Lactobacillus mucilaginosus BL-SWN1 and application thereof

Abstract

The invention provides fermented lactobacillus mucus BL-SWN1 and application thereof, the fermented lactobacillus mucus BL-SWN1 is screened from naturally fermented soybean clear water, is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 6, 2025, is named as Limosilicilla fermentum in taxonomy, has a preservation number of CGMCC No.33307, and belongs to the technical field of microorganisms. The lactobacillus mucus BL-SWN1 has good acid production capacity, gastrointestinal fluid and cholate tolerance, bacteriostatic activity and nitrite degradation capacity, the pH value of a culture medium can be reduced to about 4.3 within 24 h of culture, and the nitrite degradation rate reaches 95% or above. The fermentation lactobacillus mucus BL-SWN1 can be applied to vegetable fermentation products such as pickled Chinese cabbages and pickled vegetables, can also be suitable for the fermentation process of dairy products and meat products, has a wide application prospect in the food industry, and particularly has remarkable advantages in the aspect of development of healthy fermentation foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a fermented Lactobacillus mucosae BL-SWN1 and its application. Background Art

[0002] Lactic acid bacteria are a group of Gram-positive bacteria that can utilize fermentable carbohydrates to produce a large amount of lactic acid, and are widely used in fields such as food fermentation, medicine and health care, and animal feed. Lactic acid bacteria can not only produce metabolites such as organic acids, carbon dioxide, and ethanol through lactic acid fermentation, but also regulate the balance of the intestinal microecology and enhance the body's immune function, having important probiotic effects. In recent years, with the increasing demand for healthy diets, the screening and functional research of lactic acid bacteria have gradually become a research hotspot in the food field. During the processing of soy products, soy whey (also known as yellow slurry water), as a by-product of tofu production, about 4-5 tons of soy whey will be produced for every 1 ton of soybeans processed. Soy whey is rich in nutrients such as protein, oligosaccharides, isoflavones, calcium, phosphorus, potassium, iron, monosaccharides, B vitamins, amino acids, and organic acids. However, due to its high chemical oxygen demand (COD) and easy acidification characteristics, soy whey is considered a high-load organic wastewater that is difficult to treat. It not only causes environmental pollution but also wastes rich nutritional resources. With the global emphasis on sustainable development, the food industry's demand for green and environmentally friendly processes is increasing day by day. Recent studies have shown that the nutrients rich in soy whey provide ideal conditions for the growth of lactic acid bacteria. By fermenting soy whey, not only can the discharge of organic wastewater be effectively reduced and environmental pollution be reduced, but also lactic acid bacteria with excellent fermentation performance can be screened for use in the food processing field to develop new healthy fermented products. Using soy whey for lactic acid bacteria screening can not only realize the recycling of waste resources and reduce environmental pollution, but also provide high-quality strain resources for the food fermentation field. Currently, the screening of lactic acid bacteria is usually based on different environmental samples, and strains adapted to specific fermentation conditions are isolated and screened. However, the screening research on lactic acid bacteria for this special matrix of soy whey is relatively less, and there is still a lack of systematic exploration. Soy whey is rich in various nutrients, providing ideal conditions for the growth and functional characteristics research of lactic acid bacteria. By screening and identifying lactic acid bacteria from naturally fermented soy whey, not only can this by-product resource be efficiently utilized, but also lactic acid bacteria strains with excellent performance can be provided for the food fermentation field, having important research value and application potential. Summary of the Invention

[0003] Technical problem to be solved: Aiming at the above technical problems, the object of the present invention is to provide a Limosilactobacillus fermentum BL-SWN1 and its application. Limosilactobacillus fermentum BL-SWN1 is screened from naturally fermented soybean whey water, and has been deposited in the China General Microbiological Culture Collection Center on January 6, 2025. Its taxonomic name is Limosilactobacillus fermentum, and the deposit number is CGMCC No. 33307, belonging to the field of microbial technology. Limosilactobacillus fermentum BL-SWN1 in the present invention has good acid-producing ability, gastrointestinal fluid and bile salt tolerance ability, antibacterial activity and nitrite degradation ability. It can reduce the pH value of the culture medium to about 4.3 within 24 hours of cultivation, and the degradation rate of nitrite reaches more than 95%. The Limosilactobacillus fermentum BL-SWN1 of the present invention can be not only applied to vegetable fermented products such as pickled Chinese cabbages and pickles, but also applicable to the fermentation processes of dairy products and meat products, and has a wide application prospect in the food industry, especially has significant advantages in the development of healthy fermented foods.

[0004] Technical solution: A Limosilactobacillus fermentum BL-SWN1, the deposit number of the Limosilactobacillus fermentum BL-SWN1 is CGMCC No. 33307, the deposit date is January 6, 2025, the deposit unit is the China General Microbiological Culture Collection Center, and the taxonomic name is Limosilactobacillus fermentum. Furthermore, the screening method of the Limosilactobacillus fermentum BL-SWN1 includes the following steps: S1. Naturally ferment the soybean whey water under airtight conditions to obtain naturally fermented soybean whey water; S2. Prepare MRS solid medium containing 5% CaCO3; S3. Perform 10-fold serial dilutions on the naturally fermented soybean whey water to obtain a dilution of the naturally fermented soybean whey water; S4. Spread the dilution of the naturally fermented soybean whey water on the MRS solid medium containing 5% CaCO3 and culture; S5. Primary screening of lactic acid bacteria: ① Preliminarily screen high acid-producing strains according to the size of the calcium dissolution circle of the MRS solid medium, and repeatedly streak and isolate to obtain purified strains; ② Screen to obtain a collection of lactic acid bacteria strains by analyzing the Gram staining and catalase activity of the purified strains; S6. Secondary screening of lactic acid bacteria: Perform multiple screenings on the isolated strains for acid-producing ability, antibacterial activity, acid tolerance, salt tolerance and nitrite degradation ability to obtain Limosilactobacillus fermentum BL-SWN1. Furthermore, the conditions for natural fermentation in step S1 are a fermentation temperature of 35 - 40 °C and a fermentation time of 40 - 50 h. Further, the culture conditions in step S4 are a culture temperature of 35-40°C and a culture time of 20-25h. Further, the screening conditions for the lactic acid bacteria strain collection in step S5 are Gram-positive staining and negative catalase test. A microbial fermentation inoculant, which contains Limosilactobacillus fermentum BL-SWN1 as described in claim 1. Further, the application of Limosilactobacillus fermentum BL-SWN1 and the microbial fermentation inoculant in food fermentation. Further, the application is for the fermentation of vegetable products. Beneficial effects: 1. The present invention screened Limosilactobacillus fermentum BL-SWN1 from natural fermented soybean whey water, which was deposited in the China General Microbiological Culture Collection Center on January 6, 2025, and its deposit number is CGMCC No. 33307; Limosilactobacillus fermentum BL-SWN1 of the present invention has good acid production ability, fast acid production speed, and can reduce the pH value of the culture medium from 6.5 to 4.30 within 24h; it has good antibacterial activity, has a significant antibacterial effect on common pathogenic bacteria such as Escherichia coli, and the antibacterial experiment results show that the diameter of the antibacterial zone reaches 16.5±0.5mm; it has good gastrointestinal fluid tolerance, and its survival rates are 72% and 80% respectively after culturing in simulated gastric juice (pH2.5) for 3h and simulated intestinal juice (containing 0.3% bile salt) for 6h; it has certain antioxidant ability, and its DPPH free radical scavenging rate is 31.84% and ABTS+ free radical scavenging rate is 17.29%; it has high hydrophobicity and self-aggregation ability, its hydrophobic rate to xylene is 48.5%, and the self-aggregation experiment results show that the self-aggregation rate is 45.7%, which is beneficial to its attachment in the intestine and the formation of biofilms, thereby enhancing its probiotic effect and competitiveness against harmful bacteria. 2. The method for screening lactic acid bacteria in the present invention uses an improved MRS medium combined with the screening method of CaCO3 calcium dissolution circle. The technical process is relatively simple, suitable for standardized operation, easy to realize automated production, and by controlling the fermentation process and strain screening through automated equipment, the yield and production efficiency can be significantly improved. 3. Limosilactobacillus fermentum BL-SWN1 screened by the present invention is not only suitable for fermented vegetable products such as pickled vegetables and kimchi, but also suitable for the fermentation process of dairy products and meat products; the screening method based on natural fermented soybean whey water has the characteristics of rich raw materials, simple and controllable process, easy realization of fermentation conditions, and resource recycling, and has broad market application prospects and good technical promotion potential. Description of the Drawings Figure 1pH value change curves of Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 in Example 3; Figure 2 Acid production change curves of Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 in Example 3; Figure 3 Phylogenetic tree of Lactobacillus L1; Figure 4 Phylogenetic tree of Lactobacillus L2; Figure 5 Acid tolerance change curves of Lactobacillus L1 and Lactobacillus L2 in Example 5; Figure 6 Salt tolerance change curves of Lactobacillus L1 and Lactobacillus L2 in Example 5; Figure 7 Results of nitrite degradation by Lactobacillus L1 and Lactobacillus L2 in Example 6; Figure 8 DPPH radical scavenging rates of Lactobacillus L1 and Lactobacillus L2 in Example 7; Figure 9 For Lactobacillus L1 and Lactobacillus L2 in Example 7, ABTS + Radical scavenging rate; Figure 10 Bile salt tolerance of Lactobacillus L1 and Lactobacillus L2 in Example 8; Figure 11 Hydrophobicity of Lactobacillus L1 and Lactobacillus L2 cultured for 3 h in Example 9; Figure 12 Hydrophobicity of Lactobacillus L1 and Lactobacillus L2 cultured for 6 h in Example 9; Figure 13 Autoaggregation of Lactobacillus L1 and Lactobacillus L2 in Example 9; Figure 14 pH value change curve of Lactobacillus L1 powder in Example 10; Figure 15 Acid production change curve of Lactobacillus L1 powder in Example 10; Figure 16 Growth curve of Lactobacillus L1 powder in Example 10. Detailed implementation mode The present invention will be further described below in conjunction with embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments: The lactic acid bacterium L1 in the specification was identified as Limosilactobacillus fermentum, named Limosilactobacillus fermentum BL-SWN1, and was deposited in the China General Microbiological Culture Collection Center on January 6, 2025, with the deposit number CGMCC No. 33307. Example 1 Screening of target strains of lactic acid bacteria S1. Fill the self-made soybean clear water in the laboratory into a sterilized glass jar, seal it and store it in an incubator at 37°C for natural fermentation for 48 h; S2. Prepare MRS solid medium containing 5% CaCO3; S3. Under aseptic conditions, mix 25 mL of naturally fermented soybean clear water with 225 mL of sterile normal saline, use a constant-speed oscillator to oscillate at a speed of 150 rpm for 30 to ensure uniform mixing of the samples; then take 1.0 mL of the mixed solution and add it to 9.0 mL of sterile normal saline, and perform 10-fold serial dilutions in sequence until a suitable bacterial solution concentration is obtained; coat each dilution gradient sample on the MRS solid medium containing 5% CaCO3, and place it in an incubator at 37°C for 24 h; Primary screening of lactic acid bacteria ① Initially screen high-acid-producing strains according to the size of the calcium carbonate dissolution circle in the MRS solid medium. Because when lactic acid bacteria produce lactic acid, CaCO3 in the MRS medium dissolves to form a transparent circle, and high-acid-producing strains are initially screened based on the size of the calcium carbonate dissolution circle, and purified strains are obtained by repeated streak isolation; ② Inoculate the purified strains on the MRS medium plate by the method of streaking, incubate at 37°C, and observe and record the colony morphology of the strains; ③ Perform Gram staining on the strains, observe and record the morphological characteristics of the bacterial cells of the strains to be tested under an oil immersion lens in an optical microscope, and at the same time use the standard method for determining the activity of catalase, with 3% hydrogen peroxide solution as the substrate, and judge the catalase activity by observing the generation of bubbles, and screen strains that are Gram-positive and negative in the catalase test. Three lactic acid bacteria that meet the conditions are obtained, namely lactic acid bacterium L1, lactic acid bacterium L2, and lactic acid bacterium L3. The colony morphology on the agar medium, the morphological description of the bacterial cells under the microscope after Gram staining, the Gram staining results, and the catalase test results are shown in Table 1; ④ According to the test methods in the "Common Bacterial System Identification Manual" and the "Bergey's Manual of Determinative Bacteriology", inoculate the screened strains into biochemical identification tubes, and observe and record the phenomena after 18 - 72 h, as shown in Table 2. Lactic acid bacteria L1 - L3 are initially identified as Lactobacillus plantarum. Table 1 Morphological characteristics of strains L1, L2, and L3 Strain number Colony characteristics Gram stain Catalase Cell characteristics L1 Round, white, translucent, with a bulge in the middle + - Rod-shaped L2 Round, white, opaque, smooth with a bulge + - Rod-shaped L3 Round, white, opaque, small colonies + - Rod-shaped Note: + indicates positive and - indicates negative. Physiological and biochemical identification results of strains L1, L2, and L3 in Table 2 Note: + indicates positive and - indicates negative. Example 2 Determination of acid production ability of Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 (1) Determination of acid production rate Freshly cultured Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 were inoculated into MRS liquid medium and cultured at 37°C for 24 h. The MRS liquid medium without inoculation was used as a blank control. The pH value was measured with a pH meter every 4 h. The pH value can reflect the acid production ability of the strain. (2) Determination of acid production amount Freshly cultured Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 were inoculated into MRS liquid medium and cultured at 37°C for 24 h. The MRS liquid medium without inoculation was used as a blank control. The total titratable acidity (TTA) of Lactobacillus was measured every 4 h with reference to the national standard GB 12456-2021 "National Food Safety Standard - Determination of Total Acidity in Foods". The TTA can reflect the acid production amount of the strain. From Figure 1 and Figure 2 it can be seen that after 24 h of culture, the pH of the medium of Lactobacillus L1, Lactobacillus L2, and Lactobacillus L3 decreased from 6.5 to 4.3, 4.3, and 4.5 respectively. And the acid production amounts of Lactobacillus L1 and Lactobacillus L2 after 12 h of culture were 10.68 mg / kg, 11.12 mg / kg, and 9.34 mg / kg respectively. However, the acid production amount of Lactobacillus L3 was the lowest during the 24 h of culture. Therefore, the acid production ability of Lactobacillus L3 is lower than that of Lactobacillus L1 and Lactobacillus L2. Thus, Lactobacillus L1 and Lactobacillus L2 were screened based on the strength of acid production ability. Example 3 Specific species identification of Lactobacillus L1 and Lactobacillus L2 The 16S rDNA molecular identification method was used to determine the specific species of Lactobacillus L1 and Lactobacillus L2 as follows: (1) DNA extraction: Use E.Z.N.A TM Mag-Bind Soil DNAKit to extract DNA from the provided samples; (2) PCR amplification and sequencing: Use Robust PCR Master Mix for PCR amplification ① The reaction system for the first round of PCR amplification was 50 μL: including Robust PCR Master Mix 15 μL, Bar-PCR primer F 1 μL, Primer R 1 μL, template DNA (10 - 20 ng), add corresponding volume according to the amount of template, make up to 30 μL with dd H2O; The reaction conditions are: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 45 °C for 20 s, extension at 65 °C for 30 s, for a total of 5 cycles; Then perform 20 cycles in the second stage, with the conditions: denaturation at 94 °C for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 30 s, and finally extend at 72 °C for 5 min, and finally keep warm at 10 °C; The amplified PCR products are detected by 2% agarose gel electrophoresis, electrophoresed at 120 V for 20 min, and observed under a gel imaging system, record the results and take pictures for analysis; ② In the second round of amplification, Illumina bridge PCR compatible primers are introduced, and the reaction system is 50 μL: including Robust PCR Master Mix 15 μL, Primer F 1 μl, Index-PCR Primer R 1 μL, 20 - 30 ng of the first-round PCR product, make up to 30 μL with dd H2O; The reaction conditions are: pre-denaturation at 95 °C for 3 min, denaturation at 94 °C for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 30 s, for a total of 5 cycles; Finally extend at 72 °C for 5 min, and finally keep warm at 10 °C; ③ Sequence analysis: The sequencing results are used for similarity alignment analysis by Blast in NCBI; Use Contig Express software to splice and manually correct the sequences to generate a Fasta format file, and use Mega 11.0 software to construct a phylogenetic tree by clustering according to the Neighbor-Joining method. From Figure 3 、 Figure 4 it can be seen that the homology of Lactobacillus L1 and Limosilactobacillus fermentum strain CIP 1029805 can reach 100%; the homology of Lactobacillus L2 and Lactiplantibacillus fermentum strain NBRC 15885 can reach 99%. Example 4 Determination of the antibacterial ability of Lactobacillus L1 and Lactobacillus L2 Using the Oxford cup quantitative diffusion method, take 150 μL of the bacterial strain fermentation broth in the Oxford cup, use Escherichia coli as the indicator bacterium, culture at 37 °C for 24 h, and measure the diameter of the antibacterial circle. Table 3 The antibacterial circle diameters of Lactobacillus L1 and Lactobacillus L2 Strain number Inhibition zone size (mm) L1 10.933±0.1155 L2 10.833±0.2887 As can be seen from Table 3, the lactic acid bacteria L1 and L2 are ranked according to the size of the inhibition zone, indicating that the lactic acid bacteria L1 and L2 have certain antibacterial abilities. Example 5 Tolerance analysis of lactic acid bacteria L1 and L2 (1) Acid tolerance analysis of strains Respectively, draw 1% inoculum of the activated lactic acid bacteria broth and inoculate it into MRS liquid medium with pH values of 3, 3.5, 4, 4.5, and 5. Incubate it in a constant temperature shaking incubator at 37°C and 200 r / min for 24 h, and measure the OD 600nm / 10 value. Draw the acid tolerance change curve; (2) Salt tolerance analysis of strains Respectively, draw 1% inoculum of the activated lactic acid bacteria broth and inoculate it into MRS liquid medium with NaCl contents of 2%, 4%, 6%, 8%, and 10%. Incubate it in a constant temperature shaking incubator at 37°C and 200 r / min for 24 h, and measure the OD 600nm / 10 value and draw the salt tolerance change curve. As Figure 5 can be seen, when the pH value decreases from 5.0 to 4.0, both lactic acid bacteria L1 and L2 can survive stably. However, as the pH value decreases to 3.0, lactic acid bacteria L1 rapidly inactivates, indicating that lactic acid bacteria L1 has poor tolerance to strong acid environments. As Figure 6 can be seen, when the NaCl content is in the range of 2-4%, both lactic acid bacteria L1 and L2 can exist stably. However, as the NaCl content increases to 8%, both lactic acid bacteria L1 and L2 are completely inactivated. Example 6 Determination of nitrite degradation by lactic acid bacteria L1 and L2 Inoculate the activated strains into 20 mL of sterilized MRS liquid medium containing 10 mg / L sodium nitrite at an inoculum amount of 0.15%. Incubate it at 37°C for 24 h. Take 2 mL of the fermentation broth for centrifugation every 4 h, and transfer 1 mL of the supernatant to a 50 mL stoppered colorimetric tube. Refer to the national standard GB5009.33-2016 "National Food Safety Standard Determination of Nitrate and Nitrite in Foods" to determine the sodium nitrite content. As Figure 7 can be seen, the degradation effects of lactic acid bacteria L1 and L2 on nitrite are both above 95%. Example 7 Antioxidant activities of lactic acid bacteria L1 and L2 Activate lactic acid bacteria L1 and L2 in MRS broth medium for 2 generations respectively to make the cell concentration reach 109 CFU / mL. Centrifuge to discard the supernatant, wash 3 - 4 times with PBS, then add PBS, shake and mix well to prepare a bacterial suspension, and adjust the bacterial concentration to reach 1.0×10 8 CFU / mL to obtain a bacterial suspension. (1) Determination of DPPH free radical scavenging rate Take 2 mL of the bacterial suspension, add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, and react in the dark at room temperature for 30 min; after centrifuging at 3000×g for 10 min, take the supernatant and measure the absorbance As at 517 nm. Use a 3 μg / mL vitamin C solution as a positive control to calculate the DPPH free radical scavenging rate; the calculation formula for the DPPH scavenging rate is: DPPH free radical scavenging rate (%) = [1 - (A s - A0) / A i ×100%, where: A s is the absorbance of 2 mL of DPPH anhydrous ethanol solution + 2 mL of bacterial suspension, A0 is the absorbance of 2 mL of anhydrous ethanol + 2 mL of bacterial suspension, and A i is the absorbance of 2 mL of DPPH anhydrous ethanol solution + 2 mL of distilled water. (2) ABTS + free radical scavenging rate determination By mixing the prepared ABTS + solution (7 mmol / L) with potassium persulfate solution (2.45 mmol / L) to prepare an ABTS + stock solution, and after standing in the dark for 12 - 16 h, dilute this solution with 70% ethanol solution to an absorbance value of 0.7 (734 nm) to prepare an ABTS + working solution; mix 0.15 mL of the sample solution with 2 mL of ABTS + working solution, react in the dark at room temperature for 6 min, and then measure the absorbance at 734 nm; the calculation formula for the ABTS + free radical scavenging rate is: ABTS + free radical scavenging rate (%) = (A1 - A2) / A0×100%, where: A1 is the absorbance of 0.15 mL of bacterial suspension + 2 mL of ABTS + working solution; A2 is the absorbance of 2 mL of anhydrous ethanol + 2 mL of bacterial suspension; A0 is the absorbance of 2 mL of ABTS + working solution + 2 mL of distilled water. From Figure 8 and Figure 9 it can be seen that Lactobacillus L1 and Lactobacillus L2 have effects on DPPH free radicals and ABTS +All free radicals have a certain scavenging ability. The results show that both strains of lactic acid bacteria have a certain antioxidant ability, and the antioxidant activity of L1 is more significant. Example 8 Determination of the tolerance of lactic acid bacteria L1 and L2 to gastrointestinal fluid and bile salts (1) Survival rate under simulated gastric juice Take 1 mL of the bacterial suspension and add it to 9 mL of simulated gastric juice. After mixing well, incubate it statically at 37 °C for 3 h. Sampling is carried out at 0 h and 3 h respectively, diluted and spread on MRS agar medium, and plate counting is carried out after culturing at 37 °C for 48 h to calculate the survival rate. (2) Survival rate under simulated intestinal fluid Take 1 mL of the bacterial suspension that has reacted in gastric juice and add it to 9 mL of simulated intestinal fluid. After mixing well, incubate it statically at 37 °C for 6 h. Sampling is carried out at 0 h and 6 h respectively, diluted and spread on MRS agar medium, and plate counting is carried out after culturing at 37 °C for 48 h to calculate the survival rate. Survival rate of lactic acid bacteria (%) = Nm / Nn × 100%, where: Nm and Nn represent the viable bacteria counts before and after digestion with simulated gastric juice or intestinal fluid / (CFU / mL). Table 4 Tolerance of lactic acid bacteria L1 and L2 to gastrointestinal fluid As can be seen from Table 4, the survival rate of lactic acid bacteria L2 is higher than that of lactic acid bacteria L1 during simulated gastric juice digestion, and the survival rate of lactic acid bacteria L1 is higher than that of lactic acid bacteria L2 during simulated intestinal fluid digestion. (3) Determination of bile salt tolerance Activate the strain in MRS medium for 2 generations to make the cell concentration reach 10 9 CFU / mL, and inoculate it into MRS medium containing 0.3% porcine bile salt at an inoculation amount of 0.15%. Incubate it at 37 °C for 24 h, using MRS medium without bile salt as a control, and measure its OD 600nm absorbance value. From Figure 10 it can be seen that lactic acid bacteria L1 has the strongest bile salt tolerance compared with lactic acid bacteria L2. Example 9 Determination of the hydrophobicity and autoaggregation of lactic acid bacteria L1 and L2 (1) Hydrophobicity Activate the strain in MRS medium for 2 generations to make the cell concentration reach 10 9CFU / mL, centrifuge at 6000 r / min for 10 min. After the precipitate is washed with sterile PBS (pH = 7.20), resuspend it in PBS, adjust the absorbance of the bacterial suspension within the range of 0.80 ± 0.05. Add 9 mL of the bacterial suspension to 3 mL of organic solvents (xylene, n - hexane, and ethyl acetate), react at 37 °C for 10 min for temperature equilibration, shake well, and incubate statically at 37 °C for 3 h. Carefully aspirate the lower aqueous phase to measure the OD 600nm of the absorbance value, calculate the hydrophobicity rate according to formula (3), and use sterile PBS as the blank control. Hydrophobicity rate (%) = [1 - (A t - A0)] × 100%, where: A t is the absorbance value after static incubation for 3 h; A0 is the absorbance value at 0 h. (2) Auto - aggregation Activate the strain for 2 generations in MRS medium to make the cell concentration reach 10 9 CFU / mL, centrifuge at 6000 r / min for 10 min. After the precipitate is washed with sterile PBS (pH = 7.20), resuspend it in PBS, adjust the absorbance of the bacterial suspension within the range of 0.80 ± 0.05, shake well, and incubate statically at 37 °C for 6 h. Carefully aspirate the upper phase at 3 h and 6 h respectively to measure the OD 600nm of the absorbance value, calculate the auto - aggregation rate, measure it in parallel three times, and use sterile PBS as the blank control. Auto - aggregation rate (%) = [1 - (A j - A i )] × 100%, where: A j is the absorbance value after static incubation for 3 h and 6 h; A i is the absorbance value at 0 h. As can be seen from Figure 11 and Figure 12 , the overall hydrophobicity of Lactobacillus L1 is higher than that of Lactobacillus L2, and it shows the best performance in xylene, indicating that L1 may have stronger survival and adaptation abilities in a more hydrophobic environment. With the increase of time (6 h compared with 3 h), the hydrophobicity of both strains has increased, indicating that the adsorption process of the strains in the organic phase is a dynamic process, which may be affected by cell - surface proteins or exopolysaccharides; in n - hexane and ethyl acetate, the hydrophobicity of Lactobacillus L1 and L2 is relatively low, which may be related to the polarity or compatibility of these solvents. As can be seen from Figure 13It can be seen that the auto-aggregation ability of Lactobacillus L1 is significantly higher than that of Lactobacillus L2. Especially after 4 h, its auto-aggregation rate increases substantially, while the auto-aggregation rate of Lactobacillus L2 is relatively low and the change is not obvious. This indicates that Lactobacillus L1 may have higher cell surface hydrophobicity or auto-aggregation characteristics mediated by specific surface proteins, thereby enhancing its colonization ability in the host intestine and facilitating the exertion of probiotic effects. The lower auto-aggregation ability of Lactobacillus L2 may make it more suitable for applications such as fermented foods or probiotic preparations that require lower aggregation. In summary, although Lactobacillus L2 is superior to Lactobacillus L1 in some individual tolerance indicators such as gastric juice tolerance, acid tolerance, and salt tolerance, considering all the experimental data, Lactobacillus L1 is more prominent in multiple key probiotic characteristics and fermentation performance and has stronger comprehensive application potential. First, the antioxidant ability of Lactobacillus L1 is significantly better than that of Lactobacillus L2, with higher DPPH and ABTS + radical scavenging rates, which is helpful for its antioxidant application in functional foods. Second, Lactobacillus L1 has stronger bile salt tolerance and survives more stably in the complex intestinal environment. In addition, Lactobacillus L1 exhibits higher hydrophobicity and auto-aggregation ability, meaning that it is more likely to form a biofilm on the intestinal wall, improving its colonization ability and competitive advantage against harmful bacteria. More importantly, Lactobacillus L1 and L2 perform similarly in terms of acid production ability, antibacterial activity, nitrite degradation rate, etc., indicating that its basic fermentation ability is not inferior to that of Lactobacillus L2. In the actual food fermentation and probiotic preparation applications, this multi-dimensional characteristic coordination and intestinal adaptability are more crucial. Therefore, finally selecting L1 as the target strain has sufficient experimental basis and practical significance. And Lactobacillus L1 is named Limosilactobacillus fermentum BL-SWN1, which was deposited in the China General Microbiological Culture Collection Center on January 6, 2025, and its deposit number is CGMCC No. 33307. Example 10 A preparation method for preparing microbial powder using Limosilactobacillus fermentum BL-SWN1 includes the following steps: S1. Inoculate Limosilactobacillus fermentum BL-SWN1 after two generations of activation culture into MRS liquid medium and anaerobically culture it at 37 °C for 24 h. The OD 600 value of the culture solution is about 1.2; S2. Centrifuge the bacterial solution at 8000 r / min for 10 min, discard the supernatant, collect the bacterial cell precipitate, and wash the bacterial cells 3 - 4 times with PBS buffer (pH 7.2) to remove residual medium components. After washing, centrifuge again to collect the bacterial cells for subsequent freeze-drying treatment; S3. Add a protective agent mixture prepared in a mass ratio of 1:3 to the bacteria, wherein the protective agent consists of 5% γ-cyclodextrin, 3% trehalose, 3% glycine, and 2% mannitol. After fully mixing, spread the mixed bacterial mud on a sterile culture dish with a thickness of about 1 cm; pre-freeze it at -80°C for 12 hours, then place it in a vacuum freeze dryer, and freeze-dry it under vacuum conditions for 48 hours to obtain fermented mucus Lactobacillus BL-SWN1 bacterial powder. Table 5 Survival rate of lactic acid bacteria L1 before and after preparation of bacterial powder From Table 5, Figure 14 , Figure 15 and Figure 16 It can be seen that after the freeze-dried bacterial powder was revived and inoculated into fresh MRS liquid medium and cultured at 37°C for 24 hours, the pH dropped from 6.40 to 4.24, and the acid production capacity was basically consistent with the original liquid bacterial solution. The number of live bacteria in the bacterial powder was determined by the plate count method to be about 0.84×10 11 CFU / g, and the survival rate was 94.38%, indicating that the freeze-dried bacterial powder had good stability, recovery ability and application potential, and was suitable for use as a food fermentation agent and probiotic preparation. The bacterial powder was stored at 4°C after packaging. Embodiment 11 The method for preparing sauerkraut by fermenting Lactobacillus mucilaginosus BL-SWN1 comprises the following steps: Step 1. Take 500g of fresh Chinese cabbage, wash and cut into strips, blanch in boiling water for 30s to remove surface bacteria, remove and cool naturally to room temperature; Step 2. Add 75g of salt, mix in layers and put into a clean fermentation container in a "layer of cabbage and layer of salt" manner to prepare pretreated Chinese cabbage; Step 3. Dissolve the freeze-dried and revived fermented Lactobacillus mucoides BL-SWN1 powder in an appropriate amount of sterile water to prepare a bacterial solution (the concentration of live bacteria is about 1×109 CFU / mL), inoculate 25 mL of the fermented Lactobacillus mucoides BL-SWN1 bacterial solution into the pretreated Chinese cabbage and mix thoroughly; after mixing, seal the container, place it in an environment of 25°C and ferment for 7 days to obtain sauerkraut. The initial pH of Chinese cabbage was 6.2, dropped to 4.5 on the second day, and stabilized at 3.9 on the seventh day, indicating that Lactobacillus mucosae BL-SWN1 has good acid-producing ability and pH control performance; moreover, the pickled Chinese cabbage prepared in Example 11 had good sensory quality, presenting a light yellowish-green color overall, with natural color, firm tissue structure, a typical sour aroma of lactic acid fermentation, and no putrid smell; in terms of taste, the pickled Chinese cabbage prepared in Example 11 had a moderate sour taste, pure flavor, no astringency, and good crispness and resilience; at the same time, no abnormal fermentation phenomena such as strange smell or tissue softening were detected, indicating that Lactobacillus mucosae BL-SWN1 has the ability to regulate fermentation flavor and improve tissue texture, and is suitable for application in the preparation of plant-based foods such as pickled Chinese cabbage. As described above, it is only the preferred embodiment of the present invention, and there is no limitation to the present invention in any form. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still belong to the scope of protection of the technical solution of the present invention.

Claims

1. A fermented Lactobacillus mucosae BL-SWN1, characterized in that: The preservation number of the Limosilactobacillus fermentum BL-SWN1 is CGMCC No. 33307, the preservation date is January 6, 2025, the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the taxonomic name is Limosilactobacillus fermentum.

2. The screening method of Lactobacillus mucosae BL-SWN1 according to claim 1, characterized in that, It includes the following steps: S1. Naturally ferment the soybean clear water under airtight conditions to obtain naturally fermented soybean clear water. S2. Prepare an MRS solid medium containing 5% CaCO3. S3. Perform 10-fold serial dilutions on the naturally fermented soybean clear water to obtain a diluted solution of the naturally fermented soybean clear water. S4. Spread the diluted solution of the naturally fermented soybean clear water on the MRS solid medium containing 5% CaCO3 and culture it. S5. Primary screening of lactic acid bacteria: ① Initially screen high acid-producing strains based on the size of the calcium carbonate dissolution circle in the MRS solid medium, and repeatedly streak and isolate to obtain purified strains; ② Screen to obtain a collection of lactic acid bacteria strains by analyzing the Gram staining and catalase activity of the purified strains. S6. Secondary screening of lactic acid bacteria: Perform multiple screenings on the isolated strains for acid production ability, antibacterial activity, acid resistance, salt tolerance, and nitrite degradation ability to obtain Limosilactobacillus fermentum BL-SWN1.

3. The screening method of Lactobacillus mucosae BL-SWN1 according to claim 2, characterized in that: The conditions for natural fermentation in step S1 are a fermentation temperature of 35-40 °C and a fermentation time of 40-50 h.

4. The screening method of Lactobacillus mucosae BL-SWN1 according to claim 2, characterized in that: The conditions for culturing in step S4 are a culture temperature of 35-40 °C and a culture time of 20-25 h.

5. The screening method of Lactobacillus mucosae BL-SWN1 according to claim 2, characterized in that: The screening conditions for the collection of lactic acid bacteria strains in step S5 are positive Gram staining and negative catalase test.

6. A microbial fermentation inoculant, characterized in that, The microbial fermentation inoculant contains the Limosilactobacillus fermentum BL-SWN1 as described in claim 1.

7. Use of the Limosilactobacillus fermentum BL-SWN1 as described in claim 1 or the microbial fermentation inoculant as described in claim 6 in food fermentation.

8. Use of the Limosilactobacillus fermentum BL-SWN1 as described in claim 1 or the microbial fermentation inoculant as described in claim 6 in the fermentation of vegetable products.

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