final concentration of 4.0%, and the mixture was incubated overnight at 4.0℃. The precipitated protein was removed by centrifugation at 12000g for 30.0 min at 4.0℃. 95.0% (w / v) pre-cooled ethanol was added to the supernatant, and the mixture was allowed to stand overnight. The mixture was then centrifuged at 12000g for 30.0 min at 4.0℃, and this process was repeated twice. The precipitate was redissolved in deionized water. After complete dissolution, the mixture was dialyzed at 4.0℃ using a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 48.0 h, with water changed every 8.0 h. After dialysis, the precipitate was freeze-dried. Extracellular polysaccharides were determined using the phenol-sulfuric acid method.

[0212] The results showed that after 24 hours of fermentation, the extracellular polysaccharide content produced by Lactobacillus plantarum L23104 was 2460.12±1.46 mg / L, which can act as a stabilizer to enhance the stability of fermented products and improve their texture and taste.

[0213] Example 17: Preparation of a high-yield mixed microbial inoculant for extracellular polysaccharides

[0214] 1. Enrichment Culture. Two loops of activated *Wickham's abnormal* yeast W23087 were inoculated into YPD medium and cultured at 28.0℃ and 200.0 rpm for 48.0 h. 100.0 μL of the culture was then transferred to YPD fermentation medium and cultured for enrichment at 28.0℃ and 200.0 rpm for 48.0 h. Two loops of activated *Lactobacillus plantarum* L23104 were inoculated into MRS medium and cultured at 37.0℃ for 24.0 h. 100.0 μL of the culture was then transferred to MRS fermentation medium and cultured at 37.0℃ for 24.0 h.

[0215] 2. Centrifugation and washing. The fermentation broths of *Saccharomyces cerevisiae* W23087 and *Lactobacillus plantarum* L23104 obtained from the enrichment culture were poured into centrifuge tubes and centrifuged at 3000 rpm for 15.0 min. After discarding the supernatant, the broths were washed twice with sterile water. The resulting precipitates were resuspended in sterile distilled water to prepare *Lactobacillus plantarum* L23104 and *Saccharomyces cerevisiae* W23087 suspensions, respectively, for later use.

[0216] 3. Bacterial ratio. A suspension of *Lactobacillus plantarum* L23104 and a suspension of yeast W23087 were mixed at a ratio of 1:0.8 to obtain a mixed bacterial solution.

[0217] 4. Add freeze-drying protectant. Trehalose was selected as the protectant. A 10% v / v trehalose solution was prepared, sterilized at 121.0℃ for 20.0 min, and then mixed with an equal volume of the mixed bacterial solution.

[0218] 5. Freeze-drying. After thoroughly mixing the bacterial culture with added trehalose preservative, pour the mixture into agar plates. Seal the plates with plastic wrap and quickly pre-freeze them in an ultra-low temperature freezer at -80℃ for 3 hours. Immediately transfer the pre-frozen plates to a freeze dryer for freeze-drying. Once freeze-dried, quickly transfer the plates to a clean bench and seal them with plastic wrap and sealing film. Store the freeze-dried powder at -20℃. The total viable count in the freeze-dried powder should reach 10. 8 CFU / g.

[0219] Example 18: Performance Testing of Microbial Agents

[0220] 1. Polysaccharide yield detection

[0221] The bacterial agent prepared in Example 17 was inoculated into mMRS medium at 2% v / v to bring the total bacterial concentration in the medium to 10. 5 CFU / mL, cultured at 37.0℃ for 24.0 h. Single strains L23104 and W23087 at the same concentration were used as control groups (bacterial concentration 10). 5 The extracellular polysaccharide (CFU / mL) was prepared according to the method in Example 16, and the content of the extracellular polysaccharide was detected.

[0222] The results are shown in Table 5. The results indicate that the extracellular polysaccharide produced by the compound bacterial agent was 2713.33±2.71 mg / L, which was higher than the polysaccharide content of Lactobacillus plantarum L23104 (9.33%).

[0223] Table 5. Extracellular polysaccharide yield

[0224] 2. Aroma testing

[0225] The bacterial agent prepared in Example 17 was inoculated into YPD liquid medium at 2% v / v to bring the total bacterial concentration in the medium to 10. 5 CFU / mL, cultured at 28.0℃ for 16 h. Single strains L23104 and W23087 at the same concentration were used as control groups (bacterial concentration 10). 5 The amount of ester produced over 16 hours was determined according to the method described in Example 4 (CFU / mL).

[0226] The results are shown in Table 6. The results indicate that the ester production of the compound bacterial agent was 23.54±0.57 g / L, which was 18.22% higher than that of abnormal Wickham yeast W23087.

[0227] Table 6 Ester Production

[0228] Example 19: Application of compound microbial inoculants in fermented bread

[0229] Dough fermentation and bread preparation method: High-gluten flour, sucrose, sodium chloride, and drinking water were used as the main raw materials, mixed in a ratio of 10:0.6:0.2:0.6. 0.25g of a compound microbial agent (prepared in Example 5) and 0.25g of a commercial compound leavening agent were added, with a mass ratio of agent to flour of 1:100. The mixture was soaked in warm water for 30 minutes, formed into dough, and then placed in a proofing box at 35.0℃ for 3.0 hours. After fermentation, the dough was baked in an oven at 190.0℃ (top heat) and 200.0℃ (bottom heat) for 25.0 minutes. After baking, the bread was equilibrated at 25.0℃ and stored in an airtight container for further analysis. Control group 1 was supplemented with 0.25g of Lactobacillus plantarum L23104 and 0.25g of commercial compound starter culture; control group 2 was supplemented with 0.25g of Wickham yeast W23087 and 0.25g of commercial compound starter culture; and control group 3 was supplemented with 0.5g of commercial compound starter culture. The remaining methods were the same as those for the experimental groups.

[0230] The textural properties of bread were determined using a texture analyzer; the quality evaluation of bread was conducted according to the "GB / T 35869-2018 Grain and Oil Inspection: Rapid Baking Method for Evaluation of Wheat Flour Bread Baking Quality"; the content of extracellular polysaccharides in bread was determined according to Example 1; and the determination of volatile flavor compounds in bread was conducted according to Example 4.

[0231] The results are shown in Table 7. The experimental group showed normal physicochemical indicators, normal bread crust color, and a white, soft, and elastic bread core. The polysaccharide content in the bread fermented with the added compound microbial agent was more than 40% higher than that in the three control groups. Volatile flavor compound analysis showed that the main flavor compounds were alcohols and esters, exhibiting high aroma activity and contributing significantly to the overall flavor of the bread, with a relative content exceeding 90.0%. The contents of ethyl acetate (4.26 μg / L), phenylethanol (3.79 μg / L), propyl acetate (0.65 μg / L), and isoamyl alcohol (0.58 μg / L) were significantly higher than those in control group 3. In control group 3, the contents of ethyl acetate (1.07 μg / L), phenylethanol (0.68 μg / L), propyl acetate (0.16 μg / L), and isoamyl alcohol (0.10 μg / L) increased by 75.0%, and the contents of alcohols such as ethanol and isoamyl alcohol increased by 82.0%.

[0232] Table 7 Bread Testing Data

[0233] Example 20: Application of compound microbial inoculants in fermented steamed buns

[0234] Take 100.0g wheat flour, 0.5g compound microbial inoculant, 0.5g commercial compound fermentation agent, and 50.0mL water into a dough mixer. First, mix slowly for 3.0min, then mix quickly for 3.0min. Divide the dough into portions, knead until smooth, and place in a proofing box at 38.0℃ and 85.0% relative humidity for 50.0min. Steam for 20.0min, then turn off the heat and let it sit in the pot for 5.0min. Remove the steamed buns and let them sit at room temperature for 1.0h to obtain the test sample. The products were divided into three groups: control group 1 was supplemented with 0.5g of *Lactobacillus plantarum* L23104 and 0.5g of commercial compound starter; control group 2 was supplemented with 0.5g of *Wickham's yeast* W23087 and 0.5g of commercial compound starter; and control group 3 was supplemented with 1.0g of commercial compound starter. The remaining methods were the same as those for the experimental groups. The textural properties of the steamed buns were determined by a texture analyzer. The extracellular polysaccharide content in the steamed buns was determined according to Example 1. The determination of volatile flavor compounds in the steamed buns was carried out according to Example 4.

[0235] The results, as shown in Table 8, indicate that compared to the control group, the compound microbial inoculant demonstrated stronger sugar utilization, producing 35.0% more gas during fermentation, causing the dough to expand and resulting in a larger specific volume, lower chewiness, and a more suitable texture. The polysaccharide content in the steamed buns fermented with the compound microbial inoculant was more than 29.6% higher than that in the control group. A total of 52 volatile compounds were identified, including 16 alcohols, 11 aldehydes, 9 ketones, 9 esters, 6 acids, and 1 other type of compound. The total volatile flavor compound content was more than 27.7% higher than that of the three control groups, resulting in a richer aroma in the steamed buns.

[0236] Table 8. Steamed Bun Test Data

[0237] Example 21: Application of compound microbial inoculants in fermented rice wine

[0238] Washed, cooked, and cooled glutinous rice was poured into a glass bottle. Water and glutinous rice were mixed evenly according to a material-to-liquid ratio of 1:4. After pulping with a pulping machine, α-amylase, 0.5% w / w compound microbial agent, and 0.5% w / w commercial yeast were added. Fermentation was carried out in a constant temperature incubator at 28.0℃. After fermentation, the fermented raw wine was obtained by preliminary filtration. Control group 1 was supplemented with 0.5% w / w Lactobacillus plantarum L23104 and 0.5% w / w commercial yeast. Control group 2 was supplemented with 0.5% w / w Wickham yeast W23087 and 0.5% w / w commercial yeast. Control group 3 was supplemented with 1.0% w / w commercial yeast. The other methods were the same as those of the experimental group. Total acid, total ester, and alcohol content were determined according to GB / T 10781.3-2006 Rice Aroma Baijiu. Volatile flavor substances were determined according to Example 4.

[0239] The results, as shown in Table 9, indicate that the rice wine fermented with the compound microbial agent was clear and transparent with no suspended matter, and had an alcohol content of 26.0% vol. Control group 1 had 22.0% vol, control group 2 had 21.0% vol, and control group 3 had 24.0% vol. The rice wine fermented with the compound microbial agent had a high total content of flavor compounds. A total of 57 volatile substances were detected in the rice wine, including 17 esters, 15 alcohols, 10 acids, 5 aldehydes and ketones, and 7 other substances. The rice wine had a pure rice aroma and a mellow body.

[0240] Table 9 Rice Wine Testing Data

[0241] Example 22: Application of compound microbial agents in fermented wine

[0242] Fully ripe grapes were selected, crushed, and placed into containers, with the raw material not exceeding 3 / 4 of the container's capacity. The inoculum amount of both compound microbial inoculant and commercial compound fermentation agent was 2.5% w / w. Sulfur dioxide was added at 30.0 mg / L, and pectinase at 0.01–0.03 g / L. Cold maceration was performed for 24–48 hours, with a control group not using the compound microbial inoculant. Sugar content and specific gravity were measured daily during fermentation, and the cap was pressed three times. Fermentation ended when the sugar content dropped to 4.0 g / L and the specific gravity to 0.8 g / mL. The juice was then clarified and bottled. Physicochemical indicators such as total acid (calculated as tartaric acid), volatile acid (calculated as acetic acid), total sugar (calculated as glucose), alcohol content, total SO2, free SO2, and pH value in the wine were determined using GB / T15038-2006, General Analytical Methods for Wines and Fruit Wines. The determination of volatile flavor compounds followed the same procedure as in Example 4.

[0243] The results showed that the alcohol content of the wine produced by the compound microbial agent was 14.0% vol, while that of the control group was 12.0% vol. The total acid and volatile acid content were 15.0% and 21.0% higher than the control group, respectively, resulting in a refreshing taste. Other physicochemical indicators were normal. Among the volatile flavor compounds, there were 15 types of alcohols, 13 types of esters, and 5 other types of aroma compounds. The total ester content in the experimental group was 5.03 g / L, while that in the control group was 4.51 g / L, with the experimental group showing a 10.34% higher total ester content. The ethyl acetate content was 0.36 μg / L, compared to 0.28 μg / L in the control group, indicating a more intense fruity aroma in the experimental group.

[0244] Example 23: Application of compound microbial agents in fermented apple cider vinegar

[0245] Fresh apples were washed and cut into chunks. An equal amount of pH 4.0 citric acid solution and 0.1% w / w pectinase were added, and the mixture was soaked for 10.0 min, then mashed into apple puree. The puree was enzymatically hydrolyzed at 45.0℃ for 2.0 h, followed by enzyme inactivation at 80.0℃ for 10.0 min. After cooling, the juice was extracted and filtered for later use. The initial sugar content of the pulp was adjusted to 15.0°Brix, and the mixture was inoculated with 2.5% w / w compound microbial inoculant and 2.5% w / w commercial compound fermentation agent for alcoholic fermentation for 24.0 h.

[0246] After alcoholic fermentation, 2.5% w / w acetic acid bacteria (purchased from Taisto Biotechnology, catalog number TS349550) were inoculated for acetic acid fermentation to obtain acetic acid fermentation broth. The broth was centrifuged for 10.0 min using a high-efficiency centrifuge, and the supernatant was collected and pasteurized for 20.0 min to obtain the finished apple cider vinegar product. A commercial compound fermentation agent was used as a control group. The physicochemical properties of the apple cider vinegar produced by the compound microbial agent were determined according to GB / T 30884-2014 Apple Cider Vinegar Beverages.

[0247] The results showed that the compound microbial inoculant apple cider vinegar was clear in color, free of suspension, and had normal physicochemical indicators. The total ester content in the experimental group was 4.13 g / L, while that in the control group was 2.03 g / L. Among the volatile flavor compounds, the contents of phenylacetaldehyde (4.31 μg / L), ethyl isovalerate (4.17 μg / L), phenethyl acetate (2.77 μg / L), and phenylethanol were significantly higher in the experimental group (37.0%) than in the control group. The polysaccharide content in the experimental group was 2.22 g / L, while that in the control group was 1.04 g / L, giving the apple cider vinegar a rich fruity aroma and a sweet and sour taste.

[0248] Example 24: Application of compound microbial inoculants in fermented chili sauce

[0249] Fresh, undamaged chili peppers were selected, washed, stems removed, and dried. They were then crushed and weighed for later use. The fermentation tank was boiled in water for 15.0 minutes and then dried for later use. 5.0% w / w salt was added, and 1.0% w / w compound microbial inoculant was inoculated and mixed thoroughly. An equal amount of *Wickham's yeast* W23087 and *Lactobacillus plantarum* L23104 were used as control groups. Fermentation was carried out at 30.0℃ for 2.0 days.

[0250] pH value determination: determined according to the method in GB 5009.237-2016; capsaicin content determination: determined according to the method in GB / T 30389-2013 Determination of total capsaicin content in chili peppers and their oleoresins; Organic acid content determination: 5.0 g of chili pepper sample was weighed and dried in an oven at 60.0℃ until the moisture content was below 15.0%, then 25.0 mL of methanol and tetrahydrofuran (1:1) solution was added. The sample was sealed with plastic wrap and extracted by ultrasonic shaking in a water bath at 60.0℃ for 1.0 h. After filtration, the above operation was repeated twice. The filtrates were combined and rotary evaporated at 70.0℃ to 10.0 mL, and then diluted to 50.0 mL. The solution was filtered through a 0.45 μm filter membrane for chromatographic analysis. Chromatographic conditions: Column: Thermo Fisher C18 (4.6×250mm, 5μm); Mobile phase: Methanol:Water = 80:20 (V:V); Flow rate: 0.8mL / min; Injection volume: 20.0μL; Oven temperature: 30.0℃; Detection wavelength: 280.0nm;

[0251] The determination of volatile flavor compounds was carried out according to Example 4. The results showed that the pH changes between the experimental group and the control group were not significant; before fermentation, the capsaicin content was the highest at 0.68 mg / g, and after fermentation, the capsaicin content decreased significantly to 0.28 mg / g; the polysaccharide content in the experimental group was 2.38 g / L, while the polysaccharide content in the control group was 1.74 g / L. The experimental group had a milder spicy taste in the chili sauce, making it more palatable.

[0252] The results are shown in Table 10. The total amount of organic acids initially increased and then decreased during fermentation, which may be due to the utilization of aromatic esters by microorganisms in the later stages of fermentation. A total of 152 volatile components were detected in the chili sauce during fermentation, including 45 alcohols, 35 esters, 19 aldehydes, 11 acids, 17 ketones, 9 alkenes, 3 phenols, 11 alkanes, and 2 others. The total ester content in the experimental group was 7.61 g / L, while that in the control group was 4.33 g / L, which was 43.10% higher than that in the control group. The experimental group had ethyl acetate content of 2.74 μg / L, isoamyl acetate content of 2.49 μg / L, and phenylethanol content of 1.12 μg / L. Therefore, mixed fermentation of *Lactobacillus plantarum* L23104 and *Wickham's abnormal* W23087 can enrich the flavor and taste of the chili sauce.

[0253] Table 10 Changes in organic acid content of compound microbial inoculants in fermented chili sauce

[0254] Example 25: Application of compound microbial inoculants in fermented stretchy yogurt

[0255] Take pure milk, preheat it at 450℃ for 10.0 min, add 1.0% w / w of extracellular polysaccharide produced by Lactobacillus plantarum L23104, pasteurize at 68.0℃ for 30.0 min, let it stand and cool, inoculate with 0.1% w / w of compound microbial agent and 0.1% w / w of commercial compound fermentation agent, ferment in an incubator at 40.0℃ for 10.0 h, and store it in a refrigerator. Control group 1 was supplemented with 0.1% w / w *Lactobacillus plantarum* L23104 and 0.1% w / w commercial compound starter culture; control group 2 was supplemented with 0.1% w / w *Wickham's abnormal* W23087 and 0.1% w / w commercial compound starter culture; control group 3 was supplemented with 0.2% w / w commercial compound starter culture. All other methods were the same as the experimental groups. pH value was determined according to the method in GB 5009.237-2016; total acid was determined according to the method in GB 12456-2021 National Food Safety Standard - Determination of Total Acid in Food; total bacterial count was determined according to GB... The determination of total bacterial count in yogurt was carried out according to the method in "National Food Safety Standard for Microbiological Examination of Food" (4789.2-2022). The textural properties of yogurt were determined by a texture analyzer, including indicators such as hardness, elasticity, resilience, cohesion, viscosity, and chewiness. The stringiness was determined by a texture analyzer by squeezing the yogurt through a piston probe and then allowing it to rise upwards at a certain speed; the distance of the rise reflected the stringiness. The determination of volatile flavor compounds in stringy yogurt was carried out according to Example 4.

[0256] The results are shown in Table 11. The stringiness of the experimental group was 17.23 mm, while that of control group 1 was 12.11 mm, control group 2 was 11.45 mm, and control group 3 was 10.55 mm. The addition of extracellular polysaccharides produced by *Lactobacillus plantarum* L23104 improved the viscosity and adhesiveness of the stringy yogurt, resulting in a smoother texture. The experimental group contained 40 volatile flavor compounds, including ethyl acetate (23.78%), isoamyl acetate (19.92%), and acetaldehyde (19.76%). The control group contained 34 volatile flavor compounds, including ethyl acetate (20.21%), isoamyl acetate (15.53%), and acetaldehyde (14.50%). The experimental group exhibited a richer flavor profile and a more pronounced milky aroma.

[0257] Table 11 Detection data for stretchy yogurt

[0258] Example 26: Application of compound microbial agents in fermented multigrain cakes

[0259] Based on the dry flour weight, 20.0% w / w buckwheat flour, 20.0% w / w barley flour, 35.0% w / w milk, 30.0% w / w whole egg liquid, 5.0% w / w white sugar, 0.6% w / w salt, and 1.0% w / w exopolysaccharide-producing microbial agent and commercial compound leavening agent (experimental group) were mixed and kneaded in a bowl until the dough was about to form a thin membrane. Then 8.0% w / w butter was added and kneaded until the membrane was thin and transparent. The dough was then placed in a proofing box at 28.0℃ and 75.0% humidity for the first proofing, and then placed in a proofing box at 35.0℃ and 75.0% humidity for the second proofing. After proofing, the surface was brushed with egg liquid and baked in an oven at 180℃ for 15.0 minutes. After cooling, the dough was refrigerated. The control group consisted of no exopolysaccharide-producing microbial agent and only 1.0% w / w commercial compound leavening agent. The textural properties of the multigrain cake were determined using a texture analyzer; the cake quality was evaluated according to the "GB / T 35869-2018 Grain and Oil Inspection: Rapid Baking Method for Evaluation of Wheat Flour Bread Baking Quality"; the determination of volatile flavor compounds in the multigrain cake was carried out according to Example 4.

[0260] The results showed that the experimental group had a polysaccharide content of 5.12 g / L, while the control group had a polysaccharide content of 3.54 g / L. This resulted in the experimental group having the lowest hardness and chewiness of the multigrain cake, and a better texture evaluation. The experimental group had a total ester content of 9.21 g / L, while the control group had a total ester content of 4.48 g / L. 69 and 58 volatile flavor compounds were detected in the experimental and control groups, respectively, including alcohols, aldehydes, esters, acids, ketones, aromatic heterocyclic compounds, and alkanes and alkenes. The ester content in the experimental group was 20.1%, higher than the 25.5% in the control group, contributing to the cake's rich aroma.

[0261] Example 27: Application of compound microbial inoculants in fermented flavored fermented bean curd

[0262] Take 25.0g of soybeans, clean and remove impurities, soak for 10.0h, drain and grind into a slurry, filter the slurry through 4 layers of gauze, boil in 100℃ boiling water for 5.0min, add brine to coagulate the slurry, and press it into white curd. Inoculate the white curd with a suspension of Rhizopus by spraying, and then cultivate in a constant temperature room at 25.0℃ for 48h to obtain raw curd. Take 10.0g of raw curd, add salt, and inoculate with 1.0% w / w compound microbial agent (experimental group) and 1.0% w / w commercial compound fermentation agent (control group) for 7 days. After pickling, drain the curd, fill it with broth into jars, vacuum seal and place it at 25.0℃ for later fermentation to mature into fermented bean curd. The total bacterial count was determined according to the method in GB 4789.2-2022 National Food Safety Standard for Microbiological Examination of Food - Determination of Total Bacterial Count; the textural properties of fermented bean curd were determined by a texture analyzer, including indicators such as hardness, elasticity, resilience, cohesion, viscosity, and chewiness; the determination of volatile flavor substances in fermented bean curd was carried out according to Example 4.

[0263] The results showed that the polysaccharide content in the experimental group was 3.84 g / L, while that in the control group was 1.77 g / L. The total ester content in the experimental group was 8.32 g / L, while that in the control group was 5.56 g / L, with the experimental group showing a 63% higher content. Fermented bean curd contained 98 volatile components, including 31 esters, 20 alcohols, 18 aldehydes, and 29 other volatile substances. The highest content was ethyl octanoate at 5.11 μg / L (23.1%) and ethyl palmitate at 4.38 μg / L (16.2%), which contribute to the rich, wine-like aroma of the fermented bean curd.

[0264] The ITS rDNA sequence (SEQ ID NO.1) of *Saccharomyces cerevisiae* W23087.

[0265] Lactobacillus plantarum L23104 16S rDNA sequence (SEQ ID NO.2):

Claims

1. A type of Wickerhamomyces anomalus W23087, characterized in that, The aforementioned abnormal Wickham yeast was deposited at the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29669.

2. A strain of *Lactiplantibacillus plantarum* L23104, characterized in that, The Lactobacillus plantarum L23104 was deposited with the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29668.

3. A compound microbial agent, characterized in that, It contains Lactiplantibacillus plantarum L23104 and Wickerhamomyces anomalus W23087; The Lactobacillus plantarum L23104 was deposited at the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC No. 29668. The aforementioned abnormal Wickham yeast W23087 was deposited on January 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29669.

4. The compound microbial agent according to claim 2, characterized in that, In the compound microbial agent, the cell count ratio of *Lactobacillus plantarum* and *Wickham's yeast anomala* is 1:0.8–1.2; the cell count per gram or milliliter of the compound microbial agent is not less than 1.0 × 10⁻⁶. 8 CFU.

5. The compound microbial agent according to any one of claims 3 to 5, characterized in that, The compound microbial agent includes live cells of *Lactobacillus plantarum* L23104 and *Wickham's yeast* W23087, or freeze-dried cells or immobilized cells, or strains containing *Lactobacillus plantarum* L23104 and *Wickham's yeast* W23087 in any other form.

6. The application of the compound microbial agent according to any one of claims 3 to 5 in fermented noodle products, fermented wines, fermented fruit juices, fermented dairy products or fermented condiments.

7. The application according to claim 6, characterized in that, The fermented dough products include bread, steamed buns, and cakes.

8. The application according to claim 6, characterized in that, The fermented alcoholic beverages include rice wine and wine; the fermented dairy products include stringy yogurt and cheese.

9. The application according to claim 6, characterized in that, The fermented fruit juice includes apple cider vinegar and prickly pear juice; the fermented condiments include chili sauce, tomato sauce, and fermented bean curd.

10. A microbial preparation, characterized in that, The microbial preparation contains the abnormal Wickham yeast W23087, abnormal Wickham yeast W23087 metabolites, or its fermentation broth as described in claim 1.

11. The microbial preparation according to claim 10, characterized in that, The amount of *Wickham's abnormal yeast* W23087 in each gram or milliliter of the microbial preparation is not less than 1 × 10⁻⁶. 8 CFU.

12. The microbial preparation according to claim 10 or 11, characterized in that, The microbial preparation contains live cells of the abnormal Wickham yeast W23087 as described in claim 1, or freeze-dried cells or immobilized cells, or the abnormal Wickham yeast W23087 strain existing in any other form.

13. The use of the abnormal Wickham yeast W23087 of claim 1 or any of the microbial preparations of claims 2 to 4 in fermented noodle products, alcoholic beverages, fermented drinks, condiments or fermented feed.

14. The application according to claim 5, characterized in that, The flour products include bread or steamed buns.

15. The application according to claim 5, characterized in that, The alcoholic beverages mentioned include small-batch liquor or wine.

16. The application according to claim 5, characterized in that, The fermented beverage includes mango juice or apple cider vinegar.

17. The application according to claim 5, characterized in that, The condiments include sweet bean sauce or soy sauce.

18. A method for enhancing the flavor of fermented products, characterized in that, The method involves adding the abnormal Wickham yeast W23087 of claim 1 or any of the compound microbial agents of claims 10-12 during the preparation of the fermented product; the flavor is an ester or alcohol substance.

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