Lactobacillus acidophilus LA88 metabiotic composition and application thereof in preparation of products for regulating vaginal microecology

Abstract

The invention relates to the technical field of probiotics and application thereof, in particular to a lactobacillus acidophilus LA88 metagen composition and application thereof in preparation of products for regulating vaginal microecology. According to the scheme, a microecological regulator with a synergistic interaction effect is constructed by adding a prebiotic combination in a specific proportion based on a lactobacillus acidophilus LA88 (the preservation number is CGMCC No.24109), and the microecological regulator is particularly suitable for regulating the microecological environment of the female vagina. According to the present invention, the adopted metabiotics are in the form of lactobacillus subjected to inactivation treatment, such that the safety and the stability of the product are improved, the metabiotics contain rich bioactive components, and can directly regulate the local immune environment of the vagina, and the prebiotics combination can promote the rapid proliferation of the beneficial bacteria through nutrition supply and metabolism regulation. According to the technical scheme, the technical problems of unsatisfactory safety and curative effect of the probiotics for regulating the vagina micro-ecosystem can be solved, and ideal application and popularization prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics and their applications, and in particular to a Lactobacillus acidophilus LA88 postbiotic composition and its application in preparing products for regulating vaginal microecology. Background Art

[0002] The vaginal microbiome is a crucial barrier to female reproductive health. This dynamic equilibrium, comprised of diverse microorganisms, plays a key role in maintaining vaginal health. Under normal circumstances, beneficial vaginal bacteria (such as Lactobacilli) effectively inhibit the colonization and proliferation of pathogens through space-occupying protection and biological antagonism, maintaining an optimal vaginal pH and alkalinity, thereby maintaining a healthy vaginal environment. This microecological balance is crucial for preventing various gynecological diseases.

[0003] However, the vaginal microbiome is not static. Environmental changes, antibiotic use, hormonal fluctuations, and altered host immune status can all lead to changes in the structure of the vaginal microbiome, potentially causing an imbalance in the microbiome. For example, frequent use of broad-spectrum antibiotics can inadvertently disrupt beneficial vaginal flora, allowing drug-resistant or opportunistic pathogens to proliferate, leading to diseases such as bacterial vaginosis and candidal infections.

[0004] Although live probiotics are widely believed to help restore the balance of vaginal flora, their application has certain limitations and potential risks. First, the storage conditions of live bacterial products are relatively demanding, requiring the maintenance of specific temperature and humidity to ensure the activity of the strains. This not only increases production costs but also places higher demands on storage and transportation. Second, even after the successful introduction of live probiotics 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 weakened immune systems. In addition, the efficacy of probiotics alone in restoring the balance of vaginal flora is limited. Relying solely on probiotics makes it difficult to establish a stable microecological environment and cannot achieve the ideal microecological regulation effect. Therefore, when designing products to restore the balance of the vaginal microecological environment, in addition to considering how to effectively introduce beneficial microorganisms, it is also important to provide these microorganisms with the necessary nutritional support to ensure that they can function stably and long-term in the body.

[0005] In summary, the vaginal microbiome is crucial to female reproductive health, and its dynamic balance is influenced by a variety of internal and external factors. Although live probiotics are considered a promising tool for restoring vaginal microbiome balance, their practical application faces challenges such as harsh storage conditions, challenges with survival in vivo, and limited efficacy when used alone. Therefore, the development of postbiotic-based microbiome regulators has significant clinical value and application prospects. This intervention approach is not only safer but also gentler than drug treatment, offering a new solution for maintaining female vaginal health. Summary of the Invention

[0006] The purpose of the present invention is to provide a Lactobacillus acidophilus LA88 postbiotic composition to solve the technical problems of the prior art of the safety and unsatisfactory efficacy of probiotics for regulating the vaginal microecological system.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A Lactobacillus acidophilus LA88 postbiotic composition, comprising a Lactobacillus acidophilus LA88 postbiotic and a prebiotic combination; the prebiotic combination comprises at least one of acarbose, oligofructose, oligoxylose, and inulin; the preservation number of Lactobacillus acidophilus LA88 is CGMCC No. 24109.

[0008] Furthermore, the total bacterial concentration of Lactobacillus acidophilus LA88 postbiotics was 1×10 11 -3×10 11 / g; preferably 1×10 11 pcs / g.

[0009] Furthermore, the mass ratio of the Lactobacillus acidophilus LA88 postbiotic and prebiotic combination is 80:20-99:1 (preferably 85:15); the prebiotic combination consists of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin.

[0010] Furthermore, the prebiotic combination consists of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin in a mass ratio of 1-3:1-3:1-3:1-3; Preferably, the mass ratio of acarbose, fructooligosaccharides, xylo-oligosaccharides and inulin is 1:2:2:3.

[0011] The present technical solution also provides a method for preparing a Lactobacillus acidophilus LA88 postbiotic, which comprises adding a prebiotic composition to the Lactobacillus acidophilus LA88 postbiotic, and mixing to obtain a postbiotic with added prebiotics; the prebiotic composition comprises acarbose, oligofructose, oligoxylose, and inulin.

[0012] This technical solution also provides an application of a Lactobacillus acidophilus LA88 postbiotic composition in the preparation of products for regulating vaginal microecology, wherein the products include sanitary napkins, sanitary pads, sanitary paper towels or vaginal washes; the products are used to promote the proliferation of beneficial bacteria and inhibit the proliferation of harmful bacteria.

[0013] Furthermore, the product for regulating vaginal microecology is a sanitary napkin; Lactobacillus acidophilus LA88 postbiotics is mixed with carboxymethyl cellulose solution and then loaded on the surface layer of the sanitary napkin; the loading amount is 10 4 pieces / cm 2 .

[0014] In summary, the technical principle of this technical solution is: The core of this invention is to provide a postbiotic (provided by Micro-Con Microorganisms) prepared based on Lactobacillus acidophilus LA88 (preservation number CGMCC No. 24109), and to add a prebiotic combination in a specific proportion through scientific compatibility to construct a functional microecological regulator with synergistic effects, which is particularly suitable for regulating the female vaginal microecological environment.

[0015] Lactobacillus acidophilus is one of the main lactic acid bacteria naturally present in the human body, and plays an important role in maintaining the stability of vaginal pH and inhibiting the growth of pathogenic microorganisms. Although traditional live bacterial preparations can improve the problem of vaginal microecological imbalance to a certain extent, they have high requirements for storage and transportation conditions, and there are problems such as low survival rate in the body and high immune risk. The present invention uses Lactobacillus acidophilus LA88 postbiotics as inactivated bacterial powder, which avoids the uncontrollable ectopic colonization of live bacteria. While improving safety, it still has good immune regulation, antibacterial, anti-inflammatory and adhesion protection functions, and can directly participate in the regulation of the local microenvironment of the vagina.

[0016] To enhance the biological functions of postbiotics, the present invention further introduces a prebiotic combination consisting of acarbose, oligofructose, oligoxylose, and inulin. This combination not only provides nutritional support for beneficial bacteria (such as Lactobacillus and Bifidobacterium) residing in the vagina, promoting their colonization and proliferation, but also achieves multiple synergistic effects. Through structural complementarity and metabolic pathway synergy, the prebiotics form a "prebiotic network," significantly improving overall regulatory efficiency and contributing to the establishment of a stable and healthy vaginal microecological system.

[0017] The present invention combines the Lactobacillus acidophilus LA88 postbiotic with the aforementioned prebiotic combination, achieving a shift from "passive regulation" to "active construction." Postbiotics provide immediate antibacterial, anti-inflammatory, and immunomodulatory signals; prebiotics provide continuous nutritional support for the host's existing beneficial bacteria, promoting their rapid proliferation. The synergistic effect of the two can effectively restore the microecological structure dominated by lactobacilli in the vagina, reconstruct an acidic environment dominated by lactic acid, lower the pH value, and inhibit the growth of pathogens. This "postbiotic + prebiotic" composite strategy breaks through the limitations of traditional single live bacterial preparations, taking into account safety, effectiveness, and sustainability, and represents the development direction of a new generation of microecological regulators.

[0018] The beneficial effects of this technical solution are: (1) High safety: The inactivated Lactobacillus acidophilus LA88 postbiotics do not contain active microorganisms, avoiding potential risks such as infection, drug-resistant gene transfer or abnormal immune response caused by live bacteria entering the body. It is particularly suitable for women with low immune function or in special physiological periods (such as pregnancy and menstruation).

[0019] (2) Strong regulatory ability: Postbiotics have complete cell structures 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 microecological system.

[0020] (3) Significant synergistic effect: The synergistic effect between multiple prebiotics can enhance the selective promotion of lactobacilli, forming a regulatory system with superimposed functions and long-lasting effects, which is significantly better than the effect of using a single prebiotic or probiotic.

[0021] (4) Wide range of application scenarios: The postbiotics can be applied to a variety of gynecological care products such as sanitary napkins, panty liners, paper towels, lotions, etc. By being loaded on the surface of the product, continuous intervention can be achieved during daily use, which is convenient, practical and easy to accept.

[0022] In summary, the present invention optimizes the combination ratio of postbiotics and prebiotics to construct a new type of vaginal microecological regulator that is safe, efficient, stable, controllable, and highly adaptable, filling the gap in the existing technology regarding postbiotics in the field of female intimate care, and has important scientific research value and industrial application prospects. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0024] Example 1: Preparation of Lactobacillus acidophilus LA88 postbiotics containing a prebiotic composition Lactobacillus acidophilus LA88 postbiotics were purchased from Weikang Probiotics (Suzhou) Co., Ltd. and are inactivated microbial cells and their components of Lactobacillus acidophilus LA88, in the form of dried bacterial powder. The trade name of Lactobacillus acidophilus LA88 postbiotics is: Probiotic Powder (Bactericidal Type), and the product specifications are: 1×10 11 The product is packed with inner PE bag and outer oxygen and water barrier aluminum foil bag. The product quality inspection report shows that the number of lactic acid bacteria in the product is 1.0×10 11 The probiotic powder (bactericidal type) is essentially Lactobacillus acidophilus LA88 postbiotics. When used, the bacterial concentration can be 1×10 11 / g-3×10 11 The probiotic powder (bactericidal type) used in the experimental research is uniformly referred to as Lactobacillus acidophilus LA88 postbiotics in the following text. The subsequent experimental research uses a lactic acid bacteria concentration of 1×10 11 Lactobacillus acidophilus LA88 postbiotics per gram.

[0025] Among them, Lactobacillus acidophilus LA88 (Lactobacillus acidophilus Lactobacillus acidophilus The LA88 strain is owned by Weikang Probiotics (Suzhou) Co., Ltd. (see patent CN114591875B). Originally collected from Dali Bai Autonomous Prefecture, Yunnan Province, it is currently deposited at the General Microbiology Center of the China National Culture Collection Administration under the deposit number CGMCC No. 24109. This technical proposal purchased the company's probiotic powder (sterilized), a conventional heat-inactivated product of Lactobacillus acidophilus LA88, for further research and development of new products.

[0026] At a lactic acid bacteria concentration of 1×10 11 The prebiotic composition is added to a Lactobacillus acidophilus LA88 postbiotic containing 100 mg / g of Lactobacillus acidophilus LA88 and mixed thoroughly to obtain a postbiotic containing prebiotics. The prebiotic composition consists of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin. To ensure product quality, acarbose, fructooligosaccharides, xylooligosaccharides, and inulin are sterilized by conventional irradiation. The ratio of sterilized probiotic powder to prebiotics is 80:20-99:1 (preferably 85:15). The mass ratio of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin is 1-3:1-3:1-3:1-3 (preferably 1:2:2:3).

[0027] More specifically, the prebiotic composition is introduced as follows: The systematic name of acarbose is: 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; Molecular formula: C 25 H 43 NO 18 ; Molecular weight: 645.6 g / mol; CAS number: 56180-94-0.

[0028] Fructooligosaccharides (FOS): a type of functional oligosaccharide composed of 2-9 fructose molecules connected by β(2→1) glycosidic bonds, with glucose as the terminal unit (GFn type) Xylooligosaccharides (XOS): are composed of 2-7 xylose sugars connected by β(1→4) glycosidic bonds.

[0029] Inulin: It is composed of 10-60 fructose units connected by β(2→1) glycosidic bonds, with glucose as the terminal unit (GFn type).

[0030] The above substances can be obtained through commercial means and are known in the prior art.

[0031] Experimental Example 1: Acid tolerance of Lactobacillus acidophilus LA88 postbiotics Take Lactobacillus acidophilus LA88 postbiotics (probiotic powder (bactericidal type) of Micro-Kang probiotics) and the postbiotics containing prebiotics (bactericidal probiotic powder + prebiotic composition) prepared according to the optimal technical parameters of Example 1. Place Lactobacillus acidophilus LA88 postbiotics or postbiotics containing prebiotics in sterile physiological saline and resuspend to obtain a resuspension (bacterial concentration of 1×10 9CFU / mL), 1.0 mL of the resuspended solution was added to 9.0 mL of sterile PBS at different pH values ​​(pH 2.0, pH 3.0, pH 4.0, and pH 5.0). The suspension was allowed to stand at room temperature. Samples were collected at the beginning (0 h) and after 3 h of treatment. The bacterial count was determined by hemocytometer, and the tolerance rate was calculated using the following formula: Tolerance rate (%) = N1 / N0 × 100%, where N1 is the bacterial count after 3 h of treatment with sterile PBS at different pH values; N0 is the bacterial count at 0 h. The test results are shown in Table 1. It can be seen that the Lactobacillus acidophilus LA88 postbiotics and postbiotics containing prebiotics described in this invention have excellent acid tolerance. The tolerance rate remained essentially 100% in an environment with a pH of 3.0-5.0 for 3 h. The normal vaginal pH of healthy women is approximately 4.0. This indicates that the Lactobacillus acidophilus LA88 postbiotics have good acid tolerance and can normally colonize the vagina.

[0032] Table 1: Test results of acid tolerance of Lactobacillus acidophilus LA88 postbiotics

[0033] Experimental Example 2: Adhesion of Lactobacillus acidophilus LA88 postbiotics to vaginal epithelial cells Experimental studies were conducted using Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder) and a postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic combination) prepared according to the optimal technical parameters of Example 1. Lactobacillus acidophilus LA88 postbiotics (number 2 in Table 2), a postbiotic containing prebiotics (prebiotics comprising a combination of acarbose, oligofructose, oligoxylose, and inulin, number 1 in Table 2; alternatively, a combination of oligofructose, oligoxylose, and inulin, number 4 in Table 2; alternatively, acarbose alone, number 5 in Table 2), and commercially available live Lactobacillus acidophilus (obtained from frozen culture via conventional activation, ATCC4356, number 3 in Table 2) were resuspended in RPMI 1640 medium (Gibco) to a bacterial concentration of 1×10 8100 mg / ml (sample resuspension) for measuring vaginal epithelial cell adhesion. Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder) were purchased. A postbiotic containing prebiotics (where the prebiotics are a combination of acarbose, oligofructose, oligoxylose, and inulin) was prepared according to the optimal technical parameters of Example 1, representing the optimal preparation method. A postbiotic containing prebiotics (where the prebiotics are a combination of oligofructose, oligoxylose, and inulin) was prepared as follows: To the Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder), prebiotics were added at a ratio of 85:15, with the prebiotics consisting of oligofructose, oligoxylose, and inulin in a ratio of 2:2:3. This means that, based on the optimal preparation method for the postbiotic containing prebiotics, the combination of four prebiotics was replaced with a combination of three prebiotics in equal amounts. The preparation method of the postbiotic containing prebiotics (in the case where the prebiotic is acarbose) is: based on the optimal preparation method of the postbiotic containing prebiotics, the composition formed by four prebiotics is replaced with acarbose in equal amounts.

[0034] Vaginal epithelial cells used were human vaginal epithelial cells VK2 / E6E7 (ATCC-CRL2616), which were suspended in 10 ml RPMI l640 and cultured in a cell culture incubator at 37°C and 5% CO2. The concentration of vaginal epithelial cells was adjusted to 1×10 5 The sample resuspension solution was adjusted to a final concentration of 2×10 7 1 mL of bacterial suspension was then mixed with an equal amount of vaginal epithelial cell suspension and incubated at 37°C with shaking for 24 hours. The suspension was centrifuged at 1000 rpm for 5 minutes and washed three times with PBS buffer. Bacteria not adhered to the vaginal epithelial cells were discarded. The precipitate was smeared, dried, and Gram-stained. Fifty epithelial cells were randomly counted under an oil immersion lens. The number of bacteria adhered to the epithelial cells was calculated, and the average and adhesion index were calculated. The adhesion index was calculated as: total number of bacteria adhered to 50 epithelial cells / 50. The experimental results are detailed in Table 2. The results show that the Lactobacillus acidophilus postbiotics and live bacteria have similar adhesion to vaginal epithelial cells, and exhibit stronger adhesion than commercially available Lactobacillus acidophilus.

[0035] Table 2: Adhesion test results of different samples of vaginal epithelial cells (n=10, VK2 / E6E7 cell adhesion coefficient is the average of 10 tests)

[0036] The experimental data shown in Table 2 indicate that the Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder) used in this protocol significantly outperformed commercially available Lactobacillus acidophilus in its ability to adhere to human vaginal epithelial cells (Group 2 vs. Group 3, t-test, p < 0.05). Compared to live bacterial formulations, this postbiotic avoids potential safety concerns associated with the introduction of live bacteria and is more convenient for storage and transportation.

[0037] Based on this, the inventors further added prebiotic ingredients to the bactericidal probiotic powder to enhance its performance. Experimental results showed that when the prebiotic composition consisted of acarbose, oligofructose, oligoxylose, and inulin, the resulting postbiotic-containing prebiotic exhibited the best adhesion-promoting effect (Group 1). Compared with the LA88 postbiotic (bactericidal probiotic powder) without prebiotics, it significantly improved the adhesion of Lactobacillus acidophilus postbiotics to human vaginal epithelial cells (Group 1 vs. Group 2, t-test, p < 0.05).

[0038] Further analysis revealed no significant difference in adhesion between the postbiotic formulation containing only acarbose (Group 5) and the Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder, Group 2, without prebiotics) (Group 5 vs. Group 2, t-test, p>0.05), indicating that acarbose alone does not enhance adhesion. A prebiotic combination of fructooligosaccharides, xylooligosaccharides, and inulin (Group 4) did improve adhesion to some extent (relative to Group 2), but the improvement was very limited.

[0039] The total prebiotic dosage was consistent in Groups 1, 4, and 5. Theoretically, the combined use of acarbose and the three oligosaccharides should have yielded an adhesion effect in Group 1 that was intermediate between Groups 4 and 5. However, actual data showed that Group 1 (using the four prebiotic ingredients combined) exhibited significantly higher adhesion than not only Group 4 but also Group 5 (Group 1 vs. Group 4, t-test, p<0.05; Group 1 vs. Group 5, t-test, p<0.05). This result demonstrates a significant synergistic effect between acarbose, fructooligosaccharides, xylooligosaccharides, and inulin. The combined use of these four prebiotics significantly enhanced the adhesion of Lactobacillus acidophilus postbiotics to human vaginal epithelial cells, producing an unexpected technical benefit.

[0040] In summary, the prebiotic combination of acarbose, fructooligosaccharides, xylooligosaccharides, and inulin demonstrated a significant synergistic effect in enhancing the adhesion of Lactobacillus acidophilus LA88 postbiotics to human vaginal epithelial cells. Although acarbose alone had no significant effect on adhesion, and the ternary combination of fructooligosaccharides, xylooligosaccharides, and inulin only exhibited a limited promoting effect, the combined effect of the four components significantly outperformed the individual components or partial combinations.

[0041] The Lactobacillus acidophilus LA88 postbiotic provided in this solution exhibits superior adhesion to human vaginal epithelial cells compared to commercially available lactobacilli. This adhesion is further enhanced by the addition of prebiotics. This means that the Lactobacillus acidophilus LA88 postbiotic is able to more stably adhere and function in the vaginal environment. This strong adhesion helps form a protective barrier, reducing the chances of pathogenic microorganisms attaching, thereby effectively alleviating symptoms associated with vaginal microecological imbalance.

[0042] Experimental Example 3: Anti-inflammatory and anti-allergic effects of Lactobacillus acidophilus LA88 postbiotics (1) Effects on cellular inflammatory factor levels Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder) were used to prepare a postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition consists of acarbose, oligofructose, oligoxylose, and inulin) according to the optimal technical parameters of Example 1. Raw264.7 mouse macrophages in the logarithmic phase were collected and inoculated into 6-well plates, with 1×10 per well. 5 The cells were treated after reaching 80-90% confluence. The control group used DMEM medium, and the other groups used DMEM medium supplemented with 1 μg / mL LPS to culture the cells. After 1 hour, the strain intervention groups continued to add DMEM medium with different bacterial suspensions (the final bacterial concentration in the culture system was 1×10 4 Cells were expressed in 400 cells / mL. Equal volumes of DMEM were added to the control and model groups. The intervention groups received Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder), a postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic combination), and commercially available Lactobacillus acidophilus, respectively. After 24 hours of treatment, cell culture supernatants were collected from each intervention group and assayed for TNF-α, IL-6, IL-10, and NO inflammatory cytokines according to the ELISA kit instructions. The experimental data are detailed in Table 3. These results demonstrate that the Lactobacillus acidophilus LA88 postbiotics disclosed herein exhibit excellent anti-inflammatory efficacy, and the addition of prebiotics further enhances this anti-inflammatory effect.

[0043] Table 3: Anti-inflammatory efficacy test results of different samples (unit: pg / mL; 3 replicates were set for each group, and the data in the table are the average of 3 replicates)

[0044] (2) Effect of tryptase levels 2-day-old wild-type AB strain zebrafish were randomly selected and plated in a 24-well plate, with 10 zebrafish treated per well. The intervention group received a 500 μg / mL aqueous solution of the sample (based on the bacterial powder concentration), while the positive control, sodium cromoglycate, was 1000 μg / mL. A normal control group and a model control group were also established. Six parallel experiments were conducted. Samples included Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder), a postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic combination; the prebiotic combination consisted of acarbose, oligofructose, oligoxylose, and inulin), and commercially available Lactobacillus acidophilus (in powder form). The Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder) were prepared using the optimized technical parameters of Example 1 to obtain the postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic combination).

[0045] Except for the normal control group, all other experimental groups were given C48 / 80 in water to establish the zebrafish allergy model. All experimental groups were given BAPNA in water. After 1 day of co-treatment with BAPNA, the liquid was transferred to a 96-well plate at 250 μL / well. The OD value of the tryptase expression level of each experimental group was measured using a multifunctional microplate reader. 405 The anti-allergic efficacy of the sample was evaluated based on the statistical analysis results of this index. The experimental data are shown in Table 4. The data results show that the Lactobacillus acidophilus LA88 postbiotics and postbiotics containing prebiotics involved in the present invention have excellent anti-allergic efficacy.

[0046] Table 4: Experimental results of the effects of different samples on tryptase levels

[0047] Experimental Example 4: Safety of Lactobacillus acidophilus LA88 postbiotics (1) Cytotoxicity Raw264.7 mouse macrophages in the logarithmic phase were collected, 100 μL of culture medium was added, and then cultured in a 96-well plate. The concentration of the cell suspension was adjusted to 10 cells per well. 4After observing that the cells were completely attached to the wall, different concentrations of Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder) suspension were added. Culture medium was added as a sample control, and the 96-well plate was transferred to 37°C and incubated for 24 hours. The culture medium was centrifuged to remove the culture medium, and 20 μL of 0.5% MTT solution was added to each well and incubated for 1 day. After removing the supernatant, 150 μL of DMSO solution was added to each well and shaken. After observing that the blue-purple formazan crystals were completely dissolved, the absorbance was measured using a microplate reader at a wavelength of 490 nm. Three replicates were set for each concentration, and a well without bacterial suspension or culture medium served as a blank control. The cell survival rate formula is as follows: Cell survival rate = (Ai - A0) / (Aj - A0) × 100%, where A0 is the absorbance of the blank control; Ai is the absorbance of the sample control; and Aj is the absorbance of the sample control. The results showed that the concentration of Lactobacillus acidophilus LA88 postbiotic was less than 1×10 12 When the concentration of 5-64kJ / mL was 400 μg / mL, the cell viability was above 98% and it had no cytotoxicity to Raw264.7 mouse macrophages.

[0048] (2) Multiple skin irritations Multiple skin irritation tests were conducted in accordance with the "Safety Technical Specifications for Cosmetics" (2015 edition). Four female, standard-grade New Zealand White rabbits were used as experimental animals. Animals were acclimated to the experimental animal room environment for 3 days prior to the experiment. Approximately 24 hours before the test, the fur on both sides of the animal's spine was clipped, leaving a 3 cm x 3 cm area for application to a 2.5 cm x 2.5 cm area. 0.5 ml of a suspension of the test bactericidal probiotic powder was applied to one side of the skin, with the other side serving as a control. This application was repeated once daily for 14 days. Starting on the second day, the fur was clipped before each application. Residual test substance was removed with water, and the results were observed one hour later. Scoring was performed according to the scoring criteria outlined in Chapter 6, Skin Irritation / Corrosion Test, of the "Safety Technical Specifications for Cosmetics" (2015 edition). Both the control and test areas were treated identically. The average score for each animal was calculated daily using the formula listed above, and the skin irritation intensity was determined. The average skin irritation score for each animal over 14 days was 0.00. The experimental results show that the test substance is non-irritating to the skin of New Zealand white rabbits after multiple exposures.

[0049] (3) Multiple vaginal mucosal irritation The experiment was conducted in accordance with the "Technical Specifications for Disinfection" (2002 edition). Four standard-grade New Zealand White rabbits (female, non-pregnant) were used as the test material. A 5g sample was dissolved in 20mL of distilled water and used as the test material. A control group received normal saline. A 2mL syringe was connected to a blunt-tipped flexible tube approximately 8cm long. The syringe and catheter were filled with the test solution (a suspension of bactericidal probiotic powder) and ready for use. Each set of tubes was prepared for each animal. The animal was positioned supine, with the perineum and vaginal opening exposed. A catheter moistened with the test solution or control solution was gently inserted into the vagina (4-5cm). 2mL of the test solution was slowly injected using a syringe. The catheter was removed to complete the infection. Infection was repeated every 24 hours for 5 consecutive days. Animals in the control group were treated similarly with normal saline. Twenty-four hours after the last infection, the animals were sacrificed by gas embolism. The intact vagina was removed by laparotomy and longitudinally incised. Visual inspection was performed for signs of congestion and edema for reference during pathological sampling. The vagina was then fixed in 10% formalin solution for at least 24 hours. Tissue sections were prepared from the two ends and three central locations of the vagina and stained with HE for histopathological examination. The experimental results showed that the test substance showed no irritation to the vaginal mucosa of New Zealand white rabbits after multiple exposure.

[0050] Application Example 1 Lactobacillus acidophilus LA88 postbiotics (bactericidal probiotic powder) or postbiotics containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition consists of acarbose, oligofructose, oligoxylose and inulin) are dissolved in a finishing agent and fully mixed to obtain a postbiotic mixture, which is added to a sanitary napkin to prepare a postbiotic sanitary napkin. The mass ratio of Lactobacillus acidophilus LA88 postbiotics to the finishing agent is 1:10000, or the mass ratio of the postbiotics containing prebiotics to the finishing agent is 1:10000. The finishing agent is specifically a 2% carboxymethyl cellulose solution. The postbiotic mixture is 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 non-woven fabric of the surface layer is 10 4 pieces / cm 2 about.

[0051] Experimental Example 5: Experimental Study on Promoting the Proliferation of Beneficial Bacteria Take the postbiotic sanitary napkin samples prepared in Application Example 1 (samples AD and control group), cut out a 1cm 2 Under a sterile environment, the fragments of different sizes were placed in 100 mL of sterilized MRS broth for sterilization. This experimental example also studied different formulations and prepared corresponding sanitary napkin samples containing postbiotics, as follows: Sample A: Lactobacillus acidophilus LA88 postbiotic (bactericidal probiotic powder), prepared according to the optimized technical parameters of Example 1, was used to prepare postbiotic sanitary napkin samples. For the preparation of Lactobacillus acidophilus LA88 postbiotics, see Example 1. For the preparation of postbiotic sanitary napkin samples, see Application Example 1.

[0052] Sample B: A postbiotic containing prebiotics (bactericidal probiotic powder + prebiotic composition; the prebiotic composition consists of acarbose, oligofructose, oligoxylose, and inulin), prepared according to the optimized technical parameters of Example 1. This was used to prepare a postbiotic sanitary napkin sample. The preparation method for the postbiotic containing prebiotics is described in Example 1, and the preparation method for the postbiotic sanitary napkin sample is described in Application Example 1.

[0053] Sample C: Similar to Sample B, except for the composition of the prebiotic composition. Sample C consists of oligofructose, oligoxylose, and inulin in a mass ratio of 2:2:3, replacing the four substances in Sample B in equal amounts. See Example 1 for the preparation of postbiotics containing prebiotics, and Application Example 1 for the preparation of postbiotic sanitary napkin samples containing postbiotics.

[0054] Sample D: Similar to Sample B, except for the composition of the prebiotic composition. For Sample D, the prebiotic composition in Sample B was replaced with an equal amount of acarbose. For the preparation of the postbiotic containing prebiotics, see Example 1. For the preparation of the postbiotic-containing sanitary napkin sample, see Application Example 1.

[0055] Control group: non-woven fabric on the surface of the sanitary napkin without any coating.

[0056] This technical solution studies the effects of the sample on two common vaginal probiotics. Lactobacillus gasseri ) and Lactobacillus crispatus ( Lactobacillus crispatus) are key members of the vaginal microbiome and play a vital role in maintaining female reproductive health. These two lactobacilli produce lactic acid through metabolism, helping to maintain an acidic vaginal environment. This acidic environment is unfavorable for 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 secrete antimicrobial substances such as bacteriocins and other small molecules that can inhibit or kill potentially harmful microorganisms, including some common bacteria and fungi that cause vaginitis. They also help the body resist infection by enhancing local vaginal immune responses. A healthy vaginal flora helps maintain the integrity of the vaginal mucosa, reduces inflammation, and may influence the repair and regeneration of epithelial cells. Among them, Lactobacillus crispatus is often considered one of the most ideal dominant bacterial species in the vagina because it is particularly adept at maintaining vaginal health. If the microbial agent and sanitary napkin products prepared in this protocol can effectively promote the proliferation of Lactobacillus gasseri and Lactobacillus crispatus, they will play a significant role in maintaining female health.

[0057] According to the groups, the activated indicator bacteria (Lactobacillus gasseri ATCC19992; Lactobacillus crispatus ATCC33820) suspension (10 8 A 2% (v / v) inoculum of the activated indicator bacteria suspension (CFU / mL) was inoculated into MRS broth containing sanitary napkin fragments and incubated at 37°C for 18 hours. A 10-fold serial dilution was then performed, and three appropriate dilutions were evenly plated onto solid culture medium and incubated at 37°C for 48 hours. Colony growth on the plates at each dilution was observed and counted. A control group consisted of a 2% (v / v) inoculum of the activated indicator bacteria suspension in MRS broth without sanitary napkins. The plates were incubated at 37°C for 18 hours, and then counted. The proliferation rate was calculated as (number of colonies in the experimental group - number of colonies in the control group) / number of colonies in the control group × 100%. The results are the average of the proliferation rates at the three dilutions and are shown in Table 5.

[0058] Table 5: Test results of proliferation rate of different samples

[0059] Sample B added a prebiotic composition to Sample A, which significantly improved the effect of promoting the growth of probiotics (there were significant differences in 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 non-woven fabric remained consistent (approximately 10 4 pieces / cm 2), and the addition ratio of the prebiotic composition is the same as that of the bactericidal probiotic powder (85:15), so the total amount of prebiotics used in samples B to D remains consistent. Under this unified condition, the efficacy of the prebiotic compositions in samples B to D is well comparable. Specifically, sample C uses a prebiotic composition composed of oligofructose, oligoxylose and inulin, which has a proliferation promotion rate of 94.87% for Lactobacillus gasseri and a proliferation promotion rate of 103.08% for Lactobacillus crispatus; sample D uses acarbose as a prebiotic, which has a proliferation promotion rate of 93.25% for Lactobacillus gasseri and a proliferation promotion rate of 101.58% for Lactobacillus crispatus; and sample B uses a compound composed of acarbose, oligofructose, oligoxylose and inulin. The prebiotic system promoted the growth of Lactobacillus gasseri by 97.05% (sample B vs. sample A, t-test, p < 0.05); the growth of Lactobacillus crispatus reached a high rate of 111.23%, significantly different from samples C, D, and A (sample B vs. sample C, t-test, p < 0.05; sample B vs. sample D, t-test, p < 0.05; sample B vs. sample A, t-test, p < 0.05). Theoretically, assuming the same total dosage of the prebiotic system, the effect of sample B should be between samples C and D. However, experimental results showed that sample B actually outperformed samples C and D. This indicates that the four prebiotic components in sample B exhibit a stronger synergistic effect in promoting the growth of both Lactobacillus gasseri and Lactobacillus crispatus, demonstrating a significant synergistic effect.

[0060] These research results have important biological significance and application value: Lactobacillus gasseri has the ability to regulate intestinal flora, enhance immunity, and inhibit pathogens, and it also helps maintain local microecological balance in the vaginal environment. Lactobacillus crispatus is one of the most common and representative dominant bacterial species in the vagina of healthy women and is considered a key "guardian" against infections such as bacterial vaginosis. Sample A, through the use of Lactobacillus acidophilus LA88 postbiotics, and Sample BD, through the rational combination of multiple prebiotics and Lactobacillus acidophilus LA88 postbiotics, can not only more effectively support the proliferation of these two important probiotics, but also help establish a more stable and healthy vaginal microecological system. This synergistic effect provides a theoretical basis and technical support for the development of functional products that efficiently and targetedly regulate the structure of the vaginal flora, and has broad application prospects.

[0061] Experimental Example 6: Antibacterial Performance Testing was conducted according to the method provided in "5.1.3 Carrier Antibacterial Test" of "Methods for Evaluating Antibacterial and Antibacterial Effects" of WS / T650-2019, using the same sampling method as in Experimental Example 5. The experimental results are shown in Table 6. As can be seen from the data, the Lactobacillus acidophilus LA88 postbiotics and prebiotic-added products of the present invention exhibit excellent pathogenic bacteria inhibition efficacy when added to sanitary napkins.

[0062] Table 6: Antibacterial performance test results of different samples

[0063] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A Lactobacillus acidophilus LA88 postbiotic composition, characterized in that: The invention comprises a combination of Lactobacillus acidophilus LA88 postbiotics and prebiotics; the prebiotic combination comprises at least one of acarbose, oligofructose, oligoxylose and inulin; the preservation number of Lactobacillus acidophilus LA88 is CGMCC No. 24109.

2. A Lactobacillus acidophilus LA88 postbiotic composition according to claim 1, characterized in that: The total bacterial concentration of Lactobacillus acidophilus LA88 postbiotics was 1×10 11 -3×10 11 pcs / g.

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

4. A Lactobacillus acidophilus LA88 postbiotic composition according to claim 3, characterized in that: The prebiotic combination consists of acarbose, fructooligosaccharides, xylooligosaccharides and inulin in a mass ratio of 1-3:1-3:1-3:1-3.

5. A method for preparing a Lactobacillus acidophilus LA88 postbiotic composition according to any one of claims 1 to 4, characterized in that: A prebiotic composition is added to a Lactobacillus acidophilus LA88 postbiotic, and the mixture is mixed to obtain a postbiotic with added prebiotics; the prebiotic composition comprises acarbose, oligofructose, oligoxylose and inulin.

6. Use of a Lactobacillus acidophilus LA88 postbiotic composition according to any one of claims 1 to 4 in the preparation of a product for regulating vaginal microecology.

7. Use of a Lactobacillus acidophilus LA88 postbiotic composition according to claim 6 in preparing a product for regulating vaginal microecology, characterized in that: The product includes a sanitary napkin, a sanitary pad or a vaginal wash; 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 postbiotic composition according to claim 7 in preparing a product for regulating vaginal microecology, characterized in that: The product for regulating vaginal microecology is a sanitary napkin; Lactobacillus acidophilus LA88 postbiotics are mixed with carboxymethyl cellulose solution and then loaded on the surface layer of the sanitary napkin; the loading amount is 10 4 pieces / cm 2 .

Images