Preparation method of metagen composition as well as product and application of metagen composition

Abstract

The invention provides a preparation method, a product and application of a postbiotic composition, and belongs to the technical field of biological medicine. The preparation method of the postbiotic composition provided by the invention comprises the following steps: sequentially inoculating activated plant lactobacillus YBR-01 and lactobacillus crispatus YBR-01 into a culture medium, carrying out fermentation culture under the action of beta-galactosidase, and carrying out thermal inactivation to obtain the postbiotic composition. The metagen composition prepared by the method can improve vaginitis by regulating microecological balance of vagina, resisting oxidation and inflammation, enhancing metabolism and improving immunity. Meanwhile, the postbiotic composition also can overcome the drug resistance of antibiotics, has better safety, and provides a new thought for preparing products for improving vaginitis.

Description

Technical Field

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

[0002] Vaginal flora disorder refers to the abnormal increase of microorganisms that normally exist in the vagina, such as Escherichia coli, Gardnerella vaginalis, Staphylococcus aureus, Streptococcus, Porphyromonas, Mobiluncus, Mycoplasma hominis and Candida, while the lactobacillus that produces hydrogen peroxide decreases or disappears, causing a group of clinical syndromes with abnormal increase in vaginal secretions as the main manifestation.

[0003] The treatment generally uses anti-anaerobic drugs such as metronidazole, tinidazole, and clindamycin, but antibiotic resistance, side effects caused by antibiotics, easy recurrence, and superinfection have limited the treatment effect. Currently, vaginal lactobacillus capsules have appeared in the clinic to improve vaginal flora imbalance, but their clinical application is limited because their specific mechanism is still unclear and there are safety issues. Therefore, exploring the specific mechanism of lactobacillus in improving vaginal inflammation and finding new methods that can improve the symptoms of vaginal inflammation, overcome antibiotic resistance, and improve safety performance are scientific issues that need to be solved urgently.

[0004] Postbiotics are a completely 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, studies have shown that postbiotics have many effects on the intestine, such as reducing inflammation, promoting the passage of waste through the digestive tract, enhancing metabolic function, increasing calcium absorption, improving immune function, and preventing type II diabetes. However, there are few studies on the effect of postbiotics on the vagina, and they overcome the shortcomings of probiotics, such as low safety, the need for bacterial growth or colonization, population restrictions, and short and unstable shelf life. Therefore, this study uses postbiotics as an "advanced alternative" to probiotics through vaginal administration for the treatment of vaginitis caused by vaginal flora disorders.

[0005] Normal vaginal microbiota contains Lactobacillus plantarum and Lactobacillus crispatus, which are of great significance for improving vaginitis and restoring the balance of vaginal microecology. Therefore, developing a preparation method and product of a postbiotic composition of Lactobacillus 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 in the prior art, the present invention aims to provide a method for preparing a postbiotic composition, and its products and applications. The method for preparing a postbiotic composition provided by the present invention sequentially inoculates activated Lactobacillus 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 by heat inactivation. The postbiotic composition prepared by the method improves vaginitis by regulating the balance of vaginal microecology, anti-oxidation, 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 that improve vaginitis.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a postbiotic composition, the preparation method comprising 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 medium at an inoculum amount of 3.5%-4% v / v, adding β-galactosidase, and continuing fermentation culture to obtain a fermentation liquid; 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, and its preservation number is CGMCC No.26680.

[0008] 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.

[0009] Preferably, the culture medium in steps S2-S3 is MRS culture medium.

[0010] Specifically, the fermentation culture in step S2 is cultured at 35-39° C. for 4-8 hours.

[0011] Preferably, the fermentation culture in step S2 is cultured at 37° C. for 6 hours.

[0012] Specifically, the fermentation culture in step S3 is cultured at 35-39° C. for 8-12 hours.

[0013] Preferably, the fermentation culture in step S3 is cultured at 37° C. for 10 h.

[0014] Specifically, the inoculation ratio of Lactobacillus plantarum to Lactobacillus crispatus is 1:1.15-2.

[0015] Preferably, the ratio of the inoculum amount of Lactobacillus plantarum to that of Lactobacillus crispatus is 1.75.

[0016] Preferably, the inoculation amount of Lactobacillus plantarum is 2% v / v.

[0017] Preferably, the inoculation amount of Lactobacillus crispatus is 3.5% v / v.

[0018] Specifically, the liquid used for resuspension in step S4 includes: one or more of sterile water for injection, sterile physiological saline, and sterile PBS.

[0019] Preferably, the liquid used for resuspension in step S4 is sterile PBS.

[0020] Specifically, the inactivation condition in step S4 is heating at 80-120° C. for 10-20 min.

[0021] Preferably, the inactivation condition in step S4 is heating at 80° C. for 20 min.

[0022] In a second aspect, the present invention provides a postbiotic composition prepared by the above preparation method.

[0023] In a third aspect, the present invention provides the use of the above-mentioned postbiotic composition in preparing a product, wherein the product includes any of the following products: (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.

[0024] Specifically, the product contains ≥1×10 8 CFU / g of postbiotic composition.

[0025] Preferably, the product contains 1×10 8 -1×10 12 CFU / g of postbiotic composition.

[0026] In a fourth aspect, the present invention provides a drug for preventing, treating and / or assisting in the treatment of vaginitis, wherein the drug comprises the above-mentioned postbiotic composition.

[0027] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0028] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

[0029] Specifically, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form and a parenteral dosage form according to the administration method.

[0030] Preferably, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0031] Preferably, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, skin dosage forms, mucosal dosage forms and cavity dosage forms.

[0032] In a fifth aspect, the present invention provides a female private care product for improving vaginitis, wherein the female private care product comprises the above-mentioned postbiotic composition.

[0033] Specifically, the female private care products include but are not limited to cleaning products, care lotions, care gels, sanitary napkins, tampons or skin care products.

[0034] In a sixth aspect, the present invention provides a health product for anti-inflammatory, anti-oxidant, immunity-enhancing and / or microbiota balance regulating effects, wherein the health product comprises the above-mentioned postbiotic composition.

[0035] Specifically, the health care product also includes nutritional additives that are nutritionally acceptable.

[0036] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0037] Specifically, the dosage forms of the health care products include tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0038] The beneficial effects of the present invention are: The present invention provides a preparation method of a postbiotic composition, a product and an application thereof, and belongs to the field of biomedical technology. The preparation method of the postbiotic composition provided by the present invention sequentially inoculates activated Lactobacillus 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 by heat inactivation. The postbiotic composition prepared by the method improves vaginitis by regulating the balance of vaginal microecology, anti-oxidation, 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 to improve vaginitis.

[0039] Collection Instructions Name of biological material: Lactobacillus plantarum ( Lactiplantibacillus plantarum )YBR-01; Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum ; Storage time: February 27, 2023; Deposit number: CGMCC No.26679; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0040] Collection Instructions Name of biological material: Lactobacillus crispatus ( Lactobacillus crispatus )YBR-01; Classification name: Lactobacillus crispatus Lactobacillus crispatus ; Storage time: February 27, 2023; Deposit number: CGMCC No.26680; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : The growth curve and microscopic morphology of Lactobacillus plantarum -YBR-01; A in the figure is the growth curve of Lactobacillus plantarum -YBR-01; B is the microscopic morphology of Lactobacillus plantarum -YBR-01.

[0042] Figure 2 The growth curve and microscopic morphology of Lactobacillus crispatus-YBR-01; A in the figure is the growth curve of Lactobacillus crispatus-YBR-01; B is the microscopic morphology of Lactobacillus crispatus-YBR-01.

[0043] Figure 3 The results of acid resistance and bile resistance of Lactobacillus plantarum-YBR-01 are shown in FIG. 1 . A in the figure is the result of acid resistance, and B is the result of bile resistance.

[0044] Figure 4 The results of acid resistance and bile resistance of Lactobacillus crispatus-YBR-01 are shown in FIG. 1 . A in the figure is the result of acid resistance, and B is the result of bile resistance.

[0045] Figure 5 This is a hemolytic test of Lactobacillus plantarum-YBR-01; the left side of the figure represents the Staphylococcus aureus-positive control; the right side represents Lactobacillus plantarum-YBR-01.

[0046] Figure 6 This is a hemolytic test of Lactobacillus crispatus-YBR-01; the left side of the figure represents Staphylococcus aureus-positive control; the right side represents Lactobacillus crispatus-YBR-01.

[0047] Figure 7 This is the drug resistance test of Lactobacillus plantarum-YBR-01; CTR in the figure 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 midenomycin; and SXT represents co-trimoxazole.

[0048] Figure 8 This is the drug resistance test of Lactobacillus crispatus-YBR-01; CTR in the figure 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 midenomycin; and SXT represents co-trimoxazole.

[0049] Fig. 9 Flow chart of animal experiments.

[0050] Fig.10 is the activity of MPO, the relative expression of IL-1β and IL-10; A in the figure is the activity of MPO; B is the relative expression of IL-1β; C is the relative expression of IL-10; the data are expressed as mean ± standard deviation; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0052] Example 1 Preparation of MRS culture medium MRS medium: the main ingredients are peptone 10.0g, beef powder 5.0g, yeast powder 4.0g, glucose 20.0g, Tween80 1.0mL, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, agar powder 15.0g, deionized water 1000mL; weigh the above ingredients in sequence, add deionized water and sterilize at 121℃ high temperature and high pressure for 15min-20min. It is mainly used for the cultivation of lactic acid bacteria.

[0053] Example 2 Microscopic morphology and growth curve of Lactobacillus plantarum-YBR-01 1. Microscopic morphological observation: 1. Preparation: Take 200 μL bacterial suspension (1×10 8 CFU / mL) on a glass slide, and then fix it with the flame of an alcohol lamp. During this process, the coated surface needs to face up and pass through the flame several times. Touch the glass slide with your hand until it feels slightly hot. Prevent overheating, which will deform the bacteria or affect the staining reaction. 2. Staining: Add ammonium oxalate crystal violet for 1 minute, wash with water, and dry naturally. 3. Observation: Place the slide under an optical microscope, first find the field of view under a low-power microscope, and then observe the bacterial morphology under a high-power microscope. You can also drop cedar oil and observe under an oil microscope.

[0054] 2. Growth curve determination: 200 μL of Lactobacillus plantarum-YBR-01 (1×10 8 CFU / mL) were inoculated into 10 mL MRS medium and cultured in a shaking incubator at 37°C. The optical density (OD) at a wavelength of 600 nm was measured every 2 hours for 24 hours. The experiment was repeated three times.

[0055] First, the optical density of the bacterial suspension of Lactobacillus plantarum-YBR-01 was tested for 24 hours. The results showed that the strain entered the logarithmic growth phase after about 2 hours and entered the stable growth phase after 8 hours. Figure 1 A in the figure). Under a high-power optical microscope, the morphology of Lactobacillus plantarum-YBR-01 is straight or curved rods, existing singly, sometimes in pairs or chains ( Figure 1 B in the figure).

[0056] Example 3 Microscopic morphology and growth curve of Lactobacillus crispatus-YBR-01 1. Microscopic morphological observation: 1) Preparation: Take 200 μL bacterial suspension (1×10 7 CFU / mL) on a glass slide, and then fix it with the flame of an alcohol lamp. During this process, the coated surface needs to face up and pass through the flame several times. Touch the glass slide with your hand until it feels slightly hot. Prevent overheating, which will deform the bacteria or affect the staining reaction; 2) Staining: Add ammonium oxalate crystal violet for 1 minute, wash with water, and dry naturally; 3) Observation: Place the slide under an optical microscope, first find the field of view under a low-power microscope, and then observe the bacterial morphology under a high-power microscope. You can also drop cedar oil and observe under an oil microscope.

[0057] 2. Growth curve determination: 200 μL of Lactobacillus crispatus-YBR-01 (1×10 7 CFU / mL) were inoculated into 10 mL MRS medium and cultured in a shaking incubator at 37°C. The optical density (OD) at a wavelength of 600 nm was measured every 2 hours for 24 hours. The experiment was repeated three times.

[0058] First, the optical density of the bacterial suspension of the strain was tested for 24 hours. The results showed that the strain entered the logarithmic growth phase after about 6 hours and gradually entered the stable growth phase after 14 hours ( Figure 2 A in the figure). Under a high-power optical microscope, L. crispatus appears as an elongated, curved, and slender bacillus ( Figure 2 B in the figure).

[0059] Example 4 Acid and bile salt resistance test 1. Acid resistance test (1) Cultivate the preserved Lactobacillus crispatus-YBR-01 or Lactobacillus plantarum-YBR-01 in MRS medium in a constant temperature incubator at 37°C for 24-36 hours, and retain the strain for later use; (2) Prepare PBS buffers with pH values ​​of 2, 3, 4, 5, and 7. Take 1 mL of bacterial culture solution that has been cultured for 24-36 hours, centrifuge at 3000 rpm for 10 minutes, discard the supernatant, wash twice with PBS, add 1 mL of PBS with different pH values ​​to resuspend, place in a 37°C constant temperature incubator for 4 hours, then dilute and plate, and count the viable bacteria. Repeat three times for each pH PBS buffer.

[0060] 2. Bile salt tolerance test (1) Cultivating the preserved Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 in an MRS medium in a constant temperature incubator at 37°C for 24-36 hours, and retaining the strain for later use; (2) Take 10mL of the bacterial culture that has been cultured for 24 hours, centrifuge at 3000rpm for 10 minutes, discard the supernatant, wash twice with PBS, and resuspend the bacteria in 5mL of MRS culture medium. Add different concentrations of bile salts to prepare MRS culture medium containing 0%, 0.1%, 0.2%, and 0.3%. Take 200ul of the resuspended bacterial solution and put it into the prepared 10mL culture medium and culture it at 37℃ for 24-36 hours, then dilute and plate for counting. Repeat three times for each bile salt concentration.

[0061] The results of the test are as follows Figure 3-Figure 4 As shown in the figure, Lactobacillus plantarum-YBR-01 has strong tolerance to acid and bile salts and can still grow well in an acidic environment of 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 of pH = 3 and 0.1% bile salts ( Figure 4 ).

[0062] Example 5 Hemolysis Test After subculturing and activating Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 in MRS medium, 4 μL was inoculated on a sterile defibrinated sheep blood plate, and cultured in an anaerobic incubator at 37°C for 24 hours to observe whether a hemolytic ring appeared around the colony. Staphylococcus aureus ATCC25923 was used as a positive control in this experiment. (If a grass-green ring is formed around the colony due to incomplete rupture of red blood cells, it is α hemolysis; if a clear-cut and completely transparent hemolytic ring is formed around the colony due to complete rupture of red blood cells, it is β hemolysis; if the culture medium around the colony does not change, it is γ hemolysis, that is, no hemolysis.

[0063] The results of the hemolytic test of Lactobacillus plantarum-YBR-01 are as follows Figure 5 As shown, Figure 5 The left side represents Staphylococcus aureus. Figure 5 The right side represents Lactobacillus plantarum-YBR-01. The results showed that the diameter of the hemolysis zone of Staphylococcus aureus could reach about 1-2 cm, while no obvious hemolysis zone was observed for Lactobacillus plantarum-YBR-01.

[0064] The results of the hemolysis test of Lactobacillus crispatus-YBR-01 are as follows Figure 6 As shown, Figure 6 The left side represents Staphylococcus aureus. Figure 6 The right side represents Lactobacillus crispatus-YBR-01. The results showed that the diameter of the hemolysis zone of Staphylococcus aureus could reach about 1-2 cm, while no obvious hemolysis zone was observed for Lactobacillus crispatus-YBR-01.

[0065] Example 6 Drug resistance The Kirby-Bauer (KB) disk method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2018 edition) was used to determine the sensitivity of Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 to 10 commonly used antimicrobial drugs, including CTR-cefotaxime, CIP-ciprofloxacin hydrochloride, PEN-penicillin, AMP-ampicillin, GEN-gentamicin, C-chloramphenicol, TET-tetracycline, E-polymyxin E, MY-midramycin, and SXT-compound sulfamethoxazole. Take 100 μL of Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 (1×10 7 CFU / mL) was spread on the culture medium, and then the filter paper containing the drug was placed on it to observe the size of the inhibition zone.

[0066] The drug resistance results of Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01 are shown in Figure 7 and Figure 8 It was found that Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01 were sensitive to most antibiotics, while CIP, GEN and MY had weak killing ability against Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01.

[0067] Example 7 Preparation of postbiotic composition 1. Activation of strains Lactobacillus plantarum-YBR-01 was inoculated into MRS medium for activation and cultured at 37°C until the viable count of Lactobacillus plantarum-YBR-01 was 1×10 5 CFU / mL, for future use.

[0068] 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 was 1×10 5 CFU / mL, for future use.

[0069] 2. Fermentation culture The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at a rate of 2% v / v, and then the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at a rate of 3.5% v / v. 0.275% w / v of β-galactosidase was added, and the culture was fermented at a rate of 37°C for 10 h. The fermentation broth was centrifuged at 3000 rpm for 15 min, and then prepared into 1×10 8 CFU / mL of composite bacterial suspension.

[0070] 3. Preparation of postbiotic composition: The composite bacterial suspension was heated at 80° C. for 20 min to prepare a heat-killed bacterial solution, which was the postbiotic composition.

[0071] Example 8 Preparation of postbiotic composition 1. Activation of strains Lactobacillus plantarum-YBR-01 was inoculated into MRS medium for activation and cultured at 37°C until the viable count of Lactobacillus plantarum-YBR-01 was 1×10 5 CFU / mL, for future use.

[0072] 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 was 1×10 5 CFU / mL, for future use.

[0073] 2. Fermentation culture The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at a rate of 3% v / v, and then the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at a rate of 3.5% v / v. 0.25% w / v β-galactosidase was added, and the culture was fermented at 37°C for 10 h. The fermentation broth was centrifuged at 3000 rpm for 15 min and then prepared into 1×10 8 CFU / mL of composite bacterial suspension.

[0074] 3. Preparation of postbiotic composition: The composite bacterial suspension was heated at 80° C. for 20 min to prepare a heat-killed bacterial solution, which was the postbiotic composition.

[0075] Example 9 Preparation of postbiotic composition 1. Activation of strains Lactobacillus plantarum-YBR-01 was inoculated into MRS medium for activation and cultured at 37°C until the viable count of Lactobacillus plantarum-YBR-01 was 1×10 5 CFU / mL, for future use.

[0076] 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 was 1×10 5 CFU / mL, for future use.

[0077] 2. Fermentation culture The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at a rate of 2% v / v, and then the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at a rate of 4% v / v. 0.3% w / v β-galactosidase was added, and the culture was fermented at a rate of 37°C for 10 h. The fermentation broth was centrifuged at 3000 rpm for 15 min and then prepared into 1×10 8 CFU / mL of composite bacterial suspension.

[0078] 3. Preparation of postbiotic composition: The composite bacterial suspension was heated at 80° C. for 20 min to prepare a heat-killed bacterial solution, which was the postbiotic composition.

[0079] Comparative Example 1 Preparation of postbiotic composition The difference between Comparative Example 1 and Example 7 is only that: "2. Fermentation culture" is different. Activated Lactobacillus plantarum-YBR-01 was inoculated at a rate of 2% v / v and activated Lactobacillus crispatus-YBR-01 was inoculated at a rate of 3.5% v / v into MRS medium, 0.275% w / v of β-galactosidase was added, and the culture was fermented at 37°C for 16 hours. The fermentation liquid was centrifuged at 3000 rpm for 15 minutes and then prepared with sterile PBS to 1×10 8 CFU / mL of composite bacterial suspension.

[0080] Comparative Example 2 Preparation of postbiotic composition The difference between Comparative Example 2 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at an inoculum amount of 2% v / v, and fermented at 37°C for 6 hours. Then, the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at an inoculum amount of 3.5% v / v, and 0.275% w / v lactase was added. The fermentation was continued at 37°C for 10 hours. After the fermentation liquid was centrifuged at 3000 rpm for 15 minutes, it was prepared with sterile PBS to 1×10 8 CFU / mL of composite bacterial suspension.

[0081] Comparative Example 3 Preparation of postbiotic composition The difference between Comparative Example 3 and Example 7 is only that: "2. Fermentation culture" is different. Activated Lactobacillus plantarum-YBR-01 was inoculated at a rate of 2% v / v and activated Lactobacillus crispatus-YBR-01 was inoculated at a rate of 3.5% v / v into MRS medium, 0.275% w / v lactase was added, and the culture was fermented at 37°C for 16 hours. The fermentation liquid was centrifuged at 3000 rpm for 15 minutes and then prepared into 1×10 8 CFU / mL of composite bacterial suspension.

[0082] Comparative Example 4 Preparation of postbiotic composition The difference between Comparative Example 4 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at an inoculum amount of 3.5% v / v, and fermented at 37°C for 6 hours. Then, the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at an inoculum amount of 2% v / v, and 0.275% w / v of β-galactosidase was added, and fermented at 37°C for 10 hours. After the fermentation liquid was centrifuged at 3000 rpm for 15 minutes, it was prepared with sterile PBS to 1×10 8 CFU / mL of composite bacterial suspension.

[0083] Comparative Example 5 Preparation of postbiotic composition The difference between Comparative Example 5 and Example 7 is only that: "2. Fermentation culture" is different. The activated Lactobacillus plantarum-YBR-01 was inoculated into MRS medium at an inoculum amount of 3.5% v / v, and fermented at 37°C for 6 hours. Then, the activated Lactobacillus crispatus-YBR-01 was inoculated into MRS medium at an inoculum amount of 3.5% v / v, and 0.35% w / v of β-galactosidase was added. The fermentation was continued at 37°C for 10 hours. After the fermentation liquid was centrifuged at 3000 rpm for 15 minutes, it was prepared with sterile PBS to 1×10 8 CFU / mL of composite bacterial suspension.

[0084] Experimental Example 1 Determination of Hydroxyl Radical Scavenging Ability 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 into the culture medium of Lactobacillus plantarum-YBR-01 (1×10 8 CFU / mL) supernatant, Lactobacillus crispatus-YBR-01 culture medium (1×10 8 CFU / mL) supernatant, 1 mL of the postbiotic composition prepared in Examples 7-9 or Comparative Examples 1-5. The mixture was allowed to stand for 30 min at room temperature, and deionized water was used as a blank control. The absorbance at 510 nm was measured, and the scavenging rate of hydroxyl radicals was calculated: hydroxyl radical scavenging rate = [1-A510 (sample) / A510 (blank)] × 100%. The results are shown in Table 1.

[0085] Table 1 Hydroxyl radical scavenging ability

[0086] The measurement results show that the hydroxyl radical scavenging rate of the postbiotic composition prepared in Examples 7-8 of the present invention can reach 52.27%-55.40%.

[0087] Experimental Example 2 Effect of postbiotic composition on vaginitis caused by vaginal flora disturbance 1. Purchase and living environment of experimental animals Animals were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. 48 BALB / c female mice (SPF level, 6-8 weeks old, weighing 20-22g). Mice had free access to water and food during the experiment. Animals were fed with standard pellet feed every day. Cleaned regularly every day. Mice were housed in sterile polypropylene cages, 3-5 per cage, under specific pathogen-free conditions. They were maintained in a room with controlled temperature (22±2°C) and humidity (50±15%) with a light-dark cycle (L:D, 12:12h), and 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 housing conditions.

[0088] 2. Establishment of animal model BALB / c female mice were subcutaneously injected with 0.05 mL of 2 mg / mL estradiol phthalate injection for 3 consecutive times, once every 2 days. On the 6th day, the vagina of the mice was first flushed with sterile PBS solution (pH 8.5) 3 times (5 minutes between each flushing), and beta-hemolytic streptococci, Staphylococcus aureus, and Escherichia coli O157 were prepared at a volume ratio of 1:1:2 to a concentration of 1×10 9 CFU / mL of mixed bacterial solution; cut the medical absorbable gelatin sponge into 0.5cm×0.5cm size, inject 20μL of infected bacterial solution into the sponge, and plug the sponge injected with bacteria into the mouse vagina (inserted into the mouse vagina about 1cm-1.5cm, the injection amount of each bacteria is 0.025mL / 100g, 1 time / d); give estradiol benzoate injection in the same way, and on the 6th day, plug the surgical absorbable hemostatic sponge injected with 20μL of normal saline into the mouse vagina as a blank control group.

[0089] 3. Animal model grouping and drug treatment (1) Animal model groups: The mice were grouped according to their weight using the variance analysis method of random group design, and a total of 6 groups were formed, with 8 mice in each group, including: ① healthy group, ② model group, ③ Lp group: model + Lactobacillus plantarum, ④ Lc group: model + Lactobacillus crispatus, ⑤ Lp&Lc group: model + Lactobacillus plantarum + Lactobacillus crispatus, ⑥ p-Lp&p-Lc group: model + the postbiotic composition of Example 7.

[0090] (2) Drug treatment: Healthy group (C): 100 μL of normal saline was used to rinse the vagina once a day for 14 consecutive days; Model group (M): The vagina was flushed with 100 μL of normal saline, once a day for 14 consecutive days; Lp group (Lp): 1×10 8 The vagina was flushed with CFU concentration of Lactobacillus plantarum-YBR-01 once a day for 14 consecutive days.

[0091] Lc group (Lc): 1×10 8 The vagina was flushed with CFU concentration of Lactobacillus crispatus-YBR-01 once a day for 14 consecutive days.

[0092] Lp&Lc group (Lp&Lc): 1×10 8 CFU Lactobacillus plantarum-YBR-01 and 1 × 10 8 CFU Lactobacillus crispatus-YBR-01 was administered once a day for 14 consecutive days to flush the vagina.

[0093] Postbiotic composition group (p-Lp & p-Lc): The postbiotic composition of Example 7 was used to flush the vagina, once a day, for 14 consecutive days. (Postbiotic treatment elements: Place the medical absorbent gelatin sponge block in the bacterial solution to make it fully contact. After the medical absorbent gelatin sponge is fully infiltrated, hold the mouse tail in the left hand to turn it upside down, gently wipe the skin around the mouse vagina with 75% alcohol, and gently insert the medical absorbent gelatin sponge containing heat-killed bacterial solution into the uterine cavity through the vagina with tweezers, and keep the mouse upside down for 1-2 minutes.) Animal experiment flow chart Fig. 9 .

[0094] 4. Sample collection and testing indicators during the experiment When the mouse vagina was obviously congested, swollen and accompanied by a large amount of purulent secretions, it indicated that the mouse BV model was successfully established, and the vaginal opening and vaginal secretions were scored. The scoring criteria are as follows: 0 points: no redness or swelling at the vaginal opening, and the vaginal opening is slightly open; 1 point: the vaginal opening is slightly red and swollen, and there is a small amount of secretions adhering to the vaginal opening; 2 points: the vaginal opening is red and swollen with purulent secretions.

[0095] The scoring results are shown in Table 2. The scoring results of vaginal opening lesions and vaginal secretions of mice showed that compared with the healthy group, the model group had redness and swelling of the vaginal opening accompanied by purulent secretions. The treatment with the postbiotic composition not only alleviated the redness and swelling of the vaginal opening, but also made the vaginal secretions transparent.

[0096] Table 2 Vaginal lesion scores of mice

[0097] 5. Collection and testing indicators of killed mouse specimens (1) Mouse dissection Before the anatomical experiment, all anatomical instruments were sterilized in advance, cryotubes and specimen bottles were prepared, groups and samples to be loaded were marked in advance, and 75% alcohol and 4% paraformaldehyde were prepared for use. Vaginal lavage fluid was collected 4 days after drug withdrawal, and the mice were killed by removing their eyeballs and taking blood the next day. The blood was collected into a centrifuge tube, and the serum was collected by centrifugation at 3000 rpm for 10 minutes after standing at 37°C for 1 hour and then stored at -80°C. After that, the mice were killed by dislocating the cervical vertebrae, fixing the limbs of the mice, disinfecting the mouse skin with alcohol, cutting open the mouse abdomen, and removing the vagina and uterus and storing them in a -80°C refrigerator.

[0098] (2) Determination of MPO activity The biochemical kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog number A044-1-1) was used to detect the activity of MPO in vaginal tissue (n=3).

[0099] The results of MPO activity assay were as follows Fig.10 As shown in A, the MPO activity in the model group was significantly higher than that in the healthy group, and treatment with the postbiotic composition could significantly reduce the MPO activity.

[0100] (3) qPCR detection The reverse transcription and qPCR reaction system kit (purchased from TaKaRa, catalog number RR047A) was used to detect the levels of IL-1β and IL-10 in vaginal tissue by qPCR according to the instructions (n=3). The specific process is as follows: 1) Weigh the same amount of vaginal tissue, add 1 mL of Trizol, cut into pieces with ophthalmic scissors, and then homogenize with a tissue homogenizer. This process is performed on ice. After sufficient homogenization, centrifuge at 5000 rpm for 10 minutes and transfer the supernatant to a new EP tube; 2) Add 160 μL chloroform, shake thoroughly and centrifuge at 13,000 rpm for 15 min at 4°C. After centrifugation, aspirate the supernatant into a new EP tube, taking care not to aspirate the protein layer; 3) Add an equal volume of isopropanol obtained in step 2), mix thoroughly by inverting, centrifuge at 13,000 rpm for 10 min at 4°C, and discard the supernatant; 4) Add 1 mL of 75% ethanol, wash thoroughly, centrifuge at 13,000 rpm for 10 min in a 4°C centrifuge, and discard the supernatant; 5) Add 1 mL of anhydrous ethanol, shake thoroughly to wash, centrifuge at 13,000 rpm in a 4°C centrifuge for 10 min, and discard the supernatant; 6) Dry at room temperature for 20 minutes; 7) Add 160 μL RNase-Freed Water and place in a 55°C water bath to promote RNA dissolution; 8) Determine RNA concentration; 9) Composition of the reverse transcription system: Step 1: (Genomic DNA Removal Step) Step 1: Add reagents and their amounts, and reaction conditions are shown in Table 3: Table 3 Step 1 Addition of reagents and their amounts, reaction conditions

[0101] Step 2: (Reverse transcription step) Step 2: Add reagents and their amounts, and reaction conditions are shown in Table 4: Table 4 Step 2 Addition of reagents and their amounts, reaction conditions

[0102] After reverse transcription of mRNA into cDNA, it was diluted 3-fold with nuclease-free water and used immediately.

[0103] 10) Use the obtained cDNA as a template for q-PCR reaction.

[0104] The reaction system of q-PCR is as shown in Table 5: Table 5 q-PCR reaction system

[0105] The total reaction system per well was 20.0 μL. The reaction conditions were shown in Table 6. The entire q-PCR process lasted about 71 min.

[0106] Table 6 q-PCR reaction conditions

[0107] The results of qRT-PCR are shown in Fig.10 In Figures B and C, compared with the healthy group, the expression level of IL-1β in the model group was significantly increased, and the postbiotic composition could significantly reduce the expression of these pro-inflammatory factors. The expression of the anti-inflammatory factor IL-10 showed an opposite trend.

[0108] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

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 medium at an inoculum amount of 3.5%-4% v / v, adding β-galactosidase, and continuing fermentation culture to obtain a fermentation liquid; 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, and its preservation number is CGMCC No.26680.

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 preparation method according to claim 1, characterized in that: The amount of β-galactosidase added in step S3 is 0.25%-0.3% w / v.

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

6. Use of the postbiotic composition according to claim 5 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.

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

8. 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 5.

9. A female private care product for improving vaginitis, characterized in that: The female private care product comprises the postbiotic composition according to claim 5.

10. 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 5.

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