Method for preparing lactobacillus leavening by using whey permeate

Abstract

The invention relates to the technical field of preparation of natural food preservatives, in particular to a method for preparing a lactobacillus fermentation product by using whey permeate. According to the present invention, the lactic acid bacteria fermentation product is prepared by using the specific plant lactobacillus (Lactobacillus plantarum) LW11 with the preservation number of CGMCC No.34365, such that the good sterilization and antibacterial ability is provided, and the growth of microorganisms can be effectively inhibited; the bacteriostasis test method of the whey permeate leavening can effectively and rapidly test the anticorrosion inhibition effect of the whey permeate leavening.

Description

Technical Field

[0001] This invention relates to the field of natural food preservative preparation technology, specifically to a method for preparing lactic acid bacteria fermentation products using whey permeate. Background Technology

[0002] Food is susceptible to microbial spoilage, which can lead to a decline in the product's color, taste, texture, and nutritional value, and shorten its shelf life. Worse still, many food spoilage bacteria, such as Penicillium and Aspergillus, as well as pathogenic bacteria like Staphylococcus aureus, Salmonella, and Helicobacter pylori, can produce biotoxins, posing serious health risks. In recent years, food preservatives have been widely used in the food industry because they effectively prevent microbial spoilage, thus extending shelf life. Currently, chemical preservatives, such as benzoic acid and its sodium salt, and sorbic acid and its potassium salt, are commonly used in my country's food industry. While chemical preservatives can be directly added to products to extend shelf life, the biological risks of these compounds to humans remain unclear. Long-term consumption of foods containing chemical additives may burden or harm the body and may even lead to the development of bactericide-resistant superbugs. With the continued development of clean labeling and consumers' increasing pursuit of healthy lifestyles, natural and safe food preservatives are gaining popularity.

[0003] Currently, while some natural plant extract preservatives offer a degree of safety, their antibacterial effects do not match those of chemical preservatives. Some natural preservatives, such as nisin, while exhibiting good antibacterial properties, are expensive and costly. Therefore, selecting a highly efficient, safe, and low-cost natural preservative is a pressing technical challenge for the food industry. Whey permeate, a byproduct of whey protein concentrate and whey protein isolate production, has low industrial utilization and contains high concentrations of lactose, easily leading to lactose intolerance. Existing technologies lack methods to transform whey permeate into a high-value product with potent antibacterial properties and low risk of lactose intolerance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing lactic acid bacteria fermentation products using whey permeate, thereby solving the problems mentioned in the background art. Lactic acid bacteria (LAB) are ideal candidates for use as antimicrobial agents in food. During fermentation, LAB can induce rapid acidification of raw materials, producing organic acids, antifungal peptides, volatile compounds, and other antifungal compounds that inhibit fungal growth. Using lactic acid bacteria with antimicrobial activity to ferment whey permeate to prepare lactic acid bacteria fermentation products has good application prospects, possessing advantages such as simple production process, better antibacterial effect, and reduced production costs. Furthermore, since whey permeate has a high lactose content, when using a combined enzymatic hydrolysis and stepwise fermentation method to prepare lactic acid bacteria fermentation products, the addition of lactase and protease can decompose the lactose in the raw materials into glucose and galactose, and hydrolyze proteins into small molecule peptides and amino acids. This not only avoids the inhibitory effect of high-concentration nutrients on microorganisms, allowing them to maintain good growth and metabolic activity, thereby improving fermentation efficiency, but also reduces the risk of lactose intolerance when using milk-based materials as fermentation substrates, while simultaneously improving the quality of the fermentation product. By continuously supplementing nutrients, the growth period and product synthesis time of microorganisms are extended, thereby increasing the yield of antimicrobial metabolites. The lactic acid bacteria fermentation product described in this invention has good bactericidal and bacteriostatic abilities and can be used to replace commonly used chemical preservatives on the market.

[0005] This invention provides a strain of Lactiplantibacillus plantarum LW11, with accession number CGMCC No: 34365, which was deposited at the China General Microbiological Culture Collection Center on April 28, 2025.

[0006] The present invention also provides a microbial preparation containing the aforementioned Lactiplantibacillus plantarum LW11.

[0007] In one embodiment, the microbial preparation contains at least 1 × 10⁻⁶ Lactiplantibacillus plantarum LW11 cells. 8 CFU / mL or 1×10 8 CFU / g.

[0008] The present invention also provides products containing the aforementioned Lactiplantibacillus plantarum LW11 or products containing the aforementioned microbial preparations.

[0009] In one embodiment, the product includes food, medicine, or health products.

[0010] In one embodiment, the food product includes fermented dairy products; preferably, the fermented dairy products include fermented whey permeate.

[0011] This invention also provides a method for preparing fermented whey permeate, comprising fermenting the whey permeate with *Lactobacillus plantarum* LW11. Fermented whey permeate liquid can be obtained after fermentation by *Lactobacillus plantarum*.

[0012] In one implementation, the method includes the following steps:

[0013] S1. Activation of bacterial strain: Inoculate Lactiplantibacillus plantarum LW11 into MRS medium at a volume of 2% of the medium. Activate to the 3rd generation at a temperature of 35-37°C. The activation time for each generation is 22-24 hours, for a total activation time of 70-72 hours.

[0014] S2. Preparation of expansion medium: Prepare a whey permeate solution with a mass concentration of 5-10% using whey permeate powder. After enzymatic hydrolysis with 50-100 U / mL lactase and 200-400 U / mL neutral protease at 35-45℃ for 2-4 hours, the temperature is raised to 90℃ to inactivate the enzymes for 10 minutes to obtain the treated whey permeate solution.

[0015] S3. Inoculation: Inoculate the activated Lactiplantibacillus plantarum LW11 obtained in step 1 at a rate of 1×10⁻⁶. 8 ~1×10 9 CFU / mL was inoculated into the whey permeate solution obtained in step S2.

[0016] S4. Stepwise fermentation: After the inoculum obtained in step S3 is sealed and fermented in a fermenter for 12-24 hours, the whey permeate solution obtained in step S2 is added again at 1 / 2 to 1 / 3 of the original fermentation liquid volume, and fermented again for 12-24 hours to obtain fermented whey permeate. The fermentation conditions are 35-40℃, and the fermentation process can usually be static culture. The total fermentation time is 24-48 hours.

[0017] S5. Sterilization: The fermented whey permeate obtained in step S4 is sterilized by centrifugation and microfiltration. The centrifugation speed is 3000-8000 rpm for 10-15 min, and the microfiltration uses a filter membrane with a pore size of 0.2-0.3 μm.

[0018] S6. Drying: The fermented whey permeate obtained in step S5 can be dried and powdered using conventional methods in the art, such as freeze-drying or spray drying. The resulting whey permeate fermentation product is obtained.

[0019] As a further description of the above technical solution: the whey penetrant powder contains 8-10% protein, 0.2-0.5% fat, and 80-85% lactose.

[0020] As a further description of the above technical solution: the plant lactobacillus can be cultured using conventional methods in the art, such as MRS or M17 culture medium.

[0021] As a further description of the above technical solution: the whey permeate fermentation broth of the present invention contains the fermentation product of Lactiplantibacillus plantarum LW11.

[0022] The inventors have discovered that the whey permeate fermentation broth prepared by the method of this invention has excellent antibacterial properties.

[0023] Based on the above research, this invention also provides the application of Lactiplantibacillus plantarum LW11, with accession number CGMCC No. 34365, in the preparation of lactic acid bacteria fermentation broth from whey permeate.

[0024] The present invention also provides a method for increasing the antibacterial activity of whey permeate by inoculating the Lactobacillus plantarum LW11 into a solution of whey permeate for fermentation.

[0025] The present invention also provides a method for reducing the risk of lactose intolerance in whey permeate by inoculating the Lactobacillus plantarum LW11 into a whey permeate solution for fermentation.

[0026] The present invention also provides the use of the Lactobacillus plantarum LW11 or the microbial preparation in the preparation of antibacterial agents.

[0027] This invention provides a novel method for preparing lactic acid bacteria fermentation products using fermentation. Compared to other natural biological preservatives, the whey permeate fermentation method provided in this invention is environmentally friendly, low-cost, and simple in process, suitable for large-scale production, reducing equipment investment, saving energy, and being environmentally friendly. Experiments have shown that the whey permeate fermentation broth prepared by this method has better antibacterial properties and can be used for food preservation.

[0028] The beneficial effects of this invention are as follows:

[0029] The lactic acid bacteria strain used in this invention is *Lactobacillus plantarum* LW11, which exhibits high growth activity and antibacterial activity at high substrate concentrations. *Lactobacillus plantarum* LW11 can be fermented in conventional culture media to obtain products with antibacterial activity, and it can also be fermented in whey permeate solutions to obtain ferments with high antibacterial activity.

[0030] This invention uses a single raw material, whey permeate powder, to ferment and prepare lactic acid bacteria fermentation products, resulting in low production costs and cleaner labels. The combined enzymatic hydrolysis of raw materials and the step-by-step fermentation process enhance antibacterial properties, and the fermentation products exhibit good heat resistance and solubility, making them widely applicable.

[0031] The fermented whey permeate of this invention has high activity against some food spoilage fungi (Aspergillus flavus, Penicillium chrysogenum) and foodborne pathogens (such as Staphylococcus aureus, Escherichia coli, Salmonella, Helicobacter pylori, etc.).

[0032] Preservation of biological materials:

[0033] Lactiplantibacillus plantarum LW11, taxonomically named Lactiplantibacillus plantarum, was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34365. Attached Figure Description

[0034] Figure 1 Schematic diagram of lactic acid bacteria screening results; double-layer plate method for screening lactic acid bacteria strains with inhibitory activity against Aspergillus flavus and Penicillium chrysogenum, numbers 1-5 represent control, strain LW11 (screened from kimchi mother water), Lactobacillus plantarum Lp90 (purchased from Weikang Biotechnology), Lactobacillus plantarum VJLHD16L1 (laboratory preservation), and strain LW28 (screened from kimchi mother water) (A); inhibition zone diameters of the four lactic acid bacteria against Aspergillus flavus and Penicillium chrysogenum (B); inhibition rate of the four lactic acid bacteria against Aspergillus flavus and Penicillium chrysogenum determined by 96-well microplate method (C);

[0035] Figure 2 This is a schematic diagram of the results of lactic acid bacteria identification;

[0036] Figure 3 The growth curve of Lactobacillus plantarum LW11 in whey permeate;

[0037] Figure 4 Flowchart of whey permeate fermentation process;

[0038] Figure 5 This is a schematic diagram illustrating the effect of the antifungal effect of the lactic acid bacteria fermentation product of the present invention;

[0039] Figure 6 This is a schematic diagram illustrating the effect of the antifungal effect of the lactic acid bacteria fermentation product of the present invention;

[0040] Figure 7 This is a schematic diagram illustrating the effect of heat treatment on the antibacterial stability of lactic acid bacteria fermentation products according to the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement this invention. However, the embodiments are not intended to limit the invention. Modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are all within the scope of this invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0042] The Lactiplantibacillus plantarum LW11 used below, with accession number CGMCC No.34365, was deposited at the China General Microbiological Culture Collection Center on April 28, 2025.

[0043] The Lactobacillus plantarum Lp90 strain used below is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 10453, deposited on January 27, 2015, and has been published in patent CN104928208A.

[0044] The whey penetrant powder (sweet whey powder) used in the following examples was purchased from Xi'an Lavia Biotechnology Co., Ltd., product number wicecpb3; the whey penetrant powder has a protein content of 8-10% (w / v), fat content of 0.2-0.5% (w / v), and lactose content of 80-85% (w / v).

[0045] Example 1: Screening and Identification of Strains

[0046] S1. Strain Screening: Target lactic acid bacteria strains were screened from traditional kimchi mother brine, laboratory-preserved strains, and commercially available lactic acid bacteria strains. First, the kimchi brine primer was serially diluted and spread onto MRS solid medium, incubated at 37°C for 24 hours. Then, single colonies were picked and inoculated into MRS liquid medium, incubated at 37°C for 24 hours for activation, and subcultured twice. Commercially available strains and laboratory-preserved strains were subcultured twice under the same conditions. 2% of the activated second-generation culture was inoculated into the fermentation broth and incubated at 37°C for 48 hours, with a final fermentation concentration of 1×10⁻⁶. 9 ~1×10 10 The concentration was CFU / mL, then centrifuged at 8000 r / min for 10 min, and filtered through a 0.22 μm filter membrane to obtain cell-free supernatant (CFS).

[0047] Preliminary screening was performed using a double-layer plate method. The lower layer of the plate was prepared using solid MRS agar medium. Two thin lines, approximately 3 cm long, were streaked onto the lower layer plate with activated lactic acid bacteria, and the culture was continued until all lactic acid bacteria had grown. A PDA semi-solid medium was then prepared, containing 1×10⁻⁶ bacteria. 6 Pour a spore concentration of spores / mL of mold spores into this culture medium to prepare an upper plate. After solidification, incubate the plate at 30℃ for 3–5 days and observe whether an inhibition zone appears around the growth bands of lactic acid bacteria. Repeat the experiment three times for each sample and take the average value to determine the antibacterial effect.

[0048] Further screening was performed using the 96-well microplate method. 100 μL of cell-free supernatant and 100 μL of a final concentration of 1×10⁻⁶ were added to each well of a sterile 96-well microplate. 6 A suspension of indicator bacteria with spore counts / mL was prepared. After thorough mixing and incubation at 30°C for 48 hours, the absorbance was measured at 580 nm using a microplate reader, with MRS medium as a control. The antibacterial activity of lactic acid bacteria can be expressed as the inhibition rate:

[0049] Antibacterial rate (%) = (1-OD) LAB / OD Control )×100(1).

[0050] Where: OD LAB OD580 nm indicates the OD580 nm of the indicator bacteria after 48 h of culture in cell-free lactic acid bacteria supernatant (CFS); ODcontrol indicates the OD580 nm of the indicator bacteria after 48 h of culture in MRS medium.

[0051] S2: Identification of Strains: The screened lactic acid bacteria underwent physiological and biochemical identification, including colony morphology observation, Gram staining, microstructure observation, catalase test, VP test, gelatin liquefaction test, and carbohydrate utilization experiment, and molecular identification was also performed. Effective probiotic isolates were identified by 16S rRNA gene sequencing. LAB strains were grown on MRS agar plates at 37℃ for 24 h, and colony morphology was observed. Cell morphology was observed using Gram staining (magnification = 1000×). Lactic acid bacteria cells were harvested by centrifugation (4℃, 6000×g, 10 min), washed three times with phosphate-buffered saline (PBS, pH 7.2), and fixed overnight with 2.5% (v / v) glutaraldehyde at 4℃. Next, the cells were washed three times with 0.1M PBS (15 min each time), dehydrated with a series of 30%, 50%, 70%, 80%, and 90% ethanol for 15 min each time, and then dehydrated twice with 100% ethanol for 15 min each time. One drop of the final bacterial suspension was aspirated onto a clean silicon wafer using a dropper, air-dried, vacuum-sputtered with gold, and the sample was morphologically observed using a scanning electron microscope. LAB strain was cultured in MRS liquid medium at 37°C for 24 h. 1 mL of the bacterial suspension was centrifuged (6000×g, 3 min), the supernatant was discarded, 1 mL of sterile water was added, and the mixture was thoroughly mixed and centrifuged again (6000×g, 3 min), discarding the supernatant. This process was repeated twice. LAB was resuspended in 1 mL of sterile water and used as a template for bacterial identification. Bacterial DNA was extracted, and genomic DNA of the isolates was extracted and amplified using universal PCR primers (forward 27F (AGTTTGATCMTGGCTCAG) primer) and reverse PCR primer 1492R (GGTTACCTTGTTACGACTT). The purified products were sequenced using 16S rDNA, and the resulting sequences were used to construct a phylogenetic tree using Mega11.0 software.

[0052] The results are as follows:

[0053] After screening, one commercially available *Lactobacillus plantarum* Lp90 (purchased from Microcon Biotechnology), one laboratory-preserved *Lactobacillus plantarum* VJLHD16L1, and two isolated lactic acid bacteria strains showed effective antifungal activity against *Aspergillus flavus* and *Penicillium chrysogenum* pathogens, exhibiting an inhibition zone >30 mm around the lactic acid bacteria. Figure 1 A) Two isolated lactic acid bacteria were derived from Chinese pickled vegetables and named LW11 and LW28, respectively. These four strains were used for further research, while strains showing less inhibition were discarded. Among them, LW11 exhibited the greatest inhibitory effect against *Aspergillus flavus* and *Penicillium chrysogenum*, with inhibition limits of 47.028 mm and 48.515 mm, respectively. Figure 1 B). Experiments using the 96-well microplate method revealed that LW11 CFS exhibited the highest inhibition rate against Aspergillus flavus and Penicillium chrysogenum. Figure 1 C).

[0054] The LW11 strain was identified through colony morphology observation, carbohydrate metabolism analysis, and 16S rDNA gene sequencing. Figure 2 As shown in Figure A, after culturing strain LW11 on MRS medium for 48 hours, the colonies exhibited a milky white color, smooth surface, and regular edges. LW11 is a Gram-positive bacillus, with a cell morphology of short rods arranged in short chains. Figure 2 B). Furthermore, SEM showed that strain LW11 had a short, strip-like shape and intact cell morphology (B). Figure 2 C) This is consistent with the observations under an optical microscope. Table 1 shows some biochemical indicators of strain LW11, which, combined with its physiological characteristics, can be classified as *Lactobacillus*. Similarity analysis using the BLAST program with reported bacterial 16S rDNA gene sequences in the National Center for Biotechnology Information (NCBI) database, and phylogenetic tree analysis, confirmed that the nucleotide sequence of strain LW11 is 100% homologous to the standard strain *Lactiplantibacillus plantarum* from the National Culture Collection (NCC). Therefore, strain LW11 is confirmed as *Lactiplantibacillus plantarum*.

[0055] Table 1. Biochemical indicators of strain LW11

[0056]

[0057] Example 2: Growth curve of Lactobacillus plantarum LW11 in whey permeate

[0058] Lactiplantibacillus plantarum LW11 was inoculated into MRS medium at an inoculum size of 2%, and activated up to the third generation at 35–37°C. Each generation was activated for 22–24 hours, for a total activation time of 70–72 hours. The OD600 nm was adjusted to 0.5. A 10% whey permeate solution was prepared using whey permeate powder, and then enzymatically hydrolyzed with 100 U / mL lactase and 400 U / mL neutral protease at 45°C for 2 hours. The solution was then heated to 90°C for 10 minutes to inactivate the enzymes, yielding the treated whey permeate solution. Take 2 mL of the activated bacterial solution and add it to 100 mL of enzyme-inactivated whey permeate solution (lactose content is about 80 g / L). Incubate at 37°C. Take samples every 2 hours from 0 to 24 hours and every 4 hours from 24 to 48 hours. Measure the OD600 nm. Use Lactobacillus plantarum strain Lp90 as a control.

[0059] The results are as follows Figure 3As shown, Lactobacillus plantarum LW11 has good tolerance to high sugar environment. After 5 hours of culture, the OD600 nm value began to increase significantly. At 24 hours, the OD600 nm value of LW11 was 6.201, which was higher than the OD600 nm value of Lp90 (4.599).

[0060] Example 3: Preparation of lactic acid bacteria fermentation product using whey permeate

[0061] This embodiment provides a method for preparing lactic acid bacteria fermentation products using whey permeate, such as... Figure 4 As shown, it includes the following steps:

[0062] S1. Activation of bacterial strain: Inoculate Lactiplantibacillus plantarum LW11 into MRS medium at a volume of 2% of the medium. Activate to the 3rd generation at a temperature of 35-37°C. The activation time for each generation is 22-24 hours, for a total activation time of 70-72 hours.

[0063] S2. Preparation of expansion medium: Prepare a 5% whey permeate solution using whey permeate powder, and then enzymatically hydrolyze it with 50 U / mL lactase and 100 U / mL neutral protease at 35℃ for 4 hours. After that, heat the solution to 90℃ to inactivate the enzymes for 10 minutes to obtain the treated whey permeate solution.

[0064] S3. Inoculation: Inoculate the activated Lactiplantibacillus plantarum LW11 obtained in step 1 at a rate of 1×10⁻⁶. 9 CFU / mL was inoculated into the whey permeate solution obtained in step S2.

[0065] S4. Stepwise fermentation: After the inoculum obtained in step S3 is fermented in a fermenter at 35°C for 24 hours, the whey permeate solution obtained in step S2 is added again at 1 / 2 the volume of the original fermentation liquid, and the fermentation is carried out again at 37°C for 24 hours. The fermentation process can usually be static culture.

[0066] S5. Sterilization: The fermented whey permeate obtained in step S4 is sterilized by centrifugation and microfiltration. The centrifugation speed is 5000 rpm for 12 min, and the microfiltration uses a microporous membrane with a pore size of 0.2 μm.

[0067] S6. Drying: The sterilized fermented whey permeate from step S5 is dried and powdered by freeze drying to obtain whey permeate fermentation product.

[0068] Example 4: Preparation of lactic acid bacteria fermentation product using whey permeate

[0069] Based on the above embodiments, a method for preparing lactic acid bacteria fermentation products using whey permeate includes the following steps:

[0070] S1. Activation of bacterial strain: Inoculate Lactiplantibacillus plantarum LW11 into MRS medium at a volume of 2% of the medium. Activate to the 3rd generation at a temperature of 35-37°C. The activation time for each generation is 22-24 hours, for a total activation time of 70-72 hours.

[0071] S2. Preparation of expansion medium: Prepare a 7% whey permeate solution using whey permeate powder, and then enzymatically hydrolyze it with 80 U / mL lactase and 150 U / mL neutral protease at 40℃ for 3 hours. After that, heat the solution to 90℃ to inactivate the enzymes for 10 minutes to obtain the treated whey permeate solution.

[0072] S3. Inoculation: Inoculate the activated Lactiplantibacillus plantarum LW11 obtained in step 1 at a rate of 1×10⁻⁶. 9 CFU / mL was inoculated into the whey permeate solution obtained in step S2.

[0073] S4. Stepwise fermentation: The inoculum obtained in step S3 is sealed and fermented in a fermenter at 37°C for 12 hours. Half of the original fermentation liquid volume of the whey permeate solution obtained in step S2 is added again, and the mixture is fermented again at 39°C for 12 hours. The fermentation process can usually be static culture.

[0074] S5. Sterilization: The fermented whey permeate obtained in step S4 is sterilized by centrifugation and microfiltration. The centrifugation speed is 3000 rpm for 15 min, and the microfiltration uses a microporous membrane with a pore size of 0.3 μm.

[0075] S6. Drying: The sterilized fermented whey permeate from step S5 can be dried and powdered using conventional methods in the art, such as freeze drying or spray drying, to obtain the whey permeate fermentation product.

[0076] Example 5: Preparation of lactic acid bacteria fermentation product using whey permeate

[0077] Based on the above embodiments, a method for preparing lactic acid bacteria fermentation products using whey permeate includes the following steps:

[0078] S1. Activation of bacterial strain: Inoculate Lactiplantibacillus plantarum LW11 into MRS medium at a volume of 2% of the medium. Activate to the 3rd generation at a temperature of 35-37°C. The activation time for each generation is 22-24 hours, for a total activation time of 70-72 hours.

[0079] S2. Preparation of expansion medium: Prepare a 10% whey permeate solution using whey permeate powder. After enzymatic hydrolysis with 100 U / mL lactase and 200 U / mL neutral protease at 45℃ for 2 hours, the temperature is raised to 90℃ to inactivate the enzymes for 10 minutes to obtain the treated whey permeate solution.

[0080] S3. Inoculation: Inoculate the activated Lactiplantibacillus plantarum LW11 obtained in step 1 at a rate of 1×10⁻⁶. 8 CFU / mL was inoculated into the whey permeate solution obtained in step S2.

[0081] S4. Stepwise fermentation: The inoculum obtained in step S3 is sealed and fermented in a fermenter at 35°C for 18 hours. Then, the whey permeate solution obtained in step S2 is added again at 1 / 3 of the original fermentation liquid volume. The fermentation conditions are 40°C for a second fermentation for 12 hours. The fermentation process can usually be static culture.

[0082] S5. Sterilization: The fermented whey permeate obtained in step S4 is sterilized by centrifugation and microfiltration. The centrifugation speed is 8000 rpm for 10 min, and the microfiltration uses a microporous membrane with a pore size of 0.2 μm.

[0083] S6. Drying: The fermented whey permeate after step S5 can be dried and powdered using conventional methods in the art, such as freeze drying or spray drying, to obtain the whey permeate fermentation product.

[0084] Comparative Example 1: Effect of bacterial strains on the antibacterial activity of whey permeate fermentation products

[0085] This comparative example provides a method for preparing lactic acid bacteria fermentation products using whey permeate. The only difference between this method and Example 3 is that Lactobacillus plantarum LW11 is replaced with Lactobacillus plantarum Lp90 strain.

[0086] Comparative Example 2: Effect of non-stepwise fermentation on the antibacterial activity of whey permeate fermentation product

[0087] This comparative example provides a method for preparing lactic acid bacteria fermentation products using whey permeate. The difference between this method and Example 3 is that no secondary culture medium is added for fermentation; instead, a single fermentation is used. Specifically, step S4 involves sealing and fermenting the inoculum obtained in step S3 in a fermenter at 35°C for 48 hours.

[0088] Comparative Example 3: Effect of no lactase and protease treatment on the antibacterial activity of whey permeate fermentation product

[0089] This comparative example provides a method for preparing lactic acid bacteria fermentation products using whey permeate. The difference between this method and Example 3 is that the whey permeate solution is not treated with lactase and protease in step S2.

[0090] Example 6: Antibacterial effect of whey osmotic fermentation broth

[0091] This embodiment verifies the antibacterial effect of whey permeate fermentation broth, and the test method is as follows.

[0092] 1. Antibacterial effect of fermented whey exudate

[0093] (1) Test strains: Staphylococcus aureus ATCC 25923, Pseudomonas fluorescens ATCC 13525, Escherichia coli ATCC 35218, Salmonella ATCC 14028, Helicobacter pylori ATCC 43504.

[0094] (2) Preparation of bacterial suspension:

[0095] The tested bacterial strains were activated and cultured at 37°C for 24 hours in their respective conventional culture media. Single colonies were picked and placed in sterile physiological saline, and the absorbance was measured. The OD600 nm value was adjusted to 0.5 (approximately 1 × 10⁻⁶ colonies). 8 (CFU / mL)

[0096] (3) Determination of inhibition zones:

[0097] Using the Oxford cup method, 100 μL of the cultured bacterial indicator suspension was aspirated onto LB agar, spread evenly with a spreader, and placed in an autoclaved Oxford cup. 200 μL of sterile fermented whey permeate supernatant was pipetted into an Oxford cup (8 mm × 6 mm × 10 mm). 100 μL of cell-free supernatant from *Lactobacillus plantarum* LW11 fermentation on MRS medium and cell-free supernatant from *Lactobacillus plantarum* Lp90 fermentation on MRS medium were used as controls. The cell-free supernatant from the fermentation on MRS medium was obtained by microfiltration after centrifuging *Lactobacillus plantarum* on MRS medium for 48 h at 8000 rpm for 10 min. After the operation was completed, the sample was incubated at 37℃ for 16 hours. The size of the inhibition zone was observed and measured. Each sample was repeated three times, and the average value was used to determine the antibacterial effect. The culture medium used for Helicobacter pylori was brain heart extract blood agar medium. The sample was incubated in a three-gas incubator at 37℃ for 3 days with a three-gas ratio of 5% oxygen, 10% carbon dioxide, and 85% nitrogen.

[0098] The results are shown in Table 2. The fermented whey penetrant exhibited significant inhibitory effects against Staphylococcus aureus, Salmonella, Pseudomonas fluorescens, Escherichia coli, and Helicobacter pylori, forming distinct inhibition zones. The inhibition zones against Staphylococcus aureus and Pseudomonas fluorescens were particularly large, with inhibition zone diameters exceeding 20 mm. Comparative Example 1 shows that using commercially available Lactobacillus plantarum Lp90 as the fermentation strain resulted in smaller inhibition zones, indicating that the LW11 strain possesses superior characteristics in producing antibacterial metabolites during fermentation. Comparative Example 2 shows that the inhibition zone decreased after non-stepwise fermentation, suggesting that stepwise fermentation allows for a greater concentration of antibacterial components in the fermentation product. Comparative Example 3 shows that without enzymatic pretreatment, the antibacterial activity of the fermentation product was significantly reduced, indicating that enzymatic treatment promoted an increase in antibacterial components in the fermentation product. Furthermore, when *Lactobacillus plantarum* LW11 and *Lactobacillus plantarum* Lp90 fermented MRS medium, the supernatant of the fermentation broth showed similar antibacterial effects against various pathogenic bacteria. However, *Lactobacillus plantarum* Lp90 fermented whey permeate but failed to produce a strong antibacterial effect. These results indicate that *Lactobacillus plantarum* LW11 provided by this invention can ferment whey permeate to prepare a product with strong antibacterial activity, which is not a common feature of the same strain.

[0099] Table 2 Comparison of antibacterial effects of fermentation products

[0100]

[0101] The fermented whey permeate powders obtained in Examples 3-5 and Comparative Examples 1-3 were first prepared into a 5g / 100g reconstituted water solution with water. The lactose content was determined by high performance liquid chromatography according to the first method in GB 5413.5. The results are shown in Table 3.

[0102] Table 3. Lactose content of reconstituted fermented whey permeate solution

[0103]

[0104] 2. Antifungal effect of fermented whey penetrant

[0105] (1) Mix 5% (v / v) fermented whey permeate with hot PDA medium, pour it onto a petri dish, and let it solidify. Add 5 μL of spore suspension (1×10⁻⁶) to the center of the petri dish. 6 (Spores / mL), and an equal volume of sterilized but unfermented whey permeate solution was added to PDA medium as a control. All plates were incubated at 30°C. The antibacterial effect of the fermented whey permeate was observed, and the diameter of colony hyphae was measured.

[0106] The results are as follows Figure 5As shown, the diameter of Aspergillus flavus in the embodiment is less than 16 mm, while the diameter of colonies in the comparative examples is all above 24 mm. The diameter of Penicillium chrysogenum colonies is about 9 mm, while the diameter of colonies in the comparative examples is greater than 15 mm, indicating that the whey permeate fermentation broth of the embodiment has a better antifungal effect.

[0107] (2) Add 1 mL of the fermented whey permeate obtained in Example 3 to a final concentration of 1×10 6 Spores were mixed with *Aspergillus flavus* and *Penicillium chrysogenum* spores at 140 rpm for 4 hours with shaking. After centrifugation, the supernatant was removed, with PBS as a control. FDA and PI were added to the spore pellet at concentrations of 50 mg / L and 20 mg / L, respectively, and then stained in the dark for 10 minutes. Analysis was performed using flow cytometry at 25 psi sheath pressure. FDA and PI fluorescence were excited by an argon ion laser (488 nm) and detected using HQ bandpass filters at 530 / 540 nm and 625 / 626 nm, respectively. The cell density of each sample was maintained at approximately 1 × 10⁻⁶. 6 Spore count / mL. A total of 10,000 cells were measured for each sample.

[0108] The results are as follows Figure 6 As shown, the proportion of viable Aspergillus spores decreased from 92.2% to 37.5% and the proportion of viable Penicillium spores decreased from 94.8% to 17.3% after treatment with whey penetrant fermentation broth, indicating that fermented whey penetrant has a significant antifungal effect.

[0109] 3. Effect of heat treatment on the antibacterial stability of fermented whey penetrant

[0110] Heat treatment is a common process in food processing. The fermented whey permeate prepared in Example 3 was treated at 65℃, 80℃, 100℃, and 121℃ for 30 min, respectively, with an untreated fermented whey permeate as a control. Antibacterial experiments were conducted on the tested bacteria to determine the heat stability of its antibacterial effect. The results of its effect on the stability of the extract are shown in […]. Figure 7 .

[0111] Depend on Figure 7 It can be seen that after heat treatment at 65℃, 80℃, 100℃ and 121℃, the diameter of the inhibition zone of the fermented whey permeate in Example 3 decreased slightly, indicating that the fermented whey permeate has some heat-sensitive metabolites. Therefore, low-temperature sterilization is gentler and has less impact on the antibacterial substances.

[0112] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A strain of Lactiplantibacillus plantarum LW11, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34365.

2. A microbial preparation containing Lactobacillus plantarum LW11 as described in claim 1.

3. The microbial preparation as described in claim 2, characterized in that, The microbial preparation contains at least 1 × 10⁻⁶ LW11 bacterial cells. 8 CFU / mL or 1×10 8 CFU / g.

4. A product containing Lactobacillus plantarum LW11 as described in claim 1 or a microbial preparation as described in claim 2 or 3.

5. The product as described in claim 4, characterized in that, The products include food, medicine, or health products.

6. The product as described in claim 4, characterized in that, The food product includes fermented dairy products; preferably, the fermented dairy products include fermented whey permeate.

7. A method for preparing fermented whey permeate, characterized in that, The Lactobacillus plantarum LW11 of claim 1 was added to a substrate containing whey permeate for fermentation.

8. The method as described in claim 7, characterized in that, Includes the following steps: (1) Preparation of expansion medium: Prepare a whey permeate solution with a mass concentration of 5-10% using whey permeate powder. After enzymatic hydrolysis with 50-100 U / mL lactase and 200-400 U / mL neutral protease at 35-45℃ for 2-4 hours, the temperature is raised to 90℃ to inactivate the enzyme for 10 minutes to obtain the treated whey permeate solution. (2) Inoculation: The activated Lactobacillus plantarum LW11 was inoculated at a rate of 1×10⁻⁶. 8 ~1×10 9 The solution was inoculated with a concentration of CFU / mL into the whey permeate solution obtained in step (1) to obtain the inoculation solution; (3) Stepwise fermentation: After the inoculum obtained in step (2) is sealed and fermented in a fermentation tank for 12 to 24 hours, the whey permeate solution obtained in step (1) is added again at 1 / 2 to 1 / 3 of the original fermentation liquid volume, and fermented for 12 to 24 hours to obtain fermented whey permeate; the fermentation conditions are 35 to 40℃. (4) Sterilization and drying: The fermented whey permeate obtained in step (3) is sterilized and dried to obtain whey permeate fermentation product.

9. A method for increasing the antibacterial activity of whey permeate or reducing the risk of lactose intolerance in whey permeate, characterized in that, The Lactobacillus plantarum LW11 of claim 1 was inoculated into a solution containing whey permeate for fermentation.

10. The use of the *Lactobacillus plantarum* LW11 of claim 1 or the microbial preparation of claim 2 or 3 in the preparation of antibacterial agents.

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