CN119326803B

CN119326803B - Preparation method of metagen composition, product and application thereof

Info

Publication number: CN119326803B
Application number: CN202411874426.XA
Authority: CN
Inventors: 陈廷涛; 郭念阳; 丁广鹏; 魏静
Original assignee: Jiangxi Yibairun Biotechnology Co ltd
Current assignee: Jiangxi Yibairun Biotechnology Co ltd
Priority date: 2024-12-19
Filing date: 2024-12-19
Publication date: 2025-06-27
Anticipated expiration: 2044-12-19
Also published as: CN119326803A

Abstract

The present invention provides a preparation method of a postbiotic composition and its products and applications, belonging to the field of biomedical technology. The preparation method of the postbiotic composition provided by the present invention sequentially inoculates activated plant 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 vaginal microecological balance, 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.

Description

Preparation method of metagen composition, product and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method of a metazoan composition, a product and application thereof.

Background

The vaginal flora disorder refers to a group of clinical symptoms mainly represented by abnormal vaginal secretion increase caused by abnormal increase of microorganisms such as escherichia coli, gardnerella vaginalis, staphylococcus aureus, streptococcus peptis, rhodomonas, campylobacter, mycoplasma hominis and candida which are normally present in vagina, and decrease or disappear of lactobacillus generating hydrogen peroxide.

The anti-anaerobic medicines such as metronidazole, tinidazole, clindamycin and the like are generally selected as the treatment modes, but the problems of antibiotic resistance, side effect caused by antibiotics, easy recrudescence, double infection and the like cause limited treatment effects. At present, vaginal lactic acid bacteria capsules are clinically used for improving the dysbacteriosis of the vagina, but the specific mechanism is still unclear and the safety is not guaranteed, so that the clinical application is limited. Therefore, exploring the specific mechanism of lactobacillus for improving the colpitis and searching a new method for improving the colpitis symptoms and overcoming the antibiotic resistance and improving the safety performance are the scientific problems to be solved urgently at present.

The metazoan is a brand new field and can be compared with the "fragment" components of probiotics and metabolites thereof, such as cell surface components, lactic acid, bioactive peptides and the like. At present, researches show that the metazoan acts on intestinal tracts to have various effects of reducing inflammation, promoting waste to pass through the digestive tracts, enhancing metabolic functions, increasing calcium absorption, improving immune functions, preventing type II diabetes mellitus and the like. However, the metazoan has less research on the action on the vagina and overcomes the disadvantages of low safety of the probiotic bacteria, need of bacterial growth or colonization, limited crowd and short and unstable shelf life. The present study therefore uses metazoan as a "superior alternative" to probiotics for the treatment of vaginitis caused by vaginal dysbacteriosis by means of vaginal administration.

The normal vaginal microbiota contains lactobacillus plantarum and lactobacillus crispatus, and has important significance for improving colpitis and restoring vaginal microecological balance. Therefore, a preparation method and a product of the Lactobacillus plantarum and Lactobacillus crispatus metazoan composition are developed, the effect and the specific mechanism of the composition on colpitis caused by vaginal flora disorder are researched, and a new treatment idea can be provided for improving colpitis.

Disclosure of Invention

Based on the problems and deficiencies in the prior art, the present invention is directed to a method for preparing a metacomposition, and products and applications thereof. The preparation method of the metagen composition provided by the invention sequentially inoculates activated lactobacillus plantarum YBR-01 and lactobacillus crispatus YBR-01 in a culture medium, carries out fermentation culture under the action of beta-galactosidase, and prepares the metagen composition through heat inactivation. The metagen composition prepared by the method can improve colpitis by regulating vaginal microecological balance, resisting oxidation, resisting inflammation, enhancing metabolism, and enhancing immunity. Meanwhile, the metagen composition can also overcome antibiotic resistance, has better safety, and provides a new thought for preparing products for improving colpitis.

The technical scheme of the invention is as follows:

in a first aspect, the present invention provides a method for preparing a metacomposition, said method comprising the steps of:

S1, respectively activating lactobacillus plantarum and lactobacillus crispatus;

s2, inoculating the activated lactobacillus plantarum into a culture medium according to the inoculum size of 2% -3% v/v, and fermenting and culturing to obtain a fermentation culture solution;

S3, adding activated lactobacillus crispatus into the fermentation culture solution, inoculating the lactobacillus crispatus into the culture medium according to the inoculum size of 3.5% -4% v/v, adding beta-galactosidase, and continuing fermentation culture to obtain a fermentation solution;

S4, resuspension of the fermentation liquor and inactivation are carried out to obtain a metagen composition;

The lactobacillus plantarum (Lactiplantibacillus plantarum) is lactobacillus plantarum YBR-01 with the preservation number of CGMCC No.26679, and the lactobacillus crispatus (Lactobacillus crispatus) is lactobacillus crispatus YBR-01 with the preservation number of CGMCC No.26680.

In particular, the medium described in steps S2-S3 includes one or more of MRS medium, TPY medium, MC medium, BCP medium.

Preferably, the medium described in steps S2-S3 is MRS medium.

Specifically, the fermentation culture described in step S2 is a culture at 35-39℃for 4-8 hours.

Preferably, the fermentation culture described in step S2 is a culture at 37 ℃ for 6 hours.

Specifically, the fermentation culture described in step S3 is conducted at 35-39℃for 8-12 hours.

Preferably, the fermentation culture described in step S3 is a 37 ℃ culture for 10 hours.

Specifically, the ratio of the inoculation amount of the lactobacillus plantarum to the lactobacillus crispatus is 1:1.15-2.

Preferably, the ratio of the inoculum size of the lactobacillus plantarum to the lactobacillus crispatus is 1.75.

Preferably, the inoculation amount of the lactobacillus plantarum is 2% v/v.

Preferably, the Lactobacillus crispatus is inoculated in an amount of 3.5% v/v.

Specifically, the liquid used for the resuspension in the step S4 comprises one or more of sterile water for injection, sterile physiological saline and sterile PBS.

Preferably, the liquid used for the resuspension described in step S4 is sterile PBS.

Specifically, the inactivation condition in the step S4 is heating at 80-120 ℃ for 10-20min.

Preferably, the inactivation conditions described in step S4 are heating at 80 ℃ for 20min.

In a second aspect, the invention provides a metagen composition prepared by the above preparation method.

In a third aspect, the present invention provides the use of the metacomposition described above in the preparation of a product comprising any one of the following:

(1) Preparation of a medicament for preventing, treating and/or assisting in the treatment of vaginitis, or

(2) Female personal care product for improving colpitis, or

(3) Health care products for anti-inflammatory, antioxidant, enhancing immunity and/or regulating the balance of flora.

Specifically, the product contains the metazoan composition which is more than or equal to 1 multiplied by 10 ⁸ CFU/g.

Preferably, the product contains 1X 10 ⁸-1×10¹² CFU/g metacomposition.

In a fourth aspect, the present invention provides a medicament for the prophylaxis, treatment and/or adjuvant therapy of vaginitis, said medicament comprising a metacomposition as described above.

Specifically, the medicine also comprises pharmaceutically acceptable auxiliary materials.

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

In particular, the dosage forms of the drug include, but are not limited to, parenteral dosage forms and parenteral dosage forms, depending on the mode of administration.

Preferably, the parenteral dosage form includes, but is not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, oils.

Preferably, the parenteral dosage form includes, but is not limited to, injection dosage form, respiratory tract dosage form, skin dosage form, mucosal dosage form and luminal dosage form.

In a fifth aspect, the present invention provides a feminine care article for ameliorating vaginitis, said feminine care article comprising the metacomposition described above.

In particular, the feminine care products include, but are not limited to, cleaning products, care lotions, care gels, sanitary napkins, tampons, or care products.

In a sixth aspect, the present invention provides a health product for anti-inflammatory, antioxidant, immunity enhancing and/or flora balance regulating, said health product comprising the metazoan composition described above.

Specifically, the health care product also comprises a nutrition additive which is acceptable in nutrition.

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

Specifically, the formulation of the health care product comprises tablets, capsules, soft capsules, granules, pills, gel candies, powder, oral liquid or drops.

The beneficial effects of the invention are as follows:

the invention provides a preparation method of a metazoan composition, a product and application thereof, and belongs to the technical field of biological medicines. The preparation method of the metagen composition provided by the invention sequentially inoculates activated lactobacillus plantarum YBR-01 and lactobacillus crispatus YBR-01 in a culture medium, carries out fermentation culture under the action of beta-galactosidase, and prepares the metagen composition through heat inactivation. The metagen composition prepared by the method can improve colpitis by regulating vaginal microecological balance, resisting oxidation, resisting inflammation, enhancing metabolism, and enhancing immunity. Meanwhile, the metagen composition can also overcome antibiotic resistance, has better safety, and provides a new thought for preparing products for improving colpitis.

Preservation description

Biological material name is Lactobacillus plantarum (Lactiplantibacillus plantarum) YBR-01;

classified naming, namely lactobacillus plantarum Lactiplantibacillus plantarum;

The preservation time is 2023, 02 and 27 days;

the preservation number is CGMCC No.26679;

the preservation unit is China general microbiological culture Collection center;

the preservation address is North Star Xili No. 1 and 3 in the Chaoyang district of Beijing city.

Preservation description

Biological material name is Lactobacillus crispatus (Lactobacillus crispatus) YBR-01;

The classification name is Lactobacillus crispatus Lactobacillus crispatus;

The preservation time is 2023, 02 and 27 days;

The preservation number is CGMCC No.26680;

Drawings

FIG. 1 shows the growth curve and microscopic morphology of Lactobacillus plantarum-YBR-01, wherein A shows the growth curve of Lactobacillus plantarum-YBR-01, and B shows the microscopic morphology of Lactobacillus plantarum-YBR-01.

FIG. 2 shows the growth curve and microscopic morphology of Lactobacillus crispatus-YBR-01, wherein A is the growth curve of Lactobacillus crispatus-YBR-01, and B is the microscopic morphology of Lactobacillus crispatus-YBR-01.

FIG. 3 shows the results of acid resistance and cholate resistance of Lactobacillus plantarum-YBR-01, wherein A is the result of acid resistance, and B is the result of cholate resistance.

FIG. 4 shows the results of acid resistance and cholate resistance of Lactobacillus crispatus YBR-01, wherein A is the result of acid resistance and B is the result of cholate resistance.

FIG. 5 shows a hemolysis test of Lactobacillus plantarum YBR-01, wherein the left side of the figure represents Staphylococcus aureus-positive control, and the right side represents Lactobacillus plantarum YBR-01.

FIG. 6 shows a hemolysis test of Lactobacillus crispatus YBR-01, wherein the left side of the figure represents Staphylococcus aureus-positive control and the right side represents Lactobacillus crispatus YBR-01.

FIG. 7 shows the measurement of the resistance to Lactobacillus plantarum YBR-01, wherein 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 milbemycin, and SXT represents compound neonomine.

FIG. 8 shows a drug resistance assay for Lactobacillus crispatus YBR-01, wherein 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 milbemycin, and SXT represents compound neonomine.

Fig. 9 is a flow chart of an animal experiment.

FIG. 10 shows MPO activity, relative IL-1. Beta. And IL-10 expression levels, wherein A is MPO activity, B is IL-1. Beta. Relative expression level, C is IL-10 relative expression level, data are expressed as mean.+ -. Standard deviation, P is represented by 0.05, P is represented by 0.01, and P is represented by 0.001.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of MRS Medium

The MRS culture medium comprises 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g,Tween80 1.0mL g of glucose, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 15.0g of agar powder and 1000mL of deionized water, and is mainly used for culturing lactobacillus by sequentially weighing the components and adding deionized water into 121 ℃ and sterilizing at high temperature and high pressure for 15-20 min.

EXAMPLE 2 microscopic morphology and growth Curve of Lactobacillus plantarum-YBR-01

1. The microscopic morphology observation comprises 1, tabletting, namely uniformly spreading 200 mu L of thallus suspension (1X 10 ⁸ CFU/mL) on a glass slide, then fixing the thallus suspension by using an alcohol lamp flame, wherein the process is to enable a coating surface to face upwards, the glass slide is contacted with the flame for a plurality of times, when the hand is scalded slightly, overheating is prevented, bacteria deformation or dyeing reaction is affected by overheating, 2, dyeing, namely, dropwise adding ammonium oxalate crystal violet for dyeing for 1min, washing and naturally airing, 3, observing, namely, putting the glass slide under an optical microscope, finding a visual field under a low-power mirror, observing the bacterial morphology under a high-power mirror, and observing the bacterial morphology under an oil mirror.

2. Growth curve determination 200. Mu.L of Lactobacillus plantarum YBR-01 (1X 10 ⁸ CFU/mL) was inoculated into 10: 10 mL MRS medium and shake-cultivated at 37 ℃. The Optical Density (OD) at 600nm wavelength was measured every 2 hours for 24 hours. The experiment was repeated three times.

The optical density of the lactobacillus plantarum-YBR-01 bacterial suspension is firstly detected for 24 hours, and the result shows that the bacterial strain enters a logarithmic growth phase after about 2 hours and enters a stationary growth phase after 8 hours (A in figure 1). The Lactobacillus plantarum YBR-01 is in the form of a straight or curved rod under a high-power optical microscope, and exists singly, sometimes in pairs or in a chain (B in FIG. 1).

EXAMPLE 3 microscopic morphology and growth Curve of Lactobacillus crispatus YBR-01

1. Microscopic morphology observation, namely 1) tabletting, namely uniformly spreading 200 mu L of bacterial suspension (1X 10 ⁷ CFU/mL) on a glass slide, then fixing the glass slide by using an alcohol lamp flame, wherein the process is to enable the coating to face upwards, the glass slide is contacted with the flame for a plurality of times, when the hand is slightly scalded, overheating is prevented, bacteria deformation or dyeing reaction is affected by overheating, 2) dyeing, namely dripping ammonium oxalate crystal violet for 1min, washing and naturally airing, and 3) observation, namely putting the glass slide under an optical microscope, looking for a visual field under a low-power mirror, observing the bacterial morphology under a high-power mirror, and also dripping cedar oil under an oil mirror.

2. Growth curve determination 200. Mu.L of Lactobacillus crispatus YBR-01 (1X 10 ⁷ CFU/mL) was inoculated into 10: 10 mL MRS medium and shake-cultured at 37 ℃. The Optical Density (OD) at 600 nm wavelength was measured every 2 hours for 24 hours. The experiment was repeated three times.

The optical density of the bacterial suspension of the strain is firstly tested for 24 hours, and the result shows that the strain enters a logarithmic growth phase for about 6 hours and then gradually enters a stationary growth phase after 14 hours (A in fig. 2). Under high magnification light microscope L.crispatus is a morphologically elongated, curved, slim bacillus (B in FIG. 2).

Example 4 acid and bile salt resistance experiments

1. Acid resistance test

(1) Culturing the preserved Lactobacillus crispatus-YBR-01 or Lactobacillus plantarum-YBR-01 in a 37 ℃ constant temperature incubator with MRS culture medium for 24-36h, and reserving the strain for standby;

(2) PBS buffer solution with pH value of 2, 3, 4, 5 and 7 is prepared, 1mL of bacterial solution which is cultured for 24-36h is taken, centrifugation is carried out for 10min at 3000rpm, the supernatant is discarded, PBS is used for washing twice, 1mL of PBS with different pH values is added for resuspension, a constant temperature incubator with the temperature of 37 ℃ is placed for 4h, and then a plate is diluted for viable bacteria counting. Three replicates of each pH PBS buffer.

2. Experiment of bile salt resistance

(1) Culturing the preserved Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 in a MRS culture medium at 37 ℃ for 24-36h, and reserving the strain for standby;

(2) 10mL of the bacterial liquid cultured for 24 hours was centrifuged at 3000rpm for 10 minutes, the supernatant was discarded, the bacterial liquid was washed twice with PBS, and the bacterial cells were resuspended in 5mL of MRS medium. Adding bile salts with different concentrations to prepare MRS culture solution containing 0%, 0.1%, 0.2% and 0.3%. 200ul of the resuspended bacteria liquid is put into 10mL of prepared culture liquid to be cultured for 24-36 hours at 37 ℃, and then diluted and plated for counting. Each bile salt concentration was replicated in triplicate.

The measurement results are shown in fig. 3-4, and lactobacillus plantarum-YBR-01 has strong acid and bile salt tolerance and can well grow in an acidic environment with ph=3 and 0.1% of bile salt (fig. 3). Lactobacillus crispatus YBR-01 is highly resistant to acids and bile salts and still grows in an acidic environment with ph=3 and 0.1% bile salts (fig. 4).

EXAMPLE 5 hemolysis test

After the lactobacillus plantarum-YBR-01 or lactobacillus crispatus-YBR-01 is subjected to passage activation in an MRS culture medium, 4 mu L of the strain is inoculated on a sterile defibrinated sheep blood plate, and after the strain is cultured for 24 hours in an anaerobic incubator at 37 ℃, whether hemolysis rings appear around colonies is observed. The experiment uses staphylococcus aureus ATCC25923 as a positive control. (alpha hemolysis is formed by incomplete rupture of red blood cells around a colony, beta hemolysis is formed by well-defined and completely transparent hemolysis rings formed by complete rupture of red blood cells around a colony, and gamma hemolysis is formed by no change of culture medium around a colony, namely no hemolysis.

The hemolysis test result of the lactobacillus plantarum-YBR-01 is shown in fig. 5, wherein the left side of the fig. 5 represents staphylococcus aureus, and the right side of the fig. 5 represents the lactobacillus plantarum-YBR-01. The results show that the diameter of the hemolytic circle of staphylococcus aureus can reach about 1-2 cm, while the lactobacillus plantarum-YBR-01 has no obvious hemolytic circle.

The results of the hemolysis test of Lactobacillus crispatus YBR-01 are shown in FIG. 6, wherein the left side of FIG. 6 represents Staphylococcus aureus and the right side of FIG. 6 represents Lactobacillus crispatus YBR-01. The results show that the diameter of the hemolytic circle of staphylococcus aureus can reach about 1-2 cm, while the apparent hemolytic circle of lactobacillus crispatus-YBR-01 is not seen.

EXAMPLE 6 drug resistance

The sensitivity of Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 to CTR-ceftriaxone, CIP-ciprofloxacin hydrochloride, PEN-penicillin, AMP-ampicillin, GEN-gentamicin, C-chloramphenicol, TET-tetracycline, E-polymyxin E, MY-mildy, SXT-Compound Xinnomin was determined using the Kirby-Bauer (K-B) paper sheet method recommended by the American clinical laboratory standardization institute (CLINICAL AND Laboratory Standards Institute, CLSI,2018 edition). 100 mu L of Lactobacillus plantarum-YBR-01 or Lactobacillus crispatus-YBR-01 (1X 10 ⁷ CFU/mL) is coated on a culture medium, and a filter paper sheet containing a drug is placed therein to observe the size of a bacteriostasis zone.

The results of the drug resistance of Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01 are shown in FIG. 7 and FIG. 8, respectively, and it was found that Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01 are sensitive to most antibiotics, while CIP, GEN and MY have weaker killing power against Lactobacillus plantarum-YBR-01 and Lactobacillus crispatus-YBR-01.

Example 7 preparation of metacomposition

1. Activation of strains

Inoculating lactobacillus plantarum-YBR-01 into MRS culture medium for activation, and culturing at 37 ℃ to ensure that the viable count of lactobacillus plantarum-YBR-01 is 1 multiplied by 10 ⁵ CFU/mL for later use.

Inoculating Lactobacillus crispatus-YBR-01 into MRS culture medium for activation, and culturing at 37deg.C to make the viable count of Lactobacillus crispatus-YBR-01 be 1×10 ⁵ CFU/mL for use.

2. Fermentation culture

The activated lactobacillus plantarum-YBR-01 is inoculated in MRS culture medium according to the inoculum size of 2% v/v, fermented at the constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated in MRS culture medium according to the inoculum size of 3.5% v/v, and then 0.275% w/v of beta-galactosidase is added for fermenting at the constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

3. Preparation of the metagen composition:

heating the composite bacterial suspension at 80 ℃ for 20min to prepare heat-inactivated bacterial liquid, namely the metazoan composition.

Example 8 preparation of metacomposition

1. Activation of strains

2. Fermentation culture

The activated lactobacillus plantarum-YBR-01 is inoculated in an MRS culture medium according to the inoculum size of 3% v/v, fermented at a constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated in the MRS culture medium according to the inoculum size of 3.5% v/v, and then 0.25% w/v of beta-galactosidase is added for fermenting at a constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

3. Preparation of the metagen composition:

Example 9 preparation of metacomposition

1. Activation of strains

2. Fermentation culture

The activated lactobacillus plantarum-YBR-01 is inoculated to an MRS culture medium according to the inoculum size of 2% v/v, fermented at a constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated to the MRS culture medium according to the inoculum size of 4% v/v, and then 0.3% w/v of beta-galactosidase is added to the culture medium for fermenting at a constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

3. Preparation of the metagen composition:

Comparative example 1 preparation of metacomposition

Comparative example 1 differs from example 7 only in "2, fermentation culture". The activated lactobacillus plantarum-YBR-01 is inoculated in an inoculum size of 2% v/v, the activated lactobacillus crispatus-YBR-01 is inoculated in an inoculum size of 3.5% v/v in an MRS culture medium, and then 0.275% w/v of beta-galactosidase is added for fermentation at a constant temperature of 37 ℃ for 16 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

Comparative example 2 preparation of metacomposition

Comparative example 2 differs from example 7 only in "2, fermentation culture". The activated lactobacillus plantarum-YBR-01 is inoculated in MRS culture medium according to the inoculum size of 2% v/v, fermented at the constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated in MRS culture medium according to the inoculum size of 3.5% v/v, lactase of 0.275% w/v is added, and the fermentation is carried out at the constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

Comparative example 3 preparation of metacomposition

Comparative example 3 differs from example 7 only in "2, fermentation culture". The activated lactobacillus plantarum-YBR-01 is inoculated in an inoculum size of 2% v/v, the activated lactobacillus crispatus-YBR-01 is inoculated in an inoculum size of 3.5% v/v in an MRS culture medium, lactase of 0.275% w/v is added, and the fermentation is carried out for 16 hours at a constant temperature of 37 ℃. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

Comparative example 4 preparation of metacomposition

Comparative example 4 differs from example 7 only in "2, fermentation culture". The activated lactobacillus plantarum-YBR-01 is inoculated to an MRS culture medium according to the inoculum size of 3.5% v/v, fermented at the constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated to the MRS culture medium according to the inoculum size of 2% v/v, and then 0.275% w/v of beta-galactosidase is added to the culture medium, and the culture medium is fermented at the constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

Comparative example 5 preparation of metacomposition

Comparative example 5 differs from example 7 only in "2, fermentation culture". The activated lactobacillus plantarum-YBR-01 is inoculated in an inoculum size of 3.5% v/v in an MRS culture medium, fermented at a constant temperature of 37 ℃ for 6 hours, then the activated lactobacillus crispatus-YBR-01 is inoculated in the MRS culture medium in an inoculum size of 3.5% v/v, and then 0.35% w/v of beta-galactosidase is added for fermenting at a constant temperature of 37 ℃ for 10 hours. After centrifugation at 3000rpm for 15min, the broth was formulated into a 1X 10 ⁸ CFU/mL complex bacterial suspension with sterile PBS.

Experimental example 1 determination of the hydroxyl radical scavenging ability

1ML of FeSO ₄ solution (2 mmol/L), 1mL of 6mmol/L H ₂O₂ mL, and 1mL of 6mmol/L salicylic acid were added to 1mL of the culture solution of Lactobacillus plantarum-YBR-01 (1X 10 ⁸ CFU/mL), the supernatant of the culture solution of Lactobacillus crispatus-YBR-01 (1X 10 ⁸ CFU/mL), the supernatant of the metacomposition prepared in examples 7 to 9 or comparative examples 1 to 5, respectively. Standing for 30min at room temperature, measuring absorbance at 510nm with deionized water as blank control, and calculating the clearance of hydroxyl free radical = [1-A510 (sample)/A510 (blank) ]. Times.100%. The measurement results are shown in Table 1.

TABLE 1 hydroxyl radical scavenging ability

The measurement results show that the hydroxyl radical clearance of the metagen compositions prepared in the embodiments 7-8 can reach 52.27% -55.40%.

Experimental example 2 Effect of metazoan composition on vaginitis caused by disturbance of vaginal flora

1. Experimental animals' purchase and living environment

Animals were purchased from Hunan Style Lekka laboratory animals Co. BALB/c female mice (SPF grade, 6-8 weeks size, body weight 20-22 g) 48. Mice were free to feed and eat during the experiment. Animal feeding was given standard pellet feed daily. Daily cleaning is performed regularly. Mice were housed in sterile polypropylene cages, 3-5 per cage, under specific pathogen-free conditions. And maintained in a light and dark cycle (L: D,12:12 h) in a room with controlled temperature (22.+ -. 2 ℃) and humidity (50.+ -. 15%), providing a standard diet and water. All mice used in this study were housed a week before the study began to accommodate housing conditions.

2. Animal model building

BALB/c female mice were subcutaneously injected with 2mg/mL of estradiol phthalate injection 0.05mL, 3 times in succession, 1 time for 2 days. On day 6, the vagina of the mouse was flushed 3 times (5 min each time) with sterile PBS (pH8.5), the mixed bacterial solution with concentration of 1X 10 ⁹ CFU/mL was prepared from Streptococcus mutans, staphylococcus aureus and Escherichia coli O157 in a volume ratio of 1:1:2, the medical absorptive gelatin sponge was cut into 0.5cm by 0.5cm, 20. Mu.L of the infectious bacterial solution was injected into the sponge, the sponge was plugged into the vagina of the mouse (about 1cm-1.5cm for the insertion of the mouse, each bacterial injection was 0.025mL/100g,1 time/d), and the estradiol benzoate injection was administered in the same way, and the surgical absorptive hemostatic sponge with 20. Mu.L of physiological saline was plugged into the vagina of the mouse on day 6 as a blank control group.

3. Grouping of animal models and treatment of drugs

(1) Grouping animal models:

The mice were grouped according to their body weight by means of an analysis of variance designed randomly, 6 groups of 8 mice each, ① healthy groups, ② model groups, ③ Lp groups: model+Lactobacillus plantarum, ④ Lc groups: model+Lactobacillus crispatus, ⑤ Lp & Lc groups: model+Lactobacillus plantarum+Lactobacillus crispatus, ⑥ p-Lp & p-Lc groups: model+metazoan composition of example 7.

(2) And (3) drug treatment:

Healthy group (C) in which vagina was flushed with 100. Mu.L of physiological saline, and the administration was 1 time per day for 14 consecutive days;

model group (M) vagina is flushed with 100. Mu.L of physiological saline, and the administration is carried out 1 time a day for 14 days;

Lp group (Lp) the vagina was flushed with Lactobacillus plantarum YBR-01 at a concentration of 1X 10 ⁸ CFU, administered 1 time a day for 14 consecutive days.

Lc group (Lc) vagina was irrigated with Lactobacillus crispatus YBR-01 at a concentration of 1X 10 ⁸ CFU, administered 1 time a day for 14 consecutive days.

Lp & Lc group (Lp & Lc) the vagina was rinsed with 1X 10 ⁸ CFU Lactobacillus plantarum-YBR-01 and 1X 10 ⁸ CFU Lactobacillus crispatus-YBR-01, administered 1 time a day for 14 consecutive days.

The metazoan composition group (p-Lp & p-Lc) the vagina was rinsed with the metazoan composition of example 7, administered 1 time a day for 14 consecutive days. (metazoan treatment element: a square block of a medical absorbent gelatin sponge is placed in a bacterial solution to be brought into sufficient contact therewith, after the medical absorbent gelatin sponge is sufficiently infiltrated, the mouse is held upside down by holding the mouse with the hand, the skin around the vagina of the mouse is gently rubbed with 75% alcohol, the medical absorbent gelatin sponge containing a heat-inactivated bacterial solution is gently passed through the vaginal plug into the uterine cavity with forceps, and the mouse is kept upside down for 1-2 min.)

The animal experiment flow chart is shown in figure 9.

4. Sample collection and detection index during experiment

When the mice vagina was significantly engorged with redness and swelling with a large amount of purulent secretions, it was shown that the mice BV model was successfully prepared and the vaginal orifice and vaginal secretions were scored. The scoring standard is 0 points, namely, no red swelling of the vaginal orifice, normal slight tension of the vaginal orifice, 1 point, slightly red swelling of the vaginal orifice, adhesion of a small amount of secretion on the vaginal orifice, and 2 points, red swelling of the vaginal orifice and purulent secretion.

The scoring results are shown in Table 2, and the results of the vaginal opening lesions and vaginal secretion scoring of the mice indicate that the model group showed vaginal opening reddening and swelling with purulent secretion, and that the post-natal composition treatment not only alleviated the condition of vaginal opening reddening but also the vaginal secretion became transparent, as compared with the healthy group.

Table 2 mouse vaginal orifice lesion score

5. Acquisition and detection index of sacrificed mouse specimen

(1) Dissection of mice

All dissecting instruments are sterilized in advance before the dissection test, a freezing tube and a specimen bottle are prepared, the group and the sample to be filled are marked in advance, and 75% alcohol and 4% paraformaldehyde are prepared for standby. Collecting vaginal lavage fluid after stopping drug for 4d, collecting eyeball blood from the mice on the next day, killing the mice, collecting blood into a centrifuge tube, standing at 37 ℃ for 1h, centrifuging at 3000rpm for 10min, collecting serum, and preserving at-80 ℃. The mice were then sacrificed by cervical vertebrae removal, the limbs of the mice were fixed, the skin of the mice was sterilized with alcohol, the abdomen of the mice was cut, and the vagina and uterus were peeled off and stored in a-80 ℃ refrigerator.

(2) Determination of MPO Activity

Biochemical kit (purchased from south kyo established bioengineering research, all company limited, cat No. a 044-1-1) the activity of vaginal tissue MPO was detected (n=3).

The MPO activity measurement results are shown as A in FIG. 10, and the MPO activity of the model group is obviously higher than that of the healthy group, and the MPO activity can be obviously reduced by the treatment of the metagen composition.

(3) QPCR detection

IL-1β, IL-10 levels (n=3) in vaginal tissue were detected by qPCR according to the instructions using reverse transcription and qPCR reaction system kit (purchased from TaKaRa, cat. RR 047A). The specific process is as follows:

1) The vaginal tissue with the same quality is weighed, added with 1mL of Trizol, sheared by an ophthalmic scissors, homogenized by a tissue homogenizer, fully homogenized on ice, centrifuged at 5000rpm for 10min, and the supernatant is transferred to a new EP tube;

2) Adding 160 mu L of chloroform, fully oscillating and uniformly mixing, centrifuging at 13000rpm for 15min at 4 ℃, and sucking the supernatant into a new EP tube after centrifuging, wherein the supernatant is not sucked into a protein layer;

3) Adding the isopropyl alcohol sucked in the step 2), mixing the isopropyl alcohol with the isopropyl alcohol upside down, centrifuging the mixture for 10 minutes at 13000rpm in a4 ℃ centrifuge, and pouring out the supernatant;

4) 1mL of 75% ethanol was added, after washing thoroughly, the mixture was centrifuged at 13000rpm for 10min in a 4 ℃ centrifuge, and the supernatant was decanted;

5) Adding 1mL of absolute ethyl alcohol, sufficiently oscillating and washing, centrifuging at 13000rpm in a4 ℃ centrifuge for 10min, and pouring out clear liquid;

6) Airing at room temperature for 20min;

7) Adding 160 mu L of RNase-FREED WATER into a water bath kettle at 55 ℃ to promote RNA dissolution;

8) Determining the concentration of RNA;

9) Composition of reverse transcription system:

step 1 (genomic DNA removal step)

The adding amount of the reagent and the reaction conditions in the step 1 are shown in Table 3:

TABLE 3 addition of reagents in step 1, addition amount and reaction conditions

Step 2 (reverse transcription reaction step)

Step2, adding reagent, adding amount and reaction conditions are shown in Table 4:

TABLE 4 addition amount of reagents in step 2 and reaction conditions

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

10 Q-PCR reaction using the obtained cDNA as a template.

The reaction system of q-PCR was as shown in Table 5 below:

TABLE 5 q reaction System for PCR

The total reaction system per well was 20.0. Mu.L. The reaction conditions are shown in Table 6, and the entire q-PCR process was performed for about 71min.

TABLE 6 q reaction conditions for PCR

The qRT-PCR detection results are shown as B and C in FIG. 10, and compared with a healthy group, the expression level of the model group IL-1 beta is obviously increased, and the expression of the pro-inflammatory factors can be obviously reduced by the metacomposite. The expression of the anti-inflammatory factor IL-10 appears to be the opposite trend.

The above detailed description is directed to a specific description of one possible embodiment of the invention, which is not intended to limit the scope of the invention. It should be noted that all equivalent implementations or modifications that do not depart from the spirit and scope of the present invention are intended to be included within the scope of the present invention. The scope of the invention should therefore be determined by 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 Lactobacillus plantarum is Lactobacillus plantarum YBR-01, with a preservation number of CGMCC No. 26679; the Lactobacillus crispatus is Lactobacillus crispatus YBR-01, with a preservation number of 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 comprises 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 described in step S2 is cultured at 35-39°C for 4-8 hours; and the fermentation culture described 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. The use of the postbiotic composition according to claim 4 in preparing a product, characterized in that the product comprises 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.

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

7. A drug for preventing, treating and/or assisting in the treatment of vaginitis, characterized in that the drug 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-oxidant, immunity-enhancing and/or microbiota balance regulation, characterized in that the health product comprises the postbiotic composition according to claim 4.