Lactobacillus acidophilus la88 probiotic composition and use in the manufacture of a product for modulating the vaginal microflora

Abstract

The present application relates to the technical field of probiotics and its application, and particularly relates to a lactobacillus acidophilus LA88 postbiotic composition and application in preparation of a product for regulating vaginal microecology.The present application provides a postbiotic based on lactobacillus acidophilus LA88 (preservation number: CGMCC No. 24109), and a specific proportion of prebiotic combination is added to construct a microecological regulator with synergistic effect, which is especially suitable for regulating the microecological environment of female vagina.The postbiotic is in the form of inactivated lactobacillus, which improves the safety and stability of the product, and the postbiotic contains rich bioactive components, which can directly regulate the local immune environment of the vagina, and the prebiotic combination promotes the rapid proliferation of beneficial bacteria through nutrient supply and metabolic regulation.The technical scheme can solve the technical problems of safety and unsatisfactory curative effect of probiotics for regulating the vaginal microecosystem, and has an ideal application and promotion prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics and their applications, and particularly relates to a lactobacillus acidophilus LA88 postbiotic composition and application thereof in preparing a product for regulating vaginal microecology. BACKGROUND

[0002] The vaginal microecosystem is an important barrier for female reproductive health, which is a dynamic balance system composed of multiple microorganisms and plays a key role in maintaining vaginal health. Under normal circumstances, the beneficial flora (such as lactobacilli) in the vagina can effectively inhibit the colonization and proliferation of pathogenic microorganisms through positional protection and biological antagonism, maintain the pH of the vaginal environment, and thus maintain a healthy vaginal environment. This microecological balance is crucial for preventing various gynecological diseases.

[0003] However, the microecological flora in the vagina is not constant. Environmental changes, antibiotic use, fluctuations in hormone levels, and changes in the host's immune status can all lead to changes in the structure of the vaginal microbial community, and thus cause microbial imbalance. For example, frequent use of broad-spectrum antibiotics can inadvertently destroy the beneficial flora in the vagina, allowing drug-resistant or opportunistic pathogens to proliferate, leading to diseases such as bacterial vaginosis and candida infection.

[0004] Although live probiotics are widely believed to help restore vaginal flora balance, their application has certain limitations and potential risks. First, the preservation conditions of live bacteria products are relatively harsh, requiring specific temperature and humidity to maintain strain activity, which not only increases production costs but also raises higher requirements for storage and transportation. Second, even if live probiotics are successfully introduced into the human body, these microorganisms may have difficulty surviving and functioning due to the complex environmental factors in the body, and may even cause unnecessary side effects or infection risks, especially in individuals with low immunity. In addition, the use of probiotics alone has limited effect on restoring vaginal flora balance. Simply relying on probiotics is difficult to build a stable microecological environment and achieve the desired microecological regulation effect. Therefore, when designing products for restoring vaginal microecological balance, in addition to considering how to effectively introduce beneficial microorganisms, attention should also be paid to providing necessary nutritional support for these microorganisms to ensure that they can function long-term and stably in the body.

[0005] In summary, the vaginal microecosystem is essential for female reproductive health, and its dynamic balance is affected by various internal and external factors. Although live probiotics are considered a potential tool to help restore vaginal microecological balance, their practical application faces challenges such as stringent preservation conditions, survival challenges in the body, and limited effectiveness when used alone. Therefore, the development of microecological modulators based on postbiotics has important clinical value and application prospects. This intervention is not only safer, but also more gentle than drug therapy, providing a new solution for maintaining women's vaginal health. SUMMARY

[0006] The present application aims to provide a Lactobacillus acidophilus LA88 postbiotic composition to solve the technical problems of safety and unsatisfactory efficacy of probiotics for regulating the vaginal microecosystem in the prior art.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A Lactobacillus acidophilus LA88 postbiotic composition comprises a combination of Lactobacillus acidophilus LA88 postbiotics and prebiotics; the prebiotic combination comprises at least one of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin; the preservation number of Lactobacillus acidophilus LA88 is CGMCC No. 24109.

[0009] Further, the total bacterial concentration of Lactobacillus acidophilus LA88 postbiotics is 1x10 11 -3x10 11 individuals / g; preferably 1x10 11 individuals / g.

[0010] Further, the mass ratio of the combination of Lactobacillus acidophilus LA88 postbiotics and prebiotics is 80:20-99:1 (preferably 85:15); the prebiotic combination consists of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin.

[0011] Further, the prebiotic combination consists of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin in a mass ratio of 1-3:1-3:1-3:1-3;

[0012] Preferably, the mass ratio of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin is 1:2:2:3.

[0013] The present technical solution also provides a preparation method of Lactobacillus acidophilus LA88 postbiotics, wherein a prebiotic combination is added to Lactobacillus acidophilus LA88 postbiotics, and the postbiotics with added prebiotics are obtained after mixing; the prebiotic combination comprises acarbose, fructooligosaccharides, xylooligosaccharides, and inulin.

[0014] The technical scheme also provides application of the Lactobacillus acidophilus LA88 probiotic composition in preparation of a product for regulating a vaginal micro-ecosystem, and the product comprises a sanitary napkin, a sanitary pad, a sanitary napkin or a vaginal lotion; and the product is used for promoting proliferation of beneficial bacteria and inhibiting proliferation of harmful bacteria.

[0015] Further, the product for regulating the vaginal micro-ecosystem is a sanitary napkin; the Lactobacillus acidophilus LA88 probiotic is mixed with a carboxymethyl cellulose solution, and then is loaded on a surface layer of the sanitary napkin; and the loading amount is 10 4 individual / cm 2 .

[0016] In summary, the technical principle of the technical scheme is that:

[0017] The core of the application is to provide a probiotic (micro-kang microbial preparation) prepared based on Lactobacillus acidophilus LA88 (preservation number CGMCC No. 24109), and to construct a functional micro-ecological regulator with synergistic effect by adding a specific proportion of a prebiotic combination, which is especially suitable for regulating the vaginal micro-ecological environment of women.

[0018] Lactobacillus acidophilus is one of the main lactic acid bacteria naturally existing in the human body, and plays an important role in maintaining the stability of the vaginal pH value and inhibiting the growth of pathogenic microorganisms. Although traditional live bacterial preparations can improve the problem of imbalance of the vaginal micro-ecosystem to a certain extent, they have high requirements for storage and transportation conditions, and have problems such as low in-vivo survival rate, high immune risk and the like. The probiotic of Lactobacillus acidophilus LA88 used in the application is a bacterium powder after inactivation treatment, which avoids the uncontrollability of ectopic colonization of live bacteria, improves the safety, and still has good functions of immune regulation, antibiosis, anti-inflammation and adhesion protection, and can directly participate in the regulation of the local micro-environment of the vagina.

[0019] In order to enhance the biological function of the probiotic, the application further introduces a prebiotic combination composed of acarbose, fructooligosaccharide, xylooligosaccharide and inulin. The combination not only provides nutritional support for beneficial bacteria (such as lactic acid bacteria and bifidobacteria) remaining in the vagina, promotes the colonization and reproduction of the beneficial bacteria, but also realizes multiple synergistic effects. Through structural complementation and metabolic pathway synergy, the prebiotics form a "prebiotic network", which significantly improves the overall regulation efficiency and helps to build a stable and healthy vaginal micro-ecosystem.

[0020] The present application combines Lactobacillus acidophilus LA88 probiotics with the above-mentioned prebiotics, realizing the transition from "passive regulation" to "active construction". The probiotics provide immediate antibacterial, anti-inflammatory and immune-regulating signals; the prebiotics provide sustained nutritional support for the host's original beneficial bacteria, promoting their rapid proliferation; under the synergistic action of the two, the vaginal microecological structure dominated by lactobacillus can be effectively restored, the acid environment dominated by lactic acid can be reconstructed, the pH value can be reduced, and the growth of pathogenic bacteria can be inhibited. This "probiotics + prebiotics" compound strategy breaks through the limitations of traditional single live bacterial preparations, and takes into account safety, effectiveness and sustainability, representing the development direction of the new generation of microecological regulators.

[0021] The beneficial effects of the technical solution are:

[0022] (1) High safety: the inactivated Lactobacillus acidophilus LA88 probiotics do not contain active microorganisms, avoiding potential risks such as infection, drug resistance gene transfer or abnormal immune response caused by live bacteria entering the body, and are particularly suitable for female populations with low immune function or in special physiological periods (such as pregnancy and menstrual period).

[0023] (2) Strong regulation ability: the probiotics have complete cell structure and microbial metabolites, which can directly regulate the local immune environment of the vagina and inhibit the adhesion and colonization of pathogenic bacteria; at the same time, the prebiotic combination promotes the rapid proliferation of beneficial bacteria through nutrient supply and metabolic regulation, and accelerates the recovery of the vaginal microecosystem.

[0024] (3) Significant synergistic effect: the synergistic effect of multiple prebiotics can enhance the selective promotion of lactobacillus, forming a regulation system with functional superposition and long-lasting effect, which is significantly better than the use effect of single prebiotics or probiotics.

[0025] (4) Wide application scenarios: the probiotics can be applied to various gynecological care products such as sanitary napkins, pads, paper towels and lotions, and can realize continuous intervention during daily use by being loaded on the surface of the product, which is convenient and practical and easy to accept.

[0026] In summary, the present application optimizes the combination ratio of probiotics and prebiotics, and constructs a new type of vaginal microecological regulator that is safe and efficient, stable and controllable, and has strong adaptability, filling the gap in the use of probiotics in the field of female intimate care in the prior art, and having important scientific research value and industrial application prospect. DETAILED DESCRIPTION

[0027] The present application will be further described in detail below in conjunction with examples, but the embodiments of the present application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

[0028] Example 1: Preparation of Lactobacillus acidophilus LA88 postbiotic containing prebiotic composition

[0029] Lactobacillus acidophilus LA88 postbiotic is purchased from Weikang Probiotics (Suzhou) Co., Ltd. and is a microbial cell and its components of Lactobacillus acidophilus LA88 after inactivation treatment, in the form of dry bacterial powder. The trade name of Lactobacillus acidophilus LA88 postbiotic is: Probiotic Bacterial Powder (Sterilized Type), the product specification is: 1x10 11 9 / g, 1 kg / bag, the model is Lactobacillus acidophilus LA88, and the commodity packaging form is inner PE bag and outer oxygen and water resistant aluminum foil bag packaging. The product quality inspection report shows that the number of lactic acid bacteria in the product is about 1.0x10 11 9 / g. Probiotic Bacterial Powder (Sterilized Type) is essentially Lactobacillus acidophilus LA88 postbiotic, and when used, the concentration of the bacterial powder can be 1x10 11 9 / g-3x10 11 9 / g. Hereinafter, the Probiotic Bacterial Powder (Sterilized Type) used in the experimental study is referred to as Lactobacillus acidophilus LA88 postbiotic, and the concentration of the lactic acid bacteria in the subsequent experimental study is 1x10 11 9 / g.

[0030] Among them, Lactobacillus acidophilus LA88 (Lactobacillus acidophilus Lactobacillus acidophilus LA88 strain) is a strain owned by Weikang Probiotics (Suzhou) Co., Ltd. (see patent CN114591875B), which was originally collected from Dali Bai Autonomous Prefecture, Yunnan, and is now preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 24109. The company produces a product - Probiotic Bacterial Powder (Sterilized Type) - of Lactobacillus acidophilus LA88 after conventional heat inactivation treatment, which is purchased for further research and development of new products.

[0031] In the Lactobacillus acidophilus LA88 postbiotic with a concentration of 1x10 11 9 / g, the prebiotic composition is added and mixed uniformly to obtain a postbiotic containing prebiotic. The prebiotic composition is composed of acarbose, fructooligosaccharide, xylooligosaccharide and inulin. In order to ensure product quality, acarbose, fructooligosaccharide, xylooligosaccharide and inulin are all subjected to conventional irradiation sterilization treatment. The ratio of sterilized probiotic bacterial powder to prebiotic is 80:20-99:1 (preferably 85:15). The mass ratio of acarbose, fructooligosaccharide, xylooligosaccharide and inulin is 1-3:1-3:1-3:1-3 (preferably 1:2:2:3).

[0032] More specifically, the prebiotic composition is introduced as follows:

[0033] The systematic name of acarbose is:

[0034] O-4,6-dideoxy-4[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohexen-1-yl]amino-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranoside;

[0035] Molecular formula: C 25 H 43 NO 18 ; molecular weight: 645.6 g / mol; CAS number: 56180-94-0.

[0036] Fructooligosaccharides (FOS): a class of functional oligosaccharides composed of 2-9 fructose molecules connected by β(2→1) glycosidic bonds, with glucose as the terminal unit (GFn type)

[0037] Xylooligosaccharides (XOS): composed of 2-7 xylose molecules connected by β(1→4) glycosidic bonds.

[0038] Inulin: composed of 10-60 fructose units connected by β(2→1) glycosidic bonds, with glucose as the terminal unit (GFn type).

[0039] The above substances can be obtained by commercial means and are known in the prior art.

[0040] Experimental Example 1: Acid tolerance of Lactobacillus acidophilus LA88 probiotics

[0041] Take Lactobacillus acidophilus LA88 probiotics (micro-kangyi probiotic powder (sterilization type)), and prepare the probiotics containing prebiotics (sterilization type probiotic powder + prebiotic composition) according to the optimal technical parameters of Example 1. Resuspend Lactobacillus acidophilus LA88 probiotics or probiotics containing prebiotics in sterile saline to obtain a resuspension (bacterial concentration is 1×10 9CFU / mL or so), 1.0 mL of the resuspension was taken into 9.0 mL of sterile PBS of different pH (pH 2.0, pH 3.0, pH 4.0, pH 5.0) and left at room temperature, and samples were taken at the beginning (0 h) and after treatment (3 h), respectively, and the number of bacteria was determined by hemocytometry and the tolerance rate was calculated, with the formula as follows: tolerance rate (%) = N1 / N0 x 100%, wherein N1: the number of bacteria after 3 h of treatment with sterile PBS of different pH; N0: the number of bacteria at 0 h. The test results are shown in Table 1, and it can be seen that the Lactobacillus acidophilus LA88 postbiotic and the postbiotic containing prebiotics have good acid tolerance, and the tolerance rate basically remains about 100% in the environment of pH 3.0-5.0 for 3 h. The normal healthy female vaginal pH is about 4.0, and thus it can be seen that the Lactobacillus acidophilus LA88 postbiotic has good acid tolerance and can normally colonize in the vagina.

[0042] Table 1: Test results of the acid tolerance of Lactobacillus acidophilus LA88 postbiotic

[0043]

[0044] Experimental Example 2: Adhesion of Lactobacillus acidophilus LA88 postbiotic to vaginal epithelial cells

[0045] Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder) and postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition) prepared according to the optimal technical parameters of Example 1 were taken for experimental study. Lactobacillus acidophilus LA88 postbiotic (No. 2 in Table 2), postbiotic containing prebiotics (prebiotic being a combination of acarbose, fructooligosaccharide, xylooligosaccharide and inulin, No. 1 in Table 2; or, prebiotic being a combination of fructooligosaccharide, xylooligosaccharide and inulin, No. 4 in Table 2; or, prebiotic being acarbose alone, No. 5 in Table 2), commercially available Lactobacillus acidophilus live bacteria (frozen bacteria were activated by routine method, ATCC4356, No. 3 in Table 2) were resuspended with RPMI 1640 medium (Gibco Company) and adjusted to a bacterial concentration of 1 x 10 8The Lactobacillus acidophilus LA88 probiotic powder was purchased. The probiotic containing prebiotics (the prebiotics were a combination of acarbose, fructo-oligosaccharides, xylo-oligosaccharides and inulin) was prepared according to the optimal technical parameters of Example 1, which was the optimal preparation method. The probiotic containing prebiotics (the prebiotics were a combination of fructo-oligosaccharides, xylo-oligosaccharides and inulin) was prepared as follows: on the basis of the Lactobacillus acidophilus LA88 probiotic powder, the prebiotics were added in a ratio of 85:15, wherein the prebiotics were composed of fructo-oligosaccharides, xylo-oligosaccharides and inulin in a ratio of 2:2:3. That is, on the basis of the optimal preparation method of the probiotic containing prebiotics, the combination of four prebiotics was replaced with an equal amount of a combination of three prebiotics. The preparation method of the probiotic containing prebiotics (the prebiotics were acarbose) was as follows: on the basis of the optimal preparation method of the probiotic containing prebiotics, the combination of four prebiotics was replaced with an equal amount of acarbose.

[0046] The human vaginal epithelial cells VK2 / E6E7 (ATCC-CRL2616) were suspended in 10 ml of RPMI 1640 and cultured in a cell incubator at 37°C and 5% CO2, and the concentration of the vaginal epithelial cells was adjusted to 1 x 10 5 The sample suspension was adjusted to a final concentration of 2 x 10 7 Then, 1 mL of the bacterial solution and an equal amount of the vaginal epithelial cell suspension were mixed, and cultured at 37°C for 24 h. Centrifugation was performed at 1000 r / min for 5 min, and PBS buffer was used for washing 3 times. The bacterial solution not adhered to the vaginal epithelial cells was discarded, and the precipitate was smeared, dried, Gram-stained, and observed under an oil immersion lens. The number of bacteria adhered to the epithelial cells was counted randomly in 50 epithelial cells, and the average value and the adhesion index were calculated. The adhesion index was calculated as follows: total number of bacteria adhered to 50 epithelial cells / 50. The experimental results are shown in Table 2, which shows that the adhesion ability of the Lactobacillus acidophilus probiotic to the vaginal epithelial cells was basically the same as that of the live bacteria, and was stronger than that of the commercially available Lactobacillus acidophilus.

[0047] Table 2: Test results of the adhesion of different samples to vaginal epithelial cells (n=10, the average value of 10 tests of the VK2 / E6E7 cell adhesion coefficient)

[0048]

[0049] According to the experimental data shown in Table 2, it can be seen that the adhesion ability of Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder) used in the present solution to human vaginal epithelial cells is significantly better than that of commercially available Lactobacillus acidophilus (group No. 2 vs. group No. 3, t-test, p<0.05). Compared with live bacterial preparations, the postbiotic form avoids the potential safety problems brought by the introduction of live bacteria, and is more conducive to storage and transportation.

[0050] On this basis, the inventors further added prebiotic components to the bactericidal probiotic powder in order to enhance its functional performance. The experimental results show that when the prebiotic composition is composed of acarbose, fructooligosaccharides, xylooligosaccharides and inulin, the prebiotic-containing postbiotic prepared thereby exhibits the best adhesion promotion effect (group No. 1). Compared with the LA88 postbiotic (bactericidal probiotic powder) without the addition of prebiotics (group No. 2), it significantly improves the adhesion ability of Lactobacillus acidophilus postbiotic to human vaginal epithelial cells (group No. 1 vs. group No. 2, t-test, p<0.05).

[0051] Further analysis found that there was no significant difference in adhesion effect between the postbiotic preparation with only acarbose added (group No. 5) and Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder, group No. 2, without the addition of prebiotics) (group No. 5 vs. group No. 2, t-test, p>0.05), indicating that acarbose alone does not have the effect of improving adhesion ability. The prebiotic combination composed of fructooligosaccharides, xylooligosaccharides and inulin (group No. 4) improves the adhesion performance to a certain extent (relative to group No. 2), but the improvement is very limited.

[0052] In groups No. 1, No. 4 and No. 5, the total amount of prebiotics is consistent. From the theoretical expectation, after the combination of acarbose with the above three oligosaccharides, the adhesion effect of group No. 1 should be between that of groups No. 4 and No. 5. However, the actual data shows that the adhesion ability of group No. 1 (four prebiotic components combined) is not only significantly higher than that of group No. 4, but also significantly better than that of group No. 5 (group No. 1 vs. group No. 4, t-test, p<0.05; group No. 1 vs. group No. 5, t-test, p<0.05). This result shows that there is a significant synergistic effect between acarbose and fructooligosaccharides, xylooligosaccharides and inulin, and the combination of the four can significantly enhance the adhesion ability of Lactobacillus acidophilus postbiotic to human vaginal epithelial cells, producing an unexpected technical effect.

[0053] In summary, the prebiotic combination consisting of acarbose, fructooligosaccharide, xylooligosaccharide and inulin shows a significant synergistic effect in improving the adhesion ability of Lactobacillus acidophilus LA88 postbiotic to human vaginal epithelial cells. Although acarbose alone has no significant effect on adhesion, the ternary combination of fructooligosaccharide, xylooligosaccharide and inulin also shows limited promotion, but the combination of the four shows a significantly better adhesion promotion effect than each single component or partial combination.

[0054] The Lactobacillus acidophilus LA88 postbiotic provided by the present scheme has good adhesion to human vaginal epithelial cells compared to commercially available Lactobacillus, and especially after adding prebiotics to the Lactobacillus acidophilus LA88 postbiotic, the adhesion is further improved. This means that the Lactobacillus acidophilus LA88 postbiotic can more stably adhere in the vaginal environment and play a role. This strong adhesion helps to form a protective film, reducing the opportunity for pathogenic microorganisms to adhere, and thus can effectively alleviate symptoms related to vaginal microecological imbalance.

[0055] Experimental Example 3: Anti-inflammatory and anti-allergic effects of Lactobacillus acidophilus LA88 postbiotic

[0056] (1) Effect on the level of cell inflammatory factors

[0057] Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder) was taken, and the optimal technical parameters in Example 1 were used to prepare the postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition consists of acarbose, fructooligosaccharide, xylooligosaccharide and inulin). Logarithmic phase Raw264.7 mouse macrophages were collected and inoculated into 6-well plates at 1×10 5 cells per well. After the cells reached 80-90% confluence, the treatment was started. The control group used DMEM medium, and the other groups used DMEM medium added with 1 μg / mL LPS to culture the cells. After 1 h, the intervention groups were continuously added with DMEM medium containing different bacterial suspensions (the final bacterial concentration in the culture system was 1×10 4 The intervention groups used Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder), postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition) and commercially available Lactobacillus acidophilus. After 24 h of treatment, the cell culture supernatants of each group were collected, and the concentrations of TNF-α, IL-6, IL-10 and NO inflammatory factors were detected according to the ELISA kit instructions. The experimental data are shown in Table 3. According to the data results, the Lactobacillus acidophilus LA88 postbiotic involved in the present application has excellent anti-inflammatory efficacy, and the anti-inflammatory effect is more ideal after adding prebiotics.

[0058] Table 3: Anti-inflammatory efficacy test results of different samples (unit: pg / mL; 3 replicates were set in each group, and the data in the table is the average of 3 replicates)

[0059]

[0060] (2) Influence of trypsin-like enzyme level

[0061] Randomly select 2 dpf wild type AB strain zebrafish in a 24-well plate, and treat 10 zebrafish in each well. The intervention group is given 500 μg / mL of sample (calculated as the concentration of bacterial powder), and the positive control group is given 1000 μg / mL of cromolyn sodium, while the normal control group and the model control group are set up, and 6 parallel experiments are performed. The samples include Lactobacillus acidophilus LA88 probiotics (bactericidal probiotic powder), probiotics containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition is composed of acarbose, fructooligosaccharide, xylooligosaccharide and inulin), and commercially available Lactobacillus acidophilus (powder state). Lactobacillus acidophilus LA88 probiotics (bactericidal probiotic powder) are prepared according to the optimal technical parameters of Example 1 to obtain probiotics containing prebiotics (bactericidal probiotic powder + prebiotic composition).

[0062] Except for the normal control group, the rest of the experimental groups are given C48 / 80 to establish a zebrafish allergic model. Each experimental group is given BAPNA, and after 1 day of co-treatment with BAPNA, the liquid is transferred to a 96-well plate, 250 μL / well, and the OD 405 value of each experimental group is detected by a multifunctional enzyme marker. The anti-allergic efficacy of the sample is evaluated by statistical analysis of the index. The experimental data is shown in Table 4. According to the data results, the Lactobacillus acidophilus LA88 probiotics and the probiotics containing prebiotics involved in the present application have excellent anti-allergic efficacy.

[0063] Table 4: Experimental results of the influence of different samples on the level of trypsin-like enzyme

[0064]

[0065] Experimental Example 4: Safety of Lactobacillus acidophilus LA88 probiotics

[0066] (1) Cytotoxicity

[0067] Logarithmic phase Raw264.7 mouse macrophages are collected, 100 μL of culture solution is added, and then the cells are cultured in a 96-well plate. The concentration of the cell suspension is adjusted to contain 10 4cells; after observing that the cells were completely adherent, different concentrations of Lactobacillus acidophilus LA88 probiotic (bactericidal probiotic powder) bacterial suspension were added, and culture solution was added as a sample control group. The 96-well plate was transferred to a temperature condition of 37°C and cultured for 24 hours; the culture solution was removed by centrifugation, and 20 μL of 0.5% MTT solution was added to each well and cultured for 1 day; after the supernatant was removed, 150 μL of DMSO solution was added and shaken, and after the blue-purple formazan crystals were completely dissolved, the absorbance was measured by an enzyme-labeled instrument, and the wavelength was 490 nm. Three groups in parallel were set for each concentration, and the wells containing no bacterial suspension and culture medium were used as blank groups. The cell survival rate formula is as follows: cell survival rate = (Ai-A0) / (Aj-A0) x 100%. In the formula, A0 is the absorbance of the blank group; Ai is the absorbance of the sample group; and Aj is the absorbance of the sample control group. The results show that when the concentration of Lactobacillus acidophilus LA88 probiotic is less than 1 x 10 12

[0068] (2) Multiple skin irritation

[0069] The experiment was performed according to the Multiple Skin Irritation Test in the Cosmetics Safety Technical Specifications (2015 edition). The experimental animals were 4 ordinary New Zealand white rabbits (female). Before the experiment, the animals were adapted to the environment in the experimental animal room for 3 days. About 24 hours before the experiment, the fur on both sides of the experimental animals' back spines was cut off, and the depilation area was 3 cm x 3 cm, and the application area was 2.5 cm x 2.5 cm. 0.5 ml of bacterial suspension of the test product, bactericidal probiotic powder, was applied to one side of the skin, and the other side was used as a control. The application was performed once a day for 14 consecutive days. From the second day, the fur was cut off before each application, and the residual test product was removed with water 1 hour before observation. The results were scored according to the scoring standard described in Chapter 6 of the Cosmetics Safety Technical Specifications (2015 edition) Skin Irritation / Corrosion Test. The control area and the test area were treated in the same way, and the average score of each animal was calculated every day according to the formula, and the skin irritation intensity was determined. The average skin irritation reaction score of each animal was 0.00. The experimental results show that the test product has no skin irritation on New Zealand white rabbits.

[0070] (3) Multiple vaginal mucosa irritation

[0071] ​The experiment was carried out according to the Disinfection Technical Standard (2002 edition). The experimental animals were 4 ordinary New Zealand white rabbits (female, non-pregnant and non-pregnant). 5 g of the sample was dissolved in 20 mL of distilled water to be used as the test substance, and normal saline was used in the control group. A blunt-ended soft tube with a length of about 8 cm was connected to a 2 mL syringe. The syringe and catheter were filled with the test liquid (bactericidal probiotic powder suspension) for standby. Each animal prepared a set. The animals were fixed on their backs, exposing the perineum and vaginal orifice. The catheter was gently inserted into the vagina (4-5 cm) after being moistened with the test liquid or control liquid, and 2 mL of the test liquid was slowly injected with the syringe, and the catheter was withdrawn to complete the infection, and the infection was repeated every 24 h for 5 d. The control group animals were treated with normal saline in the same way. 24 h after the last treatment, the animals were sacrificed by air embolism, the entire vagina was removed, longitudinally incised, and observed for hyperemia, edema, etc. for reference when taking pathological samples. Then the vagina was placed in 10% formalin solution for fixation for more than 24 h, and the tissues at both ends and the central 3 parts of the vagina were selected for sectioning, HE staining, and histopathological examination. From the experimental results, it can be seen that the test substance has no irritation to the vaginal mucosa of New Zealand white rabbits.

[0072] Application Example 1

[0073] The Lactobacillus acidophilus LA88 probiotic (bactericidal probiotic powder) or the probiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition consists of acarbose, fructooligosaccharide, xylooligosaccharide and inulin) was dissolved in the finishing agent and mixed thoroughly to obtain a prebiotic mixture, which was added to the sanitary napkin to prepare a prebiotic sanitary napkin. The mass ratio of Lactobacillus acidophilus LA88 probiotic to finishing agent was 1:10000, or the mass ratio of prebiotic containing prebiotic to finishing agent was 1:10000. The finishing agent was specifically a 2% carboxymethyl cellulose solution. The prebiotic mixture was evenly dispersed on the surface of the non-woven fabric of the surface layer of the sanitary napkin, so that the amount of bacteria loaded on the surface of the surface layer non-woven fabric was about 10 4 / cm 2 .

[0074] Experimental Example 5: Experimental study on promoting the proliferation of beneficial bacteria

[0075] The prebiotic-containing sanitary napkin samples (samples A-D, control group) prepared in Application Example 1 were cut into 1 cm 2 sized pieces at the center of the surface layer, and placed in 100 mL of sterilized MRS broth under sterile conditions for sterilization standby. This experimental example also studied different formulations and prepared corresponding prebiotic-containing sanitary napkin samples, as follows:

[0076] Sample A: Lactobacillus acidophilus LA88 postbiotic (sterilized probiotic bacteria powder) prepared according to the optimal technical parameters of Example 1 was used for the preparation of postbiotic-containing sanitary napkin samples. The preparation method of Lactobacillus acidophilus LA88 postbiotic is described in Example 1, and the preparation method of postbiotic-containing sanitary napkin samples is described in Application Example 1.

[0077] Sample B: Postbiotic containing prebiotic (sterilized probiotic bacteria powder + prebiotic composition; the prebiotic composition consists of acarbose, fructooligosaccharide, xylooligosaccharide and inulin) prepared according to the optimal technical parameters of Example 1 was used for the preparation of postbiotic-containing sanitary napkin samples. The preparation method of postbiotic containing prebiotic is described in Example 1, and the preparation method of postbiotic-containing sanitary napkin samples is described in Application Example 1.

[0078] Sample C: Basically the same as sample B, the difference is the composition of the prebiotic composition. For this sample C, the prebiotic composition is composed of fructooligosaccharide, xylooligosaccharide and inulin in a mass ratio of 2:2:3, which replaces the prebiotic composition in sample B. The preparation method of postbiotic containing prebiotic is described in Example 1, and the preparation method of postbiotic-containing sanitary napkin samples is described in Application Example 1.

[0079] Sample D: Basically the same as sample B, the difference is the composition of the prebiotic composition. For this sample D, the prebiotic composition in sample B is replaced by an equal amount of acarbose. The preparation method of postbiotic containing prebiotic is described in Example 1, and the preparation method of postbiotic-containing sanitary napkin samples is described in Application Example 1.

[0080] Control group: non-woven fabric of sanitary napkin surface layer without any coating.

[0081] This technical solution studies the effect of samples on two common vaginal probiotics, Lactobacillus gasseri ( Lactobacillus gasseri ) and Lactobacillus crispatus ( Lactobacillus crispatusLactobacillus gasseri and Lactobacillus crispatus are key members of the vaginal microecosystem and play an important role in maintaining women's reproductive health. These two Lactobacillus species produce lactic acid through metabolism, which helps maintain the acidic environment of the vagina, an acidic condition that is not conducive to the growth and reproduction of many pathogenic microorganisms, thereby protecting the vagina from infection. In addition to producing lactic acid, Lactobacillus gasseri and Lactobacillus crispatus can also secrete antibacterial substances such as bacteriocins and other small molecule compounds that can inhibit or kill potential harmful microorganisms, including some common bacteria and fungi that cause vaginitis. They also help the body resist infection by enhancing the local immune response in the vagina. A healthy vaginal flora helps maintain the integrity of the vaginal mucosa, reduces the inflammatory response, and can affect the repair and regeneration process of epithelial cells. Among them, Lactobacillus crispatus is often considered one of the most ideal dominant species in the vagina because it is particularly good at maintaining vaginal health. If the microbial agent and sanitary napkin product prepared in this scheme can effectively promote the proliferation of Lactobacillus gasseri and Lactobacillus crispatus, it can play a good promoting role in maintaining women's health.

[0082] According to the grouping, the bacterial suspension (10 8 CFU / mL) of the activated indicator bacteria (Lactobacillus gasseri ATCC19992; Lactobacillus crispatus ATCC33820) was inoculated in MRS broth containing sanitary napkin fragments at an inoculation amount of 2% (v / v), and cultured at 37°C for 18h. Then, 10-fold serial dilutions were performed, and three appropriate dilutions were selected and uniformly plated on solid culture medium, and cultured at 37°C for 48h. After the culture ended, the colony growth on the plates of different dilutions was observed, and the colonies after culture were counted. The control group was the bacterial suspension of the activated indicator bacteria inoculated in MRS broth without sanitary napkin at an inoculation amount of 2% (v / v), and then cultured at 37°C for 18h before plate counting. The proliferation promotion rate = (number of colonies in the experimental group - number of colonies in the control group) / number of colonies in the control group x 100%. The experimental results were the average values of the proliferation promotion rates at three dilutions, and the results are shown in Table 5.

[0083] Table 5: Detection results of proliferation promotion rates for different samples

[0084]

[0085] Sample B added a prebiotic composition based on sample A, thereby significantly improving the effect of promoting the growth of probiotics (there were significant differences for both Lactobacillus gasseri and Lactobacillus crispatus, sample B group vs. sample A group, t test, p<0.05). In samples B to D, the amount of bacteria loaded on the surface of the top layer of non-woven fabric remained consistent (about 10 4 CFU / cm 2), while the proportion of the addition of the prebiotic composition is the same as that of the bacteriocidal probiotic powder (85:15), so the total amount of the prebiotic used in samples B to D remains consistent. Under this uniform condition, the efficacy of the prebiotic composition in samples B to D is compared, which has good comparability. Specifically, sample C uses a prebiotic composition composed of fructooligosaccharides, xylooligosaccharides and inulin, and the proliferation promotion rate of Lactobacillus gasseri is 94.87%, and the proliferation promotion rate of Lactobacillus crispatus is 103.08%; sample D uses acarbose as a prebiotic, and the proliferation promotion rate of Lactobacillus gasseri is 93.25%, and the proliferation promotion rate of Lactobacillus crispatus is 101.58%; while sample B uses a complex prebiotic system composed of acarbose, fructooligosaccharides, xylooligosaccharides and inulin, and the proliferation promotion rate of Lactobacillus gasseri reaches 97.05% (sample B group vs. sample A group, t test, p<0.05); the proliferation promotion rate of Lactobacillus crispatus is as high as 111.23%, which is significantly different from samples C, D, A (sample B group vs. sample C group, t test, p<0.05; sample B group vs. sample D group, t test, p<0.05; sample B group vs. sample A group, t test, p<0.05). Theoretically, under the premise that the total amount of the prebiotic composition is consistent, the effect of sample B should be between samples C and D. However, the experimental results show that the actual effect of sample B is better than that of samples C and D. This shows that, whether for Lactobacillus gasseri or Lactobacillus crispatus, the four prebiotic components in sample B show stronger synergistic effect in promoting the growth of target strains in bacteriocidal probiotic powder, which reflects obvious synergistic effect.

[0086] The above research results have important biological significance and application value: Lactobacillus gasseri has the ability to regulate intestinal flora, enhance immunity and inhibit pathogenic bacteria, and is also helpful to maintain local microecological balance in the vaginal environment; Lactobacillus crispatus is one of the most common and representative dominant bacteria in the vagina of healthy women, and is considered to be a key "guardian" against bacterial vaginosis and other infections. Sample A uses Lactobacillus acidophilus LA88 postbiotic, and samples B-D use a reasonable combination of multiple prebiotics and Lactobacillus acidophilus LA88 postbiotic, which not only can more effectively support the proliferation of these two important probiotics, but also helps to establish a more stable and healthy vaginal microecosystem. This synergistic promotion provides a theoretical basis and technical support for the development of functional products that can efficiently and directionally regulate vaginal flora structure, and has broad application prospects.

[0087] Experimental Example 6: Bacteriostatic performance

[0088] The test is carried out according to the method provided in "5.1.3 Carrier Bacteriostatic Test" of "WS / T650-2019 Antibacterial and Bacteriostatic Effect Evaluation Method", and the sampling mode is the same as that in Experimental Example 5. The experimental results are shown in Table 6. According to the data results, the Lactobacillus acidophilus LA88 bioinoculant and the product added with probiotics involved in the present application have excellent pathogenic bacteria inhibition effect after being added to sanitary napkins.

[0089] Table 6: Bacteriostatic performance test results of different samples

[0090]

[0091] The above-mentioned is only an embodiment of the present application, and the well-known specific structure and characteristics in the scheme are not described too much. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should also be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A Lactobacillus acidophilus LA88 probiotic composition, characterized in that: It comprises a probiotic composition of Lactobacillus acidophilus LA88 and a prebiotic combination; the prebiotic combination comprises at least one of acarbose, fructo-oligosaccharide, xylo-oligosaccharide, inulin; the preservation number of Lactobacillus acidophilus LA88 is CGMCC No. 24109.

2. The Lactobacillus acidophilus LA88 probiotic composition according to claim 1, characterized in that: The total bacterial concentration of the Lactobacillus acidophilus LA88 probiotic was 1 x 10 11 -3 x 10 11 individuals / g.

3. A Lactobacillus acidophilus LA88 probiotic composition according to claim 2, characterized in that: The mass ratio of the probiotic composition of Lactobacillus acidophilus LA88 and the prebiotic combination is 80:20-99:1; the prebiotic combination consists of acarbose, fructo-oligosaccharide, xylo-oligosaccharide, and inulin.

4. The Lactobacillus acidophilus LA88 probiotic composition according to claim 3, characterized in that: The prebiotic combination consists of acarbose, fructo-oligosaccharide, xylo-oligosaccharide, and inulin with a mass ratio of 1-3:1-3:1-3:1-3.

5. A method of preparing a Lactobacillus acidophilus LA88 probiotic composition according to any one of claims 1 to 4, characterised in that: The prebiotic combination is added to the probiotic composition of Lactobacillus acidophilus LA88, and after mixing, a prebiotic with added prebiotics is obtained; the prebiotic combination comprises acarbose, fructo-oligosaccharide, xylo-oligosaccharide, and inulin.

6. Use of a probiotic composition of Lactobacillus acidophilus LA88 in the preparation of a product for regulating the vaginal micro-ecology according to any one of claims 1-4.

7. Use of a Lactobacillus acidophilus LA88 probiotic composition according to claim 6 for the preparation of a product for modulating the vaginal microflora, characterized in that, The product comprises sanitary napkins, sanitary pads, or vaginal lotions; the product is used to promote the proliferation of beneficial bacteria and inhibit the proliferation of harmful bacteria. The product comprises sanitary napkins, sanitary pads, or vaginal lotions; the product is used to promote the proliferation of beneficial bacteria and inhibit the proliferation of harmful bacteria.

8. Use of a Lactobacillus acidophilus LA88 probiotic composition according to claim 7 for the preparation of a product for modulating the vaginal microflora, characterized in that: The product for regulating vaginal micro-ecology is a sanitary napkin; Lactobacillus acidophilus LA88 probiotics are mixed with carboxymethyl cellulose solution, and then loaded on the surface layer of the sanitary napkin; the loading amount is 10 4 / cm 2 .

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