A composite microbial agent derived from sourdough and its application in food

Abstract

The invention discloses a composite microbial agent derived from sour dough and its application in food, and belongs to the technical field of food fermentation. The invention improves the content of extracellular polysaccharides in fermented food and enhances the flavor of fermented food by compounding aroma-producing yeast and sugar-producing plant lactobacillus, provides a composite microbial agent with excellent performance, and is applied to the fermentation process of flour products (bread, steamed buns and cakes), wines (rice wine and wine), beverages (apple vinegar and sea buckthorn juice), yogurt and condiments (chili sauce, tomato sauce and fermented bean curd) for strengthening, so as to enhance the quality of fermented food and increase the content of volatile flavor substances in fermented food, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to a composite microbial agent derived from sour dough and application thereof in food, belonging to the technical field of food fermentation. Background Art

[0002] As a direct and efficient means to improve the quality of fermented foods, combined fermentation of yeast and lactic acid bacteria is increasingly favored by the market. The interaction mechanism between lactic acid bacteria and yeast in fermented foods has a decisive influence on the quality of fermented foods. Yeasts are usually unable to digest polysaccharides and proteins, while Gram-positive lactic acid bacteria usually secrete enzymes to digest some polymers and high molecular weight compounds, and the resulting digestion products (monomers or low molecular weight compounds) can be used by yeasts. In general, the growth of lactic acid bacteria requires some nutrients, such as amino acids, unsaturated fatty acids or vitamins. In a malnourished environment, nutrient-deficient lactic acid bacteria will not be able to grow. On the contrary, in mixed cultures, yeasts will provide some nutrients to lactic acid bacteria to support bacterial growth. Therefore, in yeast-lactic acid bacteria co-culture, these complementary characteristics will be combined to avoid the decrease in pH caused by the accumulation of lactic acid by lactic acid bacteria, thereby supporting the growth of lactic acid bacteria.

[0003] Microbial exopolysaccharides (EPS) are a class of mucus compounds secreted into the extracellular matrix by microorganisms during their growth. Compared with plant polysaccharides, microbial polysaccharides have the advantages of short production cycle and no restrictions on geographical and seasonal conditions, and have broad development prospects. Lactic acid bacteria exopolysaccharides are usually used as a natural substitute for commercial stabilizers, thereby increasing the viscosity of the fermentation broth, enhancing stability, improving physical and chemical properties such as emulsification and gel properties, and achieving the purpose of improving the rheological properties, texture and taste of the fermentation broth. Therefore, it can be foreseen that plant lactobacillus exopolysaccharides, as a new, efficient and safe physiological function regulator, will have theoretical research significance and practical application value.

[0004] Fermented products have a unique environment that makes the strains have higher characteristics. People screen out microorganisms with better aroma production from fermented foods to further enhance the aroma of fermented foods. Aroma-producing lactic acid bacteria are a hot topic in the study of aroma-producing microorganisms. There are many screening methods for aroma-producing lactic acid bacteria, mainly based on aroma-producing substances and sensory evaluation methods. Qiu et al. discovered a strain of Saccharomyces cerevisiae YS219 that produces ethyl hexanoate and has excellent resistance to organic acids. The effects of this strain on the microbial composition and flavor metabolites during the fermentation of Luzhou-flavor liquor were studied. The results showed that the fermentation of this strain can increase the content of ethanol and ethyl hexanoate (https: / / doi.org / 10.1016 / j.fbio.2024.103925). Zeng Wanli et al. used a culture medium that simulated the growth environment of tobacco leaves to screen and isolate aroma-producing lactic acid bacteria in tobacco leaves and obtained a methyltrophic Bacillus, the content of esters and aldehydes in its volatile components was significantly increased (DOI: 10.13417 / j.gab.039.000636). The above technologies all have problems such as single effect and weakness.

[0005] Therefore, preparing a composite microbial agent that produces both polysaccharides and aroma has extremely high practical and economic value. Summary of the invention

[0006] In order to solve the above problems, the present invention increases the content of extracellular polysaccharides in fermented foods and enhances the flavor of fermented foods by compounding aroma-producing yeast and sugar-producing plant lactobacillus, provides a composite microbial agent with excellent performance, and is applied to the fermentation process of flour products (bread, steamed buns and cakes), wines (rice wine and grape wine), beverages (apple vinegar and sea buckthorn juice), wire-drawn yogurt and condiments (chili sauce, tomato sauce and fermented bean curd) for strengthening, so as to enhance the quality of fermented foods and increase the content of volatile flavor substances in fermented foods, and has broad application prospects.

[0007] The first object of the present invention is to provide a composite microbial agent, characterized in that it contains Lactobacillus plantarum L23104 and Wickerhamomyces anomalus W23087.

[0008] The Lactobacillus plantarum L23104 has been deposited with the General Microbiology Center of China Microbiological Culture Collection Administration on January 18, 2024, with the deposit number of CGMCC No. 29668;

[0009] The abnormal Wickerhamomyces anomalus W23087 has been deposited with the General Microbiology Center of China Microbiological Culture Collection Administration on January 18, 2024, with the deposit number of CGMCC No.29669;

[0010] The plant lactobacillus L23104 and the abnormal Wickham yeast W23087 of the present invention have the following characteristic properties:

[0011] (1) Lactobacillus plantarum L23104 produces high levels of exopolysaccharides, which can be used as a stabilizer to enhance the stability of fermented products, thereby improving product texture and taste;

[0012] (2) Abnormal Wickham yeast W23087 produces high levels of esters, which can enhance the flavor characteristics of fermented foods.

[0013] The first object of the present invention is to provide a composite microbial agent, comprising Lactiplantibacillus plantarum L23104 and Wickerhamomycesanomalus W23087;

[0014] The Lactobacillus plantarum L23104 has been deposited with the General Microbiology Center of China Microbiological Culture Collection Administration on January 18, 2024, with the deposit number of CGMCC No. 29668;

[0015] The abnormal Wickerhamomyces anomalus W23087 was deposited with the General Microbiology Center of China National Microbiological Culture Collection Administration on January 18, 2024, with the deposit number CGMCC No.29669.

[0016] In one embodiment, the composite microbial agent has a cell number ratio of Lactobacillus plantarum to Wickham's yeast of 1:0.8-1.2, which is obtained by enrichment culture, centrifugal washing, proportional addition and freeze drying; and the cell number per gram or per milliliter of the composite microbial agent is not less than 1.0×10 8 CFU.

[0017] In one embodiment, the composite microbial agent includes living cells of Lactobacillus plantarum L23104 and Wickham's yeast W23087 or freeze-dried dry cells or immobilized cells, or strains containing Lactobacillus plantarum L23104 and Wickham's yeast W23087 in any other form.

[0018] The second object of the present invention is to provide a product, which includes any of the above-mentioned composite microbial agents; the product includes fermentation preparations, soil improvement preparations, sewage treatment preparations, and feed additives.

[0019] The third object of the present invention is to provide the use of any of the above-mentioned composite microbial agents in fermented flour products, fermented wines, fermented fruit juices, fermented dairy products or fermented condiments.

[0020] In one embodiment, the fermented flour products include but are not limited to bread, steamed buns, and cakes.

[0021] In one embodiment, the fermented alcoholic beverages include but are not limited to rice wine and grape wine.

[0022] In one embodiment, the fermented juice includes but is not limited to apple cider vinegar and sea buckthorn juice.

[0023] In one embodiment, the fermented dairy product includes but is not limited to yogurt and cheese.

[0024] In one embodiment, the fermented condiments include but are not limited to chili sauce, tomato sauce and fermented bean curd.

[0025] In one embodiment, the bread fermentation is obtained by inoculating the composite 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 main raw materials, mixing them in a ratio of 10:0.6:0.2:0.6, and undergoing fermentation and baking processes.

[0026] In one embodiment, the steamed bread fermentation is obtained by inoculating the composite microbial agent, the commercial composite leavening agent, and the flour into the flour at a mass ratio of 1 to 5:1 to 5:200, and then fermenting and steaming the flour.

[0027] In one embodiment, the multi-grain cake fermentation is obtained by inoculating the composite microbial agent and flour into mixed flour at a mass ratio of 1 to 5:100, and performing two proofing, fermentation and baking processes.

[0028] In one embodiment, the rice wine fermentation is obtained by inoculating the composite microbial agent into glutinous rice at a ratio of 0.5% to 2% w / w, and performing processes such as washing, removing impurities, soaking, beating, liquefying, saccharifying, fermenting and filtering the glutinous rice raw materials.

[0029] In one embodiment, the wine fermentation is obtained by inoculating the composite microbial agent into grapes at a ratio of 2.5% to 5% w / w, and performing processes such as washing, crushing, cold soaking and fermentation.

[0030] In one embodiment, the apple cider vinegar fermentation is obtained by using the composite microbial agent as a fermentation agent and performing processes such as washing, pulping, adjusting ingredients, fermenting and filtering apple raw materials.

[0031] In one embodiment, the roxburghii juice fermentation is obtained by inoculating the composite microbial agent into roxburghii at a ratio of 2.0% to 5.0% w / w, and performing processes such as washing, crushing and fermentation.

[0032] In one embodiment, the wire-drawn yogurt is fermented by using the composite microbial agent as a starter, and then pasteurizing and fermenting it.

[0033] In one embodiment, the application is for chili sauce fermentation.

[0034] In one embodiment, the chili sauce is fermented by inoculating the high-yield extracellular polysaccharide plant lactobacillus L23104 starter at a rate of 1.0% w / w into the chili peppers boiled in boiling water, adding 5.0% w / w salt, and fermenting at 30° C. for 2 days to obtain the sauce.

[0035] In one embodiment, the tomato sauce fermentation is obtained by inoculating the composite microbial agent into tomatoes at a ratio of 1.0% to 5.0% w / w, and performing processes such as washing, blanching, crushing, pasteurization and fermentation.

[0036] In one embodiment, the fermented bean curd is obtained by inoculating the composite microbial agent into soybean blank at a ratio of 1.0% to 5.0% w / w, and performing processes such as water contamination, filling, vacuuming and fermentation.

[0037] The fourth object of the present invention is to provide a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) L23104, which has been deposited with the General Microbiology Center of China Microorganism Culture Collection Administration on January 18, 2024, with a deposit number of CGMCC No.29668.

[0038] The fifth object of the present invention is to provide a strain of abnormal Wickerham yeast (Wickerhamomycesanomalus) W23087, which has been deposited with the General Microbiological Center of China Microbiological Culture Collection Administration on January 18, 2024, with a deposit number of CGMCC No.29669.

[0039] Beneficial Effects

[0040] The invention increases the content of extracellular polysaccharides in fermented foods and enhances the flavor of fermented foods by compounding aroma-producing yeast and sugar-producing plant lactobacillus, providing a composite microbial agent with excellent performance for application in strengthening the fermentation process of flour products (bread, steamed buns and cakes), alcoholic beverages (rice wine and grape wine), beverages (apple vinegar and sea buckthorn juice), wire-drawn yogurt and condiments (chili sauce, tomato sauce and fermented bean curd), so as to enhance the quality of fermented foods and increase the content of volatile flavor substances in fermented foods, and has broad application prospects.

[0041] Specific:

[0042] (1) The present invention provides a composite microbial agent, comprising a high-exopolysaccharide-producing Lactobacillus plantarum L23104 and a high-ester-producing abnormal Wickham yeast W23087; under the same conditions, the exopolysaccharide yield of the composite agent is increased by 9.33% compared with that of L23104; and the ester yield is increased by 8.22% compared with that of W23087;

[0043] (2) When the composite microbial agent provided by the present invention is used to ferment flour products such as bread and steamed buns, compared with commercially available fermentation agents, the polysaccharide content of the composite microbial agent after fermentation is higher than that of the control group by more than 40% and more than 22.67%, and the volatile flavor substances are higher than that of the control group by more than 34.64% and more than 27.71%;

[0044] (3) When the composite microbial agent provided by the present invention is used to ferment alcoholic beverages such as rice wine and grape wine, compared with commercially available fermentation agents, the total ester content of rice wine produced by the composite microbial agent is 0.44 g / L, which is 18.2% higher than that of the control group; the alcohol content of grape wine produced by the composite microbial agent is 14.0% vol, while that of the control group is 12.0% vol; the total acid and volatile acid contents are 15.0% and 21.0% higher than those of the control group;

[0045] (4) When the composite microbial agent provided by the present invention is used to ferment beverages such as apple cider vinegar and roxburghii juice, compared with commercially available fermentation agents, the content of phenylacetaldehyde in high-yield extracellular polysaccharide apple cider vinegar is significantly higher than that in the control group by 37.0%, making the apple cider vinegar rich in fruit flavor and sweet and sour. The total ester content in roxburghii juice is 17.5% higher than that in the control group;

[0046] (5) When the composite microbial agent provided by the present invention is used to ferment the drawn yogurt, the drawing effect is obvious, the viscosity and adhesiveness of the drawn yogurt are improved, and the yogurt has a soft taste. There are 40 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 milk flavor is strong;

[0047] (6) When the composite microbial agent provided by the present invention is used to ferment condiments such as chili sauce, tomato sauce and fermented bean curd, the content of capsaicin in the composite microbial agent chili sauce is significantly reduced to 0.28 mg / g compared with the commercially available fermentation agent, which softens the spicy taste of the chili sauce. The total amount of volatile flavor substances in tomato sauce is 7.10% higher than that in the control group, making the tomato sauce have a good odor coordination, rich sauce flavor and delicious taste. There are more volatile flavor substances in the fermented bean curd, and the fermented bean curd has a mellow wine aroma.

[0048] Biological deposit materials

[0049] The Lactobacillus plantarum provided by the present invention is classified and named as Lactobacillus plantarum L23104, which has been deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No. 29668, and a deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0050] The abnormal Wickerhamomyces anomalus provided by the present invention is classified and named as abnormal Wickerhamomyces anomalus W23087, which has been deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No. 29669, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the Gram staining picture of Lactobacillus plantarum L23104 of the present invention.

[0052] Figure 2 It is a phylogenetic tree diagram of Lactobacillus plantarum L23104 of the present invention.

[0053] Figure 3 It is the Gram staining picture of abnormal Wickham yeast W23087 of the present invention.

[0054] Figure 4 This is a curve diagram of ester production by abnormal Wickham yeast W23087 of the present invention. DETAILED DESCRIPTION

[0055] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0056] Detection of physical and chemical indicators of fermented food: pH value determination is determined according to the method of GB 5009.237-2016. Organic acid content is determined by high performance liquid chromatography (HPLC). Sample texture is determined by texture analyzer. Total acid is determined according to the method of GB12456-2021 National Food Safety Standard Determination of Total Acid in Food; nitrite is determined by spectrophotometry in GB5009.33-2016 National Food Safety Standard Determination of Nitrite and Nitrous Acid in Food; total acid, total ester and alcohol content in rice wine are determined according to GB / T 10781.3-2006 Rice-flavored Liquor. Physical and chemical indicators such as total acid (measured in tartaric acid), volatile acid (measured in acetic acid), total sugar (measured in glucose) and alcohol content in wine are determined according to the method of GB / T15038-2006 General Analysis Method for Grape and Fruit Wine. The physical and chemical indicators of apple cider vinegar were determined according to GB / T 30884-2014 Apple Cider Vinegar Beverage. Volatile flavor substances were determined by gas chromatography-mass spectrometry (GC-MS).

[0057] Experimental raw materials

[0058] Sourdough was obtained from: Dezhou, Shandong, and Bozhou, Anhui;

[0059] Commercial koji was purchased from Angel flagship store, product name: Angel Jinxiangba compound functional bacteria;

[0060] Commercial compound starter was purchased from Angel flagship store, product name: Angel yeast + high-activity dry yeast powder;

[0061] Commercial yogurt starter was purchased from Angel Flagship Store, product name: Angel Yogurt Starter;

[0062] Source of acetic acid bacteria: Testobacillus, product number TS349550.

[0063] Example 1, Isolation and Identification of Lactobacillus plantarum L23104

[0064] 1. Separation and purification

[0065] Weigh 5.0 g of sourdough sample and add it to 45.0 mL of sterile saline (0.85%, m / v). Vortex until the sample is thoroughly and evenly suspended. Take 1.0 mL of the suspension and dilute it 10-fold to 10. -1 ~10 -7. Use a sterilized pipette tip to draw 100.0μL of the dilution solution and apply it to the MRS plate medium. After culturing in an anaerobic incubator at 37.0℃ for 24.0h, carefully observe the colony morphology and pick out single colonies with good growth and yellowing around the colonies. Inoculate the picked single colony into liquid MRS medium, culture it at 37.0℃ for 24.0h, and then streak culture on solid MRS medium to observe the colony purity. Repeat this process three times until there are no foreign bacteria under microscopic examination. The purified colonies were contacted with enzymes and Gram stains, and the catalase-negative and Gram-positive strains were preliminarily identified as lactic acid bacteria. After culturing the final single colony in MRS liquid medium for 48.0h, it was divided into aliquots, added to sterilized 25.0% glycerol protective agent, and stored at -80℃.

[0066] 2. Morphological characteristics of Lactobacillus plantarum L23104

[0067] Streak the strain on an MRS solid plate, incubate at 37.0℃ for 24.0h, and observe its colony morphology. Pick a single colony for Gram staining and observe its cell morphology under a microscope. The colony of this strain is convex, milky white, round and smooth, translucent, with neat edges; the bacteria are rod-shaped and Gram-positive. Figure 1 shown.

[0068] 3. Physiological characteristics of Lactobacillus plantarum L23104

[0069] The results are shown in Table 1.

[0070] Table 1 Physiological and biochemical characteristics of Lactiplantibacillus plantarum L23104

[0071]

[0072] Note: "+" indicates that the strain reacts positively; "-" indicates that the strain reacts negatively;

[0073] 4. Strain identification

[0074] The genomic DNA of lactic acid bacteria was extracted using a DNA extraction kit, and its OD value (A260) and concentration were determined. The qualified purity samples were diluted to about 100 ng / μL for PCR amplification.

[0075] The universal primers for 16S rDNA were: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′ SEQ ID NO. 3), 1492R (5′-GGTTACCTTGTTACGACTT-3′ SEQ ID NO. 4);

[0076] The PCR amplification system (50.0 μL) was: 25 μL 2×Phanta Max Buffer, 0.5 μL 10 mM dNTP Mix, 0.5 μL upstream primer (10.0 μM) and downstream primer (10.0 μM), 0.4 μL high-fidelity DNA polymerase, 5.0 μL template DNA, and sterile water to a total volume of 50.0 μL; the PCR amplification program was: pre-denaturation at 95.0°C for 3.0 min, denaturation at 95.0°C for 15.0 s, annealing at 58.0°C for 15.0 s, extension at 72.0°C for 90.0 s, 35 cycles, and complete extension at 72.0°C for 5.0 min.

[0077] After 1% agarose gel electrophoresis, the 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 closest sequence of known species. The homology of 99.0% or more can directly identify the strain to be tested. By constructing a phylogenetic tree, such as Figure 2 As shown, the lactobacillus was identified as Lactobacillus plantarum and was named Lactobacillus plantarum L23104.

[0078] 5. Wire drawing experiment of Lactobacillus plantarum L23104

[0079] Take 1 drop of freshly prepared 4.0% KOH aqueous solution on a clean glass slide, use an inoculation loop to take a small amount of freshly prepared colonies on the solid culture medium, stir and mix in the KOH solution, and lift the inoculation loop every few seconds to observe whether a thread can be drawn. Repeat the operation for 4 to 5 colonies. If a thread can be drawn with the inoculation loop, it is positive, and if it is still a suspension, it is negative. Measure the maximum length of the colony thread (cm), and the results are expressed as an average value. The initial screening obtains lactic acid bacteria with thread-drawing characteristics. The results show that L23104 has the longest thread drawing. The thread drawing results are shown.

[0080] 6. Strain preservation

[0081] The Lactobacillus plantarum L23104 strain was deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No. 29668, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The Lactobacillus plantarum 16S rDNA sequence provided by the present invention is shown in SEQ ID NO.1.

[0082] Example 2: Extraction and determination of extracellular polysaccharides of Lactobacillus plantarum L23104

[0083] The EPS-producing strain obtained in the initial screening was activated for two generations and inoculated into 150.0 mL mMRS (MRS with 5.0% sucrose) liquid medium (bacterial concentration was 10 5 CFU / mL), cultured at 37.0℃ for 24.0h. Place the test tube in a boiling water bath for 10.0min and then cool to room temperature. Centrifuge at 12000g for 30.0min at 4.0℃ to remove the precipitate. Add 80.0% (m / v) trichloroacetic acid (TCA) solution to the supernatant to a final concentration of 4.0%, and incubate at 4.0℃ overnight. Centrifuge at 12000g for 30.0min at 4.0℃ to remove the precipitated protein. Add 95.0% (w / v) pre-cooled ethanol to the supernatant and let stand overnight. Centrifuge at 12000g for 30.0min at 4.0℃ and repeat the operation twice. Redissolve the precipitate in deionized water. After fully dissolved, dialyze at 4.0℃ using a dialysis bag with a molecular weight cutoff of 8000-14000Da for 48.0h, changing the water every 8.0h. Freeze-dry after dialysis. Extracellular polysaccharides were determined by the phenol-sulfuric acid method.

[0084] It was determined that after 24 hours of fermentation, the extracellular polysaccharide content of Lactobacillus plantarum L23104 was 2460.12±1.46 mg / L, which can be used as a stabilizer to enhance the stability of the fermented product and achieve the purpose of improving the texture and taste of the product.

[0085] Example 3: Isolation and Identification of Abnormal Wickham Yeast W23087

[0086] 1. Separation and purification

[0087] Weigh 5.0 g of sourdough sample and add it to 45.0 mL of sterile saline (0.85%, m / v). Vortex until the sample is thoroughly and evenly suspended. Take 1.0 mL of the suspension and dilute it 10-fold to 10. -1 ~10 -7 . Use a sterilized pipette tip to draw 100.0μL of the dilution solution and apply it to the YPD plate culture medium. After culturing in a shaker incubator at 28.0℃ for 48.0h, carefully observe the colony morphology and pick out single colonies with good growth. Inoculate the picked single colony into liquid YPD culture medium, culture it at 28℃ for 48.0h, and then streak it on solid YPD culture medium to observe its colony purity. Repeat this process three times until there are no foreign bacteria under microscopic examination. The strain with obvious spores was preliminarily identified as yeast by microscopic examination. After culturing the final single colony in YPD liquid culture medium for 48.0h, it was divided into aliquots, added to sterilized 25.0% glycerol protective agent and stored at -80℃.

[0088] 2. Morphological characteristics of abnormal Wickham yeast W23087

[0089] Streak the strain on a YPD solid plate and culture it at 28.0℃ for 48.0h. Observe the colony morphology of the aroma-producing yeast strain and take photos to record. When observing its cell morphology under a microscope, dilute 1.0mL of the bacterial solution appropriately, take 1 drop on a glass slide, and place it on the stage of an optical microscope for microscopic examination. Perform a preliminary classification based on the colony morphology and cell morphology of the strain.

[0090] On the YPD plate, cultured at 28.0℃ for 48.0h, the colony of aroma yeast W23087 was milky white, 1.0-2.0mm in diameter, spherical, with protrusions, smooth surface, neat edges, and opaque. Observed under an optical microscope, the cells of this strain were spherical or ellipsoidal, surrounded by obvious conidia, which clearly met the typical characteristics of yeast. Therefore, W23087 was preliminarily determined to be yeast. Figure 3 shown.

[0091] 3. Strain identification

[0092] A DNA extraction kit (purchased from Qingdao Haibo Biological, model HBIG11) was used to extract yeast genomic DNA, and its OD value (A260) and concentration were determined. The qualified purity samples were diluted to about 100 ng / μL for PCR amplification.

[0093] ITS rDNA universal primers were: ITS1 (5′-TCCTCCGCTTATTGATATGC-3′SEQ ID NO. 5), ITS4 (5′-TCCGTAGGTGAACCTGCGG-3′SEQ ID NO. 6);

[0094] The PCR amplification system (50.0 μL) was: 25 μL 2×Phanta Max Buffer, 0.5 μL 10 mM dNTP Mix, 0.5 μL upstream primer (10.0 μM) and downstream primer (10.0 μM), 0.4 μL high-fidelity DNA polymerase, 5.0 μL template DNA, and sterile water was added to the total volume of 50.0 μL; the PCR amplification program was: pre-denaturation at 94.0°C for 5.0 min, denaturation at 94.0°C for 30.0 s, retardation at 58.0°C for 45.0 s, extension at 72.0°C for 45.0 s, 35 cycles, and complete extension at 72.0°C for 10.0 min.

[0095] After 1% agarose gel electrophoresis, the 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 closest known species. The homology of 99% or more can directly identify the strain to be tested. By constructing a phylogenetic tree, such as Figure 2As shown, the yeast was identified as Wickham's yeast and named as Wickham's yeast W23087.

[0096] 4. Strain preservation

[0097] The abnormal Wickerhamomyces anomalus W23087 strain was deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No. 29669, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The abnormal Wickerhamomyces anomalus ITS sequence provided by the present invention is shown in SEQ ID NO.2.

[0098] Example 4: Analysis of aroma production characteristics of abnormal Wickham yeast W23087

[0099] 1. Ester production curve

[0100] The activated abnormal Wickham yeast W23087 bacterial solution was inoculated at a volume of 2.0% v / v in 10 mL YPD liquid medium (bacterial concentration was 10 5 CFU / mL), cultured at 28.0℃ for 18h, and sampled every two hours. The total ester content was determined by saponification reflux method. The culture broth of the strain was centrifuged to obtain 1.0mL of the fermentation broth supernatant, diluted with water to an appropriate concentration, and then an appropriate amount of phenolphthalein was added. It was titrated with 0.1mol / L NaOH until it turned slightly red, and then an excess of 0.1mol / L NaOH solution was added. The saponification was refluxed in a boiling water bath for 30.0min. After cooling to room temperature, it was immediately titrated with 0.1mol / L HCl solution until the red color just disappeared. The volume of HCl consumed was recorded, and each group was repeated 3 times in parallel. The results are as follows Figure 4 As shown, the ester production of abnormal Wickham yeast W23087 began to level off from 6.0 h, and the ester production reached a maximum of (19.25±0.35) g / L at 12 h.

[0101] 2. Analysis of volatile flavor substances

[0102] (1) Sample preparation

[0103] Take 5.0mL~10.0mL of bacterial solution and put it into the headspace bottle, cover it tightly and mix it evenly. Use 50 / 30μmDVB / CAR / PDMS extraction head to extract volatile flavor substances in the sample, with extraction time of 40.0min, extraction temperature of 60.0℃, thermal desorption time of 5.0min, and then perform GC-MS analysis.

[0104] (2) GC conditions

[0105] The injection was carried out in splitless mode, the carrier gas was helium, the flow rate was 1.0 mL / min, the injection port temperature was 250°C, the capillary column was SH-Rtx-Wax column (30.0 m×0.25 mm, 0.25 μm), and the column oven temperature program was an initial temperature of 40.0°C, maintained for 1.0 min, then increased to 160.0°C at 6°C / min, and then increased to 250.0°C at 3.0°C / min, and retained for 5.0 min.

[0106] (3)MS conditions

[0107] The electron energy was 70.0 eV, the injection hole temperature was 250.0 °C, the emission current was 200.0 μA, the ion source temperature was 200.0 °C, the solvent delay time was 2.0 min, the detection method was full ion scanning, and the mass scanning range was m / z 33-495. Shimadzu software was used for flavor data collection and qualitative and quantitative analysis, and the compound comparison used the NIST11 database.

[0108] There are 46 volatile flavor substances in the bacterial liquid of abnormal Wickham yeast W23087, including 14 alcohols, 12 esters, 5 acids, 5 ketones, 4 hydrocarbons, 2 phenols, 2 pyrazines, and 2 pyrroles. The main flavor substances are alcohols and esters, and the total peak area of ​​the two accounts for more than 90.0% of the total flavor substances, that is, the total relative content of alcohols and esters is as high as more than 90.0%.

[0109] Example 5: Preparation of a high-yield extracellular polysaccharide mixed microbial agent

[0110] 1. Enrichment culture

[0111] The activated abnormal Wickham yeast W23087 was inoculated into the YPD medium with two loops picked with an inoculation loop, and cultured in a shaker at 28.0°C and 200.0rpm for 48.0h; 100.0μL of the culture solution was transferred into the YPD fermentation medium, and enriched and cultured in a shaker at 28.0°C and 200.0rpm for 48.0h. The activated plant lactobacillus plantarum L23104 was inoculated into the MRS medium with two loops picked with an inoculation loop, and cultured in an incubator at 37.0°C for 24.0h; 100.0μL of the culture solution was transferred into the MRS fermentation medium, and cultured in an incubator at 37.0°C for 24.0h.

[0112] 2. Centrifugal washing

[0113] The abnormal Wickham yeast W23087 fermentation liquid and the plant lactobacillus L23104 fermentation liquid obtained by enrichment culture were poured into centrifuge tubes respectively, and centrifuged at 3000r / min for 15.0min. After the supernatant was poured out, it was washed twice with sterile water. The obtained precipitate was resuspended with sterile distilled water to prepare plant lactobacillus L23104 bacterial suspension and yeast W23087 suspension respectively, and then used for standby;

[0114] 3. Bacteria ratio

[0115] The Lactobacillus plantarum L23104 suspension and the yeast W23087 suspension were mixed at a ratio of 1:0.8 of viable bacteria to obtain a mixed bacterial solution;

[0116] 4. Add freeze-drying protective agent

[0117] Trehalose was selected as a protective agent, and a 10% v / v trehalose solution was prepared, sterilized at 121.0°C for 20.0 min, and then mixed with an equal volume of the mixed bacterial solution;

[0118] 5. Freeze-drying

[0119] Mix the mixed bacterial solution with trehalose protective agent evenly and pour it into the plate. Seal the plate with plastic wrap and quickly put it into -80℃ ultra-low temperature refrigerator for pre-freezing for 3 hours. Quickly transfer the pre-frozen plate to the freeze dryer for freeze drying. Quickly transfer the freeze-dried plate to the clean bench, seal it with plastic wrap and sealing film, and store the freeze-dried powder at -20℃. The total number of viable bacteria in the freeze-dried powder reaches 10 8 CFU / g.

[0120] Example 6: Bacterial Agent Performance Testing

[0121] 1. Polysaccharide yield detection

[0122] The bacterial agent prepared in Example 5 was inoculated into mMRS medium at 2% v / v to make the total bacterial concentration in the medium 10 5 CFU / mL, cultured at 37.0℃ for 24.0h. The same concentration of single strains L23104 and W23087 were used as the control group (bacterial concentration was 10 5 CFU / mL), and exopolysaccharide was prepared according to the method of Example 2, and the exopolysaccharide content was detected.

[0123] The results are shown in Table 2. The results show that the exopolysaccharide produced by the composite bacterial agent was 2713.33±2.71 mg / L, which was 9.33% higher than the polysaccharide content of Lactobacillus plantarum L23104.

[0124] Table 2 Extracellular polysaccharide production

[0125] <![CDATA[Inoculated strain (10 5 CFU / mL)]]> Compound Bacterial Agent L23104 W23087 Extracellular polysaccharide production (mg / L) 2713.33±2.71 2460.12±1.46 733.80±0.24

[0126] 2. Fragrance production detection

[0127] The bacterial agent prepared in Example 5 was inoculated into YPD liquid medium at 2% v / v to make the total bacterial concentration in the medium 10 5 CFU / mL, cultured at 28.0℃ for 16h. The same concentration of single strains L23104 and W23087 were used as the control group (bacterial concentration was 10 5 CFU / mL), and the ester production was detected for 16 h according to the method of Example 4.

[0128] The results are shown in Table 3. The results show that the ester production of the composite bacterial agent was 23.54±0.57 g / L, which was 18.22% higher than that of the abnormal Wickham yeast W23087.

[0129] Table 3 Ester production

[0130] <![CDATA[Inoculated strain (10 5 CFU / mL)]]> Compound Bacterial Agent L23104 W23087 Ester production (g / L) 23.54±0.57 5.29±0.01 19.25±0.35

[0131] Example 7: Application of composite microbial agent in fermented bread

[0132] Dough fermentation and bread preparation method: high-gluten flour, sucrose, sodium chloride and drinking water are used as main raw materials, mixed in a ratio of 10:0.6:0.2:0.6 by mass, 0.25g of composite microbial agent (prepared in Example 5) and 0.25g of commercial composite fermentation agent are taken, and the mass ratio of agent to flour is 1:100, soaked in warm water for 30 minutes, made into dough and placed in a 35.0℃ proofing box for 3.0 hours. After fermentation, the dough is baked in an oven at 190.0℃ on the upper fire and 200.0℃ on the lower fire for 25.0 minutes. After baking, the bread was equilibrated at 25.0°C and sealed for further analysis, wherein control group 1 was added with 0.25 g Lactobacillus plantarum L23104 and 0.25 g commercial composite leavening agent, control group 2 was added with 0.25 g abnormal Wickham yeast W23087 and 0.25 g commercial composite leavening agent, control group 3 was added with 0.5 g commercial composite leavening agent, and the rest of the methods were the same as those of the experimental group.

[0133] The texture characteristics of bread were measured by a texture analyzer; the quality evaluation of bread was determined according to GB / T 35869-2018 Grain and Oil Inspection Wheat Flour Bread Baking Quality Evaluation Rapid Baking Method; the content of extracellular polysaccharides in bread was determined according to Example 1; and the determination of volatile flavor substances in bread was carried out according to Example 4.

[0134] The results are shown in Table 4. After measurement, the physical and chemical indicators of the experimental group were normal, the bread crust color was normal, and the bread core was white, soft and elastic. The polysaccharide content in the bread fermented with the addition of composite microbial agents was more than 40% higher than that of the three control groups. The determination of volatile flavor substances showed that the main flavor substances were alcohols and esters, which showed high aroma activity and contributed to the overall flavor of bread. The relative content was as high as more than 90.0%, and the ethyl acetate content was 4.26μg / L, the phenylethanol content was 3.79μg / L, the propyl acetate content was 0.65μg / L, and the isoamyl alcohol content was 0.58μg / L. Among them, in the control group 3, the ethyl acetate content was 1.07μg / L, the phenylethanol content was 0.68μg / L, the propyl acetate content was 0.16μg / L, and the isoamyl alcohol content was 0.10μg / L. Compared with the control group, the content of esters such as ethyl acetate and propyl acetate increased by 75.0%, and the content of alcohols such as ethanol and isoamyl alcohol increased by 82.0%.

[0135] Table 4 Bread testing data

[0136]

[0137]

[0138] Example 8: Application of composite microbial agent in fermented steamed bread

[0139] Take 100.0g wheat flour, 0.5g composite microbial agent, 0.5g commercial composite leavening agent, and 50.0mL water in a dough mixer, stir slowly for 3.0min and then stir quickly for 3.0min, divide into dough, knead until the surface is smooth, place in a proofing box at a temperature of 38.0℃ and a relative humidity of 85.0% for 50.0min, steam for 20.0min, simmer in the pot for 5.0min after stopping the fire, take out the steamed buns, and place them at room temperature for 1.0h before testing The steamed bread was prepared by mixing 0.5 g Lactobacillus plantarum L23104 and 0.5 g commercial composite starter culture medium into the control group 1, 0.5 g abnormal Wickham yeast W23087 and 0.5 g commercial composite starter culture medium into the control group 2, and 1.0 g commercial composite starter culture medium into the control group 3. The other methods were the same as those of the experimental group. The texture characteristics of the steamed bread were determined by a texture analyzer. The content of extracellular polysaccharides in the steamed bread was determined by reference to Example 1. The determination of volatile flavor substances in the steamed bread was carried out by reference to Example 4.

[0140] The results are shown in Table 5. Compared with the control group, the composite microbial agent has a stronger ability to utilize sugar. During fermentation, it produces 35.0% more gas than the control group to expand the dough, making its specific volume larger, and the chewiness is also lower, and the taste is more suitable. The polysaccharide content in the steamed bread fermented with the addition of composite microbial agents is more than 29.6% higher than that of the control group. A total of 52 volatile compounds were found, including 16 alcohols, 11 aldehydes, 9 ketones, 9 esters, 6 acids and 1 other compound. The total volatile flavor substance content is more than 27.7% higher than that of the three control groups, making the steamed bread fragrant.

[0141] Table 5 Steamed bread test data

[0142]

[0143] Example 9: Application of composite microbial agent in fermentation of rice wine

[0144] The washed, cooked and cooled glutinous rice is poured into a glass bottle, water and glutinous rice are mixed evenly according to a solid-liquid ratio of 1:4, and after beating with a beater, α-amylase, 0.5% w / w composite microbial agent and 0.5% w / w commercial distiller's yeast are added, and fermentation is carried out in a constant temperature box at 28.0° C. After the fermentation is completed, the fermented original wine is obtained by preliminary filtration, wherein the control group 1 is added with 0.5% w / w Lactobacillus plantarum L23104 and 0.5% w / w commercial distiller's yeast, the control group 2 is added with 0.5% w / w abnormal Wickham yeast W23087 and 0.5% w / w commercial distiller's yeast, and the control group 3 is added with 1.0% w / w commercial distiller's yeast, and the remaining methods are the same as those of the experimental group; the total acid, total ester and alcohol content are determined with reference to "GB / T 10781.3-2006 Rice-flavored Liquor"; the volatile flavor substances are determined with reference to Example 4.

[0145] The results are shown in Table 6. The rice wine obtained by the composite microbial inoculant fermentation was clear and transparent without suspended matter, and the alcohol content was 26.0% vol, while the control group 1 was 22.0% vol, the control group 2 was 21.0% vol, and the control group 3 was 24.0% vol. The total content of flavor substances in the rice wine obtained by the composite microbial inoculant fermentation was high. A total of 57 volatile substances were detected in the obtained rice wine, including 17 ester substances, 15 alcohol substances, 10 acid substances, 5 aldehydes and ketones, and 7 other substances. The rice wine had a pure rice aroma and a mellow body.

[0146] Table 6 Rice wine testing data

[0147]

[0148] Example 10: Application of composite microbial agent in wine fermentation

[0149] Select fully mature grapes, crush them and put them into the tank. The raw materials cannot exceed 3 / 4 of the container. The inoculation amount of the composite microbial agent and the commercial composite fermentation agent is 2.5% w / w, the sulfur dioxide addition amount is 30.0 mg / L, the pectinase addition amount is 0.01-0.03 g / L, and the cold soaking is 24.0h-48.0h. The control is not added with the composite microbial agent. The sugar content and specific gravity are measured every day during the fermentation process, and the cap is pressed three times. When the sugar content drops to 4.0g / L and the specific gravity drops to 0.8g / mL, the fermentation ends, and the juice is taken for clarification and bottle storage. The total acid (in terms of tartaric acid), volatile acid (in terms of acetic acid), total sugar (in terms of glucose), alcohol content, total SO2, free SO2 and pH value in the wine are determined by "GB / T15038-2006 General Analysis Method for Grape and Fruit Wine". The determination of volatile flavor substances is the same as in Example 4.

[0150] The results showed that the alcohol content of the wine with composite microbial agents was 14.0% vol, while that of the control group was 12.0% vol. The contents of total acid and volatile acid were 15.0% and 21.0% higher than those of the control group, making the wine with composite microbial agents taste fresh, and the other physical and chemical indicators were normal. There were 15 kinds of alcohol aroma substances, 13 kinds of ester substances, and 5 kinds of other types of aroma compounds in the volatile flavor substances. The total ester content of the experimental group was 5.03g / L, and the total ester content of the control group was 4.51g / L, which was 10.34% higher than that of the control group. The ethyl acetate content was 0.36μg / L, and the ethyl acetate content of the control group was 0.28μg / L, so the fruity aroma of the experimental group was more intense.

[0151] Example 11: Application of composite microbial agent in fermentation of apple cider vinegar

[0152] Wash fresh apples and cut them into pieces. Add the same mass of pH 4.0 citric acid solution and 0.1% w / w pectinase, soak for 10.0 min, and then break them into apple puree. After enzymolysis at 45.0℃ for 2.0 h, inactivate the enzyme at 80.0℃ for 10.0 min, cool and squeeze the juice for later use. Adjust the initial sugar content of the puree to 15.0°Brix, inoculate 2.5% w / w composite microbial agent and 2.5% w / w commercial composite fermentation agent for alcohol fermentation for 24.0 h.

[0153] After the alcohol fermentation was completed, 2.5% w / w acetic acid bacteria (purchased from Testo Biotechnology, item number TS349550) was inoculated for acetic acid fermentation to obtain acetic acid fermentation liquid. After centrifugation in a high-speed centrifuge for 10.0 min, the supernatant was taken and pasteurized for 20.0 min to obtain the finished apple cider vinegar, and the commercial composite fermentation agent fermentation was used as the control group. The physical and chemical indicators of the composite microbial agent in apple cider vinegar were determined in accordance with "GB / T30884-2014 Apple Cider Vinegar Beverage".

[0154] The results showed that the composite microbial agent apple vinegar had a clear color, no suspension, and normal physical and chemical indicators. The total ester content of the experimental group was 4.13g / L, and the total ester content of the control group was 2.03g / L. The volatile flavor substances of phenylacetaldehyde 4.31μg / L, ethyl isovalerate 4.17μg / L, phenylethyl acetate 2.77μg / L, and phenylethanol content were significantly higher than the control group by 37.0%. The polysaccharide content of the experimental group was 2.22g / L, and the polysaccharide content of the control group was 1.04g / L, making the apple vinegar in the experimental group rich in fruit flavor and sweet and sour.

[0155] Example 12: Application of composite microbial agent in fermentation of roxburghii juice

[0156] Take the fresh fruit of roxburghii, peel it, mix water and roxburghii with a material-liquid ratio of 1:4, use a juicer to squeeze the juice and filter it, add 5.0% w / w white sugar, sterilize it by ultra-high temperature instantaneous sterilization (UHT), aseptically fill it, cool it for use, inoculate 3.0% w / w composite microbial agent and 3.0.% w / w commercial distiller's yeast (purchased from Angel flagship store), ferment it at 30.0°C for 24.0h, and use no composite microbial agent, only 6% w / w commercial distiller's yeast as a control. The content of extracellular polysaccharide in roxburghii juice refers to Example 1; the determination of volatile flavor substances in roxburghii juice refers to Example 4.

[0157] The results showed that the extracellular polysaccharide content of the composite microbial agent sea buckthorn juice was 10.13 μg / L, 12% higher than that of the control group. The volatile flavor substances in the composite microbial agent sea buckthorn juice were mainly aldehydes (6 kinds), alcohols (4 kinds), hydrocarbons (4 kinds) and ketones (2 kinds). The total ester content (4.17 g / L) was 17.5% higher than that of the control group (3.44 g / L), among which the content of ethyl isovalerate was the highest at 4.88 μg / L. The other physical and chemical indicators were normal. The sea buckthorn juice was sweet and mellow without any peculiar smell.

[0158] Example 13: Application of composite microbial agent in fermented chili sauce

[0159] Select fresh and intact peppers, wash them, remove the stems and dry them, crush them and weigh them for later use. Boil the fermentation tank in boiling water for 15.0 minutes and then dry them for later use. Add 5.0% w / w salt, inoculate 1.0% w / w composite microbial agent and mix them well. Inoculate the same amount of abnormal Wickham yeast W23087 and plant lactobacillus L23104 as the control group, and ferment at 30.0℃ for 2.0 days.

[0160] 1. pH value determination: refer to the method of GB 5009.237-2016;

[0161] 2. Determination of capsaicin content: Determine according to the method of "GB / T 30389-2013 Determination of total capsaicin content in pepper and its oleoresin";

[0162] 3. Determination of organic acid content: Weigh 5.0g of pepper sample and dry it in an oven at 60.0℃ until the moisture content is less than 15.0%, add methanol; 25.0mL of tetrahydrofuran (1:1) solution, seal with plastic wrap and extract in a 60.0℃ water bath with ultrasonic vibration for 1.0h, filter and repeat the above operation twice, combine the filtrate and rotary evaporate to 10.0mL at 70.0℃ and make up to 50.0mL. Filter through a 0.45μm filter membrane for chromatographic analysis. Chromatographic conditions: Chromatographic column: Thermo Fisher C18 (4.6×250mm, 5μm); Mobile phase: methanol: water = 80:20 (V:V); Flow rate is 0.8mL / min; Injection volume: 20.0μL; Column oven temperature: 30.0℃; Detection wavelength: 280.0nm;

[0163] 4. The determination of volatile flavor substances was carried out according to Example 4.

[0164] The results showed that the pH changes between the experimental group and the control group were not obvious; before fermentation, the capsaicin content was as high as 0.68 mg / g, and after fermentation, the capsaicin content was significantly reduced to 0.28 mg / g. The polysaccharide content of the experimental group was 2.38 g / L, and the polysaccharide content of the control group was 1.74 g / L. The spicy taste of the chili sauce in the experimental group was softened, making it more acceptable.

[0165] The results are shown in Table 7. The results show that the trend of total organic acid increasing first and then decreasing during the fermentation process may be due to the use of aromatic esters by microorganisms in the late fermentation period. A total of 152 volatile components of chili sauce were detected during the fermentation process, including 45 alcohols, 35 esters, 19 aldehydes, 11 acids, 17 ketones, 9 alkenes, 3 phenols, 11 alkanes and 2 other components. The total ester content of the experimental group was 7.61 g / L, and the total ester content of the control group was 4.33 g / L, which was 43.10% higher than that of the control group. The ethyl acetate content was 2.74 μg / L, the isopentyl acetate content was 2.49 μg / L, and the phenylethanol content was 1.12 μg / L. Therefore, the mixed fermentation of Lactobacillus plantarum L23104 and abnormal Wickham yeast W23087 can make the chili sauce taste and flavor richer.

[0166] Table 7 Changes in organic acid content of composite microbial agents in fermented chili sauce

[0167]

[0168] Example 14: Application of composite microbial agent in fermented tomato paste

[0169] Fresh tomatoes were washed, scalded, peeled and pulped, boiled and concentrated to a soluble solid content of 20.0.% to 40.0%, 1.0% w / w salt and 5.0% w / w crystal sugar were added and pasteurized, 1.0% w / w composite microbial agent and commercial composite fermentation agent were added after cooling, and fermented at 30.0°C for 5.0 days to obtain the tomato paste, with no composite microbial agent added and only 1.0% w / w commercial composite fermentation agent added as a control. The pH value was determined according to the method of GB 5009.237-2016; the total acid was determined according to the method of GB 12456-2021 National Food Safety Standard for Determination of Total Acid in Food; the determination of nitrite was determined according to the spectrophotometric method in GB 5009.33-2016 National Food Safety Standard for Determination of Nitrite and Nitrite in Food; the determination of volatile flavor substances in tomato sauce was carried out according to Example 4.

[0170] The results showed that the total acid content of the composite microbial agent tomato sauce was significantly higher than that of the control group, which effectively inhibited the production of nitrite by harmful microorganisms, making the nitrite content of the experimental group 3.3mg / kg, which is within the existing standard. The total ester content of the experimental group was 5.35g / L, and the total ester content of the control group was 4.97g / L. The total amount of volatile flavor substances was 7.10% higher than that of the control group, making the tomato sauce have a good odor coordination, rich sauce flavor, and delicious taste.

[0171] Example 15: Application of composite microbial inoculants in fermented wire-drawn yogurt

[0172] Pure milk was preheated at 450°C for 10.0 min, 1.0% w / w of extracellular polysaccharide produced by Lactobacillus plantarum L23104 was added, and the milk was pasteurized at 68.0°C for 30.0 min, then allowed to cool, inoculated with 0.1% w / w of composite microbial agent and 0.1% w / w of commercial composite fermentation agent, fermented in an incubator at 40.0°C for 10.0 h, and stored in a refrigerator. Control group 1 was added with 0.1% w / w Lactobacillus plantarum L23104 and 0.1% w / w commercial composite starter, control group 2 was added with 0.1% w / w abnormal Wickham yeast W23087 and 0.1% w / w commercial composite starter, and control group 3 was added with 0.2% w / w commercial composite starter. The other methods were the same as the experimental group; the pH value was determined according to the method of GB 5009.237-2016; the total acid was determined according to the method of GB 12456-2021 National Food Safety Standard Determination of Total Acid in Food; the total colony count was determined according to GB 4789.2-2022 National Food Safety Standard Food Microbiology Inspection Colony Count Determination" method is used for determination; the texture characteristics of yogurt are determined by a texture analyzer, including hardness, elasticity, resilience, cohesion, viscosity, chewiness and other indicators; the stringiness is determined by a texture analyzer, by squeezing the yogurt through a piston probe and then rising back at a certain speed. The rising distance can reflect the stringiness; the determination of volatile flavor substances in stringy yogurt is carried out with reference to Example 4.

[0173] The results are shown in Table 8. The wire drawing results of the experimental group were 17.23 mm, the control group 1 was 12.11 mm, the control group 2 was 11.45 mm, and the control group 3 was 10.55 mm. The addition of extracellular polysaccharides produced by Lactobacillus plantarum L23104 improved the viscosity and adhesiveness of the wire drawing yogurt, and the yogurt tasted soft. There were 40 volatile flavor substances in the experimental group, of which ethyl acetate accounted for 23.78%, isoamyl acetate accounted for 19.92%, and acetaldehyde accounted for 19.76%. There were 34 volatile flavor substances in the control group, of which ethyl acetate accounted for 20.21%, isoamyl acetate accounted for 15.53%, and acetaldehyde accounted for 14.50%. The flavor substances in the experimental group were richer and had a strong frankincense flavor.

[0174] Table 8 Wire-drawn yogurt test data

[0175]

[0176]

[0177] Example 16: Application of composite microbial agent in fermenting gluten-free dough

[0178] Commercially available buckwheat flour was added with 60.0% distilled water, inoculated with 1.0% w / w composite microbial agent and 1.0% w / w commercial composite leavening agent, the addition amount was based on the dry powder ratio, mixed to form gluten-free dough, and fermented in a 40.0°C proofing box for 10.0 hh, with only 1.0% w / w commercial composite leavening agent inoculated as a control. The texture characteristics of the dough were measured by a texture analyzer, including hardness, elasticity, resilience, cohesion, viscosity, chewiness and other indicators; the exopolysaccharide content in the dough was referred to Example 1; the volatile flavor substances in the dough were measured by reference to Example 4.

[0179] The results showed that the dough hardness of the experimental group was 113.1g, and the dough hardness of the control group was 153.5g. The hardness of the experimental group was 26.3% lower than that of the control group, which was conducive to the subsequent dough processing. The polysaccharide content of the experimental group (4.85g / L) was 22.27% higher than that of the control group (3.77g / L), which improved the rheological properties of the gluten-free dough. The total ester content of the experimental group was 3.79g / L, and the total ester content of the control group was 1.17g / L. The gluten-free dough contained 9 kinds of alcohols, 5 kinds of esters, 3 kinds of aldehydes, 2 kinds of ketones and other volatile compounds. The most abundant alcohol component was phenylethanol (10.9%), the types of volatile substances were 10.0% higher than those of the control group, and the phenylethanol content was 23.4% higher than that of the control group. It showed citrus and floral aromas, and a strong fermented flavor.

[0180] Example 17: Application of composite microbial agent in fermented multi-grain cake

[0181] Based on the mass of dry flour, 20.0% w / w buckwheat flour, 20.0% w / w highland 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 extracellular polysaccharide composition bacteria agent and commercial composite leavening agent (experimental group) were mixed and kneaded in a basin until the dough was about to form a hand mask, 8.0% w / w butter was added, and when the hand mask was kneaded until it was thin and transparent, it was put into a proofing box with a temperature of 28.0°C and a humidity of 75.0% for the first proofing, and then put into a proofing box with a temperature of 35.0°C and a humidity of 75.0% for the second proofing. After the proofing, a layer of egg liquid was brushed on the surface, and the dough was put into an oven and baked at 180°C for 15.0 min. After cooling, it was refrigerated and stored. The control group was not added with the extracellular polysaccharide composition bacteria agent, and only 1.0% w / w commercial composite leavening agent was added. The texture characteristics of the multi-grain cake were measured by a texture analyzer; the cake quality evaluation was measured with reference to "GB / T35869-2018 Grain and Oil Inspection Wheat Flour Bread Baking Quality Evaluation Rapid Baking Method"; the determination of volatile flavor substances in multi-grain cake was carried out with reference to Example 4.

[0182] The results showed that the polysaccharide content in the experimental group was 5.12 g / L, while that in the control group was 3.54 g / L, which made the hardness and chewiness of the multi-grain cake in the experimental group the lowest, and the texture evaluation was good. The total ester content in the experimental group was 9.21 g / L, while that in the control group was 4.48 g / L. 69 and 58 volatile flavor substances were detected in the experimental group and the control group, respectively, including alcohols, aldehydes, esters, acids, ketones, aromatic heterocyclics, and alkanes. The ester content in the experimental group was 20.1%, which was higher than 25.5% in the control group, making the cake fragrant.

[0183] Example 18: Application of composite microbial agent in fermentation of flavored fermented bean curd

[0184] Take 25.0g of soybeans, remove impurities, wash and soak for 10.0h, drain and grind, filter the pulp with 4 layers of gauze, boil in 100℃ boiling water for 5.0min, add brine to make pulp, and press it into white blank. Inoculate the Mucor suspension on the white blank by spraying, and then culture it in a constant temperature chamber at 25.0℃ for 48h to obtain the blank. Take 10.0g of the blank, add salt, and inoculate 1.0% w / w composite microbial agent (experimental group) and 1.0% w / w commercial composite fermentation agent (control group) for 7 days. Drain the pickled blank, fill the soup into the can, evacuate and place it at 25.0℃ for late fermentation to obtain fermented bean curd. The total colony count was determined according to the method of "GB 4789.2-2022 National Food Safety Standard Food Microbiology Inspection Colony Count Determination"; the texture characteristics of fermented bean curd were determined by a texture analyzer, including hardness, elasticity, resilience, cohesion, viscosity, chewiness and other indicators; the determination of volatile flavor substances in fermented bean curd was carried out according to Example 4.

[0185] 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, which was 63% higher than that in the control group. There are 98 volatile components in fermented bean curd, 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 gave the fermented bean curd a mellow wine aroma.

Claims

1. A composite microbial agent, characterized in that: Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L23104 and Wickham's yeast ( Wickerhamomyces anomalus ) W23087; wherein the bacterial cell count ratio of Lactobacillus plantarum L23104 and abnormal Wickham yeast W23087 is 1:0.8; The Lactobacillus plantarum ( Lactiplantibacillus plantarum )L23104, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 18, 2024, with the deposit number CGMCC No.29668; The abnormal Wickham yeast ( Wickerhamomyces anomalus )W23087, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 18, 2024, with the deposit number CGMCC No.29669.

2. The composite microbial agent according to claim 1, characterized in that The number of bacteria in each gram or milliliter of the composite microbial agent is not less than 1.0×10 8 CFU.

3. The composite microbial agent according to any one of claims 1 to 2, characterized in that: The Lactobacillus plantarum L23104 and the abnormal Wickham yeast W23087 in the composite microbial agent are living cells or dry bacteria obtained by freeze-drying or immobilized cells.

4. A product, characterized in that The product comprises the composite microbial agent according to any one of claims 1 to 3; the product is a fermentation preparation.

5. Use of the composite microbial agent according to any one of claims 1 to 3 in the preparation of fermented flour products, fermented wines, fermented fruit juices, fermented dairy products or fermented condiments.

6. The use according to claim 5, characterized in that: The fermented flour products include bread, steamed buns and cakes.

7. The use according to claim 5, characterized in that: The fermented alcoholic beverages include rice wine and grape wine; the fermented dairy products include wire-drawn yogurt and cheese.

8. The use according to claim 5, characterized in that: The fermented fruit juices include apple cider vinegar and sea buckthorn juice; the fermented condiments include chili sauce, tomato sauce and fermented bean curd.

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