Use and composition of postbiotic of lactobacillus paracasei et-22

Abstract

The use and composition of a postbiotic of Lactobacillus paracasei ET-22, and the use of a lysate derived from Lactobacillus paracasei ET-22 in freshening breath and / or alleviating or inhibiting halitosis. The lysate is derived from heat-inactivated bacterial cells of Lactobacillus paracasei ET-22, the lysate at least comprises 2-hydroxy-3-phenylpropionic acid, NAD and a polypeptide, and the polypeptide has a structure as represented by formula 1. The postbiotic of and the lysate derived from Lactobacillus paracasei ET-22 can significantly reduce the contents of breath volatile sulfur compounds (VSCs) and salivary volatile organic compounds (VOCs) in the oral cavity, reduce the contents of characteristic flavor compounds, such as indoles, propionic acid and hexanoic acid, and can alter the oral microflora and inhibit the growth of oral biofilms and pathogenic bacteria, thus having the effects of freshening breath and alleviating or inhibiting halitosis.

Description

Application and composition of postbiotics of Lactobacillus paracasei ET-22 Technical Field

[0001] The present application relates to the application and composition of postbiotics of Lactobacillus paracasei ET-22, and relates to the field of microbial technology. Background Art

[0002] At present, postbiotics are attracting much attention as a hot research field. In 2021, the International Scientific Association of Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics. Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to host health. As derivatives of prebiotics and probiotics, postbiotics have multiple potential benefits. They can regulate intestinal flora, enhance intestinal barrier function, regulate intestinal inflammatory responses, etc., thereby having a positive impact on human intestinal health. In addition, the biological activity of postbiotics is not limited to the intestine. It also has biological activities such as inhibiting oral pathogens and regulating lung inflammatory responses. As people's understanding of intestinal health and microbiome continues to deepen, postbiotics are considered to be a potential functional food and health management method, and have received special attention from the industry. At present, reports on the bioactivity of postbiotics mainly refer to the bioactivity of probiotics or bacteria. Lactobacillus paracasei ET-22 is a relatively typical postbiotic strain. Previous studies have found that Lactobacillus paracasei ET-22 has potential in regulating blood pressure and sleep, but the specific form and preparation method of the product will affect its performance, such as thermal stability.

[0003] Bad breath is a widespread oral health concern, impacting not only personal health but also psychological and social well-being. Research has shown that some probiotics and their postbiotics can inhibit the growth of oral pathogens and regulate the immune system. For example, Lactobacillus paracasei L9 and Bifidobacterium animalis A6 can effectively alleviate gingival bleeding, periodontal inflammation, and alveolar bone resorption, while also reducing the relative proportion of pathogenic bacteria such as Streptococcus, Fusobacterium, and Veillonella, thereby preventing periodontitis. Other studies have shown that Lactobacillus paracasei ET-22, extracted from the infant intestine, inhibits the growth of Streptococcus mutans biofilms and the synthesis of bacterial water-soluble and water-insoluble polysaccharides, effectively preventing dental caries.

[0004] Live Lactobacillus paracasei ET-22 bacteria and their postbiotics have been shown to improve bad breath. The live bacteria can adhere and colonize in the mouth, competing with bad breath-causing bacteria for attachment sites, further inhibiting their growth and improving bad breath. However, postbiotics cannot adhere and colonize, and their active ingredients are still unknown.

[0005] Summary of the Invention

[0006] The present invention provides applications and compositions of postbiotics derived from Lactobacillus paracasei ET-22, which can significantly reduce the content of volatile sulfides (VSCs) in oral breath and volatile saliva compounds (VOCs), reduce the content of characteristic flavor compounds, such as indole, propionic acid, and hexanoic acid, change oral flora, inhibit the growth of oral biofilms and pathogenic bacteria, and have the effects of freshening breath and alleviating and inhibiting bad breath.

[0007] The present invention provides a use of a Lactobacillus paracasei ET-22-derived lysate for freshening breath and / or alleviating and inhibiting bad breath. The lysate is derived from heat-inactivated cells of Lactobacillus paracasei ET-22 and comprises at least 2-hydroxy-3-phenylpropionic acid, NAD, and a polypeptide, wherein the polypeptide has a structure shown in Formula 1.

[0008] Optionally, based on the total volume of the lysate, the mass of the 2-hydroxy-3-phenylpropionic acid is not less than 145 mg / L, the mass of the NAD is not less than 0.17 mg / L, and the mass of the polypeptide is not less than 0.24 mg / L.

[0009] Optionally, the cell lysate further comprises one or more of 6-hydroxyhexanoic acid, 3-aminoglutaric acid, 2-hydroxy-3-phenylpropionic acid, 2-hydroxy-3-(4-hydroxyphenyl)propionic acid, and Asp-Phe.

[0010] Optionally, the method for preparing the cell lysate comprises the following steps: culturing Lactobacillus paracasei ET-22 in liquid, stopping fermentation when the cell grows to the logarithmic phase, and collecting the cells; resuspending the collected cells in sterile water, heat-inactivating the cells, and collecting the supernatant to obtain the cell lysate.

[0011] Optionally, the heat inactivation temperature is 95-150°C and the time is 1-15 minutes.

[0012] The present invention also provides a peptide having the effect of freshening breath and / or alleviating and inhibiting bad breath, wherein the peptide has a structure as shown in Formula 1:

[0013] Optionally, the peptide is derived from a postbiotic of Lactobacillus paracasei ET-22.

[0014] The present invention also provides a composition for freshening breath and / or alleviating and inhibiting bad breath, comprising a lysate; the lysate is derived from heat-killed cells of Lactobacillus paracasei ET-22, and the lysate comprises at least 2-hydroxy-3-phenylpropionic acid, NAD, and a polypeptide, wherein the polypeptide has a structure shown in Formula 1;

[0015] Or, it includes nicotinamide adenine dinucleotide; or, it includes 2-hydroxy-3-phenylpropionic acid; or, it includes the peptide as described above; or, it includes peptidoglycan, and the peptidoglycan is derived from the heat-killed bacteria of Lactobacillus paracasei ET-22.

[0016] Optionally, the composition is one or more of a food composition, a pharmaceutical composition, and an oral cleaning composition.

[0017] Optionally, the food composition comprises one or more of dairy drinks, tea, coffee, chewing gum, tooth-cleaning candy, compressed candy, and dried meat for pets.

[0018] Optionally, the oral cleaning composition includes one or more of toothpaste, tooth cleaning powder, mouthwash, breath freshening spray, fluoride coating, denture cleaner, toothbrush, interdental brush, dental floss, oral cotton swab, pet tooth cleaning gel, pet hair remover, and pet tooth cleaning bone.

[0019] Optionally, in the composition, the content of nicotinamide adenine dinucleotide is not less than 100 μg / mL; or, the content of 2-hydroxy-3-phenylpropionic acid is not less than 100 μg / mL; or, the content of the peptide described above is not less than 100 μg / ml.

[0020] Optionally, the composition further comprises one or more of an excipient, a diluent, a carrier, and an additive.

[0021] The present invention also provides the use of nicotinamide adenine dinucleotide in freshening breath and / or alleviating and inhibiting bad breath.

[0022] Optionally, the nicotinamide adenine dinucleotide is derived from a postbiotic of Lactobacillus paracasei ET-22.

[0023] The present invention also provides the use of nicotinamide adenine dinucleotide in reducing the content of volatile sulfides in oral breath and volatile compounds in saliva.

[0024] The present invention also provides the use of nicotinamide adenine dinucleotide in reducing the content of characteristic flavor compounds.

[0025] The present invention also provides the use of 2-hydroxy-3-phenylpropionic acid in freshening breath and / or alleviating and inhibiting bad breath.

[0026] Optionally, the 2-hydroxy-3-phenylpropionic acid is derived from a postbiotic of Lactobacillus paracasei ET-22.

[0027] The present invention also provides the use of 2-hydroxy-3-phenylpropionic acid in reducing the content of characteristic flavor compounds in the oral cavity.

[0028] The present invention also provides the use of 2-hydroxy-3-phenylpropionic acid in reducing the amount and thickness of biofilm in the oral cavity.

[0029] The present invention also provides the use of the peptide represented by Formula 1 in freshening breath and / or alleviating and inhibiting bad breath.

[0030] The present invention also provides the use of the peptide represented by Formula 1 in reducing the content of volatile organic compounds in the oral cavity and / or inhibiting the growth of microorganisms in the oral cavity.

[0031] The present invention also provides an application of peptidoglycan in freshening breath and / or alleviating and inhibiting bad breath, wherein the peptidoglycan is derived from heat-killed bacteria of Lactobacillus paracasei ET-22.

[0032] Optionally, the peptidoglycan extraction method includes the following steps: step 1, culturing Lactobacillus paracasei ET-22 in liquid, stopping fermentation and collecting the bacteria when it grows to the logarithmic phase; step 2, resuspending the collected bacteria in sterile water, heat-inactivating them, and collecting the heat-inactivated bacteria; step 3, resuspending the heat-inactivated bacteria in PBS buffer, ultrasonically disrupting the heat-inactivated bacteria, and then centrifuging and collecting the precipitate, and extracting the peptidoglycan from the precipitate.

[0033] Optionally, in step 2, the collected bacteria were resuspended in sterile water to prepare a viable bacteria concentration of 1.0×10 9 CFU / ml of bacterial solution.

[0034] Optionally, in step 2, the temperature of heat inactivation is 90-150° C. and the time is 5-15 min.

[0035] Optionally, in step 3, peptidoglycan is isolated from the precipitate, specifically comprising: resuspending the heat-killed bacterial precipitate in PBS buffer, ultrasonically disrupting it, and centrifuging to obtain the precipitate; boiling the precipitate in 4% SDS solution for 30 minutes, and then adding 10% TCA to remove the phosphate wall and protein in the heat-killed bacteria; then adding a mixed solvent including sodium acetate, chloroform and methanol for defatting; then using alkaline protease and trypsin complex enzyme solution at 37°C for 12 hours, and then boiling it with 1% SDS solution for 10 minutes, and washing the SDS to trace amounts; then dialyzing for 2 days, changing the water every 8 hours, collecting the precipitate and vacuum freeze-drying it to obtain peptidoglycan.

[0036] The present invention also provides a Lactobacillus paracasei ET-22 secretin, comprising exocytosis of Lactobacillus paracasei ET-22 and metabolites of Lactobacillus paracasei ET-22. The mass ratio of citric acid to L-methionine in the secretin is greater than 15:1. The preservation number of the Lactobacillus paracasei ET-22 is CGMCC No. 15077.

[0037] Optionally, the mass ratio of citric acid to L-methionine in the bacteriocin is 15-100:1; preferably, the mass ratio of citric acid to L-methionine in the bacteriocin is 35-45:1.

[0038] The present invention also provides a method for preparing the bacteriocin of Lactobacillus paracasei ET-22, comprising the following steps: bacteriocin extraction, wherein bacterial mud separated from a fermentation broth of Lactobacillus paracasei ET-22 is extracted with a solvent to obtain a mixed liquid; and sterilization, wherein a supernatant in the mixed liquid is separated and heat sterilized to obtain the bacteriocin.

[0039] Optionally, the extraction temperature is 0-37°C; preferably, the extraction temperature is 20-30°C; and / or, the extraction time is 0.5-3h; preferably, the extraction time is 30-120min.

[0040] Optionally, the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 0.5×10 11 -5×10 11 cfu / mL; preferably, the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

[0041] Optionally, the temperature of heat sterilization is 70-121°C; preferably, the temperature of heat sterilization is 70-100°C.

[0042] Optionally, the heat sterilization time is 5-30 min; preferably, the heat sterilization time is 10-16 min.

[0043] Optionally, the method further comprises a fermentation step of culturing Lactobacillus paracasei ET-22 in liquid culture and stopping the fermentation when the Lactobacillus paracasei ET-22 grows to a logarithmic phase;

[0044] Preferably, the fermentation step comprises: inoculating Lactobacillus paracasei ET-22 fermentation seed liquid into a culture medium, and fermenting for 12-14 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.5-6.0 to obtain Lactobacillus paracasei ET-22 fermentation liquid;

[0045] Preferably, the preparation of the Lactobacillus paracasei ET-22 fermentation seed liquid comprises: activating the Lactobacillus paracasei ET-22 strain, purifying, culturing in a first-level seed liquid, multiplying in a second-level seed liquid, culturing in a third-level seed liquid, and preparing fermentation seeds to obtain the Lactobacillus paracasei ET-22 fermentation seed liquid;

[0046] Preferably, the culture medium is MRS liquid culture medium;

[0047] Preferably, the solvent is water; preferably, the solvent is sterile water;

[0048] Preferably, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

[0049] The present invention also provides a use of the above-mentioned Lactobacillus paracasei ET-22 secretin in preparing food, wherein the food comprises at least one of a dairy product and a beverage.

[0050] Optionally, the composition includes an antioxidant composition; preferably, the composition is selected from at least one of medicines, health foods and feeds.

[0051] The application and composition of the postbiotics of Lactobacillus paracasei ET-22 provided by the present invention can significantly reduce the content of volatile sulfides (VSCs) in oral breath and volatile saliva compounds (VOCs), reduce the content of characteristic flavor compounds, such as indole, propionic acid, and hexanoic acid, and can also change the oral flora, inhibit the growth of oral biofilms and pathogenic bacteria, have the effects of freshening breath and alleviating and inhibiting bad breath, and also have an antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 shows the improvement effect of the lysate isolated from ET-22 heat-killed bacteria on VSC concentration;

[0053] Figure 2 shows the composition of volatile organic compounds after lysate intervention;

[0054] FIG3 shows the composition of volatile flavor active compounds after lysate intervention;

[0055] FIG4 is a graph showing changes in the content of characteristic flavor active compounds in the oral cavity after lysate intervention;

[0056] FIG5 is a graph showing the changes in biomass of the biofilm after cytolysate intervention;

[0057] Figure 6a is a SEM image of the biofilm of the unintervention control group in the lysate observation group;

[0058] Figure 6b is a SEM image of the biofilm after intervention with live ET-22 bacteria in the lysate observation group;

[0059] Figure 6c is a SEM image of the biofilm after intervention with ET-22 heat-killed bacteria in the lysate observation group;

[0060] Figure 6d is a SEM image of the biofilm after lysate intervention;

[0061] FIG7 a is a CLSM image of the biofilm of the unintervention control group in the lysate observation group;

[0062] Figure 7b is a CLSM image of the biofilm after intervention with live ET-22 bacteria in the lysate observation group;

[0063] Figure 7c is a CLSM image of the biofilm after intervention with ET-22 heat-killed bacteria in the lysate observation group;

[0064] Figure 7d is a CLSM image of the biofilm after lysate intervention;

[0065] Figure 8 shows the changes in biofilm thickness after cytolysate intervention;

[0066] FIG9 is a graph showing the changes in relative abundance of bad breath pathogens after cytolysate intervention;

[0067] FIG10 is a graph showing the relative proportion changes of bad breath toxicity factors after cytolysate intervention;

[0068] FIG11 shows the improvement effect of two nucleotides isolated from the lysate of ET-22 heat-killed bacteria on VSC concentration at different concentrations;

[0069] Figure 12 shows the composition of volatile organic compounds after NAD intervention;

[0070] FIG13 shows the composition of volatile flavor-active compounds after NAD intervention;

[0071] FIG14 is a graph showing changes in the content of characteristic flavor active compounds in the oral cavity after NAD intervention;

[0072] FIG15 is a graph showing the changes in biomass of biofilm after NAD intervention;

[0073] Figure 16a is a SEM image of the biofilm of the control group without intervention in the NAD observation group;

[0074] Figure 16b is a SEM image of the biofilm after intervention with ET-22 live bacteria in the NAD observation group;

[0075] Figure 16c is a SEM image of the biofilm in the NAD observation group after intervention with ET-22 heat-killed bacteria;

[0076] Figure 16d is the SEM image of the biofilm after NAD intervention;

[0077] Figure 17a is a CLSM image of the biofilm of the control group without intervention in the NAD observation group;

[0078] Figure 17b is a CLSM image of the biofilm after intervention with ET-22 live bacteria in the NAD observation group;

[0079] Figure 17c is a CLSM image of the biofilm after intervention with ET-22 heat-killed bacteria in the NAD observation group;

[0080] Figure 17d is a CLSM image of the biofilm after NAD intervention;

[0081] Figure 18 shows the changes in biofilm thickness after NAD intervention;

[0082] FIG19 is a graph showing the changes in relative abundance of bad breath pathogens after NAD intervention;

[0083] Figure 20 shows the relative proportion changes of bad breath virulence factors after NAD intervention;

[0084] FIG21 shows the improvement effect of various organic acids isolated from the lysate of ET-22 heat-killed bacteria on VSC concentration at different concentrations;

[0085] Figure 22 shows the composition of volatile organic compounds after intervention with 2-hydroxy-3-phenylpropionic acid;

[0086] FIG23 shows the composition of volatile flavor active compounds after intervention with 2-hydroxy-3-phenylpropionic acid;

[0087] FIG24 is a graph showing changes in the content of characteristic flavor active compounds in the oral cavity after intervention with 2-hydroxy-3-phenylpropionic acid;

[0088] FIG25 is a graph showing the changes in biomass of biofilm after intervention with 2-hydroxy-3-phenylpropionic acid;

[0089] Figure 26a is a SEM image of the biofilm of the unintervention control group in the 2-hydroxy-3-phenylpropionic acid observation group;

[0090] Figure 26b is a SEM image of the biofilm in the 2-hydroxy-3-phenylpropionic acid observation group after intervention with live ET-22 bacteria;

[0091] Figure 26c is a SEM image of the biofilm in the 2-hydroxy-3-phenylpropionic acid observation group after intervention with ET-22 heat-killed bacteria;

[0092] Figure 26d is a SEM image of the biofilm after intervention with 2-hydroxy-3-phenylpropionic acid;

[0093] Figure 27a is a CLSM image of the biofilm of the unintervention control group in the 2-hydroxy-3-phenylpropionic acid observation group;

[0094] Figure 27b is a CLSM image of the biofilm after intervention with live ET-22 bacteria in the 2-hydroxy-3-phenylpropionic acid observation group;

[0095] Figure 27c is a CLSM image of the biofilm in the 2-hydroxy-3-phenylpropionic acid observation group after intervention with ET-22 heat-killed bacteria;

[0096] Figure 27d is a CLSM image of the biofilm after intervention with 2-hydroxy-3-phenylpropionic acid;

[0097] Figure 28 shows the changes in biofilm thickness after intervention with 2-hydroxy-3-phenylpropionic acid;

[0098] Figure 29 shows the changes in relative abundance of bad breath pathogens after intervention with 2-hydroxy-3-phenylpropionic acid;

[0099] Figure 30 shows the relative proportion changes of bad breath toxicity factors after intervention with 2-hydroxy-3-phenylpropionic acid;

[0100] FIG31 shows the improvement effect of various polypeptide components isolated from the lysate of ET-22 heat-killed bacteria on VSC concentration at different concentrations;

[0101] FIG32 shows the composition of volatile organic compounds after intervention with polypeptide GP(HYP)GAG;

[0102] FIG33 shows the composition of volatile flavor active compounds after polypeptide GP(HYP)GAG intervention;

[0103] FIG34 shows the effect of polypeptide GP(HYP)GAG on key flavor active compounds in the oral cavity;

[0104] Figure 35 is a SEM image of the biofilm after intervention with polypeptide GP(HYP)GAG;

[0105] Figure 36 is a CLSM image of the biofilm after intervention with polypeptide GP(HYP)GAG;

[0106] Figure 37 shows the changes in biofilm thickness after intervention with polypeptide GP(HYP)GAG;

[0107] FIG38 is a graph showing the relative abundance changes of bad breath pathogens after intervention with polypeptide GP(HYP)GAG;

[0108] FIG39 is a graph showing the relative abundance changes of halitosis virulence factors after intervention with polypeptide GP(HYP)GAG;

[0109] FIG40 shows the improvement effect of peptidoglycan isolated from ET-22 heat-killed bacteria on VSC concentration;

[0110] Figure 41 shows the composition of volatile organic compounds after peptidoglycan intervention;

[0111] FIG42 shows the composition of volatile flavor-active compounds after peptidoglycan intervention;

[0112] FIG43 is a graph showing changes in the content of characteristic flavor active compounds in the oral cavity after peptidoglycan intervention;

[0113] FIG44 is a graph showing changes in biomass of biofilm after peptidoglycan intervention;

[0114] Figure 45a is a SEM image of the biofilm of the unintervention control group in the peptidoglycan observation group;

[0115] Figure 45b is a SEM image of the biofilm after intervention with live ET-22 bacteria in the peptidoglycan observation group;

[0116] Figure 45c is a SEM image of the biofilm in the peptidoglycan observation group after intervention with ET-22 heat-killed bacteria;

[0117] Figure 45d is a SEM image of the biofilm after peptidoglycan intervention;

[0118] Figure 46a is a CLSM image of the biofilm of the unintervention control group in the peptidoglycan observation group;

[0119] Figure 46b is a CLSM image of the biofilm after intervention with live ET-22 bacteria in the peptidoglycan observation group;

[0120] Figure 46c is a CLSM image of the biofilm in the peptidoglycan observation group after intervention with ET-22 heat-killed bacteria;

[0121] Figure 46d is a CLSM image of the biofilm after peptidoglycan intervention;

[0122] Figure 47 shows the changes in biofilm thickness after peptidoglycan intervention;

[0123] Figure 48 shows the relative abundance changes of Intermediate Universal Bacteria after peptidoglycan intervention;

[0124] FIG49 is a graph showing the relative proportion changes of bad breath toxicity factors after peptidoglycan intervention;

[0125] Figure 50 is a flow chart of the preparation of secretory agents from Lactobacillus paracasei ET-22 in Example 3;

[0126] Figure 51 is a physical image of the secretory activity of Lactobacillus paracasei ET-22 in Example 3;

[0127] Figure 52 is a liquid chromatogram of the bacteriocin sample obtained in Example 3;

[0128] FIG53 shows the hydroxyl radical scavenging ability of secretin of Lactobacillus paracasei ET-22 under different extraction conditions;

[0129] FIG54 shows the DPPH radical scavenging ability of secretory agents of Lactobacillus paracasei ET-22 under different extraction conditions;

[0130] Figure 55 shows the hydroxyl radical scavenging ability and DPPH radical scavenging ability of the inactivated bacteria in Comparative Example 1 at different heat sterilization temperatures. DETAILED DESCRIPTION

[0131] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0132] The Lactobacillus paracasei ET-22 strain in the embodiment of the present invention has been deposited in the China General Microorganism Culture Collection Center with a deposit number of CGMCC No. 15077.

[0133] The postbiotics in the embodiments of the present invention refer to inactivated microorganisms and / or microbial cell components, including metabolites produced during their fermentation process, active small molecules released by decomposition of fermentation substrates, dead cells and cell components after lysis.

[0134] The structure of 2-hydroxy-3-phenylpropionic acid provided in the embodiment of the present invention is shown in Formula 1-1:

[0135] Example 1 Extraction, separation and identification of nucleotides, organic acids, polypeptides and peptidoglycans from ET-22 heat-killed bacterial lysates (Lactobacillus paracasei ET-22 derived lysates) and ET-22 postbiotics

[0136] 1.1 Strain culture

[0137] The Lactobacillus paracasei ET-22 strain was purchased from the China General Microorganism Culture Collection Center with the collection number CGMCC No. 15077. It was cultured in MRS medium at 37°C in an incubator for 12 hours to recover, then diluted and plated on MRS solid medium and cultured at 37°C for 48 hours. A single colony was picked and cultured in MRS medium at 37°C for 12 hours. The morphology and staining examination showed that it was a pure culture, and the culture was then subcultured twice. The third-generation bacterial solution was used for the experiment and diluted and plated for counting.

[0138] MRS medium: 10 g / L peptone, 5 g / L beef powder, 4 g / L yeast powder, 2 g / L glucose, 1 ml / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and distilled water.

[0139] 1.2 Isolation and purification of cell lysate (preparation of postbiotic components)

[0140] Centrifuge the revived bacterial solution in 1.1 for 10 minutes at 4500 r / min. Wash the precipitate three times with sterile water and resuspend it in sterile water. Then sterilize it at 100°C for 10 minutes. Centrifuge the bacterial solution at 10000 r / min for 10 minutes. Take the precipitate as the heat-inactivated bacteria and filter the supernatant with a 0.22 μm sterile filter membrane. The filtered liquid is the lysate (lysate supernatant).

[0141] 1.3 Sample preparation

[0142] The supernatant of the cell lysate was freeze-dried and reconstituted in 1.5 mL of sterile water. 100 μL of the liquid sample was aspirated into a 1.5 mL centrifuge tube, and 400 μL of the extraction solution (acetonitrile:methanol = 1:1) was added. After vortex mixing for 30 seconds, the sample was extracted by low-temperature ultrasonication for 30 minutes (5°C, 40 kHz). The sample was placed at -20°C for 30 minutes, centrifuged at 13,000 g for 15 minutes at 4°C, and the supernatant was removed. The sample was blown dry with nitrogen and reconstituted with 100 μL of the reconstitution solution (acetonitrile:water = 1:1). The sample was extracted by low-temperature ultrasonication for 5 minutes (5°C, 40 kHz), and centrifuged at 13,000 g for 5 minutes at 4°C. The supernatant was transferred to an injection vial with an inner cannula for analysis.

[0143] LC-MS / MS Analysis: LC-MS analysis was performed using a Thermo Fisher Scientific UHPLC-Q Exactive HF-X system coupled with Fourier transform mass spectrometry. Chromatographic conditions: 2 μL of sample was separated on an HSS T3 column (100 mm × 2.1 mm ID, 1.8 μm) before detection by mass spectrometry. Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Separation gradient: 0-3.5 min, mobile phase B increased from 0% to 24.5%, flow rate 0.40 mL / min; 3.5-5 min, mobile phase B increased from 24.5% to 65%, flow rate 0.4 mL / min; 5-5.5 min, mobile phase B increased from 65% to 100%, flow rate 0.4 mL / min; 5.5-7.4 min, mobile phase B maintained at 100%, flow rate increased from 0.4 mL / min to 0.6 mL / min From 7.4 to 7.6 minutes, mobile phase B was reduced from 100% to 51.5% at a flow rate of 0.6 mL / min. From 7.6 to 7.8 minutes, mobile phase B was reduced from 51.5% to 0% at a flow rate of 0.6 mL / min to 0.5 mL / min. From 7.8 to 9 minutes, mobile phase B was maintained at 0% at a flow rate of 0.5 mL / min to 0.4 mL / min. From 9 to 10 minutes, mobile phase B was maintained at 0% at a flow rate of 0.4 mL / min. The column temperature was 40°C.

[0144] Mass spectrometry conditions: Sample mass spectrometry signals were acquired in positive and negative ion scan modes over a mass scan range of 70–1050 m / z. Sheath gas flow rate was 50 psi, auxiliary gas flow rate was 13 psi, auxiliary gas heating temperature was 425°C, ion spray voltage was set to 3500 V in positive mode and -3500 V in negative mode, ion transfer tube temperature was 325°C, and normalized collision energy was cycled from 20–40–60 V. The primary mass spectrometer resolution was 60,000, and the secondary mass spectrometer resolution was 7500. Data were acquired in DDA mode.

[0145] The composition of small molecule active substances in the lysate of Lactobacillus paracasei ET-22 was determined by non-targeted metabolomics LC-MS, revealing that they are primarily composed of peptides, organic acids, and nucleotides. See Table 1 for details.

[0146] Table 1 Core components in cytolysate

[0147] According to Table 1, the peptides in the ET-22 lysate include GPRPK, Asp-Leu, Asp-Phe, and GP(Hyp)GAG (structure shown in Formula 1); organic acids include 6-hydroxyhexanoic acid, 3-aminoglutaric acid, nonacid, 2-hydroxy-3-(4-hydroxyphenyl)propionic acid, 2-hydroxysuccinic acid, and 2-hydroxy-3-phenylpropionic acid; and nucleotides include nicotinamide adenine dinucleotide (NAD) and xanthine dinucleotide (FAD). Formula 1 is as follows:

[0148] 1.4 Isolation and purification of peptidoglycan

[0149] Peptidoglycan is obtained by inactivating the bacteria of Lactobacillus paracasei ET-22 by high-temperature heating and then is separated from the heat-inactivated bacteria.

[0150] In a specific embodiment, Lactobacillus paracasei ET-22 can be first inoculated into a solid culture medium for culture recovery, and then a single colony can be inoculated into a liquid culture medium for fermentation. After the bacteria grow to the logarithmic phase, the fermentation liquid and the bacteria are separated and the solid bacteria are collected.

[0151] Subsequently, the collected bacteria were resuspended in sterile water for heat inactivation at a temperature of 90-150°C. After inactivation for 5-15 minutes, the bacteria were taken out and cooled to room temperature, and the heat-inactivated bacteria were collected by filtration.

[0152] Finally, peptidoglycan was isolated and extracted from the heat-killed bacteria. The heat-killed bacterial precipitate was resuspended in PBS buffer, ultrasonically disrupted, and then centrifuged to obtain the precipitate. The precipitate was boiled in 4% SDS solution for 30 minutes, and then 10% TCA was added to remove the phosphate walls and proteins in the heat-killed bacteria. A mixed solvent including sodium acetate, chloroform and methanol was then added for degreasing. The product was then treated with alkaline protease and trypsin complex enzyme solution at 37°C for 12 hours, and then boiled with 1% SDS solution for 10 minutes to wash the SDS to trace amounts. The product was then dialyzed for 2 days, with the water changed every 8 hours. The precipitate was collected and freeze-dried in a vacuum to obtain peptidoglycan.

[0153] Specifically, the revived bacterial solution in 1.1 was centrifuged at 4500 rpm for 10 minutes, the precipitate was washed three times with sterile water, and resuspended in sterile water. The solution was then sterilized at 100°C for 10 minutes, and the bacterial solution was centrifuged at 10000 rpm for 10 minutes to obtain the precipitate as the heat-killed bacteria.

[0154] The heat-killed bacterial pellet was resuspended in PBS buffer, ultrasonically disrupted at 240w for 30 minutes, and then centrifuged to obtain the pellet. The pellet was then boiled in a 4% SDS solution for 30 minutes, and then 10% TCA was added. After standing for a period of time, a mixed solvent (sodium acetate: chloroform: methanol = 4:5:10 by volume) was added. The pellet was then treated with alkaline protease and trypsin complex enzyme solution at 37°C for 12 hours, and then boiled with a 1% SDS solution for 10 minutes. The SDS was washed to trace amounts, and then dialyzed for 2 days, with the water changed every 8 hours. The pellet was vacuum freeze-dried to obtain a white powder or light yellow powder, which is peptidoglycan.

[0155] Example 2: Verification of the effects of cytolysate, two nucleotides (nicotinamide adenine dinucleotide (NAD) and xanthine dinucleotide (FAD)), five organic acids, four peptides, and peptidoglycan on bad breath

[0156] Fresh saliva was collected from 10 subjects with bad breath (Halimeter index > 120). The subjects were asked not to consume any food or drink for 2 hours before donating saliva and spit saliva directly into a saliva collection tube. 5 ml of saliva was collected from each person, mixed, centrifuged at 4500 rpm for 10 minutes, and the supernatant was collected and stored at -80°C for subsequent experimental research.

[0157] (I) The effects of cytolysate, two nucleotides (nicotinamide adenine dinucleotide (NAD) and xanthine dinucleotide (FAD)), five organic acids, four peptides, and peptidoglycan on VSC concentration were determined in vitro using a Halimeter.

[0158] Lysate observation group: 100 μl of saliva was first added to a 10 ml sample bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria were inactivated) and cell lysate (1.0×10 9 100 μl of each of the 1.8 ml SHI medium (CFU / ml of viable bacteria) was inactivated and filtered, and then 1.8 ml of SHI medium was added. After nitrogen was filled, anaerobically cultured for 10 hours, and then placed in a 60°C water bath for incubation for 30 minutes. The VSC concentration was then determined using the Halimeter in vitro assay method.

[0159] Two nucleotide groups: In a 10 ml sample bottle, first add 100 μl of saliva, then add ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of viable bacteria were inactivated), 100 μl each of NAD and FAD (500 μg / ml, 100 μg / ml, 10 μg / ml), and then 1.8 ml of SHI medium was added. After nitrogen was filled, anaerobically cultured for 10 hours, and then placed in a 60°C water bath for incubation for 30 minutes. The VSC concentration was then determined using the Halimeter in vitro assay method.

[0160] Five organic acid groups: 100 μl of saliva was first added to a 10 ml sample bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of viable bacteria obtained after heat inactivation) and 100 μl of each of five organic acid components (500 μg / ml, 100 μg / ml, 10 μg / ml), were added to 1.8 ml of SHI medium, and cultured anaerobically for 10 hours after nitrogen was filled. Then, it was placed in a 60°C water bath and incubated for 30 minutes, and the VSC concentration was determined by the Halimeter in vitro determination method.

[0161] Four peptide observation groups: 100 μl of saliva was first added to a 10 ml sample bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9CFU / ml of viable bacteria obtained after heat inactivation) and 100 μl of each of the four polypeptide components (500 μg / ml, 100 μg / ml, 10 μg / ml) were added, and then 1.8 ml of SHI medium was added. After nitrogen was filled, anaerobically cultured for 10 hours, and then placed in a 60°C water bath for incubation for 30 minutes, and then the VSC concentration was determined by the Halimeter in vitro determination method.

[0162] Peptidoglycan group: 100 μl of saliva was first added to a 10 ml sample bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of viable bacteria were inactivated) and peptidoglycan (1.0×10 9 100 μl of each of the peptidoglycans extracted from viable bacteria with a CFU / ml was added to 1.8 ml of SHI medium, and the mixture was anaerobically cultured for 10 hours after nitrogen was filled. The mixture was then placed in a 60°C water bath and incubated for 30 minutes. The VSC concentration was then determined using the Halimeter in vitro assay method.

[0163] In the Examples of the present invention, the above-mentioned lysate group, two nucleotide group, five organic acid group, four polypeptide group, and peptidoglycan group all used SHI medium, whose formula composition was: 10 g / L peptone, 5 g / L trypsin protein, 5 g / L yeast extract, 2.5 g / L potassium chloride, 5 mg / L hemin, 1 mg / L vitamin K, 0.06 g / L urea, 0.174 g / L arginine, 2.5 g / L porcine gastric mucin, 5% sterile defibrinated sheep blood, 10 mg / L N-acetylmuramic acid, 1% sucrose, and distilled water.

[0164] The results of the lysate analysis are shown in Figure 1. After simulated fermentation, the CK group had the highest VSC concentration, exceeding 200 ppm, with a noticeable unpleasant odor. Compared with the control group (CK group), VSC concentrations significantly decreased after intervention with live ET-22 bacteria (ET22-L), heat-killed ET-22 bacteria (ET22-HK), and lysate (p < 0.05).

[0165] The results of the two nucleotide groups are shown in Figure 11. Under the three nucleotide concentrations, the NAD group showed a significant decrease in VSC concentration compared to the CK group at both high and medium concentrations (p < 0.05), but no significant change at low concentrations (p > 0.05). The FAD group showed no significant change compared to the CK group at any concentration (p > 0.05). Therefore, NAD was identified as the core active ingredient, and subsequent experiments were conducted.

[0166] The results of the five organic acid groups are shown in Figure 21. Under intervention with the three concentrations of the organic acid components, the high-concentration 2-hydroxy-3-phenylpropionic acid group significantly reduced VSC concentration compared to the control group (p < 0.05). At the medium-concentration intervention, the 2-hydroxy-3-phenylpropionic acid group also significantly reduced VSC concentration compared to the control group (p < 0.05). At the low-concentration intervention, no significant changes were observed in any of the components compared to the control group. Therefore, 2-hydroxy-3-phenylpropionic acid was identified as the core active ingredient, and subsequent experiments were conducted.

[0167] The results of the four peptide groups are shown in Figure 31. In Figure 31, A shows the change in VSC concentration after intervention with 500 μg / ml of peptide, B shows the change in VSC concentration after intervention with 100 μg / ml of peptide, and C shows the change in VSC concentration after intervention with 10 μg / ml of peptide. As shown in Figure 31, only GP(HYP)GAG significantly reduced VSC concentration compared to the CK group at high and medium concentrations of the peptide components under intervention (p < 0.05). Therefore, GP(HYP)GAG was identified as the core active ingredient.

[0168] The results of the peptidoglycan group are shown in Figure 40. After simulated fermentation, the CK group had the highest VSC concentration, exceeding 200 ppm, with a noticeable unpleasant odor. Compared with the control group, the ET-22 live bacteria group (designated ET22-L), the ET-22 heat-killed bacteria group (designated ET22-HK), and the peptidoglycan intervention group showed significant decreases in VSC concentration (p < 0.05).

[0169] (II) Investigate the effects of cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, GP(HYP)GAG, and peptidoglycan on VOCs

[0170] 2.1 Grouping and preparation of intervention samples

[0171] Lysate group: 100 μl of saliva was first added to the headspace bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria were inactivated) and cell lysate (1.0×10 9 100 μl of each of the 100 μl of live bacteria (CFU / ml) was inactivated and filtered, added to 800 μl of SHI medium, and anaerobically cultured for 10 hours after nitrogen was filled. The changes in the content of volatile organic compounds were tested by SPME-GC-MS.

[0172] NAD group: 100 μl of saliva was first added to the headspace bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×109 100 μl of viable bacteria (CFU / ml obtained after heat inactivation) and NAD (100 μg / ml) were added to 800 μl of SHI culture medium. After nitrogen was filled and anaerobically cultured for 10 hours, the culture medium was put into the instrument and the changes in the content of volatile organic compounds were tested by SPME-GC-MS.

[0173] 2-Hydroxy-3-phenylpropionic acid group: 100 μl of saliva was first added to the headspace bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 100 μl of viable bacteria (obtained after heat inactivation of 100 μg / ml) and 100 μl of 2-hydroxy-3-phenylpropionic acid (100 μg / ml) were added to 800 μl of SHI culture medium. After nitrogen was filled and anaerobically cultured for 10 hours, the culture medium was put into the instrument and the changes in the content of volatile organic compounds were tested by SPME-GC-MS.

[0174] GP(HYP)GAG group: 100 μl of saliva was first added to the headspace bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 100 μl of viable bacteria (obtained after heat inactivation of 100 μg / ml) and 100 μl of GP(Hyp)GAG (100 μg / ml) were added to 800 μl of SHI culture medium. After nitrogen was filled and anaerobically cultured for 10 hours, the culture medium was loaded and the changes in the content of volatile organic compounds were tested by SPME-GC-MS.

[0175] Peptidoglycan group: 100 μl of saliva was first added to the headspace bottle, followed by the addition of ET-22 live bacteria (1.0×10 9 CFU / ml), 100 μl each of ET-22 heat-killed bacteria (same as in Example 1) and peptidoglycan (same as in Example 1), 800 μl of SHI medium was added, and after anaerobically incubating for 10 hours after nitrogen was filled, the content of volatile organic compounds was tested by SPME-GC-MS.

[0176] Helium was used as the carrier gas at a rate of 1.1 mL min -1The injector temperature was set to 250°C. The oven temperature program was as follows: 40°C for 1 min, then increased to 120°C at a rate of 8°C / min, then to 230°C at a rate of 5°C / min and held for 10 min. The mass spectrometer was operated in electron ionization (EI) mode with an ion source temperature of 230°C, an interface temperature of 200°C, and an m / z scan range of 30–400. To identify specific compounds, their mass spectra were compared with those of authentic compounds listed in the NIST 17 database (V.05). The minimum match mass for both forward and reverse matches was 75 / 100. The flavor profile of each compound was determined using data from the Good Scents Company Information System (http: / / www.thegoodscentscompany.com).

[0177] In this example, a total of 241 volatile organic compounds (VOCs) were detected, including 51 alkanes, 60 esters, 27 alcohols, 20 acids, 19 ketones, 20 alkenes, 9 aldehydes, 7 phenols, and 28 other types. Results of the lysate group: Figure 1 shows the improvement effect of the lysate isolated from the ET-22 heat-killed bacteria on the VSC concentration, and Figure 2 shows the changes in the number of various compounds under the intervention of the lysate. According to Figure 2, the number of ester compounds significantly decreased and the number of alcohols significantly increased under the intervention of the lysate (p < 0.001); Results of the NAD group: Figure 12 shows the changes in the number of various compounds under the intervention of NAD, using ET22-L to represent the live bacteria intervention group and ET22-HK to represent the heat-killed bacteria intervention group. According to Figure 12, The amount of alkane compounds and acid compounds decreased significantly under NAD intervention (p < 0.01). Studies have shown that alkane compounds are products of bacterial fatty acid metabolism and decomposition. Other studies have shown that alkane substances are closely related to the production of microorganisms, and one of the causes of bad breath may be related to the degradation of fatty acids. Results of the 2-hydroxy-3-phenylpropionic acid group: Figure 22 shows the changes in the amount of various compounds after 2-hydroxy-3-phenylpropionic acid intervention. ET22-L represents the live bacteria intervention group, and ET22-HK represents The heat-killed bacteria intervention group, as shown in Figure 22, showed that the alkane compounds were significantly reduced after the intervention of 2-hydroxy-3-phenylpropionic acid (p < 0.01). Studies have shown that alkane compounds are the products of bacterial fatty acid metabolism and decomposition, and alkane substances are closely related to the production of microorganisms; The results of the GP (HYP) GAG group: Figure 32 shows the changes in the number of various compounds after the GP (Hyp) GAG intervention. ET22-L represents the live bacteria intervention group, and ET22-HK represents the heat-killed bacteria intervention group. According to Figure 32, It is known that acid compounds were significantly reduced after GP(Hyp)GAG intervention (p<0.001), and one of the causes of bad breath may be related to the degradation of fatty acids; the results of peptidoglycan: Figure 41 shows the changes in the quantity of various compounds under the intervention of peptidoglycan. According to Figure 41, the number of alkane compounds was significantly reduced (p<0.05), the number of alcohols was significantly increased (p<0.001), and the number of olefin compounds was also significantly increased (p<0.001). In addition, the number of ester compounds also increased.

[0178] Of the 241 VOCs, 71 were identified as aroma-active VOCs, which were divided into six categories (esters, alcohols, acids, ketones, phenols, and other compounds). This example further examined the changes in the content of these 71 aroma-active VOCs under the intervention of lysates. The test results are shown in Figure 3. Esters and acids are the most flavor-active VOCs, and the amount of flavor-active ester compounds was significantly reduced after intervention with ET22-L, ET22-HK, and lysates (p < 0.05). This example also further examined the changes in the content of these 71 aroma-active VOCs under the intervention of NAD. The test results are shown in Figure 13. The amount of flavor-active acid compounds was also significantly reduced after NAD intervention (p < 0.001), and acid compounds are the most flavor-active VOCs. This example further examined the changes in the content of these 71 aroma-active VOCs under the intervention of 2-hydroxy-3-phenylpropionic acid. The test results are shown in Figure 23. The number of flavor-active ester compounds and acid compounds was significantly reduced after the intervention of 2-hydroxy-3-phenylpropionic acid (p < 0.05). This example further examined the changes in the content of these 71 aroma-active VOCs under the intervention of GP(Hyp)GAG. The test results are shown in Figure 33. The number of acid compounds was also significantly reduced after the intervention of GP(Hyp)GAG (p < 0.001). This example further examined the changes in the content of these 71 aroma-active VOCs under the intervention of peptidoglycan. The test results are shown in Figure 42. Esters are the most flavor-active VOCs, and the number of flavor-active ester compounds was significantly reduced after the intervention of ET22-L, ET22-HK, and peptidoglycan (p < 0.05).

[0179] 2.2 Investigating the effects of cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, GP(Hyp)GAG, and peptidoglycan on characteristic flavor compounds

[0180] Since oral odor is not limited to VSCs, indoles and short-chain fatty acids (such as propionic acid and butyric acid) may also contribute. Indole is a compound with an unpleasant odor, hexanoic acid is a compound with a sour, cheesy odor, and propanoic acid has been shown to cause bad breath. Therefore, this example tested the effects of cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, GP(Hyp)GAG, and peptidoglycan on indole, hexanoic acid, and propanoic acid. In one embodiment, the characteristic flavor compounds include one or more of indole, hexanoic acid, and propionic acid.

[0181] The test results of the lysate are shown in Figure 4. In Figure 4, A shows the effect of the lysate on the indole concentration, B shows the effect of the lysate on the propionic acid concentration, and C shows the effect of the lysate on the hexanoic acid concentration. It can be seen that under the intervention of ET-22 live bacteria, ET-22 heat-killed bacteria, and lysate, the concentrations of indole, hexanoic acid, and propionic acid were significantly reduced (p < 0.01), and the intervention effect of the lysate on propionic acid and hexanoic acid was better than that of ET-22 live bacteria and their heat-killed bacteria.

[0182] The test results of NAD are shown in Figure 14. In Figure 14, A is the effect of NAD on indole concentration, B is the effect of NAD on propionic acid concentration, and C is the effect of NAD on hexanoic acid concentration. It can be seen that under the intervention of ET-22 live bacteria, ET-22 heat-killed bacteria and NAD, the concentrations of indole, hexanoic acid and propionic acid were significantly reduced (p<0.01), and the intervention effect of NAD was better than that of ET-22 live bacteria and its heat-killed bacteria.

[0183] The test results of 2-hydroxy-3-phenylpropionic acid are shown in Figure 24. In Figure 24, A is the effect of 2-hydroxy-3-phenylpropionic acid on the indole concentration, B is the effect of 2-hydroxy-3-phenylpropionic acid on the propionic acid concentration, and C is the effect of 2-hydroxy-3-phenylpropionic acid on the hexanoic acid concentration. It can be seen that under the intervention of ET-22 live bacteria, heat-killed bacteria and 2-hydroxy-3-phenylpropionic acid, the concentrations of indole, hexanoic acid and propionic acid were significantly reduced (p<0.01), and the inhibitory effect of 2-hydroxy-3-phenylpropionic acid was better than that of ET-22 live bacteria and heat-killed bacteria.

[0184] The test results of GP(Hyp)GAG are shown in Figure 34, where A shows the effect of GP(Hyp)GAG on indole concentration, B shows the effect of GP(Hyp)GAG on propionic acid concentration, and C shows the effect of GP(Hyp)GAG on hexanoic acid concentration. It can be seen that under the intervention of ET-22 and its heat-killed bacteria, the concentrations of indole, hexanoic acid and propionic acid were significantly reduced (p<0.01), and no hexanoic acid and propionic acid were detected under the intervention of GP(Hyp)GAG, indicating that the polypeptide GP(Hyp)GAG has a good inhibitory effect on characteristic flavor compounds.

[0185] The test results of peptidoglycan are shown in Figure 43. In Figure 43, A is the effect of peptidoglycan on the concentration of indole, B is the effect of peptidoglycan on the concentration of propionic acid, and C is the effect of peptidoglycan on the concentration of hexanoic acid. It can be seen that under the intervention of ET-22 live bacteria, ET-22 heat-killed bacteria and peptidoglycan, the concentrations of indole, hexanoic acid and propionic acid were significantly reduced (p<0.01), and the intervention effect of peptidoglycan on hexanoic acid was better than that of ET-22 live bacteria and their heat-killed bacteria.

[0186] (III) Investigating the effects of cytolysates, NAD, 2-hydroxy-3-phenylpropionic acid, and peptidoglycan on the biomass of multispecies biofilms

[0187] The relationship between biofilm biomass and the severity of bad breath was analyzed by evaluating biofilm formation under the intervention of ET-22 live bacteria, heat-killed bacteria, lysates, NAD, 2-hydroxy-3-phenylpropionic acid, and peptidoglycan. The test methods include:

[0188] (1) Cultivation in groups

[0189] Lysate group: 10 μl of saliva was added to a 96-well plate, and then ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria were inactivated) and cell lysate (1.0×10 9 100 μl of each of the CFU / ml of viable bacteria (obtained after inactivation and filtration) was added to 80 μl of SHI medium, and the plate was placed in an anaerobic box and incubated anaerobically at 37°C for 10 hours. Each group was repeated 6 times.

[0190] NAD group: 10 μl of saliva was added to a 96-well plate, and then ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 100 μl of each of CFU / ml (obtained after heat inactivation) and NAD (100 μg / ml) were added to 80 μl of SHI medium, and the plate was placed in an anaerobic box and incubated anaerobically at 37°C for 10 hours. Each group was repeated 6 times.

[0191] 2-Hydroxy-3-phenylpropionic acid group: 10 μl of saliva was added to a 96-well plate, and then ET-22 live bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 100 μl of each of viable bacteria (CFU / ml obtained after heat inactivation) and 2-hydroxy-3-phenylpropionic acid (100 μg / ml) were added to 80 μl of SHI medium, and the well plate was placed in an anaerobic box and incubated anaerobically at 37°C for 10 hours. Each group was repeated 6 times.

[0192] Peptidoglycan group: 10 μl of saliva was added to a 96-well plate, and then ET-22 live bacteria (1.0×10 9 CFU / ml), 100 μl each of ET-22 heat-killed bacteria (same as in Example 1), and peptidoglycan (same as in Example 1), were added to 80 μl of SHI medium, and the plate was placed in an anaerobic box and incubated anaerobically at 37°C for 10 hours. Each group was repeated 6 times.

[0193] (2) Gently aspirate the supernatant with a pipette tip, and then wash 1-2 times with sterile PBS buffer.

[0194] (3) Add 100 μl of 0.5% crystal violet dye to the plate and let it stand at room temperature for 20 minutes.

[0195] (4) After the dye is absorbed, wash with sterile PBS buffer for more than 3 times until the dye is rinsed clean.

[0196] (5) Place the well plate in an enzyme reader and measure the biofilm biomass at 600 nm.

[0197] The measurement results of the lysate group are shown in Figure 5. The optical density reading of the biofilm in the CK group was the highest at 595 nm. Compared with the CK group, the biomass of the biofilm decreased after intervention with ET22-L and ET22-HK (p < 0.05), and the intervention effect of the lysate on the biofilm biomass was better than that of the live ET-22 bacteria and their heat-killed bacteria.

[0198] The measurement results of the NAD group are shown in Figure 15. The optical density reading of the biofilm in the CK group was the highest. Compared with the CK group, the biomass of the biofilm decreased after intervention with ET22-L and ET22-HK (p<0.05), and the biomass of the NAD group was slightly lower than that of the CK group.

[0199] The measurement results of the 2-hydroxy-3-phenylpropionic acid group are shown in Figure 25. The optical density reading of the biofilm in the CK group was the highest. Compared with the CK group, the biomass of the biofilm decreased after intervention with ET22-L, ET22-HK, and 2-hydroxy-3-phenylpropionic acid. The biomass of the 2-hydroxy-3-phenylpropionic acid group was significantly lower than that of the CK group (p<0.05), and the intervention effect was better than that of ET-22 live bacteria and heat-killed bacteria.

[0200] The measurement results of the peptidoglycan group are shown in Figure 44. The optical density reading of the biofilm in the CK group was the highest at 595nm. Compared with the CK group, the biomass of the biofilm decreased after intervention with ET22-L and ET22-HK (p<0.05), and the biomass of the peptidoglycan group was slightly lower than that of the CK group.

[0201] (IV) The effects of live ET-22 bacteria, cell lysates, heat-killed bacteria, NAD, 2-hydroxy-3-phenylpropionic acid, GP(Hyp)GAG, and peptidoglycan on biofilm were observed using scanning electron microscopy.

[0202] Observation methods include:

[0203] (1) Cultivation in groups

[0204] Lysate group: A hydroxyapatite (HA) disk was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×109 CFU / ml of live bacteria were inactivated) and cell lysate (1.0×10 9 10 μl of each of the 100 CFU / ml of viable bacteria were inactivated and filtered, and 80 μl of SHI culture medium was added. The well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0205] NAD group: A hydroxyapatite (HA) disk was placed in a 96-well plate, 10 μl of saliva was taken and placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 10 μl of viable bacteria (CFU / ml obtained after heat inactivation) and NAD (100 μg / ml) were added, and then 80 μl of SHI culture medium was added. The well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0206] 2-Hydroxy-3-phenylpropionic acid group: A hydroxyapatite (HA) disk was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria obtained after heat inactivation) and 10 μl each of 2-hydroxy-3-phenylpropionic acid (100 μg / ml) were added, and then 80 μl of SHI culture medium was added, and the well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0207] GP(Hyp)GAG group: Hydroxyapatite (HA) discs were placed in 96-well plates, 10 μl of saliva was taken and placed in the 96-well plates, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 10 μl each of 100 μg / ml of viable bacteria (obtained after heat inactivation) and GP(Hyp)GAG (100 μg / ml) were added, and then 80 μl of SHI culture medium was added. The well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0208] Peptidoglycan group: A hydroxyapatite (HA) disk was placed in a 96-well plate, 10 μl of saliva was taken and placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), 10 μl each of ET-22 heat-killed bacteria (same as in Example 1) and peptidoglycan (same as in Example 1), and then 80 μl of SHI culture medium were added, and the well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0209] (2) Gently rinse the floating bacteria on the surface of the HA disc with sterile PBS buffer.

[0210] (3) Add 2.5% glutaraldehyde solution to the well plate for fixation and place at 4°C overnight.

[0211] (4) The fixed samples were subjected to gradient dehydration and placed in 30%, 50%, 60%, 70%, 80%, 90%, and 100% ethanol solutions in sequence, and each concentration was allowed to stand for 15 minutes.

[0212] (5) Critical point drying and gold spraying were carried out and observed under a scanning electron microscope.

[0213] The observation results of the lysate observation group are shown in Figures 6a-6d. The biofilm structure of the CK group was dense, with rod-shaped and spherical bacteria stacked and intertwined in layers, with more bacterial layers and a larger amount of bacteria; while the number of rod-shaped bacteria in the ET-22.L group increased, the number of bacterial layers decreased, and the biofilm became thinner; the biofilm of the ET-22.HK group was similar to that of the ET-22.L group, except that most of the bacteria were short rods and cocci, and almost no extracellular matrix was produced; the biofilm structure of the lysate group was looser, the number of bacterial layers also decreased, and more extracellular matrix was produced between most bacteria. Some streptococci also formed a tight island biofilm covered by a large amount of mucus or reticular structure.

[0214] The observation results of the NAD observation group are shown in Figures 16a-16d. The biofilm structure of the CK group was dense, with rod-shaped and spherical bacteria stacked and intertwined in layers, with more bacterial layers and a larger bacterial amount; while the number of rod-shaped bacteria in the biofilm of the ET-22.L group increased, the number of bacterial layers decreased, and the biofilm became thinner; the biofilm of the ET-22.HK group was similar to that of the ET-22.L group, except that most of the bacteria were short rods and cocci, and there was almost no extracellular matrix production; the biofilm structure of the NAD group had a larger number of bacterial layers, but a smaller bacterial amount.

[0215] The observation results of the 2-hydroxy-3-phenylpropionic acid group and the observation group are shown in Figures 26a-26d. The biofilm structure of the CK group was dense, with rod-shaped and spherical bacteria stacked and intertwined in layers, with a large number of bacterial layers and a large amount of bacteria; while the number of rod-shaped bacteria in the biofilm of the ET-22.L group increased, the number of bacterial layers decreased, and the biofilm became thinner; the biofilm of the ET-22.HK group was similar to that of the ET-22.L group, except that most of the bacteria were short rods and cocci, and almost no extracellular matrix was produced; the biofilm structure of the 2-hydroxy-3-phenylpropionic acid group showed a large number of mucus-like structures, and the amount of extracellular matrix was significantly reduced, the number of bacterial layers decreased, and the biofilm became thinner. GP(Hyp)GAG Group: Observation results are shown in Figure 35. The biofilm structure of the CK group was dense, with interwoven layers of various rod-shaped and spherical bacteria, a large number of bacterial layers, and a high bacterial count. In contrast, the biofilm of the ET-22.L group showed an increase in the number of rod-shaped bacteria, a decrease in bacterial layers, and a thinner biofilm. The biofilm of the ET-22.HK group was similar to the ET-22.L group, except that the majority of bacteria were short rods and cocci, and there was almost no extracellular matrix production. The biofilm of the GP(Hyp)GAG group showed almost no visible bacterial morphology, and the biofilm structure was loose, consisting mostly of densely packed microscopic extracellular matrix and some mucus-like biofilm. This suggests that the peptide GP(Hyp)GAG can effectively improve biofilm thickness.

[0216] Peptidoglycan observation group: The observation results are shown in Figures 45a-45d. The biofilm structure of the CK group was dense, with rod-shaped and spherical bacteria stacked and intertwined in layers, with more bacterial layers and a larger amount of bacteria. In the ET-22.L group, the number of rod-shaped bacteria in the biofilm increased, the number of bacterial layers decreased, and the biofilm became thinner. The biofilm of the ET-22.HK group was similar to that of the ET-22.L group, except that most of the bacteria were short rods and cocci, and almost no extracellular matrix was produced. The biofilm of the peptidoglycan group had almost no visible bacterial morphology, and the biofilm structure was relatively loose, with most of the biofilm consisting of densely packed tiny extracellular matrices and some mucus-like biofilms.

[0217] (V) The effects of live ET-22 bacteria, heat-killed bacteria, cytolysates, NAD, 2-hydroxy-3-phenylpropionic acid, polypeptide GP(Hyp)GAG (GP(Hyp)GAG), and peptidoglycan on biofilm thickness were observed using confocal microscopy (CLSM).

[0218] Observation methods include:

[0219] (1) Cultivation in groups

[0220] Lysate group: HA disc was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 viable bacteria (1.0×109 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria were inactivated) and cell lysate (1.0×10 9 10 μl of each of the 100 CFU / ml of viable bacteria were inactivated and filtered, and 80 μl of SHI culture medium was added. The well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0221] NAD group: HA disc was placed in a 96-well plate, 10 μl of saliva was taken and placed in the 96-well plate, and then 10 μl of ET-22 live bacteria (1.0×109 CFU / ml), ET-22 heat-killed bacteria (obtained after heat-killing 1.0×109 CFU / ml of live bacteria) and NAD (100 μg / ml) were added respectively, and then 80 μl of SHI medium was added, and the well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0222] 2-Hydroxy-3-phenylpropionic acid group: HA disk was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 CFU / ml of live bacteria obtained after heat inactivation) and 10 μl each of 2-hydroxy-3-phenylpropionic acid (100 μg / ml) were added, and then 80 μl of SHI culture medium was added, and the well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0223] Peptide GP (Hyp) GAG group: HA disk was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 viable bacteria (1.0×10 9 CFU / ml), ET-22 heat-killed bacteria (1.0×10 9 10 μl of each of the polypeptide GP(Hyp)GAG (100 μg / ml) was obtained by heat-inactivating viable bacteria of 100 CFU / ml, and then 80 μl of SHI medium was added. The well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0224] Peptidoglycan group: HA disc was placed in a 96-well plate, 10 μl of saliva was placed in the 96-well plate, and then ET-22 live bacteria (1.0×10 9 CFU / ml), 10 μl each of ET-22 heat-killed bacteria (same as in Example 1) and peptidoglycan (same as in Example 1), and then 80 μl of SHI culture medium were added, and the well plate was placed in an anaerobic box and anaerobically cultured at 37°C for 10 hours.

[0225] (2) Use 0.85% NaCl solution to gently rinse the planktonic bacteria on the surface of the HA disk.

[0226] (3) Use the live / dead bacteria staining kit for 646 staining. First, add 100 μl of N01 staining solution A and incubate at room temperature in the dark for 15 minutes. After washing the disc surface once with 0.85% NaCl solution, add PI staining solution B and incubate at room temperature in the dark for 15 minutes. Then, wash the disc surface once with 0.85% NaCl solution.

[0227] (4) Place the stained disc upside down on a glass slide and observe it under a confocal microscope using a ×60 oil objective lens.

[0228] (5) The experimental results were photographed and plotted using ZEN Lite 2012 software.

[0229] The observation results of the lysate observation group are shown in Figures 7a-7d. Biofilm thickness was then measured at different locations on the HA disc, and a bar graph was plotted, as shown in Figure 8. Figures 7a-7d and 8 show that compared with the CK group, both live ET-22 and heat-killed ET-22 significantly reduced biofilm thickness (p < 0.0001). Lysate also reduced biofilm thickness (p < 0.05), demonstrating a superior intervention effect on biofilm thickness compared to live ET-22 and its heat-killed counterparts.

[0230] The results of the NAD observation group are shown in Figures 17a-17d. Biofilm thickness was then measured at different locations on the HA disc and plotted as a bar graph in Figure 18. Figures 17a-17d and 18 show that both live and heat-killed ET-22 significantly reduced biofilm thickness compared to the CK group (p < 0.0001), while NAD intervention had no significant effect.

[0231] The observation results of the 2-hydroxy-3-phenylpropionic acid observation group are shown in Figures 27a-27d. Subsequently, the biofilm thickness at different locations on the HA disc was measured and plotted as a bar graph in Figure 28. Figures 27a-27d and 28 show that compared with the CK group, live ET-22 bacteria, heat-killed ET-22 bacteria, and 2-hydroxy-3-phenylpropionic acid all significantly reduced biofilm thickness (p < 0.0001), and the intervention effect of 2-hydroxy-3-phenylpropionic acid was superior to that of live ET-22 bacteria and heat-killed bacteria.

[0232] The observation results of the polypeptide GP (Hyp) GAG group, according to Figures 36 and 37, show that compared with the CK group, ET-22 live bacteria, ET-22 heat-killed bacteria, and polypeptide GP (Hyp) GAG can significantly reduce the thickness of the biofilm (p < 0.0001).

[0233] The observation results of the peptidoglycan observation group are shown in Figures 46a-46d. Subsequently, the biofilm thickness at different locations on the HA disc was measured and plotted as a bar graph in Figure 47. Figures 46a-46d and 47 show that, compared with the CK group, both live ET-22 and heat-killed ET-22 significantly reduced biofilm thickness (p < 0.0001), and peptidoglycan also reduced biofilm thickness (p < 0.05).

[0234] (6) Construct an in vitro model of oral biofilm and investigate the effects of cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, polypeptide GP(Hyp)GAG, and peptidoglycan on the biofilm.

[0235] 6.1 Construction of an in vitro oral biofilm model:

[0236] 100 μl of saliva was added to a 24-well plate. Then, 100 μl of the corresponding concentration solution was added to each experimental group. Finally, 800 μl of SHI medium was added. The plate was placed in an anaerobic chamber and incubated anaerobically at 37°C for 10 hours. The supernatant was discarded. The biofilm adhered to the plate was a multispecies biofilm. After discarding the supernatant from the incubated biofilm, the biofilm adhered to the bottom of the plate was resuspended by pipetting with sterile water and transferred to a 1.5 ml centrifuge tube for storage until the sample was sequenced.

[0237] 6.2 Investigating the effects of cytolysates, NAD, 2-hydroxy-3-phenylpropionic acid, polypeptide GP(Hyp)GAG, and peptidoglycan on halitosis-causing bacteria in multispecies biofilms

[0238] Based on the sequencing results, the bad breath pathogens in the biofilm were identified, and a total of four key bad breath pathogens were identified, namely Fusobacterium nucleatum, Streptococcus mutans, Prevotella intermedia and Solobacterium moorei. These four bacteria have been shown to be positively correlated with VSC concentrations. Prevotella intermedia is a periodontal pathogen, and it and Fusobacterium nucleatum have also been shown to be able to produce indole through the tryptophan metabolic pathway. Therefore, this example uses cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, and polypeptide GP (Hyp) GAG to intervene in the bad breath pathogens in the biofilm, and evaluates the changes in the relative abundance of the pathogens after the intervention; in addition, Intermediate Universal Bacteria are periodontal pathogens that can produce indole through the tryptophan metabolic pathway. Therefore, this example also uses peptidoglycan to intervene in Intermediate Universal Bacteria in the biofilm, and evaluates the changes in the relative abundance of Intermediate Universal Bacteria after the intervention.

[0239] The analysis results of the lysate group are shown in Figure 9. In Figure 9, A shows the effect of lysate intervention on the bad breath pathogen Prevotella intermedia, B shows the effect of lysate intervention on the bad breath pathogen Streptococcus mutans, C shows the effect of lysate intervention on the bad breath pathogen Fusobacterium nucleatum, and D shows the effect of lysate intervention on the bad breath pathogen Solobacterium moorei. In summary, ET-22.L, ET-22.HK, and lysate all inhibit the growth of bad breath pathogens, and lysate has a significant inhibitory effect on the growth of Streptococcus mutans.

[0240] The analysis results for the NAD group are shown in Figure 19 . Figure 19 shows the changes in the relative abundance of the halitosis-causing bacteria Prevotella intermedia after NAD intervention, Figure B shows the changes in the relative abundance of the halitosis-causing bacteria Streptococcus mutans after NAD intervention, Figure C shows the changes in the relative abundance of the halitosis-causing bacteria Fusobacterium nucleatum after NAD intervention, and Figure D shows the changes in the relative abundance of the halitosis-causing bacteria Solobacterium moorei after NAD intervention. As shown in Figure 19 , compared with the CK group, both live and heat-killed ET-22 bacteria significantly inhibited the growth of halitosis-causing bacteria, but the effect of NAD intervention was not significant.

[0241] The analysis results of the 2-hydroxy-3-phenylpropionic acid group are shown in Figure 29. In Figure 29, A is a graph showing the change in the relative abundance of the bad breath pathogen Prevotella.intermedia after intervention with 2-hydroxy-3-phenylpropionic acid, B is a graph showing the change in the relative abundance of the bad breath pathogen Streptococcus.mutans after intervention with 2-hydroxy-3-phenylpropionic acid, C is a graph showing the change in the relative abundance of the bad breath pathogen Fusobacterium.nucleatum after intervention with 2-hydroxy-3-phenylpropionic acid, and D is a graph showing the change in the relative abundance of the bad breath pathogen Solobacterium.moorei after intervention with 2-hydroxy-3-phenylpropionic acid. As shown in Figure 29, 2-hydroxy-3-phenylpropionic acid can significantly inhibit the growth of Solobacterium.moorei (p < 0.05), and the inhibitory effect is better than that of live ET-22 bacteria or heat-killed bacteria.

[0242] The analysis results of the polypeptide GP(Hyp)GAG group are shown in Figure 38. In Figure 38, A shows the effect of polypeptide GP(Hyp)GAG intervention on the bad breath pathogen Prevotella.intermedia, B shows the effect of polypeptide GP(Hyp)GAG intervention on the bad breath pathogen Streptococcus.mutans, C shows the effect of polypeptide GP(Hyp)GAG intervention on the bad breath pathogen Fusobacterium.nucleatum, and D shows the effect of polypeptide GP(Hyp)GAG intervention on the bad breath pathogen Solobacterium.moorei. As shown in Figure 38, GP(Hyp)GAG can significantly inhibit the growth of Solobacterium.moorei (p<0.05), and its inhibitory effect is better than that of live ET-22 bacteria or heat-killed bacteria.

[0243] The analysis results of the peptidoglycan group are shown in FIG48 . Compared with the CK group, the live ET-22 bacteria, the heat-killed ET-22 bacteria, and the peptidoglycan can significantly inhibit the growth of the bad breath pathogen E. intermedius.

[0244] 6.3 Investigating the effects of cytolysates, NAD, 2-hydroxy-3-phenylpropionic acid, polypeptide GP(Hyp)GAG, and peptidoglycan on virulence factors in multispecies biofilms

[0245] This example evaluates the effects of cytolysate, NAD, 2-hydroxy-3-phenylpropionic acid, polypeptide GP(Hyp)GAG, and peptidoglycan on oral halitosis virulence factors, including cdl, fadA, mgl, and gtfb. The gene cdl regulates the expression of L-cysteine ​​dehydrogenase, which catalyzes the degradation of protein substrates to produce VSCs, which in turn causes bad breath. Fusobacterium nucleatum secretes an amyloid-like adhesin (fadA) via a fap2-like autotransporter to enhance its toxicity. Adhesins play a key role in the pathogenicity of Fusobacterium nucleatum, and fadA refers to the mRNA encoding this adhesin. L-methionine-α-deamino-γ-mercaptomethane lyase (METase) can produce α-ketobutyrate, ammonia, and CH3SH from L-methionine. Anaerobic bacteria, through the METase encoded by mgl, can also produce large amounts of VSCs. gtfB is the most common virulence gene, encoding glucosyltransferases (Gtfs). Glycosyltransferases are involved in the adhesion of Streptococcus mutans and other bacteria to biofilms. Studies have shown that deletion of the gtfB gene in Streptococcus mutans reduces bacterial accumulation.

[0246] Test methods include:

[0247] (1) Extraction of RNA from biofilm bacteria: The extraction method of RNA from biofilm bacteria (biofilm bacterial RNA) was based on the RNAprep Pure cultured bacteria / cell total RNA extraction kit from TIANGEN.

[0248] (2) Reverse transcription

[0249] The reverse transcription system is shown in Table 2. Add the following reagents to a PCR tube in an ice bath:

[0250] Table 2 RNA reverse transcription cDNA system

[0251] Perform reverse transcription reaction on a PCR instrument according to the conditions in Table 3:

[0252] Table 3 PCR reaction procedure

[0253] (3) RT-PCR detection

[0254] After the sample RNA was reverse transcribed into cDNA, RT-PCR reaction was performed to detect biofilm-related virulence factors. The primer sequences are shown in Table 4. The experimental results were calculated using 2 -△△CT Calculation method.

[0255] Table 4 RT-PCR primer sequences for biofilm-associated virulence factors

[0256] The analysis results of the lysate group are shown in Figure 10. In Figure 10, A shows the relative proportion changes of the halitosis virulence factor cdl after lysate intervention, B shows the relative proportion changes of the halitosis virulence factor fadA after lysate intervention, C shows the relative proportion changes of the halitosis virulence factor mgl after lysate intervention, and D shows the relative proportion changes of the halitosis virulence factor gtfb after lysate intervention. As shown in Figure 10, compared with the CK group, live ET-22 bacteria, heat-killed ET-22 bacteria, and lysates all modulated the expression of virulence factors, and the lysates had a greater inhibitory effect on the expression of the virulence factor fadA than live ET-22 bacteria and heat-killed bacteria.

[0257] The analysis results of the NAD group are shown in Figure 20. In Figure 20, A is a graph showing the relative proportion changes of the bad breath virulence factor cdl after NAD intervention, B is a graph showing the relative proportion changes of the bad breath virulence factor fadA after NAD intervention, C is a graph showing the relative proportion changes of the bad breath virulence factor mgl after NAD intervention, and D is a graph showing the relative proportion changes of the bad breath virulence factor gtfb after NAD intervention. As shown in Figure 20, compared with the CK group, both ET-22 live bacteria and ET-22 heat-killed bacteria can regulate the expression of virulence factors, but the intervention effect of NAD is not obvious. In summary, NAD mainly reduces the content of characteristic flavor compounds such as indole, propionic acid, and hexanoic acid by reducing volatile sulfides (VSCs) and saliva volatile compounds (VOCs) in the oral cavity, and reducing the content of characteristic flavor compounds such as indole, propionic acid, and hexanoic acid, thereby having the effect of freshening breath and alleviating and inhibiting bad breath.

[0258] The analysis results of the 2-hydroxy-3-phenylpropionic acid group are shown in Figure 30. In Figure 30, A is a graph showing the relative proportion changes of the bad breath virulence factor cdl after intervention with 2-hydroxy-3-phenylpropionic acid, B is a graph showing the relative proportion changes of the bad breath virulence factor fadA after intervention with 2-hydroxy-3-phenylpropionic acid, C is a graph showing the relative proportion changes of the bad breath virulence factor mgl after intervention with 2-hydroxy-3-phenylpropionic acid, and D is a graph showing the relative proportion changes of the bad breath virulence factor gtfb after intervention with 2-hydroxy-3-phenylpropionic acid. As shown in Figure 30, 2-hydroxy-3-phenylpropionic acid can significantly regulate the four bad breath virulence factors (p < 0.05), and its inhibitory effect on the virulence factor fadA is better than that of ET22-L and ET22-HK.

[0259] The analysis results of the polypeptide GP(Hyp)GAG group are shown in Figure 39. In Figure 39, A shows the changes in the bad breath virulence factor cdl after the intervention of polypeptide GP(Hyp)GAG, B shows the changes in the bad breath virulence factor fadA after the intervention of polypeptide GP(Hyp)GAG, C shows the changes in the bad breath virulence factor mgl after the intervention of polypeptide GP(Hyp)GAG, and D shows the changes in the bad breath virulence factor gtfb after the intervention of polypeptide GP(Hyp)GAG. As shown in Figure 39, the polypeptide GP(Hyp)GAG can significantly regulate the four bad breath virulence factors (p<0.05), and its inhibitory effect on the virulence factors cdl and fadA is better than ET22-L and ET22-HK. Part 6.2 of the experiment has confirmed that GP(Hyp)GAG can inhibit the growth of Fusobacterium.nucleatum. We speculate that GP(Hyp)GAG may inhibit the growth of Fusobacterium.nucleatum by regulating the expression of cdl and fadA genes, thereby further reducing the production of VSC.

[0260] The analysis results of the peptidoglycan group are shown in Figure 49. In Figure 49, A is a graph showing the relative proportion changes of the bad breath virulence factor cdl after peptidoglycan intervention, B is a graph showing the relative proportion changes of the bad breath virulence factor fadA after peptidoglycan intervention, C is a graph showing the relative proportion changes of the bad breath virulence factor mgl after peptidoglycan intervention, and D is a graph showing the relative proportion changes of the bad breath virulence factor gtfb after peptidoglycan intervention. As shown in Figure 49, compared with the CK group, ET-22 live bacteria, ET-22 heat-killed bacteria, and peptidoglycan can all regulate the expression of virulence factors, but the regulatory effect is slightly worse than that of ET-22 live bacteria and its heat-killed bacteria. In summary, peptidoglycan mainly plays a role in freshening breath and alleviating and inhibiting bad breath by reducing the content of volatile sulfides (VSCs) and saliva volatile compounds (VOCs) in the oral cavity and reducing the content of characteristic flavor compounds such as indole, propionic acid, and hexanoic acid.

[0261] The above research analysis shows that:

[0262] The embodiments of the present invention provide a use and composition of a Lactobacillus paracasei ET-22-derived lysate for freshening breath and / or alleviating and inhibiting bad breath. The lysate can significantly reduce the content of volatile sulfides (VSCs) and saliva volatile compounds (VOCs) in oral breath, reduce the content of characteristic flavor compounds, and can change the oral flora, inhibit the growth of oral biofilms and pathogenic bacteria, and has the effects of freshening breath and alleviating and inhibiting bad breath.

[0263] The use and composition of nicotinamide adenine dinucleotide for breath freshening provided in the embodiments of the present invention can significantly reduce the content of volatile sulfides (VSCs) and saliva volatile compounds (VOCs) in the oral cavity, and reduce the content of characteristic flavor compounds such as indole, propionic acid, and hexanoic acid, thereby having the effects of freshening breath and alleviating and inhibiting bad breath.

[0264] The use and composition of 2-hydroxy-3-phenylpropionic acid for breath freshening provided in the embodiments of the present invention can significantly inhibit the formation of characteristic flavor compounds and reduce the concentrations of key volatile odor compounds such as indole, propionic acid, and hexanoic acid; and can also change the oral flora, especially reduce the number and thickness of biofilms, inhibit biofilm formation and the growth of pathogenic bacteria in the oral cavity, thereby having the effects of freshening breath and alleviating and inhibiting bad breath.

[0265] The embodiments of the present invention provide a breath-freshening peptide, its application, and a composition comprising the peptide. The amino acid composition of the peptide (as shown in Formula 1) is glycine-proline-hydroxyproline-glycine-alanine-glycine, which can significantly inhibit the formation of volatile organic compounds in the oral cavity and reduce the concentrations of key volatile odor compounds such as indole, propionic acid, and hexanoic acid; it can also change the oral flora, inhibit the growth of oral biofilms and pathogenic bacteria, and has the effects of freshening breath and alleviating and inhibiting bad breath.

[0266] The application and composition of Lactobacillus paracasei ET-22-derived peptidoglycan for breath freshening provided in the embodiments of the present invention can significantly reduce the content of volatile sulfides (VSCs) and saliva volatile compounds (VOCs) in oral breath, and reduce the content of characteristic flavor compounds such as indole, propionic acid, and hexanoic acid, thereby having the effects of freshening breath and alleviating and inhibiting bad breath.

[0267] The embodiment of the present invention also provides a Lactobacillus paracasei ET-22 secretin, a preparation method and an application thereof, to obtain a product with antioxidant properties.

[0268] The Lactobacillus paracasei ET-22 secretin of the embodiment of the present invention refers to a product obtained by extracting water-soluble metabolites of Lactobacillus paracasei ET-22 and exocytosis of Lactobacillus paracasei ET-22 in bacterial mud separated from Lactobacillus paracasei ET-22 fermentation broth.

[0269] An embodiment of the present invention further provides a Lactobacillus paracasei ET-22 secretin, comprising exocytosis of Lactobacillus paracasei ET-22 and metabolites of Lactobacillus paracasei ET-22, wherein the mass ratio of citric acid to L-methionine in the secretin is greater than 15:1, and the preservation number of the Lactobacillus paracasei ET-22 is CGMCC No.15077.

[0270] In the embodiment of the present invention, the bacteriocin mainly includes components such as exocytosis and bacterial metabolites. As the bacteriocin is extracted, the concentrations of citric acid and L-methionine in the bacteriocin gradually increase. Therefore, in the embodiment of the present invention, the concentrations of L-methionine and citric acid are used to characterize the bacteriocin.

[0271] The Lactobacillus paracasei ET-22 secretin in the embodiment of the present invention has an antioxidant function and can be applied to food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the secretin in the embodiment of the present invention is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

[0272] In an optional embodiment, the mass ratio of citric acid to L-methionine in the secretin is 15-100:1, specifically, it can be 15:1, 25:1, 35:1, 45:1, 55:1, 65:1, 75:1, 85:1, 100:1 or any value between 15-100:1, or any value greater than 100:1.

[0273] Preferably, the mass ratio of citric acid to L-methionine in the bacteriocin is 35-45:1.

[0274] The embodiment of the present invention also provides a method for preparing the aforementioned Lactobacillus paracasei ET-22 bacteriocin, comprising the following steps: bacteriocin extraction, wherein bacterial mud separated from Lactobacillus paracasei ET-22 fermentation broth is extracted with a solvent to obtain a mixed liquid; and sterilization, wherein a supernatant in the mixed liquid is separated and heat sterilized to obtain the bacteriocin.

[0275] In an optional embodiment, the extraction temperature is 0-37°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C or any value between 0-37°C; preferably, the extraction temperature is 20-30°C.

[0276] In an optional embodiment, the extraction time is 0.5-3 h, specifically 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h or any value between 1-3 h; preferably, the extraction time is 30-150 min.

[0277] In an optional embodiment, the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 0.5×10 11 -5×10 11 , specifically 0.5×10 11 cfu / mL, 0.7×10 11 cfu / mL, 0.9×10 11 cfu / mL, 1×10 11 cfu / mL, 2×10 11 cfu / mL, 3×10 11 cfu / mL, 4×10 11 cfu / mL, 5×10 11 cfu / mL or 0.5×10 11 -5×10 11 cfu / mL; preferably, the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

[0278] In an optional embodiment, the temperature of heat sterilization is 70-121°C, specifically 70°C, 80°C, 90°C, 100°C, 110°C, 121°C or any value between 70-121°C; preferably, the temperature of heat sterilization is 70-100°C.

[0279] In an optional embodiment, the heat sterilization time is 5-30 min, specifically 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value between 5-30 min; preferably, the heat sterilization time is 10-16 min.

[0280] In an optional embodiment, the method further includes a fermentation step of culturing Lactobacillus paracasei ET-22 in a liquid culture and stopping the fermentation when the Lactobacillus paracasei ET-22 grows to a logarithmic phase;

[0281] Preferably, the fermentation step comprises: inoculating Lactobacillus paracasei ET-22 fermentation seed liquid into a culture medium, and fermenting for 12-14 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.5-6.0 to obtain Lactobacillus paracasei ET-22 fermentation liquid;

[0282] Preferably, the preparation of the Lactobacillus paracasei ET-22 fermentation seed liquid comprises: activating the Lactobacillus paracasei ET-22 strain, purifying, culturing in a first-level seed liquid, multiplying in a second-level seed liquid, culturing in a third-level seed liquid, and preparing fermentation seeds to obtain the Lactobacillus paracasei ET-22 fermentation seed liquid;

[0283] Preferably, the culture medium is MRS liquid culture medium;

[0284] In an optional embodiment, the solvent is water; preferably, the solvent is sterile water, specifically sterile purified water;

[0285] Preferably, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

[0286] In an embodiment of the present invention, the bacteriocin obtained after sterilization is a liquid, wherein the mass ratio of citric acid to L-methionine is greater than 15:1, the L-methionine concentration is greater than 0.05 mg / ml, and the citric acid concentration is greater than 3 mg / ml. In some embodiments, the L-methionine concentration is 0.05-0.2 mg / ml and the citric acid concentration is 3-5 mg / ml.

[0287] Specifically, in the embodiments of the present invention, the separation of the bacterial sludge and the supernatant can be carried out by an existing separation method, for example, centrifugation is selected to achieve solid-liquid separation; in some embodiments, the concentration of L-methionine in the secretory agent is 0.05 mg / ml, 0.07 mg / ml, 0.09 mg / ml, 0.11 mg / ml, 0.13 mg / ml, 0.15 mg / ml, 0.17 mg / ml, 0.19 mg / ml, 0.2 mg / ml or any value between 0.05-0.2 mg / ml, or any value greater than 0.2 mg / ml; the concentration of citric acid is 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml or any value between 3-5 mg / ml, or any value greater than 5 mg / ml.

[0288] Preferably, the concentration of L-methionine in the bacteriocin is 0.05-0.1 mg / ml, and the concentration of citric acid is 3-4 mg / ml.

[0289] After the sterilization step, the bacteriocin is freeze-dried to obtain a freeze-dried bacteriocin, in which the solvent is removed, and the mass ratio of citric acid to L-methionine in the freeze-dried bacteriocin is greater than 15:1.

[0290] Specifically, in some embodiments, the secretin of Lactobacillus paracasei ET-22 is prepared by the following steps:

[0291] 1. Preparation of Tertiary Seeds

[0292] 1.1 Standard cryopreservation tubes

[0293] Prepare uniformly from the purified strains, divide into 1.5mL centrifuge tubes, no less than 50 tubes, and store in a -80℃ refrigerator with a shelf life of no more than 6 months.

[0294] 1.2 Activation of cryotubes

[0295] Take a portion of the bacterial strain stored at -80℃, thaw it at room temperature, aseptically take 200μL of bacterial solution and inoculate it into 10mL of seed liquid culture medium, and culture it at 37℃ for 8-10h.

[0296] 1.3 Primary purification

[0297] Take the cultured bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 18-20h.

[0298] 1.4 Secondary purification

[0299] Take the cultured purified bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked are uniform in size and cultured at 37°C for 18-20h.

[0300] 1.5 Primary seed preparation

[0301] Select one tube of secondary purified bacterial culture and pipette 200 μL of the bacterial culture into five tubes of 10 mL MRS liquid culture medium. Incubate at 37°C for 8-10 h.

[0302] 1.6 Secondary seed preparation

[0303] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 9-11 hours.

[0304] 1.7 Preparation of tertiary seeds

[0305] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 11-13 hours.

[0306] 1.8 Temporary storage of third-level seeds

[0307] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0308] 1.9 Process Quality Control

[0309] (1) Indicators for determining the end point of seed growth at each level: pH 3.8-4.2, OD600 ≥ 2.0.

[0310] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0311] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0312] 2. Fermentation Seed Preparation

[0313] 2.1 Vaccination

[0314] (1) Turn on the stirring paddle and temperature control program, the speed is 80 rpm, and the temperature is 36 °C.

[0315] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermentation tank to maintain positive pressure in the fermentation tank.

[0316] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0317] (4) Unscrew the top of the inoculator and pour the cultured seeds into the sterile area above the flame, with an inoculation rate of 2.5%.

[0318] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0319] (6) After inoculation, continue to introduce a small flow of nitrogen into the tank for 5-10 minutes.

[0320] (7) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0321] 2.2 Fermentation

[0322] (1) Set the fermentation parameters to 80 rpm and 36°C.

[0323] (2) The fermentation time was 11 h to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0324] (3) After fermentation is complete, the fermentation tank can be inoculated or cooled to 10-20°C for storage for no more than 6 hours.

[0325] 2.3 Process quality control

[0326] (1) Seed tank fermentation endpoint determination indicators: pH 3.8-4.2, OD600 ≥ 2.0.

[0327] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0328] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0329] 3. Fermentation Broth Culture

[0330] 3.1 Vaccination

[0331] (1) Turn on the stirring paddle and temperature control program, the speed is 80 rpm, and the temperature is 36 °C.

[0332] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0333] (3) Close the steam valve, open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation, with an inoculation amount of 2%.

[0334] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0335] 3.2 Fermentation

[0336] (1) Set the fermentation parameters to 30-40°C, 60-80 rpm, and a constant pH of 5.5-6.0.

[0337] (2) The fermentation time is 12-14 hours, and the fermentation status is monitored every 2 hours, including pH, OD600, temperature, and rotation speed.

[0338] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.

[0339] 3.3 Mushroom sludge separation

[0340] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0341] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0342] (3) Open the feed valve, and the Lactobacillus paracasei ET-22 fermentation liquid enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0343] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0344] 4. Bacteriocin Extraction

[0345] 4.1 Mud transfer

[0346] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0347] 4.2 Extraction conditions

[0348] The bacterial mud is extracted, and sterile purified water is mixed with the bacterial mud to obtain a mixed solution, wherein the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 1×1011-3×1011 cfu / mL, the extraction temperature is 0-37°C, the extraction time is 0.5-3h, and the extraction speed is 50-100 rpm.

[0349] It should be noted that in actual production process, the OD600 value of the lactobacillus paracasei ET-22 fermented liquid in the 3.2 step can be adjusted, the add-on of sterile purified water in the control extraction step is simultaneously controlled, so that the volume of the mixed liquor is 1% of the lactobacillus paracasei ET-22 fermented liquid volume, with the total colony count of lactobacillus paracasei ET-22 in the adjustment mixed liquor more easily.

[0350] 5. Centrifugal separation

[0351] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0352] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0353] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0354] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0355] (5) Transfer the centrifuged supernatant to a collection tank.

[0356] 6. Packaging and sterilization

[0357] The filled bottled supernatant was heat sterilized to obtain the bacteriocin. The sterilization conditions for each strain were as follows:

[0358] The sterilization conditions of Lactobacillus paracasei ET-22 are 70-121°C, 5-30min.

[0359] The embodiment of the present invention further provides a use of the secretin of Lactobacillus paracasei ET-22 according to any one of the aforementioned embodiments in preparing food, wherein the food comprises at least one of a dairy product and a beverage.

[0360] The embodiment of the present invention also provides a use of the secretin of Lactobacillus paracasei ET-22 according to any one of the aforementioned embodiments in preparing a composition, wherein the composition includes an antioxidant composition; preferably, the composition is selected from at least one of a medicine, a health food and a feed.

[0361] The Lactobacillus paracasei ET-22 secretin in the embodiment of the present invention has an antioxidant function and can be applied to food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the secretin in the embodiment of the present invention is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

[0362] Example 3

[0363] This embodiment provides a Lactobacillus paracasei ET-22 secretin, as shown in Figure 50, and its preparation method comprises the following steps:

[0364] 1. Preparation of Tertiary Seeds

[0365] 1.1 Standard cryopreservation tubes should be uniformly prepared from the purified strains and packaged into 1.5 mL centrifuge tubes, no less than 50 tubes, and stored in a -80°C refrigerator with a shelf life of no more than 6 months.

[0366] 1.2 Activation of cryopreserved tubes: Take a portion of the -80℃ stored bacterial strain, thaw it at room temperature, aseptically take 200μL of bacterial solution and inoculate it into 10mL of seed liquid culture medium, and incubate it at 37℃ for 8-10h.

[0367] 1.3 Primary purification: Take the cultured bacterial solution and dilute it for spreading. The dilution degree is -4, -5, and -6. Prepare two MRS solid plates for each dilution degree. Incubate them upside down at 37℃ for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked are of uniform size and incubated at 37℃ for 18-20h.

[0368] 1.4 Secondary Purification: Dilute and spread the cultured primary purified bacterial solution at dilutions of -4, -5, and -6. Prepare two MRS solid plates for each dilution. Incubate them upside down at 37°C for 48-72 hours until distinct colonies form on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10 mL MRS liquid culture medium. Ensure that the colonies are uniform in size and incubate them at 37°C for 18-20 hours.

[0369] 1.5 Preparation of primary seeds: Select one tube of secondary purified bacterial culture and pipette 200 μL of the bacterial culture into five tubes of 10 mL MRS liquid culture medium. Incubate at 37°C for 8-10 h.

[0370] 1.6 Preparation of secondary seeds: Select 4 tubes of cultured primary seeds, aspirate 4 mL of each tube and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 9-11 hours.

[0371] 1.7 Prepare the cultured secondary seeds, pour them into two bottles of 1.8LMRS liquid culture medium, and culture them at 37℃ for 11-13 hours.

[0372] 1.8 Temporary Storage of Grade 3 Seeds After preparation, grade 3 seeds can be stored at 4°C for no more than 6 hours.

[0373] 1.9 Process Quality Control

[0374] (1) Indicators for determining the end point of seed growth at each level: pH 3.8-4.2, OD600 ≥ 2.0.

[0375] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0376] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0377] Formula of three-stage seed fermentation medium

[0378] pH value: 6.2~6.4; sterilization conditions: 121℃, 15~20min.

[0379] 2. Fermentation Seed Preparation

[0380] 2.1 Vaccination

[0381] (1) Turn on the stirring paddle and temperature control program, the speed is 80 rpm, and the temperature is 36 °C.

[0382] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermentation tank to maintain positive pressure in the fermentation tank.

[0383] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0384] (4) Unscrew the top of the inoculator and pour the cultured seeds into the sterile area above the flame, with an inoculation rate of 2.5%.

[0385] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0386] (6) After inoculation, continue to introduce a small flow of nitrogen into the tank for 5-10 minutes.

[0387] (7) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0388] 2.2 Fermentation

[0389] (1) Set the fermentation parameters to 80 rpm and 36°C.

[0390] (2) The fermentation time was 11 h to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0391] (3) After fermentation is complete, the fermentation tank can be inoculated or cooled to 10-20°C for storage for no more than 6 hours.

[0392] 2.3 Process quality control

[0393] (1) Seed tank fermentation endpoint determination indicators: pH 3.8-4.2, OD600 ≥ 2.0.

[0394] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0395] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0396] Seed tank fermentation medium formula

[0397] pH value: 6.2~6.4; sterilization conditions: 121℃, 15~20min.

[0398] 3. Fermentation Broth Culture

[0399] 3.1 Vaccination

[0400] (1) Turn on the stirring paddle and temperature control program, the speed is 80 rpm, and the temperature is 36 °C.

[0401] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0402] (3) Close the steam valve, open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation, with an inoculation amount of 2%.

[0403] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0404] 3.2 Fermentation

[0405] (1) Set the fermentation parameters as follows: temperature 36°C, rotation speed 80 rpm, and constant pH 5.75.

[0406] (2) The fermentation time is 12-14 hours, and the fermentation status is monitored every 2 hours, including pH, OD600, temperature, and rotation speed.

[0407] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.

[0408] Fermentation medium formula for fermentation tank

[0409] pH value: 6.2~6.4; sterilization conditions: 121℃, 15~20min.

[0410] 3.3 Mushroom sludge separation

[0411] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0412] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0413] (3) Open the feed valve, and the Lactobacillus paracasei ET-22 fermentation liquid enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0414] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0415] 4. Bacteriocin Extraction

[0416] 4.1 Mud transfer

[0417] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0418] 4.2 Extraction conditions

[0419] The bacterial sludge was extracted and resuspended with sterile purified water to obtain a mixed solution. The total colony counts of Lactobacillus paracasei ET-22 in the mixed solution were 1×10 11 cfu / mL, 1.5×10 11 cfu / mL, 3×10 11 cfu / mL, the extraction temperatures were 4°C, 25°C, and 37°C, the extraction times were 1h, 2h, and 3h, and the extraction speed was 70rpm.

[0420] 5. Centrifugal separation

[0421] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0422] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0423] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0424] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0425] (5) Transfer the centrifuged supernatant to a collection tank.

[0426] 6. Packaging and sterilization

[0427] The filled bottled supernatant was heat sterilized to obtain the secretory agent. The sterilization conditions of Lactobacillus paracasei ET-22 were 75° C. for 10 min. The obtained secretory agent of Lactobacillus paracasei ET-22 was shown in FIG51 .

[0428] 7. Quantitative Detection

[0429] The concentrations of L-methionine and citric acid in the product obtained in Example 3 were detected, and the detection method comprised the following steps:

[0430] (1) Preparation of standard working solution

[0431] Standard stock solution: Accurately weigh appropriate amounts of standard substances (i.e., L-methionine and citric acid, accurate to 0.1 mg), dissolve in water and prepare standard stock solutions with a concentration of 5 mg / mL, and store at -20°C.

[0432] Mixed standard intermediate solution: Accurately pipette appropriate volumes of standard stock solutions, dilute to volume with water, prepare a mixed standard intermediate solution with a concentration of 500 μg / mL, and store at 4°C.

[0433] Mixed standard working solution: dilute the mixed standard intermediate solution step by step with water as needed to prepare mixed standard working solutions with concentrations of 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively. Prepare them before use.

[0434] (2) Preparation of elution solution

[0435] 0.1% phosphoric acid aqueous solution: Take 1 mL of phosphoric acid, dilute it with water and make up to 1000 mL, mix well, and use it immediately.

[0436] 0.1% phosphoric acid acetonitrile solution: Take 1 mL of phosphoric acid, dilute with acetonitrile and make up to 1000 mL, mix well, and use immediately.

[0437] (3) Preparation of test solution

[0438] Liquid sample: Mix the secretory sample of Lactobacillus paracasei ET-22 and directly pipette 1 mL of it. Centrifuge at 10,000 rpm for 10 min at 4°C. Dilute the supernatant to within the linear range and load it onto the column for analysis.

[0439] (4) Detection and analysis

[0440] The sample solution and the standard working solution were tested using high performance liquid chromatography, and the corresponding chromatogram peak areas were measured. A standard curve was plotted with the concentration of the standard working solution as the horizontal axis and the chromatogram peak area as the vertical axis. Based on the test results of the sample solution in step (3) (as shown in Figure 52, where 1 is L-methionine and 2 is citric acid), the concentrations of L-methionine and citric acid in the bacteriocin extraction step were calculated in combination with the standard curve. Figure 52 is a liquid chromatogram of the bacteriocin sample obtained in Example 3.

[0441] Chromatographic analysis conditions: An LC-20A analytical system was used, using a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A was 0.1% phosphoric acid in water; mobile phase B was 0.1% phosphoric acid in acetonitrile; the gradient elution program is shown in Table 5; the flow rate was 0.7 mL / min; the detection wavelength was 210 nm; the column temperature was 30°C; and the injection volume was 5 μL.

[0442] Table 5 Gradient elution program

[0443] The total number of Lactobacillus paracasei ET-22 colonies in the mixed solution was calculated to be 3×10 11 cfu / mL, the extraction temperature was 25℃, and the extraction time was 1h. The L-methionine concentration in the bacteriocin was 0.09mg / 100ml and the citric acid concentration was 3.68mg / 100ml.

[0444] (5) Calculate the number of colonies before inactivation of inactivated bacteria

[0445] Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

[0446] When citric acid was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 2.068x + 10.874; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0447] When L-methionine was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 74.439x-1.822; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0448] Comparative Example 1

[0449] This comparative example provides a fermented inactivated strain of Lactobacillus paracasei ET-22, the preparation method of which comprises the following steps: 1. Preparation of tertiary seeds, same as in Example 3. 2. Preparation of fermentation seeds, same as in Example 3. 3. Fermentation broth culture, same as in Example 3.

[0450] 4. Bacteria inactivation

[0451] Sterile purified water was mixed with the bacterial sludge and resuspended to obtain a mixed solution (the concentration was the same as in Example 3), and the mixed solution was heat sterilized at temperatures of 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min.

[0452] Test Example 1: 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging

[0453] 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of mixed solution (10 9 cfu / mL) as raw material for the bacteriocin or inactivated bacteria and incubate at 25°C in the dark for 30 minutes. The control group was replaced with an equal volume of PBS (pH 7.4), while the blank group was replaced with an equal volume of PBS (pH 7.4) instead of the DPPH free radical solution. After centrifugation at 2,000 × g for 10 minutes, the absorbance of the solution was measured at 517 nm. The calculation formula is as follows:

[0454] Test Example 2: Hydroxyl Radical Scavenging

[0455] A total of 1.0 mL of the mixture (10 9 cfu / mL) as the starting material for the bacteriocin or inactivated bacteria. After adding 20 mM H₂O₂ (1.0 mL), the mixture was incubated in a 37°C water bath for 90 min. The absorbance was measured at 536 nm. The hydroxyl radical scavenging activity was calculated as follows:

[0456] In the embodiment of the present invention, the components of the inactivated bacteria obtained in Comparative Example 1 were detected by LC-MS, and the peak areas greater than 10 4 The components of the bacteriocin were screened to obtain targets that characterize the inactivated bacteria components. The target screening criteria include: ① the component does not exist before heat sterilization, or the content of the component before heat sterilization is much lower than the content of the component after heat sterilization; ② within the experimental range, it can stably exist under different heat sterilization conditions (content change range ≤ 20%). It was found that 6 substances can be used as potential detection targets, namely: Proly-Alanine, L-Methionine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic peptide, GRPPK. At the same time, the 6 detection targets in the bacteriocin were detected, and it was found that only citric acid and L-methionine were present in the bacteriocin. The other four detection targets were not extracted or the content was below the detection line. Therefore, in the embodiment of the present invention, citric acid and L-methionine were used as detection targets to characterize the bacteriocin.

[0457] The test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 3 are shown in FIG53 . As can be seen from FIG53 , when the extraction concentration is 3×10 11cfu / mL, the extraction temperature was 25°C, and the extraction time was 1h, the hydroxyl radical scavenging ability of the bacteriocin was better; the test results of the DPPH radical scavenging ability of the bacteriocin obtained in Example 3 are shown in Figure 54. In Figure 54, the extraction temperature and time of K1-K9 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h and 37°C, 3h, respectively. The extraction temperatures and times for K10-K18 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively; and the extraction temperatures and times for K19-K27 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively. As shown in Figure 54, the extraction conditions had relatively little effect on DPPH radicals.

[0458] The hydroxyl radical scavenging ability and DPPH radical scavenging ability of the postbiotics obtained in Comparative Example 1 are shown in Figure 55. The DPPH radical scavenging ability of the bacteriocin does not change much with temperature at first, but decreases significantly under high temperature conditions of 121°C.

[0459] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Lactobacillus paracasei ET-22 source cell lysate in freshening breath and / or alleviating and inhibiting halitosis, characterized in that: The lysate is derived from heat-killed bacteria of Lactobacillus paracasei ET-22, and the lysate includes at least 2-hydroxy-3-phenylpropionic acid, NAD and a polypeptide, wherein the polypeptide has a structure shown in Formula 1; 2. The use according to claim 1, characterized in that: Based on the total volume of the lysate, the mass of the 2-hydroxy-3-phenylpropionic acid is not less than 145 mg / L, the mass of the NAD is not less than 0.17 mg / L, and the mass of the polypeptide is not less than 0.24 mg / L.

3. The use according to claim 1 or 2, characterized in that: The cell lysate also includes one or more of 6-hydroxyhexanoic acid, 3-aminoglutaric acid, 2-hydroxy-3-phenylpropionic acid, 2-hydroxy-3-(4-hydroxyphenyl)propionic acid, and Asp-Phe.

4. The use according to any one of claims 1 to 3, characterized in that: The method for preparing the cell lysate comprises the following steps: The Lactobacillus paracasei ET-22 was cultured in liquid, and when it grew to the logarithmic phase, the fermentation was stopped and the bacteria were collected; The collected cells were resuspended in sterile water, and after heat inactivation, the supernatant was collected to obtain the cell lysate.

5. The use according to claim 4, characterized in that: The temperature of the heat inactivation is 95-150° C. and the time is 1-15 min.

6. A peptide having breath freshening and / or halitosis relieving and inhibiting effects, characterized in that: The peptide has a structure as shown in Formula 1:

7. The peptide according to claim 6, characterized in that The peptides are derived from postbiotics of Lactobacillus paracasei ET-22.

8. A composition for freshening breath and / or alleviating and inhibiting bad breath, characterized in that: The invention comprises a lysate; the lysate is derived from heat-killed bacteria of Lactobacillus paracasei ET-22, and the lysate at least comprises 2-hydroxy-3-phenylpropionic acid, NAD and a polypeptide, and the polypeptide has a structure shown in Formula 1; or, including nicotinamide adenine dinucleotide; or, including 2-hydroxy-3-phenylpropionic acid; or, comprising the peptide of claim 6 or 7; Or, it comprises peptidoglycan, wherein the peptidoglycan is derived from the heat-killed bacteria of Lactobacillus paracasei ET-22.

9. The composition according to claim 8, characterized in that The composition is one or more of a food composition, a medical composition, and an oral cleaning composition.

10. The composition according to claim 9, characterized in that The food composition comprises one or more of dairy drinks, tea, coffee, chewing gum, tooth-cleaning candy, compressed candy, and dried meat for pets.

11. The composition according to claim 9, characterized in that The oral cleaning composition includes one or more of toothpaste, tooth cleaning powder, mouthwash, breath freshening spray, fluoride coating agent, denture cleaner, toothbrush, interdental brush, dental floss, oral cotton swab, pet tooth cleaning gel, pet hair removal paste, and pet tooth cleaning bone.

12. The composition according to claim 8, characterized in that In the composition, the content of nicotinamide adenine dinucleotide is not less than 100 μg / mL; Or, the content of 2-hydroxy-3-phenylpropionic acid is not less than 100 μg / mL; Or, the content of the peptide according to claim 6 or 7 is not less than 100 μg / ml.

13. The composition according to any one of claims 8 to 12, characterized in that The composition further comprises one or more of an excipient, a diluent, a carrier, and an additive.

14. Use of nicotinamide adenine dinucleotide in freshening breath and / or alleviating and inhibiting bad breath.

15. The use according to claim 14, characterized in that: The nicotinamide adenine dinucleotide is derived from the postbiotics of Lactobacillus paracasei ET-22.

16. Application of nicotinamide adenine dinucleotide in reducing the content of volatile sulfides in oral breath and volatile compounds in saliva.

17. Application of nicotinamide adenine dinucleotide in reducing the content of characteristic flavor compounds.

18. Use of 2-hydroxy-3-phenylpropionic acid in freshening breath and / or alleviating and inhibiting oral malodor.

19. The use according to claim 18, characterized in that The 2-hydroxy-3-phenylpropionic acid is derived from the postbiotics of Lactobacillus paracasei ET-22.

20. Application of 2-hydroxy-3-phenylpropionic acid in reducing the content of characteristic flavor compounds in the oral cavity.

21. Use of 2-hydroxy-3-phenylpropionic acid in reducing the amount and thickness of oral biofilm.

22. Use of the peptide according to claim 6 or 7 in freshening breath and / or alleviating and inhibiting bad breath.

23. Use of the peptide according to claim 6 or 7 in reducing the content of volatile organic compounds in the oral cavity and / or inhibiting the growth of microorganisms in the oral cavity.

24. Use of peptidoglycan in freshening breath and / or alleviating and inhibiting halitosis, characterized in that: The peptidoglycan is derived from the heat-killed bacteria of Lactobacillus paracasei ET-22.

25. The use according to claim 24, characterized in that The extraction method of peptidoglycan comprises the steps: Step 1, culturing Lactobacillus paracasei ET-22 in liquid, and when the Lactobacillus paracasei ET-22 grows to the logarithmic phase, stopping the fermentation and collecting the cells; Step 2, resuspending the collected bacterial cells in sterile water, performing heat inactivation, and collecting the heat-inactivated bacterial cells; Step 3: resuspend the heat-killed bacterial cells in PBS buffer, disrupt the heat-killed bacterial cells by ultrasonication, collect the precipitate by centrifugation, and extract the peptidoglycan from the precipitate.

26. The use according to claim 25, characterized in that In step 2, the collected bacteria were resuspended in sterile water to prepare a viable bacterial concentration of 1.0×10 9 CFU / ml of bacterial solution.

27. The use according to claim 25, characterized in that In step 2, the temperature of heat inactivation is 90-150° C. and the time is 5-15 min.

28. The use according to claim 25, characterized in that In step 3, peptidoglycan is isolated from the precipitate, specifically comprising: The heat-killed bacterial precipitate is resuspended in PBS buffer, ultrasonically disrupted, and then centrifuged to obtain the precipitate; the precipitate is boiled in a 4% SDS solution for 30 minutes, and then 10% TCA is added to remove the phosphate wall and protein in the heat-killed bacteria; a mixed solvent including sodium acetate, chloroform and methanol is added for degreasing; then alkaline protease and trypsin complex enzyme solution is used to treat at 37°C for 12 hours, and then the SDS is washed to trace amounts after boiling with a 1% SDS solution for 10 minutes; then dialyzed for 2 days, the water is changed every 8 hours, the precipitate is collected and vacuum freeze-dried to obtain peptidoglycan.

29. A secretin of Lactobacillus paracasei ET-22, characterized in that The invention comprises exocytosis of Lactobacillus paracasei ET-22 and metabolites of Lactobacillus paracasei ET-22. The mass ratio of citric acid to L-methionine in the exocytosis is greater than 15:

1. The preservation number of the Lactobacillus paracasei ET-22 is CGMCC No.15077.

30. The secretin of Lactobacillus paracasei ET-22 according to claim 29, characterized in that The mass ratio of citric acid to L-methionine in the bacteriocin is 15-100:1; Preferably, the mass ratio of citric acid to L-methionine in the bacteriocin is 35-45:

1.

31. A method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 29 or 30, characterized in that: The following steps are involved: Extracting the secretory factor, using a solvent to extract the bacterial mud separated from the fermentation liquid of Lactobacillus paracasei ET-22 to obtain a mixed liquid; Sterilize, separate the supernatant in the mixed solution and sterilize it by heat to obtain the bacteriocin.

32. The method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 31, characterized in that: The extraction temperature is 0-37°C; preferably, the extraction temperature is 20-30°C; And / or, the extraction time is 0.5-3h; preferably, the extraction time is 30-120min.

33. The method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 31, characterized in that: The total colony count of Lactobacillus paracasei ET-22 in the mixed solution was 0.5×10 11 -5×10 11 cfu / mL; preferably, the total colony count of Lactobacillus paracasei ET-22 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

34. The method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 31, characterized in that: The temperature of heat sterilization is 70-121°C; preferably, the temperature of heat sterilization is 70-100°C.

35. The method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 31, characterized in that: The heat sterilization time is 5-30 min; preferably, the heat sterilization time is 10-16 min.

36. The method for preparing the secretin of Lactobacillus paracasei ET-22 according to claim 31, characterized in that: The method also includes a fermentation step of: culturing Lactobacillus paracasei ET-22 in a liquid medium, and stopping the fermentation when the Lactobacillus paracasei ET-22 grows to a logarithmic phase; Preferably, the fermentation step comprises: inoculating Lactobacillus paracasei ET-22 fermentation seed liquid into a culture medium, and fermenting for 12-14 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.5-6.0 to obtain Lactobacillus paracasei ET-22 fermentation liquid; Preferably, the preparation of the fermentation seed liquid of Lactobacillus paracasei ET-22 comprises: activating, purifying, culturing the Lactobacillus paracasei ET-22 strain with a primary seed liquid, multiplying with a secondary seed liquid, culturing with a tertiary seed liquid, and preparing fermentation seeds to obtain the fermentation seed liquid of Lactobacillus paracasei ET-22; Preferably, the culture medium is MRS liquid culture medium; Preferably, the solvent is water; preferably, the solvent is sterile water; Preferably, after the sterilization step, the bacteriocin is further freeze-dried to obtain freeze-dried bacteriocin.

37. Use of the secretin of Lactobacillus paracasei ET-22 according to claim 29 or 30 in preparing food, characterized in that: The food comprises at least one of dairy products and beverages.

38. Use of the secretin of Lactobacillus paracasei ET-22 according to claim 29 or 30 in preparing a composition, characterized in that: The composition comprises an antioxidant composition; preferably, the composition is selected from at least one of medicines, health foods and feeds.

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