Weissella fusiformis JMF-006 as well as mutagenesis method and application thereof

Abstract

The invention relates to the technical field of microbial fermentation and food processing, and particularly discloses Weissella fusiformis JMF-006 as well as a mutagenesis method and application thereof. The invention provides a Weissella fusiformis JMF-006 with the exopolysaccharide yield of 20-25g / L. The Weissella fusiformis JMF-006 is obtained by breeding through a mutation breeding method of a high-yield EPS strain, and the mutation breeding method can also be used for culturing other high-yield EPS strains. The invention provides a strain composition, the strain composition comprises Weissella fusiformis JMF-006 or a high-yield EPS strain, other lactic acid bacteria strains (such as lactobacillus plantarum JMF-001 and lactobacillus casei JMF-002) can be further compounded, and the obtained strain composition has more excellent fermentation performance. When the Weissella fusiformis JMF-006, the high-yield EPS strain and the strain composition provided by the invention are applied to the fermentation process of food-derived drinks, a fermented product with stable properties and better flavor can be obtained, and the technical problems that the yield of extracellular polymeric substances is low, the fermentation performance is poor and the application range is narrow in an existing lactic acid bacteria starter are solved.

Description

Technical Field

[0001] This application relates to the fields of microbial fermentation and food processing technology, and in particular to a fusion of Weissella JMF-006 and a mutagenesis method and application. Background Technology

[0002] As a widely consumed fermented food, the textural properties and storage stability of yogurt and plant-based yogurt are key indicators determining consumer acceptance. Currently, commercially available products commonly add chemically synthesized or naturally derived colloids such as gelatin, pectin, and carrageenan to improve water retention, inhibit whey separation, and optimize sensory texture. However, with the increasing consumer demand for "clean label" products and natural, healthy foods, reducing or even completely replacing exogenous additives has become a critical issue that urgently needs to be addressed in the food fermentation industry.

[0003] Using exopolysaccharide-producing lactic acid bacteria as a starter culture to synthesize polysaccharides through in-situ microbial fermentation and thus regulate product texture is a highly promising technological approach to address the aforementioned needs. EPS can serve as a natural thickener and stabilizer, effectively increasing the viscosity of the fermentation system, optimizing mouthfeel smoothness, and enhancing water-holding capacity. However, existing lactic acid bacteria strains generally suffer from limited EPS synthesis capabilities, and the fermentation characteristics of a single strain make it difficult to achieve a synergistic balance between EPS synthesis, lactic acid production, and flavor compound accumulation. Furthermore, plant-based yogurts (such as soy-based yogurts) are more prone to problems such as coarse texture, poor system stability, and whey separation due to their protein composition and molecular structure, making the need for stabilizers even more urgent. Therefore, developing highly efficient EPS-producing lactic acid bacteria strains suitable for plant-based fermentation substrates presents a greater technological challenge.

[0004] In summary, there is an urgent need in this field to screen or breed lactic acid bacteria strains that produce high EPS, have excellent genetic stability, and are adaptable to a variety of fermentation substrates, so as to provide technical support for the green processing and quality improvement of yogurt and plant-based yogurt. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a fusion of Weissella JMF-006 and a mutagenesis method and application to solve the technical problems of low extracellular polymer yield, poor fermentation performance and narrow applicability of existing lactic acid bacteria starter cultures.

[0006] To achieve the above technical objectives, this application provides a method for mutagenesis of a high-yield EPS strain, comprising the following steps:

[0007] Step S1: Lactic acid bacteria are isolated from soy products that change from liquid to solid yogurt at room temperature, activated, and then placed on an MRS plate containing CaCO3 for purification and culture.

[0008] Step S2: Select colonies with a calcium dissolution zone diameter greater than 5 mm, and perform microwave mutagenesis, chemical mutagenesis, and high-throughput screening in sequence to obtain strains with EPS yield greater than 20 g / L.

[0009] Step S3: Subculture the strain with EPS yield greater than 20 g / L until a high-yielding EPS strain with stable EPS yield of 20-25 g / L is obtained.

[0010] This application provides a high-EPS-producing strain, obtained by mutagenesis of high-EPS-producing strains.

[0011] This application provides a fusion strain of Weissella confusa JMF-006, which was obtained by the above-mentioned mutagenesis method. The fusion strain of Weissella confusa JMF-006 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64776.

[0012] This application provides a strain composition comprising fusion Weissella JMF-006 or a high EPS-producing strain.

[0013] Furthermore, it also includes: Lactobacillus plantarum JMF-001, with accession number CCTCC NO: M2023025; and Lactobacillus casei JMF-002, with accession number CCTCC NO: M2023026.

[0014] This application provides a compound fermentation powder, comprising a fusion of Weissella JMF-006 or a high-EPS-producing strain or a combination of strains.

[0015] This application provides a method for preparing compound fermentation powder, comprising the following steps: inoculating a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains into a lactic acid bacteria liquid culture medium at a preset ratio, fermenting at a constant temperature to obtain a fermentation broth, and then freeze-drying the fermentation broth to obtain compound fermentation powder.

[0016] This application provides an application of a fusion strain of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder for the preparation of fermentation agents, dietary supplements, and for use in food fermentation.

[0017] This application provides a soy yogurt, which uses a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder as a starter to ferment a soy-based beverage to obtain soy yogurt.

[0018] This application provides a yogurt, which uses a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder as a starter culture to ferment a dairy beverage to obtain yogurt.

[0019] In summary, this application provides a strain of Fusion Weissella JMF-006, which has an extracellular polysaccharide yield of 20-25 g / L.

[0020] This application also provides a method for mutagenesis and selection of high-EPS-producing strains. Lactic acid bacteria are isolated from soy-based beverages that have been cultured at room temperature for several days. These bacteria are then subjected to microwave mutagenesis, chemical mutagenesis, high-throughput screening, and subculturing to ultimately obtain a high-EPS-producing strain. The mutagenesis and selection method provided in this application can be used to cultivate strains with highly efficient EPS synthesis capabilities.

[0021] This application provides a lactic acid bacteria composition comprising Fusion Weissella JMF-006 or the above-mentioned high EPS-producing strain, and may further be compounded with other lactic acid bacteria strains (such as Lactobacillus plantarum JMF-001 and Lactobacillus casei JMF-002), the resulting lactic acid bacteria composition having excellent fermentation performance.

[0022] Compared with existing technologies, the Fusion Weissella JMF-006, the high-EPS-producing strain and its mutagenesis and selection method, and the lactic acid bacteria composition provided in this application all have good EPS synthesis functions; when applied to the fermentation process of food-derived beverages (including animal-derived beverages and plant-derived beverages), stable fermented products can be obtained without the need for additional exogenous stabilizers. Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments in this application specification, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection claimed in this application.

[0024] In the description of this application, it should be noted that the terms "first", "second", and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] Unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections or electrical connections; they can refer to direct connections or indirect connections through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0026] The raw materials used in this invention are not particularly restricted in their source; they can be purchased on the market or prepared using conventional methods known to those skilled in the art.

[0027] This application provides a method for mutagenesis of a high-EPS-producing strain, comprising the following steps:

[0028] Step S1: Lactic acid bacteria are isolated from soy products that change from liquid to solid yogurt at room temperature, activated, and then placed on an MRS plate containing CaCO3 for purification and culture.

[0029] Step S2: Select colonies with a calcium dissolution zone diameter greater than 5 mm, and perform microwave mutagenesis, chemical mutagenesis, and high-throughput screening in sequence to obtain strains with EPS yield greater than 20 g / L.

[0030] Step S3: Subculture the strain with EPS yield greater than 20 g / L until a high-yielding EPS strain with stable EPS yield of 20-25 g / L is obtained.

[0031] In some embodiments, a microwave oven with a power range of 200~700W is used for microwave mutagenesis treatment; wherein the irradiation distance is 10~20cm and the single irradiation time is set to 40~60s.

[0032] In some specific embodiments, a microwave oven with a power range of 500W is used for microwave mutagenesis treatment; the irradiation distance is 15cm, and the single irradiation time is set to 50s.

[0033] In some embodiments, nitrosoguanidine is used as a mutagen for chemical mutagenesis; wherein the concentration of the nitrosoguanidine solution is 150~250 μg / mL, the pH is 5.5~6.5, and the mutagenesis time is 30~50 min.

[0034] In some specific embodiments, nitrosoguanidine was used as a mutagen for chemical mutagenesis; wherein the concentration of the nitrosoguanidine solution was 200 μg / mL, the pH was 6.0, and the mutagenesis time was 40 min.

[0035] In some embodiments, the number of subcultures is controlled at 5 or more; in some more preferred embodiments, the number of subcultures is controlled at 10 or more. Through stability screening during multiple subcultures, it is ensured that the final strain has the ability to continuously and stably synthesize EPS.

[0036] In some embodiments, the lactic acid bacteria include at least one of Weissella fusionis and Lactobacillus plantarum.

[0037] This application provides a high-EPS-producing strain, obtained by mutagenesis of high-EPS-producing strains.

[0038] This application provides a fusion strain of Weissella confusa JMF-006, which was obtained by the above-mentioned mutagenesis method. The fusion strain of Weissella confusa JMF-006 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64776; the deposit date is June 20, 2024; the deposit location is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0039] This application provides a strain composition comprising fusion Weissella JMF-006 or a high EPS-producing strain.

[0040] In some embodiments, it further includes: Lactobacillus plantarum JMF-001, with accession number CCTCC NO: M2023025; and Lactobacillus casei JMF-002, with accession number CCTCC NO: M2023026.

[0041] In some preferred embodiments, the bacterial composition includes Lactobacillus plantarum JMF-001, Lactobacillus casei JMF-002, and Fusion Weissella JMF-006, with a live bacterial ratio of (1~3):(1~3):(2~4).

[0042] In some specific embodiments, the bacterial composition includes Lactobacillus plantarum JMF-001, Lactobacillus casei JMF-002, and Fusion Weissella JMF-006, with a live bacteria ratio of 2:2:3.

[0043] This application provides a compound fermentation powder, comprising a fusion of Weissella JMF-006 or a high-EPS-producing strain or a combination of strains.

[0044] In some embodiments, the viable bacteria count in the compound fermentation powder is ≥1×10⁻⁶. 11 CFU / g; in some preferred embodiments, the viable count in the compound fermentation powder is greater than 3.5 × 10⁻⁶.11 CFU / g.

[0045] This application provides a method for preparing compound fermentation powder, comprising the following steps: inoculating a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains into a lactic acid bacteria liquid culture medium at a preset ratio, fermenting at a constant temperature to obtain a fermentation broth, and then freeze-drying the fermentation broth to obtain compound fermentation powder.

[0046] This application provides an application of a fusion strain of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder for the preparation of fermentation agents, dietary supplements, and for use in food fermentation.

[0047] This application provides a soy yogurt, which uses a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder as a starter to ferment a soy-based beverage to obtain soy yogurt.

[0048] This application provides a yogurt, which uses a fusion of Weissella JMF-006 or a high-yield EPS strain or a combination of strains or a compound fermentation powder as a starter culture to ferment a dairy beverage to obtain yogurt.

[0049] The applicant further provides the following specific embodiments to describe the present invention. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0050] Example 1

[0051] This embodiment provides a mutagenesis method for fusing Weissella JMF-006, including the following steps:

[0052] Step S1, preliminary screening and culture of strains: Fresh soy milk was placed at room temperature for 3 days. After 3 days, the soy milk coagulated from liquid to solid yogurt. At this time, the soy milk had no odor. The strain was isolated from the solid yogurt-like soy milk and identified as *Weissella fusionis*. The EPS (measured by dextran) yield of this strain was 15 g / L.

[0053] Step S2, strain activation and purification: The above-mentioned fused Weissella strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for primary activation; the primary activated strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for secondary activation; the strain after two activations was streaked onto MRS plates containing 1.5% CaCO3 and cultured continuously at 37°C for 18-24 hours for purification; after purification, colonies with a calcium dissolution zone diameter greater than 5 mm were picked and purified for 3 more generations to obtain the first generation strain;

[0054] Step S3, Microwave mutagenesis: Take the first-generation strain and prepare a solution with a concentration of 10... 7 The bacterial suspension was prepared at CFU / mL and placed in a 500W microwave oven. The irradiation distance was set to 15cm and the irradiation time to 50s. During the irradiation, the bacterial suspension was alternately subjected to "10s ice bath - 10s no ice bath" treatment. After the irradiation, the bacterial suspension was spread on MRS plates and incubated at 37℃ in the dark for 24h to obtain the second generation strain.

[0055] Step S4, chemical mutagenesis: The second-generation strain was dispersed in a 200 μg / mL nitrosoguanidine (NTG) solution with a pH of 6.0, and then placed in a constant temperature of 30°C and shaken for 40 min. After shaking, the mixture was placed in an ice bath for 10 min to terminate the mutagenesis reaction and obtain the third-generation strain.

[0056] Step S5, high-throughput screening: The third-generation strain was spread on MRS plates and fermented continuously at 37°C for 24 hours. Colonies with a slimy appearance and obvious elevation were picked for shake-flask fermentation. After fermentation, the EPS yield of the colonies was detected by the phenol-sulfuric acid method. Strains with an EPS yield greater than 20 g / L were selected as the fourth-generation strains.

[0057] Step S6, Genetic Stability Verification: The fourth-generation strain was passaged 10 times, and the EPS yield was measured in each generation. After the 10th generation, the fifth-generation strain with an EPS yield of 25 g / L was obtained, which is the Fusion Weissella JMF-006.

[0058] Example 2

[0059] This embodiment provides a mutagenesis method for fusing Weissella JMF-006, including the following steps:

[0060] Step S1, preliminary screening and culture of strains: Fresh soy milk was placed at room temperature for 3 days. After 3 days, the soy milk coagulated from liquid to solid yogurt. At this time, the soy milk had no odor. The strain was isolated from the solid yogurt-like soy milk and identified as *Weissella fusionis*. The EPS (measured by dextran) yield of this strain was 15 g / L.

[0061] Step S2, strain activation and purification: The above-mentioned fused Weissella strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for primary activation; the primary activated strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for secondary activation; the strain after two activations was streaked onto MRS plates containing 1.5% CaCO3 and cultured continuously at 37°C for 18-24 hours for purification; after purification, colonies with a calcium dissolution zone diameter greater than 5 mm were picked and purified for 3 more generations to obtain the first generation strain;

[0062] Step S3, Microwave mutagenesis: Take the first-generation strain and prepare a solution with a concentration of 10... 7 The bacterial suspension was prepared at CFU / mL and placed in a 400W microwave oven. The irradiation distance was set to 15cm and the irradiation time to 50s. During the irradiation, the bacterial suspension was alternately subjected to "10s ice bath - 10s no ice bath" treatment. After the irradiation, the bacterial suspension was spread on MRS plates and incubated at 37℃ in the dark for 24h to obtain the second generation strain.

[0063] Step S4, chemical mutagenesis: The second-generation strain was dispersed in a 300 μg / mL nitrosoguanidine (NTG) solution with a pH of 6.0, and then placed in a constant temperature of 30°C and shaken for 40 min. After shaking, the mixture was placed in an ice bath for 10 min to terminate the mutagenesis reaction and obtain the third-generation strain.

[0064] Step S5, high-throughput screening: The third-generation strain was spread on MRS plates and fermented continuously at 37°C for 24 hours. Colonies with a slimy appearance and obvious elevation were picked for shake-flask fermentation. After fermentation, the EPS yield of the colonies was detected by the phenol-sulfuric acid method. Strains with an EPS yield greater than 20 g / L were selected as the fourth-generation strains.

[0065] Step S6, Genetic Stability Verification: The fourth-generation strain was passaged 10 times, and the EPS yield was measured in each generation. After the 10th generation, the fifth-generation strain with an EPS yield of 24 g / L was obtained. The fifth-generation strain was Weissella fusion strain JMF-006.

[0066] Example 3

[0067] This embodiment provides a mutagenesis method for fusing Weissella JMF-006, including the following steps:

[0068] Step S1, preliminary screening and culture of strains: Fresh soy milk was placed at room temperature for 3 days. After 3 days, the soy milk coagulated from liquid to solid yogurt. At this time, the soy milk had no odor. The strain was isolated from the solid yogurt-like soy milk and identified as *Weissella fusionis*. The EPS (measured by dextran) yield of this strain was 15 g / L.

[0069] Step S2, strain activation and purification: The above-mentioned fused Weissella strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for primary activation; the primary activated strain was inoculated into MRS liquid medium and cultured continuously at 37°C for 18-24 hours for secondary activation; the strain after two activations was streaked onto MRS plates containing 1.5% CaCO3 and cultured continuously at 37°C for 18-24 hours for purification; after purification, colonies with a calcium dissolution zone diameter greater than 5 mm were picked and purified for 3 more generations to obtain the first generation strain;

[0070] Step S3, Microwave mutagenesis: Take the first-generation strain and prepare a solution with a concentration of 10... 7 The bacterial suspension was prepared at CFU / mL and placed in a 300W microwave oven. The irradiation distance was set to 15cm and the irradiation time to 50s. During the irradiation, the bacterial suspension was alternately subjected to "10s ice bath - 10s no ice bath" treatment. After the irradiation, the bacterial suspension was spread on MRS plates and incubated at 37℃ in the dark for 24h to obtain the second generation strain.

[0071] Step S4, chemical mutagenesis: The second-generation strain was dispersed in a 200 μg / mL nitrosoguanidine (NTG) solution with a pH of 6.0, and then placed in a constant temperature of 30°C and shaken for 40 min. After shaking, the mixture was placed in an ice bath for 10 min to terminate the mutagenesis reaction and obtain the third-generation strain.

[0072] Step S5, high-throughput screening: The third-generation strain was spread on MRS plates and fermented continuously at 37°C for 24 hours. Colonies with a slimy appearance and obvious elevation were picked for shake-flask fermentation. After fermentation, the EPS yield of the colonies was detected by the phenol-sulfuric acid method. Strains with an EPS yield greater than 20 g / L were selected as the fourth-generation strains.

[0073] Step S6, Genetic Stability Verification: The fourth generation strain was passaged 10 times, and the EPS yield was measured in each generation. After the 10th generation, the fifth generation strain with an EPS yield of 20~21 g / L was obtained. The fifth generation strain was Weissella fusion strain JMF-006.

[0074] Comparative Example 1

[0075] This comparative example provides a mutagenesis method for fusion Weissella JMF-006, which differs from Example 1 in that it only uses microwave mutagenesis as described in Example 1, while all other steps are the same as in Example 1. The EPS yield of the fusion Weissella obtained in this comparative example is 15 g / L.

[0076] Comparative Example 2

[0077] This comparative example provides a mutagenesis method for fused Weissella, which differs from Example 1 in that it only uses the chemical mutagenesis method of Example 1, while all other steps are the same as in Example 1. The EPS yield of the fused Weissella obtained in this comparative example is 15 g / L.

[0078] Preparation Example 1

[0079] This preparation example provides a compound fermentation powder, including the following preparation steps:

[0080] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0081] Step S2: A composite strain (with a viable count of 2.5 × 10⁻³) is prepared by mixing *Lactobacillus plantarum* JMF-001, *Lactobacillus casei* JMF-002, and *Weissella fusion* JMF-006. 9 The compound strain (CFU / mL) was inoculated into the culture medium at an inoculum of 1%, and fermented at a constant temperature of 37°C for 20 hours to obtain the fermentation broth.

[0082] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0083] Application Example 1

[0084] This application provides a yogurt without added stabilizers, which is obtained by the following preparation steps:

[0085] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0086] In step S2, the compound fermentation starter culture powder prepared in Preparation Example 1 was inoculated into the milk culture medium at an inoculation rate of 0.005%. Fermentation was carried out at a constant temperature of 30°C until the pH of the milk culture medium reached 4.6. The milk culture medium was then placed at 4°C and allowed to stand for 24 hours to obtain yogurt. The yogurt obtained in this example has a fine texture, and its viscosity is 180% higher than that of Comparative Application Example 5. Furthermore, no whey separation occurred even after 90 days of storage at 4°C. The EPS yield of the combination of *Lactobacillus plantarum* JMF-001, *Lactobacillus casei* JMF-002, and *Weisseria fusion* JMF-006 strains provided in this example is 12-15 g / kg.

[0087] Application Example 2

[0088] This application example provides a soy yogurt without added stabilizers, which is obtained through the following steps:

[0089] Step S1 involves soaking, grinding, and boiling soybeans from Northeast China to produce soy milk with 3.0% protein and 2.5% fat.

[0090] In step S2, the compound fermentation starter culture powder prepared in Example 2 was inoculated into soy milk at an inoculation rate of 0.005%. Fermentation was carried out at a constant temperature of 30°C until the pH of the soy milk reached 4.6. The soy milk was then allowed to stand at 4°C for 24 hours to obtain soy yogurt. The soy yogurt obtained in this application example has a uniform, firm, and elastic texture, a smooth mouthfeel, and a water-holding capacity of 98.5%. Furthermore, no whey separation occurred even after 90 days of storage at 4°C.

[0091] Application Example 3

[0092] This application example provides a method for preparing yogurt using JMF-006 alone for fermentation. The preparation steps are as follows:

[0093] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0094] Step S2: Inoculate the bacterial suspension containing JMF-006 (viable count 3.5 × 10⁻⁶) at a dosage of 0.6%. 10 (CFU / mL) was inoculated into milk culture medium and fermented at a constant temperature of 30°C for 48 hours to obtain yogurt. The yogurt had an acidity of 35~40°, a firm and elastic texture, and no whey separation even after storage at 4°C for 90 days, with an acidity change of less than 1%.

[0095] In this application example, the bacterial suspension containing JMF-006 is prepared from a compound fermentation powder containing JMF-006. The preparation method of the compound fermentation powder containing JMF-006 is as follows:

[0096] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0097] Step S2, fusion with Weissella JMF-006 (viable count 2.5 × 10⁶) 9 The CFU / mL concentration was inoculated into the culture medium of the strain at a rate of 1%, and fermented at a constant temperature of 37°C for 20 hours to obtain the fermentation broth.

[0098] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0099] Comparative Application Example 1

[0100] This comparative application example provides a yogurt fermented solely with Lactobacillus plantarum JMF-001. The yogurt preparation steps are as follows:

[0101] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0102] Step S2: Inoculate a bacterial suspension containing *Lactobacillus plantarum* JMF-001 (with a viable count of 3.5 × 10⁻⁶) at a concentration of 0.6%. 10 The *Lactobacillus plantarum* (CFU / mL) was inoculated into milk culture medium and fermented at 30°C for 48 hours to obtain yogurt. In this comparative application, the EPS yield of *Lactobacillus plantarum* JMF-001 was 1~2 g / L; the acidity of the yogurt reached 20°, and whey separated after storing the yogurt at 4°C for 3 days.

[0103] In this application example, the bacterial suspension containing *Lactobacillus plantarum* JMF-001 is prepared from a compound fermentation powder containing *Lactobacillus plantarum* JMF-001. The preparation method of the compound fermentation powder containing *Lactobacillus plantarum* JMF-001 is as follows:

[0104] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0105] Step S2, add Lactobacillus plantarum JMF-001 (live count 2.5 × 10⁻⁶) 9 The CFU / mL concentration was inoculated into the culture medium at a 1% inoculum and fermented at 37°C for 20 hours to obtain the fermentation broth.

[0106] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0107] Comparative Application Example 2

[0108] This comparative application example provides a yogurt fermented solely with Lactobacillus casei JMF-002. The yogurt preparation steps are as follows:

[0109] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0110] Step S2: Inoculate a bacterial suspension containing Lactobacillus casei JMF-002 (viable count 3.5 × 10⁻⁶) at a dosage of 0.6%. 10 The *Lactobacillus casei* JMF-002 was inoculated into milk culture medium and fermented at 30°C for 48 hours to obtain yogurt. In this comparative application, the EPS yield of *Lactobacillus casei* JMF-002 was 1~2 g / L; the acidity of the yogurt reached 70°, and whey separated after the yogurt was stored at 4°C for 3 days.

[0111] In this application example, the bacterial suspension containing Lactobacillus casei JMF-002 is prepared from a compound fermentation powder containing Lactobacillus casei JMF-002. The preparation method of the compound fermentation powder containing Lactobacillus casei JMF-002 is as follows:

[0112] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0113] Step S2, add Lactobacillus casei JMF-002 (2.5 × 10⁻⁶ viable cells) 9 The CFU / mL concentration was inoculated into the culture medium of the strain at a rate of 1%, and fermented at a constant temperature of 37°C for 20 hours to obtain the fermentation broth.

[0114] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0115] Comparative Application Example 3

[0116] This comparative application example provides a yogurt prepared by co-fermentation of Lactobacillus plantarum JMF-001 and Lactobacillus casei JMF-002. The preparation steps of the yogurt are as follows:

[0117] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0118] Step S2: Inoculate a bacterial suspension containing *Lactobacillus plantarum* JMF-001 and *Lactobacillus casei* JMF-002 (viable count 3.5 × 10⁻⁶) at a concentration of 0.6%. 10 The yogurt was obtained by inoculating milk culture medium with cFU / mL and fermenting at 30°C for 48 hours. The acidity of the yogurt could reach 70°, and the whey separated after storing the yogurt at 4°C for 3 days. In this comparative application example, the EPS yield of the combination of Lactobacillus plantarum JMF-001 and Lactobacillus casei JMF-002 strains was 1.5~2.5 g / L.

[0119] In this application example, the bacterial suspension containing *Lactobacillus plantarum* JMF-001 and *Lactobacillus casei* JMF-002 was prepared from a compound fermentation powder. The preparation method of the compound fermentation powder is as follows:

[0120] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0121] Step S2: A compound strain (2.5 × 10⁻⁶ viable cells) is prepared by mixing *Lactobacillus plantarum* JMF-001 and *Lactobacillus casei* JMF-002 at a viable cell ratio of 1:1.9 The compound strain (CFU / mL) was inoculated into the culture medium at an inoculum of 1%, and fermented at a constant temperature of 37°C for 20 hours to obtain the fermentation broth.

[0122] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0123] Comparative Application Example 4

[0124] This comparative application example provides a soy yogurt produced by co-fermentation of *Weisseria fusiforme* JMF-006, commercially available *Lactobacillus plantarum*, and *Lactobacillus casei*. The preparation steps of the yogurt are as follows:

[0125] Step S1 involves soaking, grinding, and boiling soybeans from Northeast China to produce soy milk with 3.0% protein and 2.5% fat.

[0126] Step S2 involves preparing a bacterial suspension containing *Westernella fusion* JMF-006, commercially available *Lactobacillus plantarum*, and commercially available *Lactobacillus casei* (with a viable count of 3.5 × 10⁻⁶). 10 The bacterial suspension (CFU / mL) was inoculated into soy milk at an inoculum of 0.6%, and fermented at a constant temperature of 30°C for 48 hours to obtain soy yogurt. In this comparative application example, the acidity of the soy yogurt was below 60°, and when the soy yogurt was stored at 4°C for 3 days, no whey separation occurred, the soy yogurt had a firm texture, and the elasticity decreased slightly. The acidity change was less than 1%. In this comparative application example, the EPS yield of the combined strain of *Lactobacillus weisseri* JMF-006, commercially available *Lactobacillus plantarum*, and *Lactobacillus casei* was 7~9 g / L.

[0127] In this application example, the bacterial suspension containing *Westernella fusion* JMF-006, commercially available *Lactobacillus plantarum*, and commercially available *Lactobacillus casei* was prepared from a compound fermentation powder. The preparation method of the compound fermentation powder is as follows:

[0128] Step S1, prepare the culture medium for the strain: Mix 94 parts of soybean extract, 2.5 parts of glucose, 2 parts of lactose, and 1.5 parts of galactose, and then sterilize at 128°C for 15 seconds using UHT. After sterilization, cool to 37°C to obtain the culture medium for the strain.

[0129] Step S2: A composite strain (with a viable count of 2.5 × 10⁻⁶ viable bacteria) is prepared by mixing *Lactobacillus fusiforme* JMF-006, commercially available *Lactobacillus plantarum*, and commercially available *Lactobacillus casei* at a ratio of 3:2:2. 9The compound strain (CFU / mL) was inoculated into the culture medium at an inoculum of 1%, and fermented at a constant temperature of 37°C for 20 hours to obtain the fermentation broth.

[0130] Step S3: Add 2.2% maltodextrin, 1.8% trehalose, and 5.5% skim milk (by mass of the fermentation broth) to the fermentation broth, mix well, and pre-freeze at -40℃. Then, freeze-dry the mixture to obtain a compound fermentation powder with a viable count of 3.5 × 10⁻⁶ cells / day. 11 CFU / g.

[0131] Comparative Application Example 5

[0132] This comparative application example provides a method for preparing yogurt using commercially available Chr. Hansen yogurt starter (containing Streptococcus thermophilus and Lactobacillus bulgaricus), with the following fermentation steps:

[0133] Step S1: Add 6% sucrose, 1.2% fructooligosaccharides, and 0.8% galactooligosaccharides by weight of milk to milk with a protein content of 3.0% and a fat content of 2.5%. Then sterilize at 95°C for 5 minutes to obtain milk culture medium.

[0134] In step S2, a commercially available Chr. Hansen yogurt starter culture was inoculated into the milk culture medium at an inoculation rate of 0.02%. Fermentation was carried out at a constant temperature of 30°C until the pH of the milk culture medium reached 4.6. The milk culture medium was then allowed to stand at 4°C for 24 hours to obtain yogurt. Although the yogurt prepared in this comparative example was solid, its structure was loose, and slight whey separation was observed on the surface. After storage at 4°C for 4 days, significant whey separation occurred, accounting for approximately 5% of the total yogurt volume. On the 30th day of storage, the yogurt formed aggregated granules, with a clear water layer on top. At this point, severe whey separation occurred, accounting for approximately 15% of the total yogurt volume.

[0135] The above are merely preferred embodiments of this application and are not intended to limit the present invention. Although this application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the foregoing examples or make equivalent substitutions for some of the technical features. However, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for mutagenesis of a high-EPS-producing strain, characterized in that, Includes the following steps: Step S1: Lactic acid bacteria are isolated from soy products that change from liquid to solid yogurt at room temperature, activated, and then placed on an MRS plate containing CaCO3 for purification and culture. Step S2: Select colonies with a calcium dissolution zone diameter greater than 5 mm, and perform microwave mutagenesis, chemical mutagenesis, and high-throughput screening in sequence to obtain strains with EPS yield greater than 20 g / L. Step S3: Subculture the strain with EPS yield greater than 20 g / L until a high-yielding EPS strain with stable EPS yield of 20-25 g / L is obtained.

2. A high-yield EPS strain, characterized in that, It was obtained using the mutagenesis method described in claim 1.

3. A fusion of Weissella confusa JMF-006, characterized in that, The fused Weissella JMF-006 was obtained by the mutagenesis method described in claim 1; the fused Weissella JMF-006 is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 64776.

4. A bacterial strain composition, characterized in that, Includes the high EPS-producing strain of claim 2 or the fusion Weissella JMF-006 of claim 3.

5. The bacterial composition according to claim 4, characterized in that, Also includes: Lactobacillus plantarum JMF-001, accession number CCTCC NO: M2023025; Lactobacillus casei JMF-002, accession number CCTCC NO: M2023026.

6. A compound fermentation microbial powder, characterized in that, The strain includes the high EPS-producing strain of claim 2, or the fusion Weissella JMF-006 of claim 3, or the strain composition of any one of claims 4 to 5.

7. A method for preparing the compound fermentation powder according to claim 6, characterized in that, Includes the following steps: The high-EPS-producing strain of claim 2, or the fusion Weissella JMF-006 of claim 3, or the strain composition of any one of claims 4 to 5, is inoculated into a lactic acid bacteria liquid culture medium at a predetermined ratio, and fermented at a constant temperature to obtain a fermentation broth. Subsequently, the fermentation broth is freeze-dried to obtain a compound fermentation powder.

8. The application of the high-EPS-producing strain of claim 2, or the fusion strain JMF-006 of claim 3, or the strain composition of any one of claims 4-5, or the compound fermentation powder of claim 6, or the compound fermentation powder obtained by the preparation method of claim 7, characterized in that, Used in the preparation of starter cultures, dietary supplements, and for food fermentation.

9. A type of soy yogurt, characterized in that, Using the high-EPS-producing strain of claim 2, or the fusion Weissella JMF-006 of claim 3, or the strain composition of any one of claims 4-5, or the compound fermentation powder of claim 6, or the compound fermentation powder obtained by the preparation method of claim 7 as a starter culture, soybean beverages are fermented to obtain soybean yogurt.

10. A type of yogurt, characterized in that, Using the high-EPS-producing strain of claim 2, or the fusion Weissella JMF-006 of claim 3, or the strain composition of any one of claims 4-5, or the compound fermentation powder of claim 6, or the compound fermentation powder obtained by the preparation method of claim 7 as a starter culture, dairy beverages are fermented to obtain yogurt.