A composite postbiotic bacterial agent for promoting vaginal microecological balance and its application

Abstract

The present invention relates to a composite postbiotic agent for promoting vaginal microecological balance and its application, wherein the strains in the composite postbiotic agent for promoting vaginal microecological balance include inactivated LA88 strain and inactivated N13 strain. The present invention has developed a new probiotic intervention strategy for promoting vaginal microecological balance, that is, inactivating Lactobacillus acidophilus Lactobacillus acidophilus LA88 strain and inactivated Lactobacillus plantarum Lactobacillus plantarum The N13 strain is compounded to form a new composite postbiotic agent, which has a good proliferation effect on a variety of beneficial vaginal bacteria, a strong inhibitory effect on a variety of vaginal pathogenic bacteria, and can promote the restoration of a healthy microbial community structure.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a composite postbiotic agent for promoting vaginal microecological balance and an application thereof. Background Art

[0002] Under normal circumstances, healthy vaginal microecology is represented by lactobacilli (such as Lactobacillus gasseri, Lactobacillus crispatus, etc.). Beneficial bacteria maintain the acidic environment of the vagina by producing lactic acid, inhibit the growth of pathogenic microorganisms, and become a key line of defense against infection. However, various factors, including but not limited to the abuse of antibiotics, changes in physiological cycles, and lifestyle factors, have seriously disturbed the balance of this microecology, leading to frequent gynecological diseases such as bacterial vaginitis and candidal infection, which seriously affect women's quality of life and mental health. In addition, during menstruation, women's estrogen and progesterone levels decrease, and the alkaline menstrual blood flushing causes the diversity of vaginal flora to increase, the beneficial bacteria to decrease, and the vaginal pathogens take advantage of the situation to enter, which further leads to a significant imbalance in the vaginal microecological environment.

[0003] At present, the strategies used to maintain the balance of vaginal microecology or treat vaginitis-related diseases include antibiotic treatment, compound drug treatment, probiotic preparation intervention, biofeedback therapy, etc. Although antibiotic treatment can effectively kill pathogens, they have the problem of broad-spectrum bactericidal, which may kill a variety of bacteria including beneficial bacteria, leading to dysbiosis of normal flora, causing secondary infection or drug resistance. Long-term or improper use may also affect the microecological balance of the intestines, vagina and other parts, causing side effects such as indigestion and yeast infection; compound drug treatment (such as the combination of antibiotics and prebiotics) attempts to balance the dual goals of bactericidal and growth promotion, but in actual application, the interaction between antibiotics and prebiotics or probiotics is difficult to precisely regulate, which may affect the effect of each other. In addition, the safety and tolerability of long-term use of compound drugs are also key considerations; biofeedback therapy requires patients to actively participate and practice continuously, and has certain requirements for patients' self-management ability. It may not be suitable for all people, and the treatment cycle may be long, and the efficacy is slow to show, requiring patients to have enough patience and perseverance. In addition, the cost and accessibility of professional biofeedback equipment and guidance are also limiting factors; the intervention of live probiotic preparations, its survival rate, etc. is one of the key issues. In comparison, the intervention of inactivated probiotic preparations is a simpler and gentler method.

[0004] Inactivated probiotics have great potential in restoring and maintaining the balance of vaginal microecology. They can be selectively used by beneficial bacteria in the vagina, thereby promoting the proliferation of beneficial bacteria. This type of beneficial bacteria competitively occupies the surface of the host vaginal epithelial cells and consumes nutrients, effectively curbing the growth of harmful vaginal bacteria, thereby achieving positive regulation of the vaginal microecology and ensuring the health of the private parts. In addition, since the probiotics have been inactivated, they will not over-reproduce in the body, are suitable for long-term use, and are not prone to infection risks. Therefore, the development of more high-standard postbiotic intervention methods is of great significance for promoting the balance of vaginal microecology. This method is not only more effective, but also safer, and can better maintain the health of women's private parts. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a composite postbiotic bacterial agent for promoting vaginal microecological balance and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a composite postbiotic agent for promoting vaginal microecological balance, wherein the strains in the composite postbiotic agent for promoting vaginal microecological balance include an inactivated LA88 strain and an inactivated N13 strain;

[0008] The deposit number of the LA88 strain is CGMCC No. 24109, its classification name is Lactobacillus acidophilus, and the deposit date is December 15, 2021;

[0009] The preservation number of the N13 strain is CGMCC No. 20496, its classification name is Lactobacillus plantarum, and the preservation time is August 6, 2020.

[0010] The present invention develops a new probiotic intervention strategy for promoting vaginal microecological balance, that is, inactivated Lactobacillus acidophilus LA88 strain and inactivated Lactobacillus plantarum N13 strain are compounded to form a new composite postbiotic agent, which has a good proliferation effect on a variety of vaginal beneficial bacteria, a strong inhibitory effect on a variety of vaginal pathogens, and can effectively restore a healthy microbial community structure, and the sanitary napkin product further prepared by the composite postbiotic agent shows a significant effect of promoting vaginal microecological balance in a population test. And when the amount of bacteria used is consistent, compared with a single LA88 strain or a single N13 strain, the compounding of the two inactivated bacteria is significantly improved in the above-mentioned efficacy, that is, the LA88 strain and the N13 strain have potential interactions, can cooperate with each other, and synergize in promoting the efficacy of vaginal microecological balance.

[0011] The LA88 strain and the N13 strain in the composite postbiotic bacterial agent involved in the present invention have been inactivated, and will not over-reproduce in the body when used, so they are suitable for long-term use and are not prone to infection risks; secondly, they have strong stability, and the inactivation treatment makes the postbiotic product not restricted by storage conditions and easy to store and transport.

[0012] Preferably, the ratio of the bacterial count of the LA88 strain to that of the N13 strain is 1:5-5:1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 1:2, 1:3, 1:4, 1:5, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0013] Based on the potential interaction between the two strains in promoting the balance of vaginal microecology, the effect is better when the two meet the above-mentioned specific ratio relationship.

[0014] Preferably, the number of bacteria in the composite postbiotic agent is not less than 1×10 8 / g or 1×10 8 Pieces / mL, for example 1×10 8 pieces / g (pieces / mL), 1×10 9 pieces / g (pieces / mL), 1×10 10 pieces / g (pieces / mL), 5×10 10 pieces / g (pieces / mL), 1×10 11 pieces / g (pieces / mL), 3×10 11 pieces / g (pieces / mL), 5×10 11 pieces / g (pieces / mL), 1×10 12 pieces / g (pieces / mL), 1×10 13Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0015] Preferably, the dosage form of the composite postbiotic bacterial agent includes a solution or a powder.

[0016] Preferably, the composite postbiotic agent is in the form of a solution, which can be prepared by the following method:

[0017] The strain is inoculated into a culture medium for activation and fermentation in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged to remove the lower layer of bacterial mud; the bacterial mud is evenly mixed with an aqueous solution containing a protective agent to form an emulsion, and then an inactivation process is adopted to form an inactivated emulsion, or optionally, different probiotic inactivated emulsions are mixed according to the ratio of bacterial cell numbers to obtain the solution.

[0018] Preferably, the composite postbiotic agent is in the form of a powder, which can be prepared by the following method:

[0019] The strain is inoculated into a culture medium for activation and fermentation in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged to remove the lower layer of bacterial mud; the bacterial mud is evenly mixed with an aqueous solution containing a protective agent to form an emulsion; the emulsion is subjected to boiling granulation, crushing and sieving to obtain a powder; or different bacterial powders are optionally mixed according to the ratio of bacterial body numbers to obtain the powder.

[0020] Preferably, the composite postbiotic bacterial agent also contains a functional auxiliary agent.

[0021] Preferably, the functional auxiliary agent includes any one of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, inulin, polydextrose, whey protein or lactoferrin, or a combination of at least two thereof.

[0022] In a second aspect, the present invention provides a use of the composite postbiotic bacterial agent for promoting vaginal microecological balance according to the first aspect in the preparation of a product for promoting vaginal microecological balance.

[0023] Preferably, the product comprises a sanitary napkin, a sanitary panty liner, a sanitary tissue or a vaginal wash.

[0024] In a third aspect, the present invention provides a use of the composite postbiotic agent for promoting vaginal microecological balance according to the first aspect in the preparation of a product for promoting the proliferation of vaginal beneficial bacteria and inhibiting vaginal pathogenic bacteria.

[0025] Preferably, the vaginal beneficial bacteria include any one of Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus iners or Lactobacillus jensenii, or a combination of at least two thereof.

[0026] The vaginal pathogenic bacteria include any one of Escherichia coli, Staphylococcus aureus, Candida albicans or Gardnerella or a combination of at least two thereof.

[0027] The products include sanitary napkins, sanitary pads, sanitary tissues or vaginal washes.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention develops a new probiotic intervention strategy for promoting vaginal microecological balance, that is, inactivated Lactobacillus acidophilus LA88 strain and inactivated Lactobacillus plantarum N13 strain are compounded to form a new composite postbiotic agent, which has a good proliferation effect on a variety of vaginal beneficial bacteria, a strong inhibitory effect on a variety of vaginal pathogenic bacteria, and can accurately regulate and restore a healthy microbial community structure. The sanitary napkin product further prepared by the composite postbiotic agent shows a significant effect of promoting vaginal microecological balance in a population test, which not only meets the privacy and convenience needs of consumers for daily care, but also provides women with a long-term, gentle and natural microecological management method.

[0030] Moreover, when the amount of bacteria used is the same, the combination of the two inactivated bacteria significantly improves the above-mentioned efficacy compared with a single LA88 strain or a single N13 strain. That is, there is potential interaction between the LA88 strain and the N13 strain, and they can cooperate with each other to synergistically enhance the efficacy in promoting the balance of vaginal microecology.

[0031] The LA88 strain and the N13 strain in the composite postbiotic bacterial agent involved in the present invention have been inactivated, and will not over-reproduce in the body when used, so they are suitable for long-term use and are not prone to infection risks; secondly, they have strong stability, and the inactivation treatment makes the postbiotic product not restricted by storage conditions and easy to store and transport.

[0032] The LA88 strain involved in the present invention is classified and named Lactobacillus acidophilus, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation time is December 15, 2021, the preservation number is CGMCC No. 24109, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] The N13 strain involved in the present invention is classified and named Lactobacillus plantarum, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation time is August 6, 2020, the preservation number is CGMCC No. 20496, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0035] The culture medium formula involved in the following examples is as follows:

[0036] MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K 2 PO 4 ·3H 2 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 0.5g / L.

[0037] The LA88 strain involved below is classified and named Lactobacillus acidophilus, the preservation date is December 15, 2021, and the preservation number is CGMCC No. 24109.

[0038] The N13 strain involved below is classified and named Lactobacillus plantarum, the preservation date is August 6, 2020, and the preservation number is CGMCC No. 20496.

[0039] The oligofructose mentioned below is a product of model 95 purchased from Quantum Hi-Tech (Guangdong) Biological Co., Ltd.

[0040] Preparation Example

[0041] The preparation method of the bacterial powder involved in the following embodiment is:

[0042] The strain was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours for activation, and the activation was repeated twice to obtain an activation solution; the activation solution was inoculated into MRS liquid culture medium at an inoculum amount of 3% (v / v), cultured at 37°C, and the bacterial concentration was monitored to obtain a fermentation liquid; the fermentation liquid was centrifuged at 5000 rpm for 5 min at 4°C, and the lower layer of bacterial mud was taken; the bacterial mud and the aqueous solution containing 20% ​​trehalose were mixed uniformly at a volume ratio of 1:1.5 to form an emulsion; the emulsion was subjected to boiling granulation and crushed through an 80-mesh sieve to obtain a bacterial cell count of 1×10 11 Pieces / g of bacterial powder.

[0043] Example 1

[0044] Evaluation of the effect of the product of the present invention on promoting the proliferation of beneficial vaginal bacteria:

[0045] (1) Indicator bacteria:

[0046] Lactobacillus crispatus (No. ATCC33820), Lactobacillus gasseri (No. ATCC19992), Lactobacillus iners (No. ATCC 55195), Lactobacillus jensenii (No. ATCC25258).

[0047] (2) Cultivation of indicator bacteria:

[0048] Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii were cultured in MRS liquid medium at 37°C until the viable count of the indicator bacteria reached 10 8 CFU / mL.

[0049] (3) Test grouping and test sample preparation:

[0050] The experiment was divided into 6 groups:

[0051] (3.1) Control group (no intervention);

[0052] (3.2) LA88 postbiotic agent intervention group (LA88 bacterial powder and oligofructose were mixed evenly at a mass ratio of 60%:40% to obtain a bacterial powder sample);

[0053] (3.3) N13 postbiotic agent intervention group (N13 bacterial powder and oligofructose were mixed evenly at a mass ratio of 60%:40% to obtain a bacterial powder sample);

[0054] (3.4) Composite postbiotic agent intervention group 1 (LA88 bacterial powder, N13 bacterial powder and oligofructose were mixed evenly at a mass ratio of 20%:40%:40% to obtain a bacterial powder sample);

[0055] (3.5) Composite postbiotic agent intervention group 2 (LA88 bacterial powder, N13 bacterial powder and oligofructose were mixed evenly at a mass ratio of 40%:20%:40% to obtain a bacterial powder sample);

[0056] (3.6) Comparison with the postbiotic agent intervention group (the commercially available Lactobacillus acidophilus ATCC4356 powder and the commercially available Lactobacillus plantarum ATCC8014 powder were mixed with oligofructose at a mass ratio of 20%:40%:40% to obtain a powder sample. The preparation method of the powder was based on the above-mentioned preparation example).

[0057] (4) Test methods:

[0058] According to the grouping, the activated indicator bacterial suspension was inoculated in MRS broth containing 0.1% of the bacterial powder samples of each group at a 2% (v / v) inoculation volume and cultured at 37°C for 24 h. Then, a 10-fold serial dilution was performed, and 3 appropriate dilutions were selected and evenly spread on the solid culture medium and cultured at 37°C for 48 h. After the culture was completed, the growth of colonies on the plates with different dilution multiples was observed and the colonies after culture were counted. The number of colonies was calculated and the growth promotion rate was analyzed. The growth promotion rate = (the number of colonies in the experimental group - the number of colonies in the control group) / the number of colonies in the control group × 100%. The results are shown in Table 1.

[0059] Table 1

[0060]

[0061] As can be seen from the data results in Table 1, each postbiotic agent intervention group showed different degrees of proliferation-promoting effects on Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii, and the effect of the composite postbiotic agent intervention group was better, indicating that the composite postbiotic agent involved in the present invention has significant proliferation-promoting properties on beneficial vaginal bacteria.

[0062] Furthermore, this embodiment also prepared a sanitary napkin product loaded with a composite postbiotic agent (the bacterial powder is the bacterial powder sample of the composite postbiotic agent intervention group 1), and the strain loading amount of the surface layer was 10 6 Pieces / cm 2 Select the center area of ​​the surface layer of the sanitary napkin product and cut out 0.5g, 1cm 2 Sterilize the small and large pieces in 100 mL MRS broth for later use.

[0063] According to the grouping, the activated indicator bacteria suspension was inoculated in MRS broth containing sanitary napkin fragments at a 2% (v / v) inoculation volume and cultured at 37°C for 24 h. Then, a 10-fold serial dilution was performed, and 3 appropriate dilutions were selected and evenly spread on the solid culture medium and cultured at 37°C for 48 h. After the culture was completed, the growth of colonies on the plates with different dilution multiples was observed and the colonies after culture were counted. The number of colonies was calculated and the growth promotion rate was analyzed. The growth promotion rate = (the number of colonies in the experimental group - the number of colonies in the control group) / the number of colonies in the control group × 100%. The results are shown in Table 2.

[0064] Table 2

[0065]

[0066] As shown in the data results in Table 2, whether the composite postbiotic agent is applied directly or loaded through sanitary napkins, it can effectively increase the number of beneficial bacteria in the female vagina, verifying its potential value as a microecological regulator.

[0067] Example 2

[0068] Evaluation of the antibacterial effect of the product of the present invention on vaginal pathogens:

[0069] (1) Indicator bacteria:

[0070] Escherichia coli (No. ATCC25922), Staphylococcus aureus (No. ATCC29213), Candida albicans (No. ATCC10231), Gardnerella (No. ATCC 14018).

[0071] (2) Cultivation of indicator bacteria:

[0072] Staphylococcus aureus, Escherichia coli, Candida albicans, and Gardnerella were cultured in LB medium, nutrient agar medium, SDA medium, and blood agar medium, respectively. The culture medium was diluted so that the number of viable indicator bacteria was 10 6 CFU / mL.

[0073] (3) Test grouping and test sample preparation:

[0074] The experiment was divided into 6 groups:

[0075] (3.1) Control group (no intervention);

[0076] (3.2) LA88 postbiotic agent intervention group (LA88 bacterial powder and oligofructose were mixed evenly at a mass ratio of 60%:40% to obtain a sample);

[0077] (3.3) N13 postbiotic agent intervention group (N13 bacterial powder and oligofructose were mixed evenly at a mass ratio of 60%:40% to obtain a sample);

[0078] (3.4) Composite postbiotic agent intervention group 1 (mix LA88 bacterial powder, N13 bacterial powder and oligofructose at a mass ratio of 20%:40%:40% to obtain a sample);

[0079] (3.5) Composite postbiotic agent intervention group 2 (mix LA88 bacterial powder, N13 bacterial powder and oligofructose at a mass ratio of 40%:20%:40% to obtain a sample);

[0080] (3.6) Comparison with the postbiotic agent intervention group (the commercially available Lactobacillus acidophilus ATCC4356 powder and the commercially available Lactobacillus plantarum ATCC8014 powder were mixed with oligofructose at a mass ratio of 20%:40%:40% to obtain a sample. The preparation method of the powder was referred to the above-mentioned preparation example).

[0081] (4) Test methods:

[0082] For the control group and the experimental group (1% sample dilution), 5 mL of the corresponding diluted indicator bacteria solution was added to 5 mL of the sample dilution solution according to the grouping, and the solution was placed in a 37°C constant temperature incubator for shaking culture for 15 min. After the culture was completed, the bacterial solution was diluted 10 times in a row with sterile PBS solution, and 100 μL of the dilution solution was evenly spread on LB solid medium, nutrient agar solid medium, SDA solid medium, and blood agar solid medium. The culture was placed in a 37°C constant temperature incubator for 18 h, and the number of colonies was recorded. The number of colonies was calculated and the inhibition rate was analyzed. The inhibition rate = (the number of colonies in the control group - the number of colonies in the experimental group) / the number of colonies in the control group × 100%. The results are shown in Table 3.

[0083] Table 3

[0084]

[0085] As can be seen from the data results in Table 3, each postbiotic agent intervention group showed different degrees of antibacterial efficacy against Escherichia coli, Staphylococcus aureus, Candida albicans and Gardnerella, and the effect of the composite postbiotic agent intervention group was better, indicating that the composite postbiotic agent involved in the present invention has significant antibacterial properties against vaginal pathogens.

[0086] Example 3

[0087] Evaluation of the efficacy of probiotic sanitary napkins among different groups:

[0088] (1) Volunteer inclusion and exclusion criteria:

[0089] (1.1) Age 20-40 years old;

[0090] (1.2) Healthy volunteers who meet the trial requirements;

[0091] (1.3) The menstrual cycle is stable, and the error between the predicted menstrual start date and the actual date is no more than 3 days;

[0092] (1.4) Fully understand the trial process, voluntarily participate in the trial and sign the written informed consent;

[0093] (1.5) Subjects with the following conditions should be excluded:

[0094] ① Patients with systemic diseases or who are receiving drug treatment (anti-tumor, antibiotics, hormones, etc.) in the past month; ② Patients with female diseases (bacterial vaginitis) and are receiving treatment (oral medications, external objects), or patients who are judged to affect the efficacy evaluation of this project; ③ Pregnant women and those who have participated in other clinical researchers in the past month; ④ Products used to clean the vulva / inner vulva other than water / shower gel / soap (without antibacterial effect) within 1 month before the trial, including related antibacterial cleaning wipes; ⑤ People with sensitive skin and allergies to chemical fiber materials are not suitable for participating in this trial; ⑥ People who are judged by the trial leader / research doctor to be unsuitable for participating in this test project.

[0095] The final number of subjects was 30.

[0096] (2) Test samples:

[0097] LA88 bacterial powder, N13 bacterial powder and oligofructose were mixed evenly at a mass ratio of 20%:40%:40% to obtain a composite postbiotic agent. 6 Pieces / cm 2 Loaded on the surface layer of sanitary napkin (total load is 5×10 8 strains), sterilized, and obtained probiotic sanitary napkins for use by 30 subjects during their menstrual period.

[0098] (3) Test time:

[0099] The trial lasted for 3 months.

[0100] (4) Sample collection process:

[0101] (4.1) Sampling requirements: Subjects are required to abstain from sexual intercourse within 48 hours before sampling, and are not allowed to excessively clean their private parts (they can take a shower normally), wipe with wet wipes, apply medication, or other behaviors that change the flora structure. They are also not allowed to use antibiotics or antifungal drugs within 30 days;

[0102] (4.2) Sampling site: 4 cm inside the vaginal opening, on the inner wall or posterior fornix;

[0103] (4.3) Sampling process: Genital secretion samples were collected before and after the intervention. The sampling steps are as follows:

[0104] Use a sterile sampling swab to insert 4 cm into the inner wall or posterior fornix of the vaginal opening, stay for 10-15 seconds, and rotate 2-3 times to collect the specimen. After sampling, the sample is recorded, stored, and transported according to one of the following situations:

[0105] ① Quickly put the sampling swab back into the sampling tube preservation solution, break off the swab handle, tighten the tube cap and seal it for storage; the swab should not touch the skin after being taken out to avoid contamination;

[0106] ②Record the relevant information of the sample (sample number, sampling time, etc.);

[0107] ③For the preservation tubes containing stabilizing liquid, if the preservation tubes can be stored and transported at room temperature according to the operating instructions, volunteers can store them at room temperature and send the samples to the recruitment unit within 1-2 days. The recruiting party will mail the collected samples within 15-20 days after the sampling time (the final time can be determined based on the instructions for the preservation time of the preservation tubes at room temperature). During the mailing process, the sample temperature should be maintained as much as possible and should be avoided from light (a layer of tin foil can be added to the outer packaging of the sample and a small amount of ice packs can be added to ensure the temperature during the mailing process).

[0108] (5) Detection method:

[0109] 16S rDNA amplicon sequencing:

[0110] (5.1) Total DNA extraction: DNA was extracted from the sample using a DNA extraction kit and stored at -20°C for further analysis. DNA concentration was measured using a DNA detection kit.

[0111] (5.2) PCR amplification and library construction: 20-50 ng DNA was used as template and a series of designed PCR primers (provided by Suzhou Jinweizhi Biotechnology Co., Ltd.) were used to amplify two highly variable regions including V3 and V4 on the 16S rDNA of prokaryotes. In addition, a linker with an index was added to the end of the PCR product of 16S rDNA by PCR for NGS sequencing. Finally, the PCR product library was electrophoresed by 1.5% agarose gel to detect the target fragment at 600 bp.

[0112] (5.3) Sequencing: Detect the library concentration using an ELISA reader, and perform PE250 / FE300 double-end sequencing according to the instruction manual of the high-throughput sequencing instrument to obtain the original sequencing data.

[0113] (6) Test results:

[0114] In this study, a total of 30 volunteers were initially included, but 29 of them completed the test process, and 1 participant withdrew for special reasons and was not included in the final evaluation. The results showed that the average relative abundance of Lactobacillus in the vagina of 23 volunteers (accounting for 79.3% of those who completed the trial) reached 98%, indicating that their vaginal microecology was in a healthy state, and the composition of the flora did not show significant differences after using the product. The detailed data of the remaining 6 volunteers in sub-healthy state after using the product are shown in Table 4 (Comparison of relative abundance of Lactobacillus after three menstrual periods), Table 5 (Comparison of relative abundance of Gardnerella after three menstrual periods) and Table 6 (Comparison of relative abundance of Staphylococcus aureus after three menstrual periods).

[0115] Table 4

[0116]

[0117] Table 5

[0118]

[0119] Table 6

[0120]

[0121] It can be seen from the results in Tables 4 to 6 that the relative abundance of lactobacilli (beneficial bacteria) in the vagina of the six sub-healthy volunteers who adopted intervention measures for three consecutive months was significantly increased (Table 4). At the same time, the relative content of major potential pathogenic bacteria such as Gardnerella and Staphylococcus aureus was also reduced (Tables 5 to 6), indicating that the probiotic product involved in the present invention has a positive effect on promoting the balance of vaginal microecology.

[0122] In summary, the three-month population efficacy evaluation study of probiotic sanitary napkins revealed that after three months of continuous use of sanitary napkins, sub-healthy subjects can significantly regulate the host vaginal microecological system, specifically by effectively increasing the relative abundance of beneficial bacteria Lactobacillus, while achieving a reduction in the abundance of pathogenic bacteria, such as Gardnerella, Staphylococcus aureus and other potential pathogenic bacteria.

[0123] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0124] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0125] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A composite postbiotic bacterial agent for promoting vaginal microecological balance, characterized in that: The strains in the composite postbiotic bacterial agent for promoting vaginal microecological balance are inactivated LA88 strain and inactivated N13 strain; The deposit number of the LA88 strain is CGMCC No. 24109, and its classification name is Lactobacillus acidophilus Lactobacillus acidophilus , the preservation time is December 15, 2021; The deposit number of the N13 strain is CGMCC No. 20496, and its classification name is Lactobacillus plantarum Lactobacillus plantarum , the preservation time is August 6, 2020; The ratio of the bacterial count of the LA88 strain to that of the N13 strain is 1:2-2:

1.

2. The composite postbiotic bacterial agent for promoting vaginal microecological balance according to claim 1, characterized in that: The number of bacteria in the composite postbiotic agent is not less than 1×10 8 / g or 1×10 8 Pieces / mL.

3. The composite postbiotic bacterial agent for promoting vaginal microecological balance according to claim 1, characterized in that: The dosage form of the composite postbiotic bacterial agent includes a solution or a powder.

4. The composite postbiotic bacterial agent for promoting vaginal microecological balance according to claim 1, characterized in that: The composite postbiotic bacterial agent also contains a functional auxiliary agent.

5. The composite postbiotic bacterial agent for promoting vaginal microecological balance according to claim 4, characterized in that: The functional auxiliary agent includes any one of fructooligosaccharide, galacto-oligosaccharide, xylooligosaccharide, isomaltooligosaccharide, inulin, polydextrose, whey protein or lactoferrin, or a combination of at least two of them.

6. Use of the composite postbiotic bacterial agent for promoting vaginal microecological balance according to any one of claims 1 to 5 in the preparation of a product for promoting vaginal microecological balance; The products include sanitary napkins, sanitary pads, sanitary tissues or vaginal washes.

7. Use of the composite postbiotic agent for promoting vaginal microecological balance according to any one of claims 1 to 5 in the preparation of a product for promoting the proliferation of vaginal beneficial bacteria and inhibiting vaginal pathogenic bacteria; The vaginal beneficial bacteria are any one of Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus iners or Lactobacillus jensenii, or a combination of at least two thereof; The vaginal pathogenic bacteria is any one of Escherichia coli, Staphylococcus aureus, Candida albicans or Gardnerella or a combination of at least two thereof; The products include sanitary napkins, sanitary pads, sanitary tissues or vaginal washes.

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