Application of saliva combined lactobacillus in preparation of product for inhibiting growth and proliferation of bacteria and / or improving flavor

Abstract

The invention discloses application of lactobacillus salivarius with bacteriostatic ability and capable of improving flavor of fermented milk in the fermented milk, and belongs to the technical field of microorganisms. The fermented milk prepared from the lactobacillus salivarius combined CCFM1332 can inhibit porphyromonas gingivalis, has a strong inhibition effect on the forming ability of salmonella typhimurium, staphylococcus aureus, pseudomonas aeruginosa and pseudomonas fluorescens biological membranes, and increases the content of essential amino acids and the content of flavor amino acids in the fermented milk; the proportion of bitter amino acids is reduced and the flavor of the fermented milk is improved. In addition, the CCFM1332 is high in acid production capacity, the pH can be rapidly reduced, the fermentation time is shortened, and the production efficiency is improved.

Description

Use of *Lactobacillus saliva-associated* in the preparation of products that inhibit bacterial growth and / or improve flavor Technical Field

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

[0002] Ligilactobacillus salivarius, a common probiotic, has had its beneficial functions extensively studied. There are existing reports of using Ligilactobacillus salivarius in fermented dairy products or other foods.

[0003] For example, an existing patent (CN118452278A) discloses the use of *Lactobacillus salivarius* fermentation to increase the niacin content in dairy products, primarily focusing on nutritional fortification. Other technical solutions mainly utilize the universal probiotic properties of *Lactobacillus salivarius* for regulating gut health (e.g., CN118165857A), or inactivate it for use in oral care products (e.g., CN105267136A).

[0004] However, the inventors discovered that not all *Lactobacillus salivarius* strains are suitable for preparing fermented milk products that possess excellent fermentation properties, significantly inhibit specific oral pathogens (such as *Porphyromonas gingivalis*), and actively improve the flavor profile of fermented milk. Currently, there is a lack of a technical solution that can simultaneously address the following issues: under conventional fermentation conditions, rapidly produce acid to complete fermentation, generating active substances that effectively inhibit *Porphyromonas gingivalis* during this process, while simultaneously optimizing the amino acid composition of the fermented milk, reducing bitterness, and enhancing umami, thereby obtaining a fermented milk product that combines oral health benefits with excellent flavor. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, this application provides the use of Ligilactobacillus salivarius in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the accession number of Ligilactobacillus salivarius is GDMCC No: 63788.

[0006] This application also provides a method for preparing fermented milk, in which Lactobacillus salivarius is inoculated into raw milk and fermented, wherein the Lactobacillus salivarius accession number is GDMCC No: 63788.

[0007] This application also provides a fermented milk prepared by the above-described preparation method.

[0008] The beneficial effects of this application include, but are not limited to: (1) This invention provides a strain of Lactobacillus salivatis 1332 with specific functional characteristics. This strain and its fermentation products show high antibacterial activity against oral pathogen Porphyromonas gingivalis (inhibition zone diameter >15mm), which is significantly better than other control strains (such as FNXYC6M7). It also has a strong inhibitory effect on the biofilm formation ability of Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas fluorescens. This makes the fermented milk prepared from it have clear potential for regulating oral health, and provides core strain resources for developing functional fermented milk with clear oral microenvironment regulation function.

[0009] (2) The application scheme provided by the present invention utilizes the strong acid production capacity of the salivary lactobacillus CCFM1332, which can quickly reduce the pH of the fermentation system, shorten the fermentation time (for example, the acidity can reach 96°T within 12 hours), improve production efficiency, and the formed curd has a uniform texture and good water retention.

[0010] (3) The present invention provides a method for applying the Lactobacillus salivatis CCFM1332 in the preparation of fermented milk. Based on the unique metabolic activity of this strain during fermentation, it can significantly increase the total amount and proportion of essential amino acids (especially histidine, threonine and lysine) in fermented milk and optimize the flavor amino acid composition, specifically by increasing the proportion of umami amino acids and decreasing the proportion of bitter amino acids, thereby fundamentally improving and enhancing the flavor quality of fermented milk products. Detailed Implementation

[0011] 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.

[0012] This application provides the use of *Ligilactobacillus salivarius* in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the accession number of *Ligilactobacillus salivarius* is GDMCC No: 63788.

[0013] In some embodiments, the product may contain fermentation products and / or culture products of Lactobacillus saliva-associated.

[0014] In some embodiments, the bacteria may be pathogenic bacteria. In some embodiments, preferably, the bacteria may be oral pathogenic bacteria. In some embodiments, more preferably, the bacteria may be *Porphyromonas gingivalis*.

[0015] In some embodiments, the Porphyromonas gingivalis may be GDM 1.851.

[0016] In some embodiments, the bacteria may include any one or more of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Pseudomonas fluorescens.

[0017] In some embodiments, the Staphylococcus aureus may be Staphylococcus aureus ATCC25923.

[0018] In some embodiments, the *Escherichia coli* may be *Escherichia coli* ATCC25922; in some embodiments, the *Salmonella typhimurium* may be *Salmonella typhimurium* TA100; in some embodiments, the *Pseudomonas aeruginosa* may be *Pseudomonas aeruginosa* ATCC9027; in some embodiments, the *Pseudomonas fluorescens* may be *Pseudomonas fluorescens* AS1.55.

[0019] In some embodiments, the product may include one or more of food, daily chemical products, health products, or pharmaceuticals. In some embodiments, preferably, the food may be a fermented food. In some embodiments, more preferably, the food may be a fermented dairy product.

[0020] In some embodiments, the product can inhibit the formation of bacterial biofilms.

[0021] In some embodiments, the product can improve the oral microbiota environment and prevent periodontal disease.

[0022] In some embodiments, the product can adjust the free amino acid composition in fermented milk and improve its flavor. Preferably, in some embodiments, the product can increase the content of essential amino acids and / or umami amino acids in fermented milk and decrease the content of bitter amino acids.

[0023] This application also provides a method for preparing fermented milk, in which Lactobacillus salivarius is inoculated into raw milk and fermented, wherein the Lactobacillus salivarius accession number is GDMCC No: 63788.

[0024] In some embodiments, the fermentation temperature can be 35–39°C. In some embodiments, the fermentation temperature can be 36–38°C. In some embodiments, the fermentation temperature can be 37–38°C. In some embodiments, preferably, the fermentation temperature can be 37°C.

[0025] In some embodiments, the fermentation time can be 10 to 14 hours. In some embodiments, the fermentation time can be 11 to 13 hours. In some embodiments, the fermentation time can be 12 to 13 hours. In some embodiments, preferably, the fermentation time can be 12 hours.

[0026] In some embodiments, the fermentation can be aerobic fermentation.

[0027] In some embodiments, the inoculation amount of *Lactobacillus saliva-associated* can be such that the number of viable bacteria in the whole milk reaches 5 × 10⁻⁶ at the time of inoculation. 6 ~5×10 7 CFU / mL. For example, the inoculation amount of the *Lactobacillus salivarius* can be such that the effective viable bacteria count in whole milk reaches 5 × 10⁻⁶ at the time of inoculation. 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 2×10 7 3×10 7 4×10 7 Or 5×10 7 CFU / mL.

[0028] In some embodiments, the raw milk may be whole milk.

[0029] In some embodiments, the whole milk may include whole milk powder, yeast extract, and water.

[0030] In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (12-18):(3-7):(70-90). In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (12.5-17.5):(3.5-6.5):(72-88). In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (13-17):(4-6):(74-86). In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (13.5-16.5):(4.5-5.5):(76-84). In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (14-16):(5-5.5):(78-82). In some embodiments, the mass ratio of whole milk powder, yeast extract, and water can be (14.5-15.5):(5-5.5):(80-82). In some embodiments, the mass ratio of whole milk powder, yeast extract and water can be (15-15.5):(5-5.5):(80-82).

[0031] In some embodiments, preferably, the mass ratio of the whole milk powder, yeast extract, and water can be 15:5:80.

[0032] This application also provides a fermented milk prepared by the above-described preparation method.

[0033] This invention provides a novel *Lactobacillus salivarius* strain CCFM1332 and its applications. One object of this invention is to provide the strain itself. Another object of this invention is to provide the application of this strain in the preparation of fermented dairy products, which effectively inhibit *Porphyromonas gingivalis* and have improved flavor characteristics.

[0034] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect, the present invention provides a strain of Ligilactobacillus salivarius CCFM1332, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 12, 2023, with the accession number GDMCC No: 63788, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0035] Secondly, the present invention provides the application of the aforementioned Lactobacillus salivarius CCFM1332 in the preparation of fermented dairy products.

[0036] Thirdly, the present invention provides a method for preparing fermented milk, comprising the step of inoculating the Lactobacillus salivarius CCFM1332 into raw milk for fermentation.

[0037] Fourthly, the present invention provides a fermented dairy product, which is obtained by fermenting raw milk with the aforementioned Lactobacillus salivarius CCFM1332.

[0038] Biological material preservation: Lactobacillus salivarius CCFM1332 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 12, 2023, with accession number GDMCC No: 63788, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0039] 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.

[0040] The strains used in this application are as follows: Staphylococcus aureus ATCC25923, purchased from CGMCC, China; Escherichia coli ATCC25922, purchased from CGMCC, China; Salmonella typhimurium TA100, purchased from Guangdong Institute of Microbiology, China; Pseudomonas aeruginosa ATCC9027, purchased from CGMCC, China; Pseudomonas fluorescens AS1.55, purchased from Mingzhou Biotechnology Co., Ltd., China; Porphyromonas gingivalis GDM 1.851, purchased from Guangdong Provincial Microbial Culture Collection Center, China; Lactobacillus salivarius CCFM1332 and Lactobacillus salivarius FNXYC6M7 were isolated from the oral cavity of healthy individuals by Jiangnan University Microbial Culture Collection Center.

[0041] 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.

[0042] 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.

[0043] BHI solid culture medium (g / L): tryptone 10.0g, beef heart extract 17.5g, sodium chloride 5.0g, yeast extract 5.0g, glucose 2.0g, disodium hydrogen phosphate dodecahydrate 2.5g, 0.5% vitamin K1 heme chloride 1mL / L, agar 20 g / L, 50mL / L sterile defibrinated sheep blood, pH 7.2~7.4.

[0044] BHI liquid culture medium (g / L): tryptone 10.0g, beef heart extract 17.5g, sodium chloride 5.0g, yeast extract 5.0g, glucose 2.0g, disodium hydrogen phosphate dodecahydrate 2.5g, 0.5% vitamin K1 heme chloride 1mL / L, pH 7.2~7.4.

[0045] LB liquid medium (g / L): Example 1: Molecular biological identification of *Lactobacillus saliva-associated*. Pure *Lactobacillus saliva-associated* bacteria were inoculated into MRS liquid 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: 3) and 1495R: 5'-CTACGGCTCCTTGTTCGA-3' (SEQ ID NO: 4). 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 *Lactobacillus*, classifying it as a species of *Lactobacillus*.

[0046]

[0047] Saliva-containing *Lactobacillus* CCFM1332 and FNXYC6M7 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 / mL 2. Preparation of whole milk: 15% whole milk powder and 5% yeast extract are mixed with distilled water and fully dissolved. The mixture is then sterilized at 95°C for 5 minutes and cooled to room temperature to obtain whole milk.

[0048] 3. Preparation of Fermented Milk: Salinomyces Lactobacillus CCFM1332 and FNXYC6M7 were inoculated at an inoculum size of 2% (v / v) with a viable count of 5 × 10⁻⁶. 6 ~5×10 7 CFU / mL, aseptically inoculated into whole milk, and cultured aerobically at 37°C for 12 hours to obtain fermented milk.

[0049] 4. The pH and acidity of fermented milk were measured using a digital pH meter.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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 concentration of CFU / mL 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 51.81% and 53.84%, respectively, indicating that the network structure of the two groups of fermented milk is relatively fine and continuous, and the stability is good. Compared with the control strain FNXYC6M7, CCFM1332 has a stronger acid-producing capacity, rapidly lowers the pH, shortens the fermentation time, and improves production efficiency.

[0054] Example 3: In vitro inhibition of Porphyromonas gingivalis by saliva-based Lactobacillus fermented milk. 1. Experimental strains: Porphyromonas gingivalis (Pg) strain stored in glycerol tubes was activated using the streak plate method. Single colonies were picked and inoculated into 5 mL of liquid BHI (heme chloride + VK) medium. After incubation at 37°C for 24 h, 2% (v / v) inoculation was added into 5 mL of liquid BHI (heme chloride + VK) medium and cultured until the logarithmic growth phase for later use.

[0055] 2. Preparation of cell-free supernatant of probiotics for sample processing: After mixing the second-generation probiotic strain, use a pipette to draw 3 mL of bacterial solution into a 5 mL EP tube, centrifuge at 8000 rpm / min for 20 min, use a disposable syringe to collect the supernatant, remove the needle, filter through a 0.22 μm microporous membrane and collect the filtrate to obtain cell-free supernatant, and store at 4 ℃ for later use.

[0056] Preparation of fermented milk supernatant: The fermented milk sample was centrifuged at 6000 r / min for 10 min at room temperature. The supernatant was collected using a disposable syringe, the syringe was removed, and the supernatant was filtered through a 0.22 μm microporous membrane and stored at 4 ℃ for later use.

[0057] 3. In vitro antibacterial activity: Place three sterile Oxford cups (8 mm) evenly on each plate. Pour BHI-H soft agar (containing the indicator strain (Porphyromonas gingivalis) with 5% sterile defibrinated sheep blood) into the plates. After the agar solidifies, remove the Oxford cups. Then, add 100 μL of a solution with a concentration of 1×10⁻⁶ to each well. 9 Cell-free supernatant and fermentation broth supernatant of probiotics were prepared using CFU / mL saliva combined with Lactobacillus CCFM1332 and FNXYC6M7. MRS medium and whole milk supernatant were used as negative controls, and 0.02% chlorhexidine was used as a positive control. The mixture was then anaerobically incubated at 37℃ for 48 h. The size of the inhibition zone was observed and measured, and the antibacterial activity of the probiotics was determined by the diameter of the inhibition zone (DIZ). Data are expressed as mean ± SD.

[0058] Judgment criteria: An inhibition zone diameter of 4mm is considered insensitive; an inhibition zone diameter of 5-10mm is considered lowly sensitive; an inhibition zone diameter of 11-15mm is considered moderately sensitive; and an inhibition zone diameter greater than 15mm is considered highly sensitive.

[0059] 4. Experimental Results Table 2 shows that the inhibition zone diameters of the cell-free supernatants of *Lactobacillus salivarius* CCFM1332 and FNXYC6M7 were both greater than 10 mm, indicating that *Porphyromonas gingivalis* was highly sensitive to CCFM1332. The antibacterial effect was enhanced after both strains were used to prepare fermented milk. Fermented milk prepared with *Lactobacillus salivarius* CCFM1332 showed the best antibacterial effect against *Porphyromonas gingivalis*, with an inhibition zone diameter reaching 19.31 mm. This indicates that fermented milk prepared with *Lactobacillus salivarius* CCFM1332 can effectively inhibit the growth of oral pathogens, improve the oral flora environment, prevent periodontal disease, and meet consumer demand for functional foods.

[0060] Table 2. Diameter of inhibition zones of different strains against *Porphyromonas gingivalis*. Example 4: In vitro reduction of biofilm formation ability of common pathogenic bacteria in fermented milk by Lactobacillus saliva-associated with CCFM1332 1. Preparation of antibiotic solution Weigh appropriate amounts of ampicillin, gentamicin sulfate, and vancomycin solid powder, dissolve them in physiological saline, and prepare working solutions of 100 μg / mL ampicillin, 150 μg / mL gentamicin sulfate, and 500 μg / mL vancomycin. After filtration and sterilization, store in a refrigerator at 4°C protected from light for later use.

[0061] 2. Effect of CCFM1332 fermented milk on the biofilm formation ability of several common pathogenic bacteria. Preparation of fermented milk supernatant: The fermented milk sample was centrifuged at 6000 r / min for 10 min at room temperature. The supernatant was collected using a disposable syringe, the needle was removed, and the supernatant was filtered through a 0.22 μm microporous membrane and stored at 4 ℃ for later use.

[0062] 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.

[0063] The biofilm formation was analyzed using the crystal violet assay. The procedure was as follows: (1) Preparation of bacterial culture: 100 μl of bacterial culture and 150 μl of CCFM1332 fermented milk supernatant, CCFM1332 cell-free supernatant, or antibiotic solution corresponding to the pathogen were added to each well of a 96-well plate for Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas fluorescens (logarithmic growth phase). The culture was incubated at 37°C for 24 h. MRS medium was used as a negative control. Ampicillin (Escherichia coli), gentamicin sulfate (Salmonella typhimurium, Pseudomonas aeruginosa, Pseudomonas fluorescens), and vancomycin (Staphylococcus aureus) were used. (2) Washing and fixation: The bacterial culture was discarded. The bacteria were gently washed three times with PBS and air-dried. The solution was fixed with 200 μl of methanol for 15 min and then air-dried. (3) Crystal violet staining: 1% crystal violet solution (200 μl / well) was added and stained for 15 min. The solution was washed with PBS until no floating color was observed and then air-dried. (4) Decolorization test: Add 95% ethanol for 3-5 min to decolorize and measure the OD570 value.

[0064] Calculation formula: Biofilm reduction (%) = (OD negative control - OD sample) / OD negative control × 1003. Experimental results: Table 3 shows that CCFM1332 fermented milk supernatant and cell-free supernatant have significant inhibitory effects on biofilm formation of the above-mentioned common pathogenic bacteria. When CCFM1332 fermented milk supernatant and cell-free supernatant were co-cultured with pathogenic bacteria, compared with the negative control group, the reduction in biofilm of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas fluorescens in CCFM1332 cell-free supernatant was greater than 70%, indicating that CCFM1332 cell-free supernatant has a strong inhibitory effect on the above five pathogenic bacteria. CCFM1332 fermented milk supernatant has a weaker effect on Escherichia coli biofilm, but still has a strong inhibitory effect on the biofilm formation ability of the other four pathogenic bacteria.

[0065] Table 3. Effects of saliva-containing lactobacillus fermented milk supernatant and cell-free supernatant on the biofilm-forming ability of various pathogenic bacteria. Example 5: Improving the Flavor of Fermented Milk by Regulating Free Amino Acid Composition with Saliva-Based Lactobacillus CCFM1332. Take 800 μL of fermented milk sample and centrifuge at 4000g for 15 min. Take 400 μL of fermented whey sample and dilute it with an equal volume of 10g / 100mL trichloroacetic acid. After standing for 1 h, centrifuge at 10000rpm for 10 min. Take the supernatant, filter it through a 0.22µm aqueous filter membrane, and transfer it to a liquid chromatography sample bottle. Use an amino acid analyzer to detect the content of various free amino acids in the fermented milk.

[0066] The results are shown in Tables 4-6. Compared with whole milk, the total amount of essential amino acids increased by 11.6% and the amount of non-essential amino acids increased by 3.5% after fermentation by the control strain FNXYC6M7. The total amount of essential amino acids increased by 14.2% after fermentation by CCFM1332, mainly in histidine, threonine and lysine, while the amount of non-essential amino acids decreased by 10%. Compared with strain FNXYC6M7, the amount of essential amino acids in fermented milk by CCFM1332 increased by 3.96%.

[0067] 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 7. With a decrease in total free amino acid content, the proportion of umami amino acids increased by 8% and the proportion of bitter amino acids decreased by 13.4% after fermentation with CCFM1332. After fermentation with FNXYC6M7, the proportion of umami amino acids increased by 5.8%, but the proportion of bitter amino acids increased by 6.3%, indicating that CCFM1332 can improve the flavor of fermented milk.

[0068] Table 4. Content and percentage of essential amino acids in different groups Table 5. Content and percentage of non-essential amino acids in different groups Table 6. Flavor-enhancing amino acid content of fermented milk in different groups 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.

[0069] 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.

[0070] 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.

[0071] 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. The use of *Ligilactobacillus salivarius* in the preparation of products that inhibit bacterial growth and / or improve flavor, wherein the accession number of *Ligilactobacillus salivarius* is GDMCC No: 63788.

2. The use as described in claim 1, characterized in that, The product contains fermentation products and / or culture products of Lactobacillus salivarius; and / or, the bacteria are pathogenic bacteria, preferably oral pathogenic bacteria, more preferably Porphyromonas gingivalis.

3. The use as described in claim 1, characterized in that, The bacteria include any one or more of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Pseudomonas fluorescens; and / or, the product includes any one or more of food, daily chemical products, health products, or pharmaceuticals, preferably, the food is a fermented food, more preferably, the food is a fermented dairy product; and / or, the product inhibits the formation of bacterial biofilms; and / or, the product improves the oral flora environment and prevents periodontal disease; and / or, the product regulates the free amino acid composition in fermented milk and improves its flavor, preferably, the product increases the content of essential amino acids and / or umami amino acids and decreases the content of bitter amino acids in fermented milk.

4. A method for preparing fermented milk, characterized in that, Lactobacillus salivarius was inoculated into raw milk and fermented. The preservation number of Lactobacillus salivarius was GDMCC No: 63788.

5. The preparation method according to claim 4, characterized in that, The fermentation temperature is 35-39°C, preferably 37°C; and / or the fermentation time is 10-14 hours, preferably 12 hours; and / or the fermentation is aerobic fermentation.

6. The preparation method according to claim 4, characterized in that, The inoculation amount of *Lactobacillus salivarius* was such that the effective viable bacteria count in the whole milk reached 5 × 10⁻⁶ at the time of inoculation. 6 ~5×10 7 CFU / mL.

7. The preparation method according to claim 4, characterized in that, The raw milk is whole milk.

8. The preparation method according to claim 7, characterized in that, The whole milk comprises whole milk powder, yeast extract, and water.

9. The preparation method according to claim 8, characterized in that, The mass ratio of whole milk powder, yeast extract and water is (12-18):(3-7):(70-90), preferably, the mass ratio of 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 4 to 9.

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