Application of phytobacterium plantarum in preparation of product for inhibiting growth and proliferation of bacteria and / or improving flavor

Abstract

The invention discloses a plant lactobacillus CCFM1296 strain for improving free amino acid content and in-vitro bacteriostatic ability of fermented milk, and belongs to the technical field of microbial lactobacillus. According to the fermented milk prepared by the plant lactobacillus CCFM1296, the content of free amino acid can be increased, the content of flavor amino acid can be increased, the loss of sweet amino acid can be reduced, and the inhibition effect on the biofilm forming ability of salmonella typhimurium, pseudomonas aeruginosa and pseudomonas fluorescens can be enhanced.

Description

Use of Lactobacillus plantarum in the preparation of products that inhibit bacterial growth and / or improve flavor Technical Field

[0001] This application relates to the field of microbial technology, and in particular to the use of *Lactobacillus plantarum* in the preparation of products that inhibit bacterial growth and / or improve flavor. Background Technology

[0002] In recent years, fermented milk has become increasingly popular among consumers due to its rich nutritional value, primarily thanks to the core role of fermentation bacteria. As the key to fermented milk preparation, fermentation bacteria utilize the carbohydrates, proteins, and fats in the raw materials through their own metabolism and enzymatic action to produce organic acids, amino acids, extracellular polysaccharides, and aromatic compounds, thereby forming the unique texture and flavor of fermented milk.

[0003] Lactobacillus plantarum is closely related to human life and is a common lactic acid bacterium found in butter, meat, and many fermented vegetable products. It can pass through the stomach and colonize the intestines to exert beneficial effects. It has a significant impact on gut microbiota and has wide applications in food fermentation, industrial lactic acid fermentation, and healthcare. However, because Lactobacillus plantarum cannot directly utilize the nutrients in milk for growth and proliferation, most research focuses on the preparation process of fermented milk using Lactobacillus plantarum. For example, patent CN116590191A discloses a strain of Lactobacillus plantarum that can be used as a single strain to ferment skim milk, and patent CN 118755609A discloses the application of a high-diacetyl-producing strain of Lactobacillus plantarum TGH in the preparation of fermented milk. However, there are no reports on multifunctional Lactobacillus plantarum fermented milk that combines external antibacterial ability and rich free amino acid content. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides the use of Lactobacillus plantarum in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the Lactobacillus plantarum has the accession number GDMCC No. 62974.

[0005] This application also provides a method for preparing fermented milk, in which *Lactobacillus plantarum* used in the above-mentioned applications is inoculated into raw milk and fermented.

[0006] This application also provides a fermented milk prepared by the above-described preparation method.

[0007] The beneficial effects of this application include, but are not limited to: the discovery that *Lactobacillus plantarum* CCFM1296 fermented milk matrix has a good ability to enhance the in vitro antibacterial activity and free amino acid content of fermented milk. This *Lactobacillus plantarum* strain CCFM1296 and its fermentation products have a strong inhibitory effect on the biofilm formation ability of *Salmonella typhimurium*, *Pseudomonas aeruginosa*, and *Pseudomonas fluorescens*, while significantly increasing the free amino acid content, increasing the umami amino acid content, and reducing the loss of sweet amino acids.

[0008] This strain of *Lactobacillus plantarum* CCFM1296 can be used in the production of fermented milk, dairy beverages, etc. This invention utilizes *Lactobacillus plantarum* CCFM1296, which enhances in vitro antibacterial activity and free amino acid content, as a starter culture to ferment whole milk, developing a multifunctional fermented milk product that combines in vitro antibacterial activity with high free amino acid content. Attached Figure Description

[0009] This application will be further illustrated by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 is a principal component analysis diagram and differential metabolite volcano plot of the fermented milk metabolome of *Lactobacillus plantarum*.

[0010] Figure 2 is a heatmap of hierarchical clustering of differential metabolites in fermented milk by Lactobacillus plantarum. Detailed Implementation

[0011] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0012] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0013] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0014] This application provides the use of Lactobacillus plantarum in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the Lactobacillus plantarum has the accession number GDMCC No. 62974.

[0015] In some embodiments, the product may include one or more of food, pharmaceuticals, health products, animal feed, or daily chemical products. In some embodiments, preferably, the food may be a fermented food. In some embodiments, more preferably, the food may be one or more of fermented milk, dairy drinks, fermented cream, milk powder, or solid beverages.

[0016] In some embodiments, the product may contain fermentation products and / or culture products of Lactobacillus plantarum.

[0017] In some embodiments, the bacteria may be pathogenic. In some embodiments, preferably, the bacteria may include one or more of Salmonella typhimurium, Pseudomonas aeruginosa, or Pseudomonas fluorescens.

[0018] In some embodiments, the Salmonella typhimurium may be Salmonella typhimurium TA100.

[0019] In some embodiments, the Pseudomonas aeruginosa may be Pseudomonas aeruginosa ATCC9027.

[0020] In some embodiments, the fluorescent Pseudomonas can be Fluorescent Pseudomonas AS1.55.

[0021] In some embodiments, the product can increase the total amount of free amino acids in fermented milk.

[0022] In some embodiments, the product can improve the flavor of fermented milk.

[0023] In some embodiments, the product can increase the content of umami amino acids in fermented milk and reduce the loss of sweet amino acids in fermented milk.

[0024] In some embodiments, the product can increase the content of essential and non-essential amino acids in fermented milk.

[0025] In some embodiments, the product can inhibit bacterial biofilm formation.

[0026] This application also provides a method for preparing fermented milk, in which *Lactobacillus plantarum* used in the above-mentioned applications is inoculated into raw milk and fermented.

[0027] In some embodiments, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be (1-4):(90-110). In some embodiments, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be (1.5-3.5):(92.5-107.5). In some embodiments, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be (2.0-3.0):(95-105). In some embodiments, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be (2.5-3.0):(97.5-102.5). In some embodiments, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be (2.5-3.0):(100-102.5).

[0028] In some embodiments, preferably, the volume ratio of *Lactobacillus plantarum* to raw milk during inoculation can be 2:100.

[0029] In some embodiments, the fermentation temperature can be 35–40°C. In some embodiments, the fermentation temperature can be 36–39°C. In some embodiments, the fermentation temperature can be 37–38°C. In some embodiments, preferably, the fermentation temperature can be 37°C.

[0030] In some embodiments, the fermentation time can be 10–15 hours. In some embodiments, the fermentation time can be 11–14 hours. In some embodiments, the fermentation time can be 12–13 hours. In some embodiments, preferably, the fermentation time can be 12 hours.

[0031] In some embodiments, the fermentation can be aerobic fermentation.

[0032] In some embodiments, the raw milk may be whole milk.

[0033] In some embodiments, the whole milk may include whole milk powder, yeast extract, and water. In some embodiments, preferably, the mass ratio of whole milk powder, yeast extract, and water is (10-20):(2-8):(70-90). In some embodiments, preferably, the mass ratio of whole milk powder, yeast extract, and water is (12-18):(3-7):(74-86). In some embodiments, preferably, the mass ratio of whole milk powder, yeast extract, and water is (13-17):(4-6):(78-82). In some embodiments, preferably, the mass ratio of whole milk powder, yeast extract, and water is (14-16):(5-6):(80-82). In some embodiments, preferably, the mass ratio of whole milk powder, yeast extract, and water is (15-16):(5-6):(80-82).

[0034] In some embodiments, more preferably, the mass ratio of the whole milk powder, yeast extract, and water can be 15:5:80.

[0035] This application also provides a fermented milk prepared by the above-described preparation method.

[0036] This application provides the following technical solution: a strain of Lactobacillus plantarum CCFM1296, which was deposited on November 13, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 62974, and the deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0037] The *Lactobacillus plantarum* CCFM1296 was isolated from the feces of healthy human beings and has the following characteristics: Under a microscope, the cells of *Lactobacillus plantarum* CCFM1296 appear as slightly irregular, round-ended, non-motile curved bacteria. After inoculation on MRS medium and cultured for 48 hours, the colonies are generally milky white, smooth, raised, and round with a diameter of 0.52 mm.

[0038] The application of *Lactobacillus plantarum* in the preparation of fermented milk.

[0039] Furthermore, the application of *Lactobacillus plantarum* in the preparation of fermented milk is carried out by the following method: the *Lactobacillus plantarum* is directly inoculated into the raw milk for fermentation.

[0040] Preferably, the raw milk is whole milk powder.

[0041] Preferably, in the above steps, the inoculation amount of *Lactobacillus plantarum* is 5 x 10⁶ to 5 x 10⁷ CFU / mL.

[0042] Preferably, in the above steps, the whole milk powder includes whole milk powder, yeast extract, and water, with the whole milk powder accounting for 15% by mass and the yeast extract accounting for 5% by mass.

[0043] Preferably, the fermentation temperature in the above steps is 37°C.

[0044] Preferably, the culture method in the above steps is aerobic culture.

[0045] Preferably, the fermentation time in the above steps is 12 hours.

[0046] This application provides the application of *Lactobacillus plantarum* CCFM1296 in the fermentation preparation of products with enhanced free amino acid content and in vitro antibacterial activity.

[0047] In one embodiment, the application involves using *Lactobacillus plantarum* CCFM1296 to ferment a whole-fat emulsion of yeast extract to prepare a product with enhanced in vitro antibacterial activity and free amino acid content.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0049] The strains used in this application are as follows: *Lactobacillus plantarum* CCFM1296 and *Lactobacillus plantarum* FSCDJY69L1 were isolated from healthy human feces by the Microbial Culture Collection Center of Jiangnan University; *Salmonella typhimurium* TA100 was purchased from Guangdong Institute of Microbiology, China; *Pseudomonas aeruginosa* ATCC9027 was purchased from CGMCC, China; and *Pseudomonas fluorescens* AS1.55 was purchased from Mingzhou Biotechnology Co., Ltd., China.

[0050] The culture media involved in the following examples are as follows: MRS solid medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L, agar 20g / L.

[0051] MRS liquid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L.

[0052] Example 1: Molecular biological identification of *Lactobacillus plantarum*. Pure *Lactobacillus plantarum* was inoculated into MRS medium tubes and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial sludge was collected, washed repeatedly with sterile water 2-3 times, and amplified using universal bacterial 16S rDNA primers 27F: 5'AGAGTTTGACCTGGCTAG-3' (SEQ ID NO: 2) and 1495R: 5'-CTACGGCTCCTTGTTCGA-3' (SEQ ID NO: 3). The amplified products were detected by 0.1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequencing results were compared with sequences in the database, showing high homology to the genus *Lactobacillus*, thus belonging to the genus *Lactobacillus*.

[0053]

[0054] *Lactobacillus plantarum* CCFM1296 and FSCDJY69L1 from glycerol storage tubes were spread onto MRS agar plates and incubated at 37°C for 48 h. Single colonies were picked and inoculated into 5 mL of MRS liquid medium. After incubation at 37°C for 24 h, a 2% (v / v) inoculum was added to another 5 mL of MRS liquid medium. The culture was incubated until the logarithmic growth phase. The culture was then centrifuged at 15,000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile distilled water and resuspended in the original culture volume of sterile distilled water to obtain the seed culture for fermentation. The concentration of the seed culture was 1 x 10⁻⁶. 9 CFU / mL2, Preparation of whole milk: 15% whole milk powder and 5% yeast extract were mixed with distilled water and thoroughly dissolved. The mixture was then sterilized at 95°C for 5 minutes and cooled to room temperature to obtain whole milk. This whole milk was used as a control group for fermented milk.

[0055] 3. Preparation of fermented milk: Lactobacillus plantarum CCFM1296 and FSCDJY69L1 were aseptically inoculated into whole milk at an inoculation amount of 2% (v / v) and cultured aerobically at 37°C for 12 hours to obtain fermented milk.

[0056] 4. The pH and acidity of fermented milk were measured using a digital pH meter.

[0057] The acidity of fermented milk was determined by acid-base titration using phenolphthalein indicator. First, 5g of fermented milk was added to each of 75mL of sterile water at room temperature, and 1mL of phenolphthalein indicator was added dropwise and mixed well. Titration was then performed with 0.1mol / L NaOH solution, with constant gentle shaking. The titration endpoint was reached when the solution turned a faint pink color that did not fade within 30 seconds. The titratable acidity of the fermented milk was calculated using formula (8).

[0058] In the formula: X2 - Acidity of the sample, in degrees (°T); C2 - Molar concentration of the NaOH standard solution, in mol / L; V2 - Volume of NaOH standard solution consumed during titration, in milliliters (mL); V0 - Volume of NaOH standard solution consumed in the blank experiment, in milliliters (mL); 100 - 100 g of sample; m2 - Mass of the sample, in grams (g); 0.1 - Molar concentration of sodium hydroxide as defined by acidity theory, in mol / L.

[0059] 5. The centrifugation conditions for water-holding capacity determination are: centrifugation speed 8000×g. Weigh the empty labeled centrifuge tube and record the mass as W0. Add 10 mL of fermented milk sample and weigh the tube, recording the mass as W1. Place the tube in a centrifuge at 15℃ for 10 min. After standing for 10 min, discard the supernatant and weigh the tube, recording the mass as W2. Perform the determination in triplicate. Calculate the water-holding capacity using the formula: In the formula: W0 is the mass of the centrifuge tube, W1 is the total mass of the centrifuge tube and the fermented milk, and W2 is the total mass of the precipitate after centrifugation and the centrifuge tube.

[0060] 6. Determination of Viable Bacteria Count After Fermentation: The determination of viable bacteria count after fermentation was performed according to GB 4789.35—2023. The viable bacteria count of the fermented milk was counted using the pour plate method. First, the fermented milk was serially diluted. 9 mL of 0.9% physiological saline was added to a centrifuge tube containing 1 mL of fermented milk sample, and the mixture was thoroughly mixed. The serial dilution was continued until a suitable gradient was reached. 1 mL of the diluted solution was transferred to a sterile petri dish. 15-20 mL of agar medium cooled to approximately 45℃ was poured into the sterile petri dish, mixed, and allowed to solidify. The dish was then incubated at 37℃. After colonies grew, colony counting was performed. Under constant temperature of 37℃, the dish was inverted and incubated for 48 h. Plates with 30-300 colonies were selected for counting. 7. Experimental Results: Table 1 shows the determination of pH, acidity, water-holding capacity, and viable bacteria count after 12 h of fermentation. The results, as shown in Table 1, indicate that GB 19302—2010, the National Food Safety Standard for Fermented Milk, stipulates that the acidity of fermented milk products should not be lower than 70°T. Table 1 shows that after 12 hours of fermentation, the acidity of both groups of fermented milk reached this standard. According to the National Food Safety Standard for Fermented Milk (GB 19302-2010), the viable bacteria count in fermented milk should be maintained at 10. 6 Both groups of fermented milk had a CFU / mL or higher viable bacteria count reaching 10⁻⁶. 9 The CFU / mL concentration meets the standard. Water-holding capacity is an important indicator for evaluating the quality of fermented milk. In the fermented milk system, casein molecules aggregate to form a network structure, which has the ability to bind small molecules such as water. The more water molecules bound, the less whey separation, and the better the quality of the fermented milk. The water-holding capacities of the two groups of fermented milk were 52.21% and 53.74%, respectively, indicating that the network structure of both groups of fermented milk is relatively fine and continuous, and has good stability.

[0061] Example 3: Non-target metabolomics analysis of fermented milk by *Lactobacillus plantarum* 1. Experimental methods (1) Processing of fermented milk sample Accurately weigh 100 μL of fermented milk sample. Metabolite extraction was performed using 400 μL of solution (V(methanol):V(distilled water) = 4:1). The mixture was precipitated at -20°C and then processed using a high-throughput tissue homogenizer at a frequency of 50 Hz for 6 min. After stirring for 30 s, the mixture was treated with ultrasound at 40 kHz at 5°C for 30 min. To precipitate proteins, the sample was then cooled at -20°C for 30 min. Centrifuged at 4°C and 13,000 g for 15 min, and the supernatant was transferred to a sample bottle for LC-MS / MS analysis.

[0062] (2) Quality control sample processing is an important step in the system calibration and quality control procedures. A pooled quality control sample (QC) is formed by mixing equal volumes of all individual samples. The QC sample is injected once every 32 samples to continuously evaluate the analytical stability.

[0063] (3) LC-MS / MS analysis: Metabolites were separated using the ExionLCTMAD system, mounted on an AC-QUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). Mobile phase: aqueous formic acid solution containing solvent A (0.1% formic acid) and solvent B (V(acetone formic acid):V(isopropanol) = 1:1). Gradient changes: 0–3 min, 95%(A):5%(B) to 80%(A):20%(B); 3–9 min, 80%(A):20%(B) to 5%(A):95%(B); 9–13 min, 5%(A):95%(B) remained constant; 13.0–13.1 min, 5%(A):95%(B) to 95%(A):5%(B); 13.1–16.0 min, 95%(A):5%(B) remained stable. Sample injection volume: 20 μL; flow rate: 0.4 mL / min; column temperature: 40 °C. Sample storage temperature during analysis: 4 °C. The UPLC system was connected to a quadrupole time-of-flight mass spectrometer, equipped with an electroinjection source (ESI) in both positive and negative modes. Optimal conditions: source temperature 500 °C; curtain gas (CUR) 207 kPa; ion source GS1 and GS2 pressures 345 kPa; electroinjection voltage (ISVF) -4000 V in negative mode and 5000 V in positive mode; declustering energy 80 V; MS / MS collision energy (CE) 20–60 V. Data acquisition mode (DDA) was selected, with a detection mass range of 50–1000 m / z.

[0064] Principal component analysis (PCA) was performed on the metabolites in the fermented milk, and the results are shown in Figure 1-A. Figure 1-A shows that the three samples were distributed in different ranges, and the three samples from the three treatment groups were relatively clustered, indicating good reproducibility within the three sample groups, while differences existed between the groups.

[0065] A volcano plot visually represents the distribution of differentially expressed metabolites between two groups of samples. The horizontal axis is typically represented by Log2 (Fold Change), with metabolites showing greater differences distributed at both ends. The vertical axis is represented by -Log10 (P-value), which is the negative logarithm of the statistical significance p-value.

[0066] The filtering parameter for the fold change P-value in the figure is the preset threshold for analysis, as shown in Figure 1. Each point in the figure represents a metabolite. The horizontal axis represents the logarithm of the fold change in the quantitative difference of a metabolite between the two samples; the vertical axis represents the logarithm of the P-value (-Log10). The larger the absolute value of the horizontal axis, the greater the fold change in the expression level of a metabolite between the two samples; the larger the value of the vertical axis, the more significant the differential expression, and the more reliable the differentially expressed metabolites are.

[0067] Metabolites (positive and negative ions combined) in the three samples were compared pairwise. After removing unidentifiable substances, the results are shown in Figures 1B, C, and D. Figure 1B shows that compared to the control group, the CCFM1296 group identified 367 differentially expressed metabolites (p < 0.05): 63 were relatively upregulated and 304 were decreased. Compared to the control group (Figure 1C), the FSCDJY69L1 group identified 461 differentially expressed metabolites, with 49 metabolites increasing in content and 412 decreasing in content. Compared to the FSCDJY69L1 group, the CCFM1296 group showed 163 upregulated metabolites and 14 downregulated metabolites (Figure 1D). Excluding unidentifiable substances, the CCFM1296 group ultimately showed 13 upregulated differentially expressed metabolites (Table 2). This result indicates that there are significant differences in the components of fermented milk produced using different strains (p < 0.05).

[0068] Cluster analysis was used to determine the metabolic patterns of metabolites under different experimental conditions. Hierarchical cluster analysis was performed on the relative values ​​of metabolites (positive and negative ions combined) in fermented milk from the CCFM1296 and FSCDJY69L1 groups at the metabolic level, and the results are represented by a heatmap. This experiment used agglomerate hierarchical clustering: each object is grouped into a class, and these classes are merged into increasingly larger objects until termination. The dataset was scaled using the Pheatmap package in R, resulting in the hierarchical clustering diagram of the relative quantitative values ​​of metabolites, as shown in Figure 2. Columns represent samples, rows represent metabolite names, and the clustering tree on the left side of the figure is the differential metabolite clustering tree. Metabolite names are not displayed if the number of metabolites exceeds 150. A darker red band indicates a stronger positive correlation between the metabolite and the sample, indicating a higher content; a darker blue band indicates a stronger negative correlation, indicating a lower content.

[0069] Table 2. Compared with the FSCDJY69L1 group, the CCFM1296 group ultimately upregulated differential metabolites. mz, mass-to-charge ratio; rt, retention time, in seconds; FC, fold change of metabolite among different groups; Log2(FC), Log2 value of the fold change of metabolite among different groups; P value, statistical p value, the smaller the value, the more significant the difference.

[0070] Table 2 shows that the metabolites of fermented milk in the CCFM1296 group underwent significant changes. Compared with the FSCDJY69L1 group, after 12 hours of fermentation, the CCFM1296 group produced a variety of amino acids, short peptides, and amino acid derivatives in the fermented milk, among which the proportion of L-proline was significantly increased, indicating that Lactobacillus plantarum CCFM1296 can enrich the content of free amino acids in fermented milk.

[0071] Example 4: Detection of Free Amino Acids in Fermented Milk by *Lactobacillus plantarum* 1. Experimental Methods (1) Sample Preparation of Fermented Milk Accurately weigh 800 μL of fermented milk sample, dilute it with an equal volume of 10 g / 100 mL trichloroacetic acid, let it stand for 1 h, and then filter it using double-layer filter paper. After filtration, take the clear filtrate and centrifuge it at 10000 rpm for 10 min. Take the supernatant, filter it through a 0.22 μm aqueous filter membrane, and transfer it to a liquid phase sample bottle for later use.

[0072] (2) Determination of Free Amino Acid Content: The content of various free amino acids in fermented milk was detected using an amino acid analyzer. The chromatographic conditions of the amino acid analyzer were set as follows: ion exchange column 4.6 mm × 60 mm, column temperature 57℃, column temperature maintained at 135℃, flow rates of pump 1 and pump 2 set to 0.45 mL / min and 0.35 mL / min respectively, and injection volume 20 μL. The ultraviolet detector was set as follows: proline was measured at 440 nm absorbance, and other amino acids were measured at 570 nm absorbance.

[0073] The experimental results are shown in Table 3-4. Compared with the control group, the essential amino acid content of the control strain FSCDJY69L1 increased by 11.83% and the non-essential amino acid content decreased by 8.19% after fermentation. The essential amino acid content of CCFM1296 increased by 9.44% and the non-essential amino acid content increased by 0.49% after fermentation, mainly due to the increase in lysine, threonine, valine, isoleucine, aspartic acid and proline.

[0074] Based on their flavor characteristics, amino acids were classified into umami amino acids (aspartic acid, glutamic acid, glycine, alanine, lysine), sweet amino acids (serine, threonine, histidine, proline), aromatic amino acids (cysteine, tyrosine, phenylalanine), and bitter amino acids (arginine, valine, methionine, isoleucine, leucine). The results are shown in Table 5. Compared with the control group, after fermentation with the control strain FSCDJY69L1, the total free amino acid content decreased by 0.35%, umami amino acids increased by 4.86%, and sweet amino acid content decreased by 34.07%. After fermentation with CCFM1296, the total free amino acid content increased by 4.00%, with umami amino acid content increasing by 7.89% and sweet amino acid content decreasing by 11.27%. This indicates that CCFM1296 fermentation can increase the total free amino acid content in fermented milk, increase the umami amino acid content, and reduce the loss of sweet amino acids.

[0075] Table 3. Content of essential amino acids in different fermented milks Table 4. Content of non-essential amino acids in different fermented milks Table 5 Total free amino acids in different fermented milks Example 5: Fermented milk from *Lactobacillus plantarum* reduces the biofilm formation ability of common pathogenic bacteria in vitro. 1. Preparation of antibiotic solution: Weigh an appropriate amount of gentamicin sulfate solid powder, dissolve it in physiological saline, and prepare a 150 μg / mL gentamicin sulfate working solution. After filtration and sterilization, store it in a refrigerator at 4°C in the dark for later use.

[0076] 2. Effects of *Lactobacillus plantarum* fermented milk on the biofilm formation ability of several common pathogenic bacteria. Preparation of fermented milk supernatant: Fermented milk samples were centrifuged at 6000 r / min for 10 min at room temperature. The supernatant was collected using a disposable syringe, the syringe tip was removed, and the supernatant was filtered through a 0.22 μm microporous membrane and stored at 4 ℃ for later use.

[0077] Preparation of cell-free probiotic supernatant: After mixing the second-generation probiotic strain, 3 mL of bacterial solution was pipetted into a 5 mL EP tube, centrifuged at 8000 rpm / min for 20 min, and the supernatant was collected using a disposable syringe. After removing the syringe, the supernatant was filtered through a 0.22 μm microporous membrane and the filtrate was collected to obtain cell-free supernatant, which was then stored at 4 ℃ for later use.

[0078] The amount of biofilm formed was analyzed using the crystal violet assay. The procedure was as follows: (1) Preparation of bacterial culture: 100 μl of bacterial culture of Salmonella Typhimurium, Pseudomonas aeruginosa, and Pseudomonas fluorescens (logarithmic growth phase) was added to each well of a 96-well plate and 150 μl of CCFM1296 fermented milk supernatant, CCFM1296 cell-free supernatant, FSCDJY69L1 fermented milk supernatant, FSCDJY69L1 cell-free supernatant, or antibiotic solution was added. The plates were incubated at 37°C for 24 h. MRS medium was used as a negative control and gentamicin sulfate was used as a positive control. (2) Washing and fixation: The bacterial culture was discarded. The plates were gently washed three times with PBS, air-dried, and fixed with 200 μl of methanol for 15 min. The plates were then air-dried. (3) Crystal violet staining: 1% crystal violet solution (200 μl / well) was added and stained for 15 min. The plates were washed with PBS until no floating color was found and then air-dried. (4) Decolorization test: Add 95% ethanol for 3-5 min to decolorize and measure the OD570 value.

[0079] Calculation formula: Biofilm reduction (%) = (OD negative control - OD sample) / OD negative control × 1003. Experimental results, as shown in Table 6, indicate that CCFM1296 fermented milk supernatant and cell-free supernatant significantly inhibited biofilm formation against the aforementioned common pathogenic bacteria. Compared to FSCDJY69L1, CCFM1296 improved the antibacterial ability against all three pathogenic bacteria after fermenting the milk. When CCFM1296 fermented milk supernatant and cell-free supernatant were co-cultured with pathogenic bacteria, the reduction in biofilm against Salmonella Typhimurium, Pseudomonas aeruginosa, and Pseudomonas fluorescens was greater than 70%. Compared with the cell-free supernatant of CCFM1296, the supernatant of CCFM1296 fermented milk increased the reduction of biofilms of Salmonella Typhimurium, Pseudomonas aeruginosa, and Pseudomonas fluorescens by 6.53%, 7.74%, and 0.69%, respectively, indicating that CCFM1296 fermented milk has a strong inhibitory effect on the biofilm formation ability of the three pathogenic bacteria, and its effect is better than that of CCFM1296 cell-free supernatant.

[0080] Table 6. Results of in vitro antibacterial experiments using *Lactobacillus plantarum* fermented milk and cell-free supernatant. The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0081] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0082] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0083] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. Use of Lactobacillus plantarum in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the Lactobacillus plantarum accession number is GDMCC No. 62974.

2. The use as described in claim 1, characterized in that, The product includes any one or more of food, medicine, health products, feed or daily chemical products. Preferably, the food is fermented food. More preferably, the food is any one or more of fermented milk, dairy drinks, fermented cream, milk powder or solid beverages.

3. The use as described in claim 1, characterized in that, The product contains fermentation products and / or culture products of *Lactobacillus plantarum*; and / or, the bacteria are pathogenic bacteria, preferably, the bacteria include any one or more of *Salmonella typhimurium*, *Pseudomonas aeruginosa*, or *Pseudomonas fluorescens*.

4. The use as described in claim 1, characterized in that, The product increases the total amount of free amino acids in fermented milk; and / or, the product improves the flavor of fermented milk; and / or, the product increases the content of umami amino acids in fermented milk and reduces the loss of sweet amino acids in fermented milk; and / or, the product increases the content of essential and non-essential amino acids in fermented milk; and / or, the product inhibits bacterial biofilm formation.

5. A method for preparing fermented milk, characterized in that, The *Lactobacillus plantarum* used in any one of claims 1 to 4 is inoculated into raw milk and fermented.

6. The preparation method according to claim 5, characterized in that, The volume ratio of *Lactobacillus plantarum* to raw milk during inoculation is (1-4):(90-110), preferably 2:

100.

7. The preparation method according to claim 5, characterized in that, The fermentation temperature is 35-40℃, preferably 37℃; and / or the fermentation time is 10-15h, preferably 12h; and / or the fermentation is aerobic fermentation.

8. The preparation method according to claim 5, characterized in that, The raw milk is whole milk.

9. The preparation method according to claim 8, characterized in that, The whole milk comprises whole milk powder, yeast extract, and water. Preferably, the mass ratio of the whole milk powder, yeast extract, and water is (10-20):(2-8):(70-90); more preferably, the mass ratio of the whole milk powder, yeast extract, and water is 15:5:

80.

10. A fermented milk, prepared by the preparation method according to any one of claims 5 to 9.

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