Lactobacillus plantarum with high yield of antioxidant peptide and application thereof

Abstract

This invention relates to the fields of microbial technology and food, and particularly to a strain of *Lactobacillus plantarum* that produces high levels of antioxidant peptides and its applications. The *Lactobacillus plantarum* strain was screened from naturally fermented soybean paste from traditional farmhouses in Northeast China (CGMCC No. 35049). It possesses the ability to produce high levels of antioxidant peptides, which can improve the nutritional composition of fermented foods and reduce oxidative spoilage, showing broad application prospects in fermented foods.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Lactobacillus plantarum* that produces high levels of antioxidant peptides and its applications. Background Technology

[0002] Lactic acid bacteria (LAB) are a class of Gram-positive bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. They exhibit rich species diversity and a wide distribution across various ecological environments, and have significant applications in the food, biomedical, and agricultural fields. LAB plays important roles in maintaining intestinal flora balance, regulating immunity, lowering serum cholesterol, improving food flavor and texture, and providing antioxidant benefits.

[0003] Lactobacillus plantarum belongs to the genus Lactobacillus. Its cells are rod-shaped and usually arranged singly, in pairs, or in short chains. Widely distributed in fermented foods, plant-based foods, and the human gut, Lactobacillus plantarum exhibits acid, salt, and bile salt tolerance, enabling it to survive and function in various food matrices. It has shown great potential in improving food flavor, enhancing nutritional value, extending shelf life, and promoting human health, and has become a hot topic in food science research.

[0004] Bioactive peptides (BPs) are small molecule peptides with biological activity. They typically consist of 2 to 50 amino acid residues and are usually obtained through the hydrolysis of precursor proteins by proteases or chemical synthesis. They possess specific biological functions, such as regulating the immune system, cell proliferation, apoptosis, and cell signal transduction. Bioactive peptides have advantages such as being natural, pollution-free, and abundant in resources, and have been proven to possess various biological activities including antioxidant, blood pressure lowering, blood sugar lowering, anti-inflammatory, and immunomodulatory effects, making them valuable in the development of functional foods and health products. Antioxidant peptides belong to the category of bioactive peptides and are small molecule natural bioactive substances with antioxidant functions. They have attracted much attention due to their "green," "highly active," and "easily absorbed" characteristics. Studies have shown that the biological activity of antioxidant peptides is affected by the amino acid sequence, molecular weight, and the content of hydrophobic amino acids. Generally, the smaller the molecular weight and the higher the proportion of hydrophobic amino acids such as proline (Pro), valine (Val), tryptophan (Trp), and phenylalanine (Phe) linked to the amino acid residues, the stronger the antioxidant activity.

[0005] Currently, the main methods for preparing antioxidant peptides include enzymatic hydrolysis, chemical synthesis, and microbial fermentation. Among these, enzymatic hydrolysis is limited in application due to its limited efficiency and tendency to leave solvent residues, while chemical synthesis is restricted by its high cost. In contrast, microbial fermentation offers advantages such as safety, reliability, diversity, high yield, ease of modification, economy, and sustainability, making it a significant source for antioxidant peptide preparation.

[0006] Nature possesses a rich diversity of microorganisms. Through targeted screening, microbial strains with unique metabolic pathways can be obtained. The antioxidant peptides produced by these strains often have advantages such as novel structures and strong activity, providing important support for the green, sustainable, and large-scale preparation of antioxidant peptides. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of *Lactobacillus plantarum* that produces high levels of antioxidant peptides and its applications. This *Lactobacillus plantarum* was screened from naturally fermented soybean paste from traditional farms in Northeast China. It possesses a high capacity for producing antioxidant peptides, which can improve the nutritional composition of fermented foods and reduce oxidative spoilage, thus showing broad application prospects in fermented foods.

[0008] One aspect of this invention relates to a strain of Lactobacillus plantarum, named Lactobacillus plantarum strain W66, which was deposited on June 30, 2025, 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. 35049.

[0009] The 16S rDNA sequence of the *Lactobacillus plantarum* strain W66 is SEQ ID NO: 1.

[0010] This invention also relates to the application of the Lactobacillus plantarum strain W66 in the production of fermented foods.

[0011] This invention also relates to the application of the Lactobacillus plantarum strain W66 in the production of biopeptides.

[0012] The biopeptide is an antioxidant peptide.

[0013] The present invention also provides a probiotic fermentation agent comprising the Lactobacillus plantarum strain W66.

[0014] The probiotic starter also includes any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Halophilic tetracoccus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

[0015] The probiotic starter contains at least 10 live bacteria of Lactobacillus plantarum strain W66. 8 CFU / g.

[0016] The present invention also provides the application of the probiotic starter in food production.

[0017] The *Lactobacillus plantarum* W66 provided by this invention has the ability to produce high levels of protease and bioactive peptides. The protease activity in its fermentation supernatant reaches 498.71±1.27 U / mL, and the peptide content reaches 30.34±0.008 mg / mL, achieving unexpected technical results.

[0018] The *Lactobacillus plantarum* W66 exhibits strong antioxidant capacity. The scavenging rates of its sterile cell supernatant, bacterial suspension, and cell lysate extract against DPPH free radicals were 79.23±1.12%, 15.31±1.03%, and 34.62±1.20%, respectively; the scavenging rates against hydroxyl free radicals were 82.70±1.03%, 23.58±2.91%, and 48.44±1.13%, respectively; and the reducing power was 89.85±1.02%, 25.32±1.25%, and 28.65±1.34%, respectively.

[0019] The antioxidant peptides produced by *Lactobacillus plantarum* W66 have strong antioxidant capacity, with scavenging rates of 69.35±1.03% and 81.27±0.25% for DPPH and ABTS free radicals, respectively.

[0020] The *Lactobacillus plantarum* W66 exhibits strong acid resistance, bile salt resistance, antibacterial activity, and adhesion. After 4 hours of culture in an acidic environment (pH 2.0), the survival rate was 40.9%; after 4 hours of culture in 0.1% bile salts, the survival rate was 55.88%; after 3 hours of treatment in simulated gastric fluid, the survival rate was 55.98%; and after 6 hours of treatment in simulated intestinal fluid, the survival rate was 35.17%. The surface hydrophobicity was 38.42%, and the self-agglutination rate was 44.26%. The inhibition zone diameters against *Escherichia coli* and *Staphylococcus aureus* reached 19.56 mm and 17.23 mm, respectively.

[0021] The bioactive peptides prepared by fermentation using *Lactobacillus plantarum* W66 provided in this invention have advantages such as safety, reliability, and sustainability. This strain can be directly applied to the production of fermented foods, improving food flavor, enhancing nutritional value, and reducing oxidative spoilage, thus possessing broad application prospects. Attached Figure Description

[0022] Figure 1 The interaction between temperature and time on peptide content;

[0023] Figure 2 The interaction between temperature and inoculum size on peptide content;

[0024] Figure 3 The interaction between temperature and pH on peptide content;

[0025] Figure 4 The interaction between time and inoculation amount on peptide content;

[0026] Figure 5 The interaction between pH and time on peptide content;

[0027] Figure 6 The interaction between pH and inoculum size on peptide content;

[0028] Figure 7 The growth curve of Lactobacillus plantarum W66;

[0029] Figure 8 Acid resistance test for Lactobacillus plantarum W66;

[0030] Figure 9 Salt tolerance test for Lactobacillus plantarum W66;

[0031] Figure 10 The survival rate of Lactobacillus plantarum W66 in simulated gastrointestinal fluid was determined. Detailed Implementation

[0032] To make the present invention clearer, the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to explain and illustrate the technical solutions of the present invention and are not intended to limit the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention shall fall within the scope of the present invention.

[0033] The Lactobacillus plantarum mentioned in this embodiment of the invention is Lactobacillus plantarum W66, which was deposited at the China General Microbiological Culture Collection Center on June 30, 2025, with accession number CGMCC No. 35049.

[0034] The culture medium used in this invention is as follows:

[0035] MRS fermentation medium: 10 g / L peptone, 3 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.575 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate monohydrate, 20 g / L glucose, 2.42 g / L trisodium citrate, 4 g / L yeast extract, 8 g / L beef extract, 1 g / L Tween 80, add distilled water to a final volume of 1 L, and adjust the pH to 7.0.

[0036] Example 1: Isolation, screening and identification of Lactobacillus plantarum W66

[0037] 1. Preliminary screening of lactic acid bacteria

[0038] Accurately weigh 1.0 g of fermented soybean paste sample collected from Shenyang, Liaoning Province, into a 15 mL sterile centrifuge tube. Dilute to 10 mL with sterile physiological saline, stir thoroughly, and let stand for 20–25 min. Transfer 1 mL of the supernatant to a new centrifuge tube and dilute with 9 mL of sterile physiological saline to a final volume of 10 mL. -4 10 -5 10 -6 100 μL of the above diluted solution was spread onto MRS solid medium and incubated at 36°C for 2–3 days. After single colonies grew, single colonies with good growth and suspected lactic acid bacteria were picked and Gram-stained. A total of 72 strains were isolated as Gram-positive bacteria. Physiological and biochemical experiments were used to confirm whether they were lactic acid bacteria, and they were named W1, W2, ..., W72. After incubation at 36°C for 2 days, the culture was purified for three generations until no contamination was observed, yielding pure colonies grown on MRS solid plates. High-quality single colonies were selected and inoculated onto MRS liquid medium, and cultured on a shaker at 120 rpm and 37°C to obtain single-cell suspensions. Finally, the strains were preserved in glycerol for later use.

[0039] 2. Secondary screening of protease-producing lactic acid bacteria

[0040] Proteases play a crucial role in the fermentation of soybean paste, breaking down proteins in raw materials into smaller peptides or amino acids. The level of protease activity is one indicator of whether a strain possesses the ability to produce high levels of antioxidant peptides.

[0041] The lactic acid bacteria isolated above were re-screened using the clear zone method to select strains with protease-producing capabilities. The preserved bacterial strains were activated and inoculated into liquid culture medium. After incubation at 37°C for 24 hours, the OD value was adjusted with 0.9% sterile physiological saline for later use. The bacterial suspension was then inoculated onto casein solid medium and incubated at 37°C for 12 hours. The diameter (D) of the clear zone was measured using calipers. The size of the clear zone reflects the protease-producing ability of the strain; a larger clear zone indicates a stronger protease-producing ability.

[0042] The results showed that among the 72 lactic acid bacteria strains obtained in the initial screening of this invention, strain W66 had the strongest protease production capacity.

[0043] 3. Strain identification

[0044] Physiological and biochemical characterization tests were conducted according to the "Classification, Identification and Test Methods of Lactic Acid Bacteria" and the "Handbook of Common Bacterial Identification". The W66 strain underwent molecular biological identification, and the PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd. After amplification, 16S rDNA sequence homology analysis was performed. The results showed that W66 is *Lactobacillus plantarum*, with a homology of 99.90%.

[0045] On June 30, 2025, the present invention deposited Lactobacillus plantarum W66 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35049.

[0046] Example 2: Determination of the protease production capacity of Lactobacillus plantarum W66

[0047] The activated Lactobacillus plantarum W66 bacterial culture was inoculated into MRS liquid medium at a volume ratio of 2% and cultured at 37°C for 24 h. The fermentation broth was centrifuged at 8000 r / min for 10 min to obtain the fermentation supernatant.

[0048] 1. Plotting the standard curve for tyrosine

[0049] Prepare tyrosine standard working solutions at concentrations of 0.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 30.0 μg / mL, 40.0 μg / mL, and 50.0 μg / mL. Pipette 1.0 mL of the tyrosine standard working solution, add 5.0 mL of sodium carbonate solution and 1.0 mL of Folin's reagent, and mix well. Place in a 40℃ water bath and react for 20 min. Measure the absorbance at 680 nm using a spectrophotometer. Plot a standard curve with tyrosine concentration on the x-axis and absorbance (OD) on the y-axis to obtain the linear regression equation. The regression coefficient (R²) is... 2 It can only be used when the value is above 0.9978; otherwise, it should be redone.

[0050] The regression equation for the obtained tyrosine standard curve is y = 189.17x - 7.2916 (R²). 2 =0.9995).

[0051] 2. Sample determination

[0052] Take an appropriate amount of the fermentation supernatant of Lactobacillus plantarum W66 and dilute it with lactate buffer. Specific operating steps are shown in the table.

[0053] Table 1 Operation Steps

[0054] Test tube A (blank control) Test tube B (sample, three parallel samples) Add 1 mL of enzyme solution Add 1 mL of enzyme solution Preheat in a water bath at 40℃±0.2℃ for 2 minutes. Preheat in a water bath at 40℃±0.2℃ for 2 minutes. Add 2 mL of trichloroacetic acid solution and mix well. Add 1 mL of casein solution and mix well. Let stand at 40℃±0.2℃ for 10 minutes Let stand at 40℃±0.2℃ for 10 minutes Add 1 mL of casein solution and mix well. Add 2 mL of trichloroacetic acid solution and mix well. Let stand for 10 minutes to filter Let stand for 10 minutes to filter Take 1 mL of filtrate Take 1 mL of filtrate Add 5 mL of sodium carbonate solution Add 5 mL of sodium carbonate solution Add 1 mL of Folin-Ciocalteu reagent and mix well. Add 1 mL of Folin-Ciocalteu reagent and mix well. Water bath at 40℃±0.2℃ for 20 minutes Water bath at 40℃±0.2℃ for 20 minutes

[0055] Enzyme activity calculation formula: Enzyme activity (U / mL) = OD 680 ×K×(4 / 10)×N.

[0056] The results showed that the fermentation supernatant of *Lactobacillus plantarum* W66 provided by this invention had high protease activity, reaching 498.71 ± 1.27 U / mL. Therefore, this strain can better decompose proteins in raw materials, produce more flavor compounds, and release bioactive peptides with specific health functions.

[0057] Example 3: Determination of the polypeptide production capacity of Lactobacillus plantarum W66

[0058] The peptide content in the fermentation supernatant of *Lactobacillus plantarum* W66 prepared in Example 2 was determined using a BCA protein concentration kit. The absorbance at 562 nm was measured using a microplate reader, and the peptide concentration was calculated based on the standard curve, following the manufacturer's instructions.

[0059] The results showed that Lactobacillus plantarum W66 had a strong ability to produce polypeptides, with the polypeptide content in its fermentation supernatant reaching as high as 30.34±0.008 mg / mL.

[0060] Example 4: Determination of the antioxidant capacity of Lactobacillus plantarum W66

[0061] 1. Sample preparation

[0062] Preparation of fermentation supernatant: Activated Lactobacillus plantarum W66 for two generations was inoculated into MRS medium at a volume fraction of 2% and cultured at 37℃ for 24h; the fermentation broth was centrifuged at 4℃ and 8000r / min for 10min to obtain sterile supernatant.

[0063] Preparation of bacterial suspension: Collect the bacterial cell pellet, wash 2-3 times with phosphate-buffered saline (PBS, pH 7.2), and resuspend in PBS to obtain a complete cell suspension. (Bacterial suspension OD = 1.000 ± 0.2)

[0064] Preparation of sterile cell fragments: The bacterial suspension was sonicated in an ice bath at a power of 300W for 2 seconds, followed by a 2.4-second pause, for a total of 10 minutes. The fragments were then centrifuged at 4°C and 8000 rpm for 10 minutes to obtain the cell fragment extract.

[0065] 2. Determination of free radical scavenging rate and reducing power

[0066] (1) Determination of DPPH free radical scavenging rate

[0067] After mixing an equal volume of sample with DPPH-anhydrous ethanol (0.2 mmol / L), the sample was stored at room temperature in the dark for 30 min. After centrifugation at 8000 r / min for 10 min, the absorbance of the supernatant was measured at a wavelength of 517 nm. The experiment was repeated 3 times.

[0068] DPPH free radical scavenging rate (%) = (1-A) 对照 / A 样品 ) × 100%.

[0069] Where A 样品 The absorbance of 2 mL of DPPH-anhydrous ethanol and 2 mL of sample; A 对照 The absorbance of distilled water is given.

[0070] The results showed that the sterile supernatant, bacterial suspension and cell lysate extract of Lactobacillus plantarum W66 all had strong DPPH free radical scavenging ability, with DPPH free radical scavenging rates of 79.23±1.12%, 15.31±1.03% and 34.62±1.20%, respectively.

[0071] (2) Determination of hydroxyl radical scavenging rate

[0072] Mix 1 mL of pH 7.4 PBS, 0.5 mL of 0.25 mmol / L o-phenanthroline solution, 0.5 mL of 2.5 mmol / L ferrous sulfate solution, and 0.5 mL of the sample thoroughly. Finally, add 0.5 mL of 20 mmol / L H2O2 and react at 37 °C for 60 min. Measure the absorbance at 536 nm.

[0073] Hydroxyl radical scavenging rate (%) = [(A 样品 -A 空白 ) / (A 对照 -A 空白 )]×100%.

[0074] Where A 对照 Using distilled water instead of hydrogen peroxide, the following are prepared: o-phenanthroline solution, ferrous sulfate, PBS, sample, and distilled water. (A) 空白 The sample contained o-phenanthroline solution, ferrous sulfate, and H2O2.

[0075] The results showed that the sterile supernatant, bacterial suspension and cell lysate extract of Lactobacillus plantarum W66 all had strong hydroxyl radical scavenging ability, with scavenging rates of 82.70±1.03%, 23.58±2.91% and 48.44±1.13%, respectively.

[0076] (3) Determination of ABTS free radical scavenging rate

[0077] Mix 7 mmol / L ABTS with 2.45 mmol / L potassium persulfate at a volume ratio of 1:1. Incubate the mixture in the dark at room temperature for 24 h, and adjust the absorbance of the ABTS solution at 734 nm to 0.700 ± 0.02 with distilled water or 95% ethanol. Mix 500 μL of the bacterial culture medium sample with 1000 μL of ABTS solution and incubate in the dark at room temperature for 10 min. Measure the absorbance at 734 nm. Perform the measurements three times.

[0078] ABTS radical scavenging rate (%) = (1-A) 样品 / A1)×100%.

[0079] Where A1 is the absorbance of the reference standard distilled water (or 95% ethanol) at a wavelength of 734 nm.

[0080] The results showed that the sterile supernatant, bacterial suspension and cell lysate extract of Lactobacillus plantarum W66 all had strong ABTS free radical scavenging ability, with ABTS free radical scavenging rates of 81.09±1.09%, 17.83±0.75% and 24.81±1.18%, respectively.

[0081] (4) Reducing power measurement

[0082] Take 1 mL of sample, add 2 mL of phosphate buffer (pH 6.6, PBS 0.2M pH 6.8), shake well, then add 2 mL of K3[Fe(CN)6] aqueous solution, mix thoroughly, and react at 50℃ for 30 min. Then add 2 mL of 10% trichloroacetic acid solution and incubate at 3000 r·min. –1 Centrifuge for 10 min (centrifugation radius 4 cm). Take 5 mL of the supernatant, add 5 mL of distilled water and 1 mL of 0.1% FeCl3 solution. After reacting for 20 min, measure the absorbance at 700 nm. Measure the sample three times and take the average value. Use distilled water as a blank control.

[0083] Restoration ability = (A 样品 –A0)×100%.

[0084] Where A 样品 A0 is the absorbance of the sample, and A0 is the absorbance of the blank group.

[0085] The results showed that the reducing power of sterile supernatant, bacterial suspension and cell lysate extract of Lactobacillus plantarum W66 was 89.85±1.02%, 25.32±1.25% and 28.65±1.34%, respectively.

[0086] The above results show that the Lactobacillus plantarum W66 provided by the present invention has a strong antioxidant capacity and has achieved unexpected technical effects.

[0087] Example 5: Preparation method of Lactobacillus plantarum W66 antioxidant peptide

[0088] 1. Sample preparation

[0089] Lactobacillus plantarum W66 was inoculated into MRS fermentation medium and activated at 37°C for 24 h. After two subcultures, the fermentation broth of the strain was obtained.

[0090] 2. Crude peptide extraction

[0091] The fermentation broth was ultrasonically disrupted in an ice bath at 300W for 2 seconds, followed by a 2.4-second pause, for a total of 10 minutes. Then, it was centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected and mixed with 1% sodium chloride and 0.3% ammonium sulfate in a 1:4 ratio, and incubated overnight at 4°C. The mixture was then centrifuged at 8000 rpm for 20 minutes at 4°C. The viscous precipitate at the bottom was collected, and Tris-HCl buffer was added in small, repeated additions. The mixture was then dialysis-treated overnight in running water using a dialysis bag. The solution was filtered through a 3kDa ultrafiltration tube and centrifuged at 4000 rpm for 20 minutes. The resulting filtrate was the crude antioxidant peptide solution.

[0092] 3. Determination of antioxidant capacity of crude peptide solution

[0093] The ability of crude peptide solution to scavenge free radicals was determined to verify its antioxidant capacity.

[0094] DPPH solution: Dissolve 6.3 mg of DPPH in 100 mL of methanol.

[0095] ABTS solution: Dissolve 7.68 mg ABTS in 2 mL of distilled water and 2.65 mg potassium persulfate in 4 mL of distilled water. Mix well and incubate at room temperature (25±1℃) in the dark for 12-16 h before dissolving in anhydrous ethanol. Adjust the concentration at 734 nm to 0.7±0.02 before use.

[0096] Determination 1: DPPH free radical scavenging ability determination method: Mix 200 μL of crude peptide solution with 2 mL of DPPH solution, incubate at room temperature in the dark for 30 min, 0.2 mg·mL-1 Vc as positive control, measure absorbance at 517 nm, and calculate according to the following.

[0097] DPPH free radical scavenging rate (%) = (1-A) 对照 / A 样品 ) × 100%.

[0098] Note: A 样品 The absorbance of 2 mL DPPH-methanol and 2 mL sample; A 对照 The absorbance of distilled water is given.

[0099] Determination 2: ABTS free radical scavenging rate determination method: 1 mL of crude peptide solution and 4 mL of ABTS solution were thoroughly mixed and incubated at room temperature in the dark for 6 min. 0.2 mg·mL-1 Vc was used as a positive control. The absorbance at a wavelength of 734 nm was measured and calculated according to the following formula.

[0100] ABTS radical scavenging rate (%) = (1-A) 样品 / A1)×100%.

[0101] Where A1 is the absorbance of the reference standard distilled water (or 95% ethanol) at a wavelength of 734 nm.

[0102] Experimental results showed that the crude peptide solution extracted from the fermentation broth of Lactobacillus plantarum W66 had a scavenging rate of 69.35±1.03% for DPPH free radicals and 81.27±0.25% for ABTS free radicals, indicating strong antioxidant capacity.

[0103] Example 6: Optimization of fermentation conditions for high-yield antioxidant peptides from Lactobacillus plantarum

[0104] 1. Response surface optimization

[0105] Based on single-factor experiments, the effects of fermentation time (12h, 18h, 24h, 30h, 36h), fermentation temperature (20℃, 25℃, 30℃, 35℃, 40℃), inoculum size (2%, 4%, 6%, 8%, 10%), and pH value (5.0, 5.5, 6.0, 6.5, 7.0) on the yield of *Lactobacillus plantarum* peptides were investigated. A regression model was established to optimize the extraction factors for high bioactive peptides, obtaining the optimal process conditions for high bioactive peptide extraction.

[0106] Fermentation temperature, fermentation time, inoculum size, and pH were selected as independent variables, and peptide yield was selected as the response surface methodology. Response surface methodology optimization was performed to obtain the regression equation for peptide content: Y = -336.339 + 7.60193A + 1.75367B + 3.3355C + 69.5297D - 0.00391667AB + 0.03475AC + 0.045AD + 0.0152083BC - 0.00333333BD + 0.2225CD - 0.13046A² - 0.0337569B² - ​​0.73475C² - 5.591D².

[0107] Where A is the fermentation temperature, B is the fermentation time, C is the inoculum size, and D is the pH.

[0108] The Design-expert 8.0 software was used to optimize and obtain the maximum peptide content under the interaction of four factors: fermentation temperature, fermentation time, inoculum size, and pH value, as well as the corresponding fermentation temperature, fermentation time, inoculum size, and pH value.

[0109] 2. Response surface methodology

[0110] Based on the principles of response surface methodology, four single factors significantly affecting peptide yield were selected from the single-factor experiments: fermentation temperature, fermentation time, inoculum size, and pH value. The peptide yield was used as the response value. A four-factor, three-level experiment was conducted, comprising 29 experimental sites and 5 central experimental sites. The levels of the response surface methodology factors are shown in the table below, as are the experimental design and results.

[0111] Table 2 Response Surface Factors and Levels

[0112]

[0113] Table 3 Experimental schemes and results of response surface design

[0114] serial number Fermentation temperature (°C) Fermentation time (h) Inoculation volume (%) pH polypeptide content (mg / mL) 1 35 24 4 7 26.54 2 25 24 2 6.5 24.16 3 30 30 4 7 28.49 4 30 18 6 6.5 27.11 5 30 24 2 6 26.84 6 35 24 2 6.5 24.53 7 30 24 4 6.5 31.08 8 30 24 4 6.5 31.1 9 25 30 4 6.5 26.45 10 30 18 2 6.5 26.15 11 30 18 4 6 28.63 12 30 24 4 6.5 31.19 13 25 24 6 6.5 24.74 14 35 30 4 6.5 27.34 15 30 24 6 7 27.04 16 30 24 4 6.5 31.04 17 25 18 4 6.5 25.58 18 30 24 6 6 27.53 19 35 24 4 6 27.48 20 30 30 4 6 29.39 21 30 18 4 7 27.77 22 25 24 4 6 26.54 23 30 24 4 6.5 31.13 24 30 30 6 6.5 28.08 25 35 18 4 6.5 26.94 26 30 30 2 6.5 26.39 27 25 24 4 7 25.15 28 35 24 6 6.5 26.5 29 30 24 2 7 25.46

[0115] 3. Regression model establishment and significance test

[0116] Response surface methodology software was used to perform analysis of variance on the data. The data was fitted to obtain a regression equation. Analysis of variance and significance tests were then performed on the regression equation to obtain the regression equation for the peptide content: Y = -336.339 + 7.60193A + 1.75367B + 3.3355C + 69.5297D - 0.00391667AB + 0.03475AC + 0.045AD + 0.0152083BC - 0.00333333BD + 0.2225CD - 0.13046A² - 0.0337569B² - ​​0.73475C² - 5.591D².

[0117] The accuracy and reliability of the model were analyzed based on the results of significance, R², AdjR², signal-to-noise ratio, lack of fit, and coefficient of variation. The regression model showed P < 0.0001, indicating that the regression model was highly significant; the lack of fit P was 0.0722 > 0.05, meaning the lack of fit was not significant; the R² coefficient was 0.9986 > 0.8, indicating that the model could explain 99.86% of the response value variation; the AdjR² coefficient of 0.993 and R² indicated a good model fit; and the signal-to-noise ratio of 90.1977 indicated high model accuracy.

[0118] The coefficient of variation (CV) was 0.3949%, less than 10%, indicating that the experimental results have high precision and reliability. In summary, the regression model has high goodness of fit, small error, and high reliability, and can be used for optimizing the content of highly bioactive peptides.

[0119] 4. Interaction Analysis

[0120] Based on the analysis of variance of the regression model, Origin software was used to plot response surface plots and contour plots according to the regression equation and the results of the analysis of variance. The results are as follows: Figure 1-6 As shown.

[0121] The optimal process conditions were determined using the Design-expert 8.0 software.

[0122] The above results show that the optimal culture conditions for producing antioxidant peptides by *Lactobacillus plantarum* W66 described in this invention are: fermentation temperature 30℃, fermentation time 24h, inoculum size 4%, and pH value 6.5.

[0123] Example 7: Growth curve determination of Lactobacillus plantarum W66

[0124] The activated Lactobacillus plantarum W66 was inoculated into MRS liquid medium (pH 6.5) at a 4% inoculation rate and fermented at 30°C for 24 hours. The OD600nm was measured and recorded every 2 hours.

[0125] Figure 7 The figure shows the growth curve of Lactobacillus plantarum W66. It can be seen from the figure that Lactobacillus plantarum W66 enters the logarithmic growth phase at 14 hours, and grows most vigorously between 16 and 18 hours. After 18 hours, its growth declines and tends to level off.

[0126] Example 8 Safety evaluation of Lactobacillus plantarum W66

[0127] 1. Antibiotic susceptibility test

[0128] The susceptibility of *Lactobacillus plantarum* to amoxicillin (25 μg / tablet), chloramphenicol (30 μg / tablet), ceftazidime (30 μg / tablet), tetracycline (30 μg / tablet), gentamicin sulfate (10 μg / tablet), and ampicillin (10 μg / tablet) was determined using the Kirby-Bauer disk diffusion method recommended by the World Health Organization. The glycerol-preserved strain was activated in sterilized MRS liquid tubes, subcultured, and cultured in a fermentation incubator for 24 hours; this was the second-generation strain. A culture of *Lactobacillus plantarum* W66 second-generation strain was spread onto MRS solid medium using a sterile cotton swab. Antibiotic disks were placed on the surface of the solid medium using sterile forceps, and the culture was incubated upside down in a 38°C fermentation incubator for 48 hours. The diameter of the inhibition zone (mm) was measured and recorded. The susceptibility of *Lactobacillus plantarum* to six common antibiotics was assessed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).

[0129] The results are shown in Table 4. Lactobacillus plantarum W66 was sensitive to six common antibiotics, including amoxicillin, chloramphenicol, ceftazidime, tetracycline, gentamicin sulfate, and ampicillin.

[0130] Table 4. Results of antibiotic susceptibility testing with Lactobacillus plantarum W66

[0131] antibiotic Sensitive S Moderately sensitive M Drug-resistant R Diameter of the inhibition zone W66 Sensitivity Amoxicillin 17–24 9–16 ≤8 18 S Chloramphenicol ≥18 13–17 ≤12 31 S Ceftazidime 17–24 9–16 ≤8 20 S tetracycline ≥15 12–14 ≤11 22 S Gentamicin sulfate ≥15 13–14 ≤12 24 S Ampicillin ≥17 9–16 ≤8 21 S

[0132] 2. In vitro antibacterial test

[0133] Two indicator bacteria (Staphylococcus aureus and Escherichia coli) were used to determine the antibacterial activity of *Lactobacillus plantarum* W66. *E. coli* and *S. aureus* preserved in glycerol were cultured in LB liquid medium for 24 hours. The bacterial suspensions of *E. coli* and *S. aureus* were then spread onto LB solid medium using sterile cotton swabs. *Lactobacillus plantarum* W66 preserved in glycerol was cultured in MRS liquid medium for 24 hours. A filter paper disc was used to pick up a suspension of *Lactobacillus plantarum* W66 and placed it onto LB solid medium using sterile forceps. The LB solid medium was placed in a 37°C incubator and incubated for 24 hours. Afterward, the presence and diameter of inhibition zones around the colonies were observed and measured.

[0134] The results are shown in Table 5. Lactobacillus plantarum W66 showed good antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0135] Table 5 Results of antibacterial experiment with Lactobacillus plantarum W66

[0136] indicator bacteria Diameter of the inhibition zone (mm) E. coli 19.56 Staphylococcus aureus 17.23

[0137] Example 9: In vitro evaluation of the probiotic function of Lactobacillus plantarum W66

[0138] 1. Acid resistance test

[0139] The pH of MRS medium was adjusted to 2.0, 3.0, and 4.0 using 0.1% hydrochloric acid, and then aliquoted into sterile test tubes. A blank control group was also prepared using untreated MRS medium, which was then sterilized. Activated *Lactobacillus plantarum* W66 was cultured at 37°C for 24 hours, then centrifuged at 5000 rpm for 10 minutes, and the OD600nm was adjusted to 0.5 with sterile PBS buffer. Then, *Lactobacillus plantarum* W66 was inoculated at 1% into MRS medium at different pH values ​​and into the blank control group medium, and the OD600nm was measured. After culturing at room temperature for 4 hours, the OD600nm was measured again. Each strain was measured three times.

[0140] Lactobacillus plantarum's acid-resistant properties enable it to survive well in the acidic environment of the stomach and colonize in the intestines to exert its probiotic effects. Therefore, the acid resistance of lactic acid bacteria is used as one of the evaluation indicators of their growth capacity.

[0141] The results are as follows Figure 8 As shown, the survival rate of Lactobacillus plantarum W66 was 40.9% after culturing in an acidic environment of pH 2.0 for 4 hours, indicating strong acid resistance.

[0142] 2. Bile salt tolerance test

[0143] MRS culture media were prepared by adding 0.1%, 0.2%, and 0.3% bile salts, and then aliquoted into sterile test tubes. Activated *Lactobacillus plantarum* W66 was cultured at 37°C for 24 hours, then centrifuged at 5000 rpm for 10 min, and the OD600nm was adjusted to 0.5 with sterile PBS buffer. Then, *Lactobacillus plantarum* W66 was inoculated at 1% into MRS culture media containing different concentrations of bile salts and a blank control group, and the OD600nm was measured. After culturing at room temperature for 4 hours, the OD600nm was measured again. Each strain was measured three times.

[0144] The survival rate is calculated using the following formula: Survival rate (%) = 100% × OD 实验组 / OD 对照组 .

[0145] The results are as follows Figure 9 As shown, Lactobacillus plantarum W66 is highly tolerant to bile salts and can survive and reproduce in low concentrations of bile salts. The survival rate was 55.88% after culturing in 0.1% bile salts for 4 hours.

[0146] 3. Simulated gastrointestinal tolerance experiment

[0147] Gastrointestinal tolerance of lactic acid bacteria is often used as one of the important evaluation indicators of their probiotic properties. Probiotic lactic acid bacteria with good gastrointestinal tolerance can be used in functional probiotic foods and functional fermented products.

[0148] 200 μL of activated *Lactobacillus plantarum* W66 bacterial suspension was added to 10 mL of MRS and incubated at 37 °C for 18 h. The suspension was then centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The bacterial suspension was washed with PBS to adjust the OD600 nm to 1. The PBS solution was adjusted to pH 3.0, sterilized, and then 0.6 g of pepsin was added for membrane filtration to simulate artificial gastric fluid. The PBS solution was then adjusted to pH 8.0, sterilized, and then 0.2 g of trypsin and 0.3% bile salts were added for membrane filtration to simulate artificial intestinal fluid. 1 mL of bacterial suspension and 9 mL of artificial gastric fluid were mixed and the OD600 nm was measured. After 3 hours, 1 mL of the mixture of bacterial suspension and artificial gastric fluid was added to 9 mL of artificial intestinal fluid, mixed, and the OD600 nm was measured again. After 6 hours, the OD600 nm was measured again.

[0149] The survival rate is calculated using the following formula: Survival rate (%) = 100% × OD 实验组 / OD 对照组 .

[0150] The results are as follows Figure 10 As shown, Lactobacillus plantarum W66 has strong tolerance to the digestive tract environment. After treatment in simulated gastric juice for 3 hours, the survival rate was 55.98%, and after treatment in simulated intestinal juice for 6 hours, the survival rate was 35.17%.

[0151] 4. Evaluation of strain adhesion ability

[0152] (1) Determination of hydrophobicity of strain surface

[0153] Centrifuge the activated *Lactobacillus plantarum* W66 bacterial suspension at 1000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Wash the suspension 2-3 times with 0.85% NaCl physiological saline to ensure the OD600nm of the bacterial suspension is between 0.5 ± 0.05, and record the initial absorbance A0 at 600nm. Mix 3 mL of the bacterial suspension and 3 mL of xylene in a 10 mL centrifuge tube, vortex for 30 s to mix thoroughly, and let the mixture stand at 37°C for 30 min. Then, collect the supernatant and measure the OD600nm (A1).

[0154] Hydrophobicity formula: Hydrophobicity (%) = (A0-A1) / A0×100%.

[0155] The results showed that the surface hydrophobicity of Lactobacillus plantarum W66 was 38.42%.

[0156] (2) Determination of self-agglomeration rate

[0157] Centrifuge the activated *Lactobacillus plantarum* W66 bacterial suspension at 1000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Wash the suspension 2-3 times with 0.85% NaCl physiological saline to ensure the OD600nm of the bacterial suspension is between 0.5 ± 0.05, and record the initial absorbance A0 at 600nm. Incubate the bacterial suspension at 37℃ for 5 hours, and measure the absorbance (A1) of the supernatant at 600nm.

[0158] Self-agglomeration formula: Self-agglomeration rate (%) = (A0-A1) / A0×100%.

[0159] The results showed that the autoagglutination rate of Lactobacillus plantarum W66 was 44.26%.

Claims

1. A type of Lactobacillus plantarum ( Lactobacillus plantarum ), characterized in that, The preservation number of the *Lactobacillus plantarum* is CGMCC No. 35049.

2. The *Lactobacillus plantarum* as described in claim 1, characterized in that, The 16S rDNA sequence of the *Lactobacillus plantarum* is SEQ ID NO:

1.

3. The application of Lactobacillus plantarum as described in claim 1 in the production of fermented foods.

4. The application of Lactobacillus plantarum as described in claim 1 in the production of biopeptides.

5. The application as described in claim 4, characterized in that, The biopeptide is an antioxidant peptide.

6. A probiotic starter, characterized in that, The probiotic fermentation agent comprises the *Lactobacillus plantarum* as described in claim 1.

7. The probiotic starter culture as described in claim 6, characterized in that, The probiotic starter also includes any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Halophilic tetracoccus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

8. The probiotic starter culture as described in claim 6 or 7, characterized in that, The probiotic starter contains no less than 10 live Lactobacillus plantarum bacteria. 8 CFU / g.

9. The use of the probiotic starter culture according to any one of claims 6-8 in food production.

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