Preparation method, product and application of a postbiotic composition

Abstract

The present invention provides a method for preparing a postbiotic composition, its product and application, belonging to the technical field of biomedicine. The method for preparing the postbiotic composition provided by the present invention inoculates the activated Lactiplantibacillus plantarum YBR-01 and Lactobacillus crispatus YBR-01 into a culture medium in sequence, and performs fermentation culture under the action of β-galactosidase, and the postbiotic composition is prepared by heat inactivation. The postbiotic composition prepared by this method can improve vaginitis by regulating the vaginal microecological balance, antioxidant, anti-inflammatory, enhancing metabolism and improving immunity. At the same time, the postbiotic composition can also overcome antibiotic resistance and has good safety, providing a new idea for preparing products for improving vaginitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method, product and application of a postbiotic composition. Background Art

[0002] Vaginal flora disorder refers to an abnormal increase in microorganisms such as Escherichia coli, Gardnerella vaginalis, Staphylococcus aureus, Peptostreptococcus, Porphyromonas, Mobiluncus, Mycoplasma hominis and Candida albicans that normally exist in the vagina, while the lactobacilli that produce hydrogen peroxide decrease or disappear, causing a group of clinical syndromes mainly manifested by abnormal increase in vaginal secretions.

[0003] The treatment methods generally choose anti-anaerobic drugs such as metronidazole, tinidazole and clindamycin. However, problems such as antibiotic resistance, side effects brought by antibiotics, easy recurrence and superinfection have limited the treatment effect. At present, vaginal lactobacillus capsules have appeared clinically to improve vaginal flora imbalance, but due to the unclear specific mechanism and safety issues, their clinical application is restricted. Therefore, exploring the specific mechanism of lactobacilli in improving vaginal inflammation and finding new methods that can not only improve vaginal inflammation symptoms but also overcome antibiotic resistance and improve safety performance are scientific problems that need to be solved urgently at present.

[0004] Postbiotics is a brand-new field, which can be compared to the "fragment" components of probiotics and their metabolites, such as cell surface components, lactic acid, bioactive peptides, etc. At present, existing studies have shown that postbiotics have various effects on the intestine, such as reducing inflammation, promoting the passage of waste through the digestive tract, enhancing metabolic function, increasing calcium absorption, enhancing immune function, and preventing type II diabetes. However, there are few studies on the action of postbiotics on the vagina, and it overcomes the disadvantages of low safety of live probiotics, the need for bacterial growth or colonization, population limitations, short and unstable shelf life. Therefore, in this study, postbiotics are used as "advanced substitutes" for probiotics and administered vaginally for the treatment of vaginitis caused by vaginal flora disorder.

[0005] The normal vaginal microbiota contains Lactiplantibacillus plantarum and Lactobacillus crispatus, which are of great significance for improving vaginitis and restoring vaginal microecological balance. Therefore, developing a preparation method and product of a postbiotic composition of Lactiplantibacillus plantarum and Lactobacillus crispatus, and studying its effect and specific mechanism on vaginitis caused by vaginal flora disorder can provide new treatment ideas for improving vaginal inflammation. Summary of the Invention

[0006] Based on the problems and deficiencies existing in the prior art, the present invention aims to provide a preparation method, product and application of a postbiotic composition. The preparation method of the postbiotic composition provided by the present invention sequentially inoculates activated Lactiplantibacillus plantarum YBR-01 and Lactobacillus crispatus YBR-01 into a culture medium, performs fermentation culture under the action of β-galactosidase, and prepares a postbiotic composition through heat inactivation. The postbiotic composition prepared by this method can improve vaginitis by regulating the vaginal microecological balance, antioxidation, anti-inflammation, enhancing metabolism and improving immunity. At the same time, the postbiotic composition can also overcome antibiotic resistance and has good safety, providing a new idea for the preparation of products for improving vaginitis.

[0007] The technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a preparation method of a postbiotic composition, and the preparation method includes the following steps:

[0009] S1. Activate Lactiplantibacillus plantarum and Lactobacillus crispatus respectively;

[0010] S2. Inoculate the activated Lactiplantibacillus plantarum into a culture medium according to an inoculation amount of 2%-3% v / v, perform fermentation culture to obtain a fermentation culture solution;

[0011] S3. Add the activated Lactobacillus crispatus to the fermentation culture solution and inoculate it into the culture medium according to an inoculation amount of 3.5%-4% v / v, add β-galactosidase, and continue fermentation culture to obtain a fermentation broth;

[0012] S4. Resuspend the fermentation broth and inactivate it to obtain a postbiotic composition;

[0013] The Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) is Lactiplantibacillus plantarum YBR-01, and the preservation number is CGMCC No. 26679; the Lactobacillus crispatus ( Lactobacillus crispatus ) is Lactobacillus crispatus YBR-01, and the preservation number is CGMCC No. 26680.

[0014] Specifically, the culture medium described in steps S2-S3 includes one or more of MRS culture medium, TPY culture medium, MC culture medium, and BCP culture medium.

[0015] Preferably, the culture medium described in steps S2-S3 is MRS culture medium.

[0016] Specifically, the fermentation culture described in step S2 is carried out at 35-39 °C for 4-8 h.

[0017] Preferably, the fermentation culture described in step S2 is carried out at 37 °C for 6 h.

[0018] Specifically, the fermentation culture described in step S3 is carried out at 35 - 39°C for 8 - 12 h.

[0019] Preferably, the fermentation culture described in step S3 is carried out at 37°C for 10 h.

[0020] Specifically, the ratio of the inoculation amounts of Lactiplantibacillus plantarum and Lactobacillus crispatus is 1:1.15 - 2.

[0021] Preferably, the ratio of the inoculation amounts of Lactiplantibacillus plantarum and Lactobacillus crispatus is 1.75.

[0022] Preferably, the inoculation amount of Lactiplantibacillus plantarum is 2% v / v.

[0023] Preferably, the inoculation amount of Lactobacillus crispatus is 3.5% v / v.

[0024] Specifically, the liquid used for resuspension in step S4 includes one or more of sterile injection water, sterile normal saline, and sterile PBS.

[0025] Preferably, the liquid used for resuspension in step S4 is sterile PBS.

[0026] Specifically, the inactivation conditions in step S4 are heating at 80 - 120°C for 10 - 20 min.

[0027] Preferably, the inactivation conditions in step S4 are heating at 80°C for 20 min.

[0028] In a second aspect, the present invention provides a postbiotic composition prepared by the above preparation method.

[0029] In a third aspect, the present invention provides the application of the above postbiotic composition in the preparation of a product, and the product includes any one of the following products:

[0030] (1) Preparing a drug for preventing, treating, and / or adjuvantly treating vaginitis; or

[0031] (2) Female personal care products for improving vaginitis; or

[0032] (3) Health products for anti - inflammation, antioxidant, immune enhancement, and / or regulating the balance of flora.

[0033] Specifically, the product contains ≥1×10 8 CFU / g of the postbiotic composition.

[0034] Preferably, the product contains 1×10 8 -1×10 12 CFU / g of the postbiotic composition.

[0035] In a fourth aspect, the present invention provides a drug for preventing, treating, and / or adjuvantly treating vaginitis, and the drug comprises the above-mentioned postbiota composition.

[0036] Specifically, the drug further comprises pharmaceutically acceptable excipients.

[0037] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizing agents, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

[0038] Specifically, the dosage form of the drug includes, but is not limited to, enteral dosage forms and parenteral dosage forms according to the administration route.

[0039] Preferably, the enteral dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0040] Preferably, the parenteral dosage forms include, but are not limited to, injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0041] In a fifth aspect, the present invention provides a female personal care product for improving vaginitis, and the female personal care product comprises the above-mentioned postbiota composition.

[0042] Specifically, the female personal care product includes, but is not limited to, cleaning products, nursing lotions, nursing gels, sanitary napkins, tampons, or skin care products.

[0043] In a sixth aspect, the present invention provides a health product for anti-inflammation, antioxidant, immune enhancement, and / or microbiota balance regulation, and the health product comprises the above-mentioned postbiota composition.

[0044] Specifically, the health product further comprises nutritionally acceptable nutritional additives.

[0045] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins.

[0046] Specifically, the dosage form of the health product includes tablets, capsules, soft capsules, granules, pills, gummy candies, powders, oral liquids, or drops.

[0047] The beneficial effects of the present invention are as follows:

[0048] The present invention provides a preparation method, product and application of a postbiotic composition, belonging to the technical field of biomedicine. In the preparation method of the postbiotic composition provided by the present invention, activated Lactiplantibacillus plantarum YBR-01 and Lactobacillus crispatus YBR-01 are sequentially inoculated into a culture medium and fermented under the action of β-galactosidase, and the postbiotic composition is prepared by heat inactivation. The postbiotic composition prepared by this method can improve vaginitis by regulating the vaginal microecological balance, antioxidant, anti-inflammatory, enhancing metabolism and improving immunity. At the same time, the postbiotic composition can also overcome antibiotic resistance and has good safety, providing a new idea for the preparation of products for improving vaginitis.

[0049] Deposit description

[0050] Biomaterial name: Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) YBR-01;

[0051] Taxonomic nomenclature: Lactiplantibacillus plantarum Lactiplantibacillus plantarum ;

[0052] Deposit time: February 27, 2023;

[0053] Deposit number: CGMCC No. 26679;

[0054] Depositary institution: China General Microbiological Culture Collection Center;

[0055] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] Deposit description

[0057] Biomaterial name: Lactobacillus crispatus ( Lactobacillus crispatus ) YBR-01;

[0058] Taxonomic nomenclature: Lactobacillus crispatus Lactobacillus crispatus ;

[0059] Deposit time: February 27, 2023;

[0060] Deposit number: CGMCC No. 26680;

[0061] Depositary institution: China General Microbiological Culture Collection Center;

[0062] Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0063] Figure 1Growth curve and microscopic morphology of Lactiplantibacillus plantarum - YBR - 01; A in the figure is the growth curve of Lactiplantibacillus plantarum - YBR - 01; B is the microscopic morphology of Lactiplantibacillus plantarum - YBR - 01.

[0064] Figure 2 Growth curve and microscopic morphology of Lactiplantibacillus crispatus - YBR - 01; A in the figure is the growth curve of Lactiplantibacillus crispatus - YBR - 01; B is the microscopic morphology of Lactiplantibacillus crispatus - YBR - 01.

[0065] Figure 3 Results of acid tolerance and bile salt tolerance determination of Lactiplantibacillus plantarum - YBR - 01; A in the figure is the result of acid tolerance determination; B is the result of bile salt tolerance determination.

[0066] Figure 4 Results of acid tolerance and bile salt tolerance determination of Lactiplantibacillus crispatus - YBR - 01; A in the figure is the result of acid tolerance determination; B is the result of bile salt tolerance determination.

[0067] Figure 5 Hemolysis test of Lactiplantibacillus plantarum - YBR - 01; on the left side in the figure represents Staphylococcus aureus - positive control; on the right side represents Lactiplantibacillus plantarum - YBR - 01.

[0068] Figure 6 Hemolysis test of Lactiplantibacillus crispatus - YBR - 01; on the left side in the figure represents Staphylococcus aureus - positive control; on the right side represents Lactiplantibacillus crispatus - YBR - 01.

[0069] Figure 7 Drug resistance determination of Lactiplantibacillus plantarum - YBR - 01; in the figure, CTR represents ceftriaxone; CIP represents ciprofloxacin hydrochloride; PEN represents penicillin; AMP represents ampicillin; GEN represents gentamicin; C represents chloramphenicol; TET represents tetracycline; E represents polymyxin; MY represents midecamycin; SXT represents co - trimoxazole.

[0070] Figure 8 Drug resistance determination of Lactiplantibacillus crispatus - YBR - 01; in the figure, CTR represents ceftriaxone; CIP represents ciprofloxacin hydrochloride; PEN represents penicillin; AMP represents ampicillin; GEN represents gentamicin; C represents chloramphenicol; TET represents tetracycline; E represents polymyxin; MY represents midecamycin; SXT represents co - trimoxazole.

[0071] Figure 9 Flow chart of animal experiment.

[0072] Figure 10They were the relative expression levels of MPO activity, IL-1β, and IL-10; A in the figure was the MPO activity; B was the relative expression level of IL-1β; C was the relative expression level of IL-10; the data were expressed as mean ± standard deviation; * represented P < 0.05; ** represented P < 0.01; *** represented P < 0.001. Detailed implementation manners

[0073] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are generally carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0074] Example 1 Preparation of MRS medium

[0075] MRS medium: The main components were 10.0 g of peptone, 5.0 g of beef powder, 4.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of ammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15.0 g of agar powder, and 1000 mL of deionized water; the above components were weighed in sequence and added to deionized water, and sterilized at 121 °C under high temperature and high pressure for 15 min - 20 min, which was mainly used for the cultivation of lactic acid bacteria.

[0076] Example 2 Microscopic morphology and growth curve of Lactiplantibacillus plantarum - YBR - 01

[0077] 1. Observation of microscopic morphology: 1. Slide preparation: Take 200 μL of the bacterial suspension (1×10 8 CFU / mL) and spread it evenly on a glass slide, then fix it with the flame of an alcohol lamp. In this process, the coated surface should be upward and passed through the flame several times. When touching the slide with the hand, it can be felt slightly hot, preventing overheating, as overheating will cause the bacteria to deform or affect the staining reaction; 2. Staining: Drop ammonium oxalate crystal violet for staining for 1 min, wash with water, and air dry naturally; 3. Observation: Place the glass slide under an optical microscope, first find the field of view under the low-power microscope, and then observe the bacterial morphology under the high-power microscope. It can also be observed under the oil immersion lens by dropping cedarwood oil.

[0078] 2. Determination of growth curve: Inoculate 200 μL of Lactiplantibacillus plantarum - YBR - 01 (1×10 8 CFU / mL) into 10 mL of MRS medium and culture it in a shaker at 37 °C. Measure the optical density (OD) at a wavelength of 600 nm every 2 hours for 24 hours. Conduct three repeated experiments.

[0079] First, the optical density of the bacterial suspension of Lactiplantibacillus plantarum - YBR - 01 was detected for 24 hours. The results showed that the strain entered the logarithmic growth phase around 2 hours and entered the stationary growth phase after 8 hours ( Figure 1 A in Figure 1 ). Under a high - power optical microscope, the morphology of Lactiplantibacillus plantarum - YBR - 01 was straight or curved rod - shaped, existing singly, sometimes in pairs or in chains (

[0080] Example 3 Microscopic Morphology and Growth Curve of Lactobacillus crispatus - YBR - 01

[0081] 1. Microscopic Morphology Observation: 1) Slide Preparation: Take 200 μL of the bacterial suspension (1×10 7 CFU / mL) and spread it evenly on a glass slide. Then fix it with the flame of an alcohol lamp. During this process, keep the smear facing up and pass it through the flame several times. When you touch the slide with your hand and feel slightly hot, it's okay. Prevent overheating as overheating will cause the bacteria to deform or affect the staining reaction; 2) Staining: Drop ammonium oxalate crystal violet for staining for 1 minute, wash with water, and air - dry naturally; 3) Observation: Place the glass slide under an optical microscope. First, find the field of view under the low - power lens, and then observe the bacterial morphology under the high - power lens. You can also observe under the oil - immersion lens by dropping cedarwood oil.

[0082] 2. Growth Curve Determination: Inoculate 200 μL of Lactobacillus crispatus - YBR - 01 (1×10 7 CFU / mL) into 10 mL of MRS medium and culture it in a shaker at 37°C. Measure the optical density (OD) at a wavelength of 600 nm every 2 hours for 24 hours. Conduct three repeated experiments.

[0083] First, the optical density of the bacterial suspension of the strain was detected for 24 hours. The results showed that the strain entered the logarithmic growth phase around 6 hours and gradually entered the stationary growth phase after 14 hours ( Figure 2 A in Figure 2 ). Under a high - power optical microscope, L. crispatus was a slender, curved, and delicate bacillus (

[0084] Example 4 Acid and Bile Salt Tolerance Experiments

[0085] 1. Acid Tolerance Experiment

[0086] (1) Culture the preserved Lactobacillus crispatus - YBR - 01 or Lactiplantibacillus plantarum - YBR - 01 in an MRS medium in a 37°C constant - temperature incubator for 24 - 36 hours, and reserve the strain for use;

[0087] (2) Prepare PBS buffers with pH values of 2, 3, 4, 5, and 7. Take 1 mL of the bacterial solution that has been cultured for 24 - 36 h, centrifuge it at 3000 rpm for 10 min, discard the supernatant, wash it twice with PBS, resuspend it with 1 mL of PBS with different pH values, place it in a constant temperature incubator at 37 °C for 4 h, then dilute and spread it on plates for viable count. Each pH PBS buffer has three replicates.

[0088] 2. Bile salt tolerance test

[0089] (1) Culture the preserved Lactiplantibacillus plantarum - YBR - 01 or Lactobacillus crispatus - YBR - 01 in MRS medium in a constant temperature incubator at 37 °C for 24 - 36 h, and reserve the strain for use.

[0090] (2) Take 10 mL of the bacterial solution that has been cultured for 24 h, centrifuge it at 3000 rpm for 10 min, discard the supernatant, wash it twice with PBS, and resuspend the bacterial cells with 5 mL of MRS medium. Add different concentrations of bile salts to prepare MRS culture solutions containing 0%, 0.1%, 0.2%, and 0.3%. Take 200 μL of the resuspended bacterial solution and put it into the prepared 10 mL culture solution, culture it at 37 °C for 24 - 36 h, then dilute and spread it on plates for counting. Each bile salt concentration has three replicates.

[0091] The measurement results are as Figures 3 - 4 shown. Lactiplantibacillus plantarum - YBR - 01 has strong tolerance to acid and bile salts and can still grow well in an acidic environment with pH = 3 and 0.1% bile salts ( Figure 3 ). Lactobacillus crispatus - YBR - 01 has strong tolerance to acid and bile salts and can still grow in an acidic environment with pH = 3 and 0.1% bile salts ( Figure 4 ).

[0092] Example 5 Hemolysis test

[0093] After subculturing and activating Lactiplantibacillus plantarum - YBR - 01 or Lactobacillus crispatus - YBR - 01 in MRS medium, take 4 μL and inoculate it on a sterile defibrinated sheep blood plate, and observe whether there is a hemolysis ring around the colonies after culturing in an anaerobic incubator at 37 °C for 24 h. In this experiment, Staphylococcus aureus ATCC25923 is used as a positive control. (If a greenish - yellow ring is formed around the colonies due to incomplete rupture of red blood cells, it is α - hemolysis; if a well - defined and completely transparent hemolysis ring is formed around the colonies due to complete rupture of red blood cells, it is β - hemolysis; if there is no change in the medium around the colonies, it is γ - hemolysis, that is, no hemolysis.)

[0094] The hemolysis test results of Lactiplantibacillus plantarum - YBR - 01 are as Figure 5 shown, Figure 5 the left side represents Staphylococcus aureus, Figure 5The right side represents Lactiplantibacillus plantarum - YBR - 01. The results show that the diameter of the hemolytic zone of Staphylococcus aureus can reach about 1 - 2 cm, while no obvious hemolytic zone is observed for Lactiplantibacillus plantarum - YBR - 01.

[0095] The hemolysis test results of Lactiplantibacillus crispatus - YBR - 01 are as Figure 6 shown. Figure 6 The left side represents Staphylococcus aureus, Figure 6 and the right side represents Lactiplantibacillus crispatus - YBR - 01. The results show that the diameter of the hemolytic zone of Staphylococcus aureus can reach about 1 - 2 cm, while no obvious hemolytic zone is observed for Lactiplantibacillus crispatus - YBR - 01.

[0096] Example 6 Drug resistance

[0097] The Kirby - Bauer (K - B) disk diffusion method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2018 edition) of the United States was used to determine the sensitivity of Lactiplantibacillus plantarum - YBR - 01 or Lactiplantibacillus crispatus - YBR - 01 to 10 commonly used antibacterial drugs, namely CTR - ceftriaxone, CIP - ciprofloxacin hydrochloride, PEN - penicillin, AMP - ampicillin, GEN - gentamicin, C - chloramphenicol, TET - tetracycline, E - polymyxin E, MY - midecamycin, and SXT - co - trimoxazole. Take 100 μL of Lactiplantibacillus plantarum - YBR - 01 or Lactiplantibacillus crispatus - YBR - 01 (1×10 7 CFU / mL) and spread it on the culture medium, then place the drug - containing filter paper in it and observe the size of the inhibition zone.

[0098] The drug resistance results of Lactiplantibacillus plantarum - YBR - 01 and Lactiplantibacillus crispatus - YBR - 01 are shown in Figure 7 and Figure 8 respectively. It was found that Lactiplantibacillus plantarum - YBR - 01 and Lactiplantibacillus crispatus - YBR - 01 are sensitive to most antibiotics, while CIP, GEN, and MY have relatively weak killing ability against Lactiplantibacillus plantarum - YBR - 01 and Lactiplantibacillus crispatus - YBR - 01.

[0099] Example 7 Preparation of postbiotic composition

[0100] 1. Activation of strains

[0101] Inoculate Lactiplantibacillus plantarum - YBR - 01 into MRS medium for activation and culture at 37°C to make the viable count of Lactiplantibacillus plantarum - YBR - 01 reach 1×10 5 CFU / mL for standby.

[0102] Inoculate Lactobacillus crispatus - YBR - 01 into MRS medium for activation, and culture it at 37°C to make the viable count of Lactobacillus crispatus - YBR - 01 reach 1×10 5 CFU / mL for standby.

[0103] 2. Fermentation culture

[0104] Inoculate the activated Lactiplantibacillus plantarum - YBR - 01 into MRS medium at an inoculation amount of 2% v / v, and ferment at a constant temperature of 37°C for 6 h. Then, inoculate the activated Lactobacillus crispatus - YBR - 01 into MRS medium at an inoculation amount of 3.5% v / v, add 0.275% w / v of β - galactosidase, and ferment at a constant temperature of 37°C for 10 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, prepare a compound bacterial suspension with a concentration of 1×10 8 CFU / mL.

[0105] 3. Preparation of postbiotic composition:

[0106] Heat the compound bacterial suspension at 80°C for 20 min to prepare a heat - inactivated bacterial solution, which is the postbiotic composition.

[0107] Example 8 Preparation of postbiotic composition

[0108] 1. Activation of strains

[0109] Inoculate Lactiplantibacillus plantarum - YBR - 01 into MRS medium for activation, and culture it at 37°C to make the viable count of Lactiplantibacillus plantarum - YBR - 01 reach 1×10 5 CFU / mL for standby.

[0110] Inoculate Lactobacillus crispatus - YBR - 01 into MRS medium for activation, and culture it at 37°C to make the viable count of Lactobacillus crispatus - YBR - 01 reach 1×10 5 CFU / mL for standby.

[0111] 2. Fermentation culture

[0112] Inoculate the activated Lactiplantibacillus plantarum - YBR - 01 into MRS medium at an inoculation amount of 3% v / v, and ferment at a constant temperature of 37°C for 6 h. Then, inoculate the activated Lactobacillus crispatus - YBR - 01 into MRS medium at an inoculation amount of 3.5% v / v, add 0.25% w / v of β - galactosidase, and ferment at a constant temperature of 37°C for 10 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, prepare a compound bacterial suspension with a concentration of 1×10 8 CFU / mL.

[0113] 3. Preparation of postbiotic composition:

[0114] The composite bacterial suspension was heated at 80 °C for 20 min to prepare a heat-inactivated bacterial solution, which is the postbiotic composition.

[0115] Example 9 Preparation of Postbiotic Composition

[0116] 1. Activation of Strains

[0117] Lactiplantibacillus plantarum - YBR - 01 was inoculated into MRS medium for activation and cultured at 37 °C until the viable count of Lactiplantibacillus plantarum - YBR - 01 reached 1×10 5 CFU / mL, and it was reserved for use.

[0118] Lactobacillus crispatus - YBR - 01 was inoculated into MRS medium for activation and cultured at 37 °C until the viable count of Lactobacillus crispatus - YBR - 01 reached 1×10 5 CFU / mL, and it was reserved for use.

[0119] 2. Fermentation Culture

[0120] The activated Lactiplantibacillus plantarum - YBR - 01 was inoculated into MRS medium at an inoculation amount of 2% v / v and fermented at a constant temperature of 37 °C for 6 h. Then, the activated Lactobacillus crispatus - YBR - 01 was inoculated into MRS medium at an inoculation amount of 4% v / v, and 0.3% w / v of β - galactosidase was added, and it was fermented at a constant temperature of 37 °C for 10 h. After the fermentation broth was centrifuged at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL with sterile PBS.

[0121] 3. Preparation of Postbiotic Composition:

[0122] The composite bacterial suspension was heated at 80 °C for 20 min to prepare a heat-inactivated bacterial solution, which is the postbiotic composition.

[0123] Comparative Example 1 Preparation of Postbiotic Composition

[0124] The difference between Comparative Example 1 and Example 7 is only that: "2. Fermentation Culture" is different. The activated Lactiplantibacillus plantarum - YBR - 01 and the activated Lactobacillus crispatus - YBR - 01 were co-inoculated into MRS medium at inoculation amounts of 2% v / v and 3.5% v / v respectively, 0.275% w / v of β - galactosidase was added, and it was fermented at a constant temperature of 37 °C for 16 h. After the fermentation broth was centrifuged at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL with sterile PBS.

[0125] Comparative Example 2 Preparation of Postbiotic Composition

[0126] The difference between Comparative Example 2 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactiplantibacillus plantarum - YBR - 01 was inoculated into MRS medium at an inoculation amount of 2% v / v, and after constant - temperature fermentation at 37°C for 6 h, the activated Lactobacillus crispatus - YBR - 01 was then inoculated into MRS medium at an inoculation amount of 3.5% v / v, 0.275% w / v of lactase was added, and fermentation was carried out at a constant temperature of 37°C for 10 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL.

[0127] Preparation of postbiotics composition in Comparative Example 3

[0128] The difference between Comparative Example 3 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactiplantibacillus plantarum - YBR - 01 and the activated Lactobacillus crispatus - YBR - 01 were co - inoculated into MRS medium at inoculation amounts of 2% v / v and 3.5% v / v respectively, 0.275% w / v of lactase was added, and fermentation was carried out at a constant temperature of 37°C for 16 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL.

[0129] Preparation of postbiotics composition in Comparative Example 4

[0130] The difference between Comparative Example 4 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactiplantibacillus plantarum - YBR - 01 was inoculated into MRS medium at an inoculation amount of 3.5% v / v, and after constant - temperature fermentation at 37°C for 6 h, the activated Lactobacillus crispatus - YBR - 01 was then inoculated into MRS medium at an inoculation amount of 2% v / v, 0.275% w / v of β - galactosidase was added, and fermentation was carried out at a constant temperature of 37°C for 10 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL.

[0131] Preparation of postbiotics composition in Comparative Example 5

[0132] The difference between Comparative Example 5 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactiplantibacillus plantarum - YBR - 01 was inoculated into MRS medium at an inoculation amount of 3.5% v / v, and after constant - temperature fermentation at 37°C for 6 h, the activated Lactobacillus crispatus - YBR - 01 was then inoculated into MRS medium at an inoculation amount of 3.5% v / v, 0.35% w / v of β - galactosidase was added, and fermentation was carried out at a constant temperature of 37°C for 10 h. After centrifuging the fermentation broth at 3000 rpm for 15 min, it was formulated into a composite bacterial suspension of 1×10 8 CFU / mL.

[0133] Experimental Example 1 Determination of Hydroxyl Radical Scavenging Ability

[0134] Take 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, and 1 mL of 6 mmol / L salicylic acid, and add them to the supernatant of Lactiplantibacillus plantarum - YBR - 01 culture solution (1×10 8 CFU / mL), the supernatant of Lactobacillus crispatus - YBR - 01 culture solution (1×10 8 CFU / mL), and 1 mL of the postbiotic composition prepared in Examples 7 - 9 or Comparative Examples 1 - 5. Let it stand at room temperature for 30 min, use deionized water as the blank control, measure the absorbance at 510 nm, and calculate the scavenging rate of hydroxyl radicals: Hydroxyl radical scavenging rate = [1 - A510 (sample) / A510 (blank)] × 100%. The measurement results are shown in Table 1.

[0135] Table 1 Hydroxyl Radical Scavenging Ability

[0136]

[0137] The measurement results show that the postbiotic compositions prepared in Examples 7 - 8 of the present invention have a hydroxyl radical scavenging rate of up to 52.27% - 55.40%.

[0138] Experimental Example 2 Effect of Postbiotic Composition on Vaginitis Caused by Vaginal Flora Disorder

[0139] 1. Purchase and Living Environment of Experimental Animals

[0140] The animals were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. 48 BALB / c female mice (SPF level, 6 - 8 weeks old, weighing 20 - 22 g). During the experiment, the mice had free access to water and food. The animals were fed standard pellet feed every day and cleaned regularly. Under specific pathogen - free conditions, the mice were kept in sterile polypropylene cages, with 3 - 5 mice in each cage. And they were maintained in a room with controlled temperature (22 ± 2°C) and humidity (50 ± 15%) under a light - dark cycle (L:D, 12:12 h), provided with standard diet and water. All mice used in this study were caged one week before the start of the study to adapt to the housing conditions.

[0141] 2. Establishment of Animal Model

[0142] Female BALB / c mice were subcutaneously injected with 0.05 mL of 2 mg / mL estradiol benzoate injection for 3 consecutive times, once every 2 days. On the 6th day, the vaginal cavity of the mice was rinsed 3 times with sterile PBS solution (pH 8.5) (with an interval of 5 minutes each time). Streptococcus hemolyticus type B, Staphylococcus aureus, and Escherichia coli O157 were mixed at a volume ratio of 1:1:2 to prepare a mixed bacterial solution with a concentration of 1×10 9 CFU / mL; absorbable gelatin sponge for medical use was cut into pieces of 0.5 cm × 0.5 cm in size, 20 μL of the infected bacterial solution was injected into the sponge, and the sponge injected with the bacterial strain was inserted into the vaginal cavity of the mice (inserted about 1 cm - 1.5 cm into the vaginal cavity of the mice, and the injection amount of each bacterium was 0.025 mL / 100 g, once a day); estradiol benzoate injection was given in the same way. On the 6th day, an absorbable hemostatic sponge for surgery injected with 20 μL of normal saline was inserted into the vaginal cavity of the mice as a blank control group.

[0143] 3. Grouping of animal models and treatment with drugs

[0144] (1) Grouping of animal models:

[0145] Using the method of analysis of variance with a randomized block design, the mice were grouped according to their body weights. A total of 6 groups were set up, with 8 mice in each group, including: ① healthy group, ② model group, ③ Lp group: model + Lactiplantibacillus plantarum, ④ Lc group: model + Lactobacillus crispatus, ⑤ Lp&Lc group: model + Lactiplantibacillus plantarum + Lactobacillus crispatus, ⑥ p-Lp&p-Lc group: model + the postbiotics composition of Example 7.

[0146] (2) Drug treatment:

[0147] Healthy group (C): The vaginal cavity was rinsed with 100 μL of normal saline once a day for 14 consecutive days;

[0148] Model group (M): The vaginal cavity was rinsed with 100 μL of normal saline once a day for 14 consecutive days;

[0149] Lp group (Lp): The vaginal cavity was rinsed with Lactiplantibacillus plantarum - YBR - 01 at a concentration of 1×10 8 CFU once a day for 14 consecutive days.

[0150] Lc group (Lc): The vaginal cavity was rinsed with Lactobacillus crispatus - YBR - 01 at a concentration of 1×10 8 CFU once a day for 14 consecutive days.

[0151] Lp&Lc group (Lp&Lc): The vaginal cavity was rinsed with Lactiplantibacillus plantarum - YBR - 01 at a concentration of 1×10 8 CFU and Lactobacillus crispatus - YBR - 01 at a concentration of 1×10 8Lactobacillus crispatus - YBR - 01 was used to irrigate the vagina, administered once a day for 14 consecutive days.

[0152] Postbiotic composition group (p - Lp&p - Lc): The postbiotic composition of Example 7 was used to irrigate the vagina, administered once a day for 14 consecutive days. (Postbiotic treatment element: Place a square of medical absorbent gelatin sponge in the bacterial solution to make it fully contact. After the medical absorbent gelatin sponge is fully infiltrated, hold the mouse's tail with the left hand to invert it, gently wipe the skin around the mouse's vagina with 75% alcohol, and gently insert the medical absorbent gelatin sponge containing heat - inactivated bacterial solution into the uterine cavity through the vagina with forceps, and keep the mouse inverted for 1 - 2 minutes.)

[0153] The flow chart of the animal experiment is shown in Figure 9 .

[0154] 4. Collection of specimens and detection indicators during the experiment

[0155] When the mouse's vagina was significantly congested, swollen and accompanied by a large amount of purulent discharge, it indicated that the BV model of the mouse was successfully prepared, and the vaginal orifice and vaginal secretions were scored. The scoring criteria were as follows: 0 point: The vaginal orifice was not swollen, and the vaginal orifice was slightly open normally; 1 point: The vaginal orifice was slightly swollen, and there was a small amount of secretion adhesion at the vaginal orifice; 2 points: The vaginal orifice was swollen and had purulent discharge.

[0156] The scoring results are shown in Table 2. The scoring results of the vaginal orifice lesions and vaginal secretions of the mice showed that compared with the healthy group, the model group had red and swollen vaginal orifices accompanied by purulent discharge. The postbiotic composition treatment not only relieved the redness and swelling of the vaginal orifice but also made the vaginal secretions become transparent.

[0157] Table 2. Scoring of vaginal orifice lesions in mice

[0158]

[0159] 5. Collection of specimens and detection indicators of sacrificed mice

[0160] (1) Mouse dissection

[0161] Before the dissection experiment, all dissection instruments were sterilized in advance. Prepare cryotubes and specimen bottles, label the groups and samples to be loaded in advance, and prepare 75% alcohol and 4% paraformaldehyde for use. After 4 days of drug withdrawal, collect the vaginal lavage fluid. The next day, sacrifice the mouse by taking blood from the eye socket, collect the blood into a centrifuge tube, let it stand at 37°C for 1 hour, then centrifuge at 3000 rpm for 10 minutes to collect the serum, and store it at - 80°C. Then sacrifice the mouse by cervical dislocation, fix the mouse's limbs, disinfect the mouse's skin with alcohol, cut open the mouse's abdomen, and dissect and preserve the vagina and uterus in a - 80°C refrigerator.

[0162] (2) Determination of MPO activity

[0163] The activity of MPO in vaginal tissues was detected using a biochemical kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., product number A044-1-1) (n = 3).

[0164] The results of MPO activity determination are shown in Figure 10 A in [Figure / Table] [X], and the MPO activity in the model group was significantly higher than that in the healthy group. Treatment with the postbiota composition could significantly reduce the MPO activity.

[0165] (3) qPCR detection

[0166] The levels of IL-1β and IL-10 in vaginal tissues were detected by qPCR using a reverse transcription and qPCR reaction system kit (purchased from TaKaRa, product number RR047A) according to the instructions (n = 3). The specific procedure is as follows:

[0167] 1) Weigh the same mass of vaginal tissue, add 1 mL of Trizol, cut it into pieces with ophthalmic scissors, and then homogenize it with a tissue homogenizer. This process is carried out on ice. After sufficient homogenization, centrifuge at 5000 rpm for 10 min, and transfer the supernatant to a new EP tube;

[0168] 2) Add 160 μL of chloroform, mix well by shaking vigorously, centrifuge at 13000 rpm for 15 min in a 4°C centrifuge. After centrifugation, aspirate the supernatant into a new EP tube, taking care not to aspirate the protein layer;

[0169] 3) Add an equal volume of isopropanol as aspirated in step 2), mix by inverting up and down, centrifuge at 13000 rpm for 10 min in a 4°C centrifuge, and pour off the supernatant;

[0170] 4) Add 1 mL of 75% ethanol, wash thoroughly, centrifuge at 13000 rpm for 10 min in a 4°C centrifuge, and pour off the supernatant;

[0171] 5) Add 1 mL of absolute ethanol, wash by shaking vigorously, centrifuge at 13000 rpm for 10 min in a 4°C centrifuge, and pour off the supernatant;

[0172] 6) Air dry at room temperature for 20 min;

[0173] 7) Add 160 μL of RNase-Freed Water and promote RNA dissolution in a 55°C water bath;

[0174] 8) Measure the RNA concentration;

[0175] 9) Composition of the reverse transcription system:

[0176] Step 1: (Genomic DNA removal step)

[0177] The reagents added in step 1, their addition amounts, and reaction conditions are shown in Table 3:

[0178] Table 3 Reagents Added in Step 1, Their Dosages, and Reaction Conditions

[0179]

[0180] Step 2: (Reverse Transcription Reaction Step)

[0181] The reagents added in Step 2, their dosages, and reaction conditions are shown in Table 4:

[0182] Table 4 Reagents Added in Step 2, Their Dosages, and Reaction Conditions

[0183]

[0184] After mRNA is reverse transcribed into cDNA, it is diluted 3-fold with nuclease-free water and used immediately.

[0185] 10) Use the obtained cDNA as a template for q-PCR reaction.

[0186] The reaction system of q-PCR is as follows in Table 5:

[0187] Table 5 Reaction System of q-PCR

[0188]

[0189] The total reaction system per well is 20.0 μL. The reaction conditions are shown in Table 6, and the entire q-PCR process takes about 71 min.

[0190] Table 6 Reaction Conditions of q-PCR

[0191]

[0192] The results of qRT-PCR detection are shown in Figure 10 B and C therein. Compared with the healthy group, the expression level of IL-1β in the model group was significantly increased, and the postbiota composition could significantly reduce the expression of these pro-inflammatory factors. The expression of the anti-inflammatory factor IL-10 showed the opposite trend.

[0193] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent should be subject to the appended claims.

Claims

1. A method for preparing a postbiotic composition, characterized in that: The preparation method comprises the following steps: S1, activate Lactobacillus plantarum and Lactobacillus crispatus respectively; S2, inoculating the activated Lactobacillus plantarum into the culture medium at an inoculum amount of 2%-3% v / v, and fermenting to obtain a fermentation culture solution; S3, adding activated Lactobacillus crispatus to the fermentation culture solution, inoculating the Lactobacillus crispatus into the culture medium at an inoculum amount of 3.5%-4% v / v, adding β-galactosidase, and continuing fermentation and culture to obtain a fermentation solution; the inoculum amount is the inoculum amount of Lactobacillus crispatus relative to the culture medium; S4, resuspending and inactivating the fermentation broth to obtain a postbiotic composition; The plant lactobacillus ( Lactiplantibacillus plantarum ) is Lactobacillus plantarum YBR-01, with a preservation number of CGMCC No.26679; the Lactobacillus crispatus ( Lactobacillus crispatus ) is Lactobacillus crispatus YBR-01, with the accession number being CGMCC No.26680; The amount of β-galactosidase added in step S3 is 0.25%-0.3% w / v.

2. The preparation method according to claim 1, characterized in that: The culture medium described in steps S2-S3 includes one or more of MRS culture medium, TPY culture medium, MC culture medium, and BCP culture medium.

3. The preparation method according to claim 1, characterized in that: The fermentation culture in step S2 is cultured at 35-39° C. for 4-8 hours; the fermentation culture in step S3 is cultured at 35-39° C. for 8-12 hours.

4. The postbiotic composition prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the postbiotic composition according to claim 4 in preparing a product, characterized in that: The products include any of the following: (1) Preparation of drugs for the prevention, treatment and / or auxiliary treatment of vaginitis; or (2) Female private care products to improve vaginitis; or (3) Health products that have anti-inflammatory, antioxidant, immunity-enhancing and / or microbial balance regulating effects.

6. The use according to claim 5, characterized in that: The product contains ≥1×10 8 CFU / g of postbiotic composition.

7. A drug for preventing, treating and / or assisting in the treatment of vaginitis, characterized in that: The medicine comprises the postbiotic composition according to claim 4.

8. A female private care product for improving vaginitis, characterized in that: The female private care product comprises the postbiotic composition according to claim 4.

9. A health product for anti-inflammatory, anti-oxidation, immunity enhancement and / or flora balance regulation, characterized in that: The health product comprises the postbiotic composition according to claim 4.

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