Lactobacillus plantarum with antioxidant and antibacterial functions and application thereof

Abstract

The present application relates to the technical field of functional microorganism screening and application, and particularly relates to a Lactobacillus plantarum with antioxidant and antibacterial functions and application thereof.The Lactobacillus plantarum is Lactobacillus plantarum WW strain, which has been preserved in the China General Microbiological Culture Collection Center on December 24, 2021, and the preservation number is CGMCC No.24189.The strain has strong acid and bile salt tolerance, outstanding antioxidant and antibacterial effects, and can be widely applied in the fields of food or medicine and the like.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a strain of *Lactobacillus plantarum* with antioxidant and antibacterial functions and its application. Background Technology

[0002] Lactic acid bacteria (LAB) are a general term for Gram-positive bacteria that produce lactic acid through metabolism, do not produce spores, and are rod-shaped or spherical in form. Lactic acid bacteria are widely distributed, inhabiting natural environments including the intestines of humans and animals, various fermented foods, and aquatic bodies. They are generally considered beneficial microorganisms and are recognized as safe food microorganisms. They have been used alone or in combination with yeast in fermented dairy products, vegetables, grains, and other foods, playing an important role in the food industry. After fermentation by lactic acid bacteria, food not only acquires a unique flavor, improves its nutritional value, and extends its shelf life, but also possesses certain health benefits after being ingested. The health benefits of lactic acid bacteria on the host are widely recognized. As probiotics, lactic acid bacteria can colonize the human intestine, playing an important role in maintaining human health and are one of the important symbiotic flora in the human body. Lactic acid bacteria in the intestines regulate gut microbiota, inhibit the growth and reproduction of pathogenic and putrefactive bacteria, lower serum cholesterol, enhance immunity, maintain microecological balance, resist mutagenesis, delay aging, improve liver function, and exhibit anti-tumor and antioxidant activities. Therefore, they exert comprehensive nutritional and health benefits on the body's physiological functions, immune responses, tumor development, aging process, and stress responses. It is evident that lactic acid bacteria are closely linked to human health.

[0003] Lactic acid bacteria are facultative anaerobes that do not produce catalase, but they have developed their own unique antioxidant systems through long-term evolution. Many previous studies have demonstrated that certain lactic acid bacteria possess excellent antioxidant activity. Enhanced antioxidant activity in lactic acid bacteria not only helps them resist the toxicity of oxygen-containing substances but also aids in self-protection and host protection within the host's intestinal environment.

[0004] Currently, research methods on the antioxidant activity of lactic acid bacteria both domestically and internationally mainly focus on two aspects: Firstly, simulating in vitro oxidation reactions to measure the free radical scavenging ability of intact lactic acid bacteria cells and cell-free extracts, as well as measuring the activity of antioxidant enzyme systems in lactic acid bacteria, thus providing a preliminary evaluation of their antioxidant activity. Secondly, feeding animals with oxidative damage using lactic acid bacteria fermentation preparations and measuring indicators such as SOD, GSH-Px, CAT activity, MDA content, and T-AOC activity in liver tissue and blood to illustrate the antioxidant activity of lactic acid bacteria, thereby demonstrating the protective effect of lactic acid bacteria on the body.

[0005] Song et al. isolated a strain of *Lactobacillus brevis* B13-2 from kimchi, which exhibited high DPPH and ABTS free radical scavenging capabilities and lipid peroxidation inhibitory activity. Even after heat treatment at 85℃ for 30 min, the bacteria retained their antioxidant activity. Zhang et al. studied the effect of *Lactobacillus plantarum* J26 fermentation on the antioxidant capacity of blueberry juice. The results showed that fermented blueberry juice significantly enhanced the DPPH, hydroxyl, and superoxide anion free radical scavenging capabilities, and significantly increased the content of phenolic substances and anthocyanins, which are closely related to antioxidant properties. Animal experiments and clinical studies have also shown that lactic acid bacteria can alleviate oxidative stress in organisms. Chen Dandie et al. found that adding an enzyme-bacterial compound to the diet significantly increased the serum IL-6 content, glutathione peroxidase (GSH-Px) and catalase (CAT) activities in broilers, enhanced liver superoxide dismutase (SOD) activity, reduced fecal uric acid content, and improved the immune and antioxidant functions of broilers. Bernini et al. found that Bifidobacterium lactis HN019 can improve inflammatory and oxidative stress markers in healthy individuals and patients with metabolic syndrome, and also showed specific antioxidant defense effects in healthy subjects.

[0006] Lactic acid bacteria have broad prospects whether applied in the food industry or as probiotics for human or livestock health. Therefore, screening out new lactic acid bacteria with antioxidant activity has application and research value. Summary of the Invention

[0007] The purpose of this invention is to provide a *Lactobacillus plantarum* with antioxidant and antibacterial functions and its applications. This *Lactobacillus plantarum* exhibits strong acid and bile salt resistance, outstanding antioxidant and antibacterial effects, and can be widely used in pharmaceuticals and other fields.

[0008] One aspect of this invention relates to a strain of Lactobacillus plantarum, which was deposited on December 24, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24189.

[0009] This invention also relates to the application of *Lactobacillus plantarum* WW in the preparation of products with antibacterial functions.

[0010] The product in question is a pharmaceutical product.

[0011] This invention also relates to the application of *Lactobacillus plantarum* WW in the preparation of pharmaceuticals with antioxidant functions.

[0012] This invention also relates to the application of *Lactobacillus plantarum* (WW) in the preparation of fermented foods.

[0013] This invention also relates to an active polypeptide derived from soybean residue, which is prepared by fermenting soybean residue with Lactobacillus plantarum WW.

[0014] The preparation method of the soybean residue active polypeptide includes:

[0015] (1) After drying the soybean residue to constant weight, pulverize it into 60 mesh, mix it with distilled water at a ratio of 1:10 (W:V), sterilize it at 121℃ for 20 min, and obtain soybean residue culture medium;

[0016] (2) Shake the activated Lactobacillus plantarum WW bacterial solution well, and then inoculate the Lactobacillus plantarum WW bacterial solution into soybean residue culture medium at a volume ratio of 1%, and culture statically at 37℃ for 36-48h to obtain soybean residue fermentation liquid;

[0017] (3) Mix the fermented soybean residue with a 15% trichloroacetic acid (TCA) solution at a volume ratio of 1:1 and let it stand at room temperature for 20 min.

[0018] (4) Centrifuge at 6000 rpm and 4℃ for 10 min and collect the supernatant;

[0019] (5) After filtering the supernatant through a 0.45μm aqueous membrane, freeze-dry it to obtain soybean residue active peptides.

[0020] This invention also relates to the application of the soybean residue active peptides in the preparation of food, health products or pharmaceuticals.

[0021] This invention also relates to the application of the soybean residue active peptides in the preparation of pharmaceuticals with antioxidant functions.

[0022] Beneficial effects:

[0023] The *Lactobacillus plantarum* WW provided by this invention has strong inhibitory effects on Salmonella, *Escherichia coli*, *Pseudomonas aeruginosa*, *Cronobacter sakazakii*, and *Shigella*, with minimum inhibitory concentrations of 64 μg / mL, 128 μg / mL, 128 μg / mL, 64 μg / mL, and 32 μg / mL, respectively. Among them, the inhibitory effect on Salmonella, *Cronobacter sakazakii*, and *Shigella* is the strongest, with inhibition zone diameters exceeding 18 mm.

[0024] Lactobacillus plantarum WW can tolerate the effects of gastric and intestinal fluids, low-acid environments, and high-bile-salt environments, maintaining high activity. After digestion with artificial gastric juice for 3 hours and artificial intestinal juice for 4 hours, the survival rate of this strain was as high as 94.1% and 107.9%, respectively, demonstrating its ability to effectively colonize the intestine and exert its functional characteristics through adverse gastrointestinal environments.

[0025] Lactobacillus plantarum WW has a strong antioxidant capacity. The enzyme activities of protease and superoxide dismutase in its fermentation supernatant are as high as 215.48 μg / mL and 187.66 μg / mL, respectively, and the scavenging rate of DPPH free radicals can reach 52.94%, which is significantly higher than that of the control Lactobacillus plantarum.

[0026] Furthermore, the soybean residue active peptides prepared by fermentation with *Lactobacillus plantarum* WW have strong antioxidant activity and can efficiently scavenge DPPH free radicals with a scavenging rate as high as 80.61%, which is significantly better than the control *Lactobacillus plantarum* ATCC8014. Moreover, the mass concentration of the soybean residue active peptides prepared by fermentation with *Lactobacillus plantarum* WW shows a linear relationship with the DPPH free radical scavenging rate. The IC50 of DPPH free radicals was calculated to be 0.22 mg / mL by fitting the equation.

[0027] Lactobacillus plantarum (WW) has broad application prospects in the production of fermented foods, health products, and pharmaceuticals. Attached Figure Description

[0028] Figure 1 Image of Lactobacillus plantarum WW colony;

[0029] Figure 2 Gram staining for Lactobacillus plantarum;

[0030] Figure 3 A phylogenetic tree of *Lactobacillus plantarum* WW based on the 16S rDNA gene sequence;

[0031] Figure 4 This image shows the antibacterial effect of Lactobacillus plantarum WW.

[0032] Figure 5 A graph showing the DPPH free radical scavenging effect of soybean residue active peptides prepared by Lactobacillus plantarum WW fermentation. Detailed Implementation

[0033] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0034] The present invention will be further described below with reference to specific embodiments.

[0035] Example 1: Isolation and Screening of Strains

[0036] 1. Sample Source

[0037] Traditional fermented soybean paste collected in Shenyang, Liaoning Province.

[0038] 2. Isolation of strains

[0039] Add 1g of fermented soybean paste to 9mL of buffered peptone, serially dilute 10-fold, and streak onto MRS agar plates at appropriate dilutions. Incubate at 37℃ for 16-18h. Select large, highly active single colonies and streak them onto MRS agar plates for purification. Repeat this process 2-3 times until the colony characteristics in the streaks are consistent. For each purified plate, select at least two single colonies for smear preparation, Gram staining, and observe under a microscope to determine if the color and cell shape are consistent, thus confirming whether the colonies in the plate are pure cultures.

[0040] Through microscopic examination, 11 strains were selected that were blue-purple, rod-shaped, and arranged individually or in chains without spores. These strains were named W1, W2, ..., W11.

[0041] 3. Secondary screening of antibacterial strains

[0042] The reference indicator strain was *Escherichia coli*, excluding interference from organic acids and hydrogen peroxide. The diameter of the inhibition zone was measured using the perforation method.

[0043] The results showed that among the 11 strains screened in this invention, strain W8 had the strongest inhibitory effect on Escherichia coli.

[0044] Example 2 Identification of strain W8

[0045] 2.1 Identification of fungal morphology

[0046] like Figure 1 and Figure 2 As shown, strain W8 is a Gram-positive, facultatively anaerobic, non-spore-forming, and non-motile bacterium; colonies are small and uniform in size, with neat edges, smooth and dense surfaces, and are white or milky white; the cells are short rod-shaped and arranged in pairs or chains.

[0047] 2.2 Identification of Physiological and Biochemical Characteristics

[0048] Table 1 Physiological and Biochemical Characteristics

[0049] Physiological and biochemical identification W8 strain Gram staining + Growth at 10℃ + Growth at 15℃ + catalase experiment - Liquefied gelatin - Reduced nitrates - Hydrolysis of arginine to produce ammonia - Arabic sugar + Aesculin + fructose + glucose + lactose + Mannose + Mannitol + Sorbitol + Ginsenosides + Melibi + Salicylic acid + sucrose + Trehalose + Xylose + Inulin + maltose + Rhamnose - Raffinose -

[0050] Note: "+" indicates a positive reaction or growth, "-" indicates a negative reaction or no growth.

[0051] 2.3 Molecular biological identification

[0052] NCBI BLAST comparison revealed that the 16S rDNA sequence of strain W8 showed the highest similarity to that of *Lactobacillus plantarum*. Furthermore, a phylogenetic tree constructed using MEGA 7.0 yielded the following results: Figure 3 As shown, strain W8 is most homologous to Lactobacillus plantarum.

[0053] In summary, based on the colony morphology, physiological and biochemical characteristics, and molecular biological identification results of strain W8, the applicant has determined that the screened strain W8 is a novel *Lactobacillus plantarum*, named it *Lactobacillus plantarum*, with strain number WW, and deposited it on December 24, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 24189.

[0054] Example 3: Experiment on the antibacterial effect of Lactobacillus plantarum WW

[0055] 1. Preparation of indicator bacteria:

[0056] Thaw cryovials containing five indicator strains of Salmonella, Escherichia coli, Pseudomonas aeruginosa, Cronobacter sakazakii, and Shigella at room temperature.

[0057] Lactobacillus plantarum WW and Lactobacillus plantarum ATCC8014 seed culture were inoculated into MRS medium at an inoculation rate of 1% (V / V) and cultured at 37°C for 24 hours.

[0058] Salmonella, Escherichia coli, Pseudomonas aeruginosa, Cronobacter sakazakii, and Shigella were inoculated into liquid culture medium at an inoculation rate of 1% (V / V) and cultured overnight at 37°C to obtain the culture medium.

[0059] 2. Antibacterial zone test

[0060] The concentrations of bacterial suspensions containing five indicator bacteria—Salmonella, Escherichia coli, Pseudomonas aeruginosa, Cronobacter sakazakii, and Shigella—were adjusted to 10 using sterile physiological saline. 6 cfu / mL.

[0061] Spread 100 μL of indicator bacterial suspension evenly onto a solid culture medium. Place a sterile Oxford cup vertically into the solid culture medium, punch holes, and then remove the cup. Add 200 μL of *Lactobacillus plantarum* WW supernatant to each well and incubate at 37°C for 48 hours. Observe for the presence of a clear zone and measure its diameter using calipers. See below for detailed results. Figure 4 .

[0062] from Figure 2It can be seen that the *Lactobacillus plantarum* WW provided by this invention has a strong inhibitory effect on Salmonella, *Escherichia coli*, *Pseudomonas aeruginosa*, *Cronobacter sakazakii*, and *Shigella*, among which the inhibitory effect on Salmonella, *Cronobacter sakazakii*, and *Shigella* is the strongest, with an inhibition zone diameter exceeding 18 mm.

[0063] 3. Determination of minimum inhibitory concentration (MIC)

[0064] The minimum inhibitory concentration of *Lactobacillus plantarum* WW against five indicator bacteria—Salmonella, *Escherichia coli*, *Pseudomonas aeruginosa*, *Cronobacter sakazakii*, and *Shigella*—was determined using the two-fold serial dilution method.

[0065] The fermentation broth of Lactobacillus plantarum WW was centrifuged at 4 ℃, 10000 r / min for 10 min, and the bacterial cells were discarded to obtain the supernatant. The supernatant was then evaporated at 45℃ and freeze-dried to obtain freeze-dried powder, which was stored for later use.

[0066] After dissolving the lyophilized supernatant of *Lactobacillus plantarum* WW in sterile water, fermentation broth of indicator bacteria was added to achieve final concentrations of 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL, respectively. A blank control group was set up without adding the lyophilized supernatant. The mixture was incubated at 37°C for 24 h.

[0067] The OD600 values ​​of indicator bacteria treated with supernatants of different concentrations were measured, and the growth of the strains was observed. The supernatant concentration at which no bacterial growth was observed to the naked eye and the OD600 value was closest to that of the blank control group was taken as the minimum inhibitory concentration (MIC). The specific results are shown in Table 2.

[0068] Table 2. Results of determination of the minimum inhibitory concentration of Lactobacillus plantarum WW

[0069]

[0070] As shown in Table 2, the minimum inhibitory concentrations of *Lactobacillus plantarum* WW against *Salmonella*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Cronobacter sakazakii*, and *Shigella* were 64 μg / mL, 128 μg / mL, 128 μg / mL, 64 μg / mL, and 32 μg / mL, respectively.

[0071] Example 4: Tolerance experiment of Lactobacillus plantarum WW to adverse environment in the digestive tract

[0072] 4.1 Acid resistance test

[0073] (1) Control group: The activated Lactobacillus plantarum WW bacterial solution was inoculated into liquid MRS medium (pH 6.7) at a ratio of 3% (V / V);

[0074] (2) Experimental group: The activated Lactobacillus plantarum WW bacterial solution was inoculated into liquid MRS medium with pH 2.0 and 3.0 at a ratio of 3% (V / V);

[0075] After incubation at 37°C for 24 hours, the bacterial culture was serially diluted 10-fold with sterile physiological saline. 200 μL of each appropriate dilution was taken for mixed bacterial counting. Each dilution was repeated 3 times. After incubation at 37°C for 24 hours, viable colonies were counted on the plates.

[0076] The number of viable bacteria in the control group is represented by N′, and the number of viable bacteria in the experimental group is represented by N″. The survival rate of Lactobacillus plantarum WW under acidic conditions was calculated, and the results are shown in Table 3.

[0077] The calculation formula is as follows:

[0078] Survival rate (%) = lg cfu N″ / lg cfu N′ × 100%.

[0079] Table 3. Tolerance of Lactobacillus plantarum WW to acidic conditions

[0080]

[0081] As can be seen from the data in Table 3, the *Lactobacillus plantarum* WW provided by this invention has a strong tolerance to acidic environments. After treatment under pH 2.0 acidic conditions for 24 hours, the survival rate is still as high as 61.5%, which is a significant effect.

[0082] 2.2 Bile Salt Tolerance Test

[0083] (1) Control group: The activated Lactobacillus plantarum WW bacterial suspension was inoculated into liquid MRS medium (pH 6.7) without ox bile salts at a ratio of 3% (V / V);

[0084] (2) Experimental group: The activated Lactobacillus plantarum WW bacterial suspension was inoculated into liquid MRS medium (pH 6.7) containing 0.3%, 0.5%, 1.0% and 1.5% ox bile salt at a ratio of 3% (V / V);

[0085] After incubation at 37°C for 24 hours, the bacterial culture was serially diluted 10-fold with sterile physiological saline. 200 μL of each appropriate dilution was taken for mixed bacterial counting. Each dilution was repeated 3 times. After incubation at 37°C for 24 hours, viable colonies were counted on the plates.

[0086] The number of viable bacteria in the control group is represented by N′, and the number of viable bacteria in the experimental group is represented by N″. The survival rate of Lactobacillus plantarum WW was calculated, and the results are shown in Table 4.

[0087] Survival rate (%) = lg cfu N″ / lg cfu N′ × 100%.

[0088] Table 4. Tolerance of Lactobacillus plantarum WW to bile salts

[0089]

[0090] As can be seen from the data in Table 4, the *Lactobacillus plantarum* WW provided by this invention has a strong tolerance to bile salts. After treatment with 1.5% bile salts for 24 hours, the survival rate is still as high as 67.2%, achieving unexpected technical results.

[0091] 4.3 Artificial gastric juice test

[0092] Take 10 mL of Lactobacillus plantarum WW fermentation broth, centrifuge at 5℃ and 5000g for 10 min to obtain bacterial sludge; wash 3 times with PBS buffer, then resuspend the bacterial sludge in 10 mL of artificial gastric fluid; digest at 37℃ for 3 h, and take samples at 0 h and 3 h to determine the viable bacterial count.

[0093] The initial viable bacterial count in the simulated gastric juice is denoted by N′, and the viable bacterial count in the simulated gastric juice after 3 hours of digestion is denoted by N″. The survival rate of *Lactobacillus plantarum* WW in the simulated gastric juice was calculated, and the results are shown in Table 5. The calculation formula is as follows:

[0094] Survival rate (%) = lg cfu N″ / lg cfu N′ × 100%.

[0095] Table 5. Tolerance of *Lactobacillus plantarum* WW to artificial gastric juice.

[0096]

[0097] As can be seen from the data in Table 5, the *Lactobacillus plantarum* WW strain provided by this invention has a strong survival ability in artificial gastric juice. After 3 hours of digestion, the number of viable *Lactobacillus plantarum* WW strains showed a slight downward trend, with a survival rate of 94.1%, demonstrating significant effectiveness.

[0098] 4.4 Artificial Intestinal Fluid Test

[0099] Take 10 mL of Lactobacillus plantarum WW fermentation broth, centrifuge at 5℃ and 5000g for 10 min to obtain bacterial sludge; wash 3 times with PBS buffer, and resuspend the bacterial sludge in 10 mL of artificial intestinal fluid; digest at 37℃ for 3 h, and take samples at 2 h and 4 h to determine the viable count.

[0100] The initial viable bacterial count in the artificial intestinal fluid is represented by N′, and the viable bacterial count in the artificial intestinal fluid after 2 hours and 4 hours of digestion is represented by N″. The survival rate of *Lactobacillus plantarum* WW in the artificial intestinal fluid was calculated, and the results are shown in Table 6. The calculation formula is as follows:

[0101] Survival rate (%) = lg cfu N″ / lg cfu N′ × 100%.

[0102] Table 6. Tolerance of *Lactobacillus plantarum* WW to artificial intestinal fluid.

[0103]

[0104] As shown in Table 6, the *Lactobacillus plantarum* WW provided by this invention exhibits strong tolerance to artificial intestinal fluid. After 2 hours and 4 hours of digestion, its survival rate not only did not decrease but also increased to 107.9%. This demonstrates that the strain can effectively proliferate in artificial intestinal fluid, achieving unexpected technical results.

[0105] The above experimental results show that the *Lactobacillus plantarum* WW provided by this invention can effectively resist the effects of gastrointestinal fluid, low acidity environment, and high bile salt environment, and maintain high activity.

[0106] Example 5: Antioxidant performance test of Lactobacillus plantarum WW

[0107] 5.1 Protease activity

[0108] The phthalaldehyde method was used to screen and evaluate the protease activity of bacterial strains. The phthalaldehyde method is a widely accepted method for determining protease hydrolysis activity. Its principle is based on the hydrolysis reaction between phthalaldehyde and β-mercaptoethanol, releasing α-amino acids, which have a strong absorption peak at 340 nm in the ultraviolet light. The proteolytic ability of lactic acid bacteria is reflected by measuring the content of α-amino acids. The specific steps are as follows.

[0109] (1) Reagent preparation

[0110] Preparation of 100 μg / mL tyrosine solution: Dry tyrosine in an oven at 105℃ to constant weight. Accurately weigh 0.1000 g of the constant-weight tyrosine and gradually add 6 mL of 1 mol / L hydrochloric acid to dissolve it. Dilute to 100 mL with 0.2 mol / L hydrochloric acid. At this point, the concentration of the tyrosine solution is 1000 μg / mL. Take 10 mL of this solution and dilute to 100 mL with 0.2 mol / L hydrochloric acid to prepare a 100 μg / mL tyrosine solution. This solution should be used promptly or stored in a refrigerator immediately after preparation.

[0111] Preparation of OPA reagent: Mix 62.5 mL of 0.1 M sodium tetraborate solution, 6.25 mL of 20% SDS solution, 100 mg of o-phthalaldehyde, 5 mL of methanol solution, and 250 μL of β-mercaptoethanol solution, and bring the volume to 250 mL to prepare OPA reagent. OPA reagent should be prepared fresh for use and stored away from light.

[0112] (2) Determination of the standard curve

[0113] Take 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of tyrosine solution (concentration of 100 μg / mL), respectively, and bring the solution to 5 mL with ddH₂O. Mix well and set aside. Take 150 μL of tyrosine solution of different concentrations, add 3 mL of LOPA reagent, gently shake to mix, react at room temperature for 2 min, and measure the OD. 340 Value: Plotted on the x-axis, OD 340 Use the ordinate to create a standard curve: Y = 0.0143X + 0.0027 (R²) 2 =0.9903).

[0114] (3) Sample determination

[0115] The activated *Lactobacillus plantarum* WW bacterial suspension was inoculated into MRS medium at a rate of 3% (V / V) and cultured at 37℃ for 24 h. 1.25 mL of the bacterial suspension was taken, and 0.3 mL of ddH₂O and 3 mL of 0.75 M trichloroacetic acid were added. After mixing thoroughly, the mixture was allowed to stand at room temperature for 10 min. The mixture was then centrifuged at 6000 r / min and 4℃ for 6 min, and the supernatant was collected. 150 μL of the supernatant was taken, and 3 mL of OPA reagent was added. The mixture was gently shaken and allowed to stand at room temperature for 2 min. The absorbance of the solution was measured at a wavelength of 340 nm. The obtained OD₂... 340 The protease activity was calculated by comparing the values ​​with the standard curve. Purchased *Lactobacillus plantarum* ATCC8014 was used as a control. Specific results are shown in Table 7.

[0116] Table 7. Protease production by Lactobacillus plantarum

[0117] strain Protease activity (μg / mL) Lactobacillus plantarum WW 215.48 Lactobacillus plantarum ATCC8014 130.45

[0118] As shown in Table 7, the protease hydrolysis activity in the fermentation supernatant of *Lactobacillus plantarum* WW provided by this invention is significantly higher than that of the control strain *Lactobacillus plantarum* ATCC8014, reaching 215.48 μg / mL.

[0119] 5.2 Superoxide dismutase

[0120] The kit employs a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using a double antibody. Sample, standard, and HRP-labeled detection antibody are added sequentially to microwells pre-coated with superoxide dismutase (SOD) antibody, followed by incubation and thorough washing. The substrate TMB is used for color development; TMB is converted to blue under the catalysis of superoxide dismutase, and then to yellow under acidic conditions. The color intensity is positively correlated with the amount of superoxide dismutase (SOD) in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the superoxide dismutase activity in the *Lactobacillus plantarum* WW fermentation supernatant. 450Plot a standard curve using the ordinate: Y = 135.03X - 12.061 (R²) 2 = 0.9946). Purchased Lactobacillus plantarum ATCC8014 was used as a control. Specific results are shown in Table 8.

[0121] Table 8. Superoxide dismutase production by Lactobacillus plantarum

[0122] strain Superoxide dismutase activity (μg / mL) Lactobacillus plantarum WW 187.66 Lactobacillus plantarum ATCC8014 121.49

[0123] As can be seen from the results in Table 8, the superoxide dismutase (SOD) hydrolysis activity in the fermentation supernatant of *Lactobacillus plantarum* WW of the present invention was significantly higher than that of the control strain *Lactobacillus plantarum* ATCC8014, reaching 187.66 μg / mL.

[0124] 5.3 DPPH free radical scavenging test

[0125] The seed cultures of *Lactobacillus plantarum* WW and the control strain *Lactobacillus plantarum* ATCC8014 were inoculated into liquid MRS medium at an inoculation rate of 1% (V / V) and cultured at 37°C for 24 h.

[0126] A 15% trichloroacetic acid (TCA) solution was prepared. The fermentation broth of *Lactobacillus plantarum* was mixed with the TCA solution at a 1:1 ratio and allowed to stand at room temperature for 20 min. The mixture was then centrifuged at 6000 rpm and 4℃ for 10 min. The supernatant was filtered through a 0.45 μm aqueous membrane and then freeze-dried to obtain crude peptides. 250 mg of crude peptides were placed in 1 mL of simulated gastric and intestinal fluids, respectively, and simulated digestion was performed at 37℃ and 120 rpm for 1 h and 2 h, respectively. The scavenging rate of *Lactobacillus plantarum* fermentation broth on DPPH free radicals after simulated digestion was measured.

[0127] Prepare 1×10 using anhydrous ethanol -4 2 mL of DPPH solution and 2 mL of the test solution were added to a test tube, vortexed, and reacted at room temperature in the dark for 30 min. The absorbance of the sample was measured at 517 nm. The DPPH free radical scavenging rate was calculated according to the following formula. The specific results are shown in Table 9.

[0128] DPPH clearance rate % = [1 - (A1 - A2) / A3] × 100%.

[0129] In the formula: A1 is the absorbance value of the sample after adding DPPH solution; A2 is the absorbance value of the sample solution; A3 is the absorbance of ethanol after adding DPPH solution.

[0130] Table 9. Scavenging rate of DPPH free radicals by Lactobacillus plantarum WW

[0131]

[0132] As shown in Table 9, the *Lactobacillus plantarum* WW provided by this invention has a strong antioxidant capacity and can effectively scavenge DPPH free radicals with a scavenging rate of 40.22%-52.94%, which is 21.8%-30.8% higher than the control strain *Lactobacillus plantarum* ATCC8014, achieving unexpected technical results.

[0133] Example 6: Application of *Lactobacillus plantarum* WW in the fermentation preparation of active peptides from soybean residue

[0134] 6.1 Preparation of soybean residue bioactive peptides

[0135] After drying the wet soybean residue to constant weight, pulverize it into a 60-mesh powder and mix it with distilled water at a ratio of 1:10 (w:v). Sterilize at 121℃ for 20 min to obtain soybean residue culture medium. Shake the activated Lactobacillus plantarum WW bacterial solution well and inoculate it into the soybean residue culture medium at a volume ratio of 1%. Incubate statically at 37℃ for 36-48 h to obtain soybean residue fermentation broth.

[0136] The soybean residue fermentation broth obtained above was mixed with a 15% trichloroacetic acid (TCA) solution at a volume ratio of 1:1 and allowed to stand at room temperature for 20 min. It was then centrifuged at 6000 rpm and 4℃ for 10 min. The supernatant was filtered through a 0.45 μm aqueous membrane and then freeze-dried to obtain soybean residue active peptides.

[0137] As a control, the same procedure was performed as described above, and Lactobacillus plantarum ATCC8014 was inoculated to prepare soybean residue bioactive peptides.

[0138] 6.2 Antioxidant activity analysis of soybean residue bioactive peptides

[0139] Prepare 1×10 using anhydrous ethanol -4 2 mL of DPPH solution and 2 mL of soybean residue active peptide solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, and 0.4 mg / mL were added to test tubes. After vortexing and mixing, the mixture was reacted at room temperature in the dark for 30 min. The absorbance of the samples was measured at 517 nm. The DPPH free radical scavenging rate was calculated according to the following formula. Specific results are shown in Table 10 and... Figure 5 .

[0140] DPPH clearance rate % = [1 - (A1 - A2) / A3] × 100%.

[0141] In the formula: A1 is the absorbance value of the sample after adding DPPH solution; A2 is the absorbance value of the sample solution; A3 is the absorbance of ethanol after adding DPPH solution.

[0142] Table 10. Scavenging rate of DPPH free radicals by soybean residue active peptides

[0143]

[0144] From Table 10 and Figure 5 It can be seen that the soybean residue active peptides prepared by fermentation with *Lactobacillus plantarum* WW in this invention have strong antioxidant activity and can efficiently scavenge DPPH free radicals with a scavenging rate of up to 80.61%, which is significantly better than the control *Lactobacillus plantarum* ATCC8014. Moreover, there is a linear relationship between the mass concentration of the soybean residue active peptides prepared by fermentation with *Lactobacillus plantarum* WW and the DPPH free radical scavenging rate. The IC50 of DPPH free radicals was calculated to be 0.22 mg / mL by fitting the equation.

[0145] In summary, the *Lactobacillus plantarum* strain WW obtained in this invention exhibits significant inhibitory effects against common Gram-negative pathogenic bacteria such as Salmonella, *Escherichia coli*, *Pseudomonas aeruginosa*, *Cronobacter sakazakii*, and *Shigella*. This strain effectively resists the effects of gastrointestinal fluids, low-acid environments, and high-bile-salt environments, maintaining high activity. The protease and superoxide dismutase (SOD) hydrolytic activities and DPPH free radical scavenging rate in the fermentation supernatant of *Lactobacillus plantarum* strain WW are significantly higher than those of the control strain *Lactobacillus plantarum* ATCC8014, demonstrating strong antioxidant capacity. This strain has broad application prospects in food and pharmaceutical production.

Claims

1. A type of Lactobacillus plantarum WW, characterized in that, The preservation number of the Lactobacillus plantarum WW is CGMCC No. 24189.

2. The use of Lactobacillus plantarum WW as described in claim 1 in the preparation of pharmaceuticals with the function of inhibiting Salmonella, Escherichia coli, Pseudomonas aeruginosa, Cronobacter sakazakii and Shigella.

3. The use of Lactobacillus plantarum WW as described in claim 1 in the preparation of pharmaceuticals with antioxidant functions.

4. The application of Lactobacillus plantarum WW as described in claim 1 in the preparation of fermented foods.

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