LAgynecological antibacterial gel for improving inflammatory response of vaginitis and vagina environment and application of LAgynecological antibacterial gel

Abstract

The invention relates to the technical field of biological medicine, in particular to # imgabs0 gynecological antibacterial gel for improving inflammatory response of vaginitis and vagina environment and application of the # imgabs0 gynecological antibacterial gel. The invention provides a postbiotic composition. The postbiotic composition comprises inactivated thalli of a probiotic composition and metabolites of the inactivated thalli; the probiotic composition is prepared from lactobacillus paracasei (Lactobacillus paracasei) ProSci-92 and lactobacillus rhamnosus ProSci-109, and the probiotic composition is prepared from lactobacillus paracasei (Lactobacillus paracasei) ProSci-92, lactobacillus rhamnosus (Lactobacillus rhamnosus) ProSci-109, lactobacillus rhamnosus (Lactobacillus rhamnosus) ProSci-109 and lactobacillus rhamnosus (Lactobacillus rhamnosus) ProSci-109. The preservation number of the lactobacillus paracasei ProSci-92 is CGMCC (China General Microbiological Culture Collection Center) No.26526; the preservation number of the lactobacillus rhamnosus ProSci-109 is CGMCC (China General Microbiological Culture Collection Center) No. 25449; the postbiotic composition can effectively slow down or treat inflammatory response of vaginitis and improve the vagina environment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for improving the inflammatory response of vaginitis and the vaginal environment. Gynecological antibacterial gel and its use. Background Art

[0002] Mixed vaginitis refers to the presence of at least two pathogens that cause an abnormal local vaginal environment separately or synergistically, that is, a local manifestation of vaginal flora imbalance. In clinical practice, infections caused by only one pathogen are rare, and most are mixed infections. Common types of vaginal infections in women of childbearing age mainly include Trichomonas vaginitis, bacterial vaginosis, and vulvovaginal candidiasis. In current clinical work, there is still a lack of understanding of mixed vaginitis and inaccurate diagnosis, which has also led to varying degrees of overtreatment or undertreatment of mixed vaginitis.

[0003] The main principles of vaginitis treatment are: eliminate the cause, choose local or systemic antifungal drugs according to the patient's condition, and use antifungal drugs in combination with anti-trichomoniasis drugs and gel disinfectants, and avoid eating irritating foods. However, while killing pathogenic bacteria, these drugs destroy the vaginal environment, such as the vaginal pH value, and kill the normal vaginal flora that plays a self-cleaning role. Therefore, only temporary relief can be achieved. Due to the loss of protection from normal flora, a new round of infection will soon appear, leading to prolonged and recurrent infections. Therefore, it is urgent to find products that can effectively slow down or treat the inflammatory response of vaginitis, improve the vaginal environment, and reduce prolonged and recurrent infections. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for improving the inflammatory response of vaginitis and the vaginal environment. Gynecological antibacterial gel and its use, the Gynecological antibacterial gel can effectively slow down or treat the inflammatory response of vaginitis, improve the vaginal environment, and reduce the persistence and recurrence of the product.

[0005] To this end, the present invention provides the following technical solutions:

[0006] The embodiments disclosed in the present invention relate to a postbiotic composition, which comprises an inactivated bacterial body of a probiotic composition and a metabolite thereof;

[0007] The probiotic composition comprises Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109; the preservation number of the Lactobacillus paracasei ProSci-92 is CGMCC No. 26526; the preservation number of the Lactobacillus rhamnosus ProSci-109 is CGMCC No. 25449.

[0008] As used herein, "metabolites" include any component, compound, substance or byproduct (including but not limited to small molecule secondary metabolites) produced by the probiotic composition described herein and having any beneficial effect disclosed in the present invention. The beneficial effects include relieving or treating symptoms such as vaginal itching, vaginal pain, abnormal vaginal discharge, shortening the duration of symptoms, effectively regulating the patient's vaginal microenvironment, reducing inflammation, etc.

[0009] In some embodiments, the total number of bacteria in the postbiotic composition is ≥1.0×10 6 More preferably, the total number of bacteria in the postbiotic composition is ≥2.0×10 11 Pieces / g.

[0010] In some embodiments, the probiotic composition is inoculated with a live bacterial count ratio of Lactobacillus paracasei to Lactobacillus rhamnosus of 1:1, and fermented to prepare the postbiotic composition.

[0011] The embodiments disclosed in the present invention relate to a method for preparing the postbiotic composition, comprising:

[0012] The live bacteria of the probiotic composition are inoculated into a fermentation medium for cultivation to obtain a fermentation liquid; and the fermentation liquid is inactivated to obtain a postbiotic composition.

[0013] In some embodiments, the inoculation amount of the live bacteria of the probiotic composition in the fermentation medium is not less than 1×10 6 CFU / mL.

[0014] In some embodiments, when the probiotic composition is inoculated, the ratio of live bacteria of Lactobacillus paracasei to Lactobacillus rhamnosus is 1:1.

[0015] In some embodiments, the total number of bacteria in the postbiotic composition is not less than 1.0×10 6 Pieces / g.

[0016] In some embodiments, the fermentation broth further comprises a drying step after being inactivated, and the drying step is spray drying, freeze drying or spray freeze drying.

[0017] In some embodiments, the fermentation medium can be a common medium, more preferably a fermentation medium widely used for lactobacillus, such as MRS liquid medium, modified BHI liquid medium, etc. The components of the fermentation medium can also be a medium containing defatted soy flour, skimmed milk powder and water.

[0018] In some embodiments, the fermentation medium comprises, by mass percentage, 0.5-2% defatted soy flour and 2-10% defatted milk powder, with the remainder being water.

[0019] Embodiments disclosed in the present invention relate to the use of the postbiotic composition including the following:

[0020] (1) Use in the preparation of products for improving or treating vaginitis;

[0021] (2) Use in the preparation of products for improving vaginal environment;

[0022] (3) Use in the preparation of products for relieving or treating vaginal itching;

[0023] (4) Use in the preparation of products for relieving or treating vaginal pain;

[0024] (5) Use in the preparation of products for relieving or treating abnormal vaginal discharge;

[0025] (6) Use in inhibiting vaginitis pathogenic bacteria or in preparing products for inhibiting vaginitis pathogenic bacteria.

[0026] In some embodiments, the vaginitis is mixed vaginitis.

[0027] In some embodiments, the product includes cosmetics, medicines, or sanitary disinfection products.

[0028] In some embodiments, the sanitary disinfection product includes a private part care solution, a private part care wipe, a private part care gel, a private part care suppository, or a private part care spray;

[0029] Or, the medicine includes external medicine or oral medicine; the external medicine includes: cream, ointment, lotion, suppository, tablet, capsule, gel or skin patch; the oral medicine includes: injection, tablet, capsule, granule or powder;

[0030] Or, the cosmetics include private parts care liquid, private parts care wipes, private parts care gel, private parts care suppository, or private parts care spray.

[0031] The embodiment disclosed in the present invention relates to a method for improving the inflammatory response of vaginitis and the vaginal environment Gynecological antibacterial gel, the product comprises the postbiotic composition.

[0032] In some embodiments, in the gynecological antibacterial gel, the addition amount of the postbiotic composition is ≥0.01% by mass, for example, 0.01%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or the range between any two of the above values.

[0033] In a preferred embodiment, the amount of the postbiotic composition added is 5-70% by mass, for example, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, 25%, 27%, 30%, 34%, 38%, 43%, 48%, 52%, 57%, 60%, 62%, 65%, 67%, 70% or the range between any two of the above values. As a preferred embodiment, the amount of the postbiotic composition added is 9-67% by mass.

[0034] In some embodiments, the The gynecological antibacterial gel also includes acceptable excipients, which may be excipients acceptable in the fields of medicines, cosmetics or sanitary disinfection products.

[0035] In some embodiments, the Gynecological antibacterial gel can be a cosmetic, medicine or sanitary disinfectant. In a preferred embodiment, the gynecological antibacterial gel includes the following raw materials in parts by weight: 50-170 parts of matrine, 60-150 parts of cnidium monnieri, 0.7-6.3 parts of biological lysozyme, 0.5-4.2 parts of lactoferrin, 57-139 parts of postbiotic composition, 3.8-10.1 parts of licorice extract, 2.6-7.4 parts of dandelion extract, 1.9-6.7 parts of honeysuckle extract, 29-73 parts of aloe, 1.1-4.5 parts of borneol, 1.7-5.2 parts of hyaluronic acid, 0.0003-0.001 parts of nanosilver, and 0.17-3.88 parts of carbomer;

[0036] Optionally, the The gynecological antibacterial gel also includes water, which is supplemented to 1000 parts by weight.

[0037] The present invention also provides a method for improving the inflammatory response of vaginitis and the vaginal environment. The preparation method of gynecological antibacterial gel comprises the following steps:

[0038] Weigh according to the formula, add or not add auxiliary materials, and prepare Gynecological antibacterial gel;

[0039] Optionally, the following steps are included:

[0040] According to the formula, add Cnidium monnieri, licorice extract, dandelion extract, and honeysuckle extract, and boil them in water for 1-4 times, the amount of water added is 5-10 times the total weight of the raw materials, the boiling temperature is 80-100° C., each time for 1-5 hours, the filtrate is combined, filtered, and concentrated to obtain a concentrated solution;

[0041] According to the formula, carbomer is added into water, soaked and dissolved, and filtered to obtain a first filtrate;

[0042] According to the formula, matrine, borneol, postbiotic composition, biological lysozyme, lactoferrin, and hyaluronic acid are mixed, dissolved in water, and filtered to obtain a second filtrate;

[0043] According to the formula, the nano silver, the liquid after the aloe vera is crushed and filtered, the first filtrate, the second filtrate and the concentrated liquid are mixed, mixed thoroughly, water is added to 1000 parts by weight, and the pH is adjusted to 5-7.

[0044] The technical solution of the present invention has the following advantages:

[0045] 1. The present invention provides a postbiotic composition, which comprises an inactivated bacterial body of a probiotic composition and its metabolites; the probiotic composition comprises Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109; the preservation number of Lactobacillus paracasei ProSci-92 is CGMCC No. 26526; the preservation number of Lactobacillus rhamnosus ProSci-109 is CGMCC No. 25449; the present invention has found that the postbiotic composition can relieve or treat symptoms such as vaginal itching, vaginal pain, abnormal vaginal discharge, etc., and at the same time, shorten the duration of symptoms, effectively regulate the patient's vaginal microenvironment such as pH value, avoid affecting the normal vaginal flora that has a self-cleaning effect, and thereby reduce the recurrence rate of vaginal inflammation. In summary, the postbiotic composition can effectively slow down or treat the inflammatory response and vaginal environment of vaginitis.

[0046] 2. The present invention provides a method for improving the inflammatory response of vaginitis and the vaginal environment. Gynecological antibacterial gel, The gynecological antibacterial gel includes the following raw materials in parts by weight: 50-170 parts of matrine, 60-150 parts of cnidium monnieri, 0.7-6.3 parts of biological lysozyme, 0.5-4.2 parts of lactoferrin, 57-139 parts of postbiotic composition, 3.8-10.1 parts of licorice extract, 2.6-7.4 parts of dandelion extract, 1.9-6.7 parts of honeysuckle extract, 29-73 parts of aloe, 1.1-4.5 parts of borneol, 1.7-5.2 parts of hyaluronic acid, 0.0003-0.001 parts of nanosilver, and 0.17-3.88 parts of carbomer; after verification by the present invention, the postbiotic composition prepared by the present invention Gynecological antibacterial gel, compared with other postbiotic compositions Gynecological antibacterial gel, prepared from the postbiotic composition of the present invention The gynecological antibacterial gel can treat or relieve vaginal itching, vaginal pain, abnormal vaginal discharge and other symptoms more significantly, can significantly shorten the duration of symptoms, effectively regulate the patient's vaginal microenvironment, and reduce inflammation, indicating that the present invention The postbiotic composition and other components in the gynecological antibacterial gel have a significant synergistic effect in improving the inflammatory response of vaginitis and the vaginal environment.

[0047] Biomaterial Deposit

[0048] Lacticaseibacillus paracasei ProSci-92, taxonomically named Lacticaseibacillus paracasei, was deposited in the General Microbiology Center of China Culture Collection Administration on February 8, 2023, with the deposit number CGMCC No. 26526, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0049] Lactobacillus rhamnosus ProSci-109, taxonomically named Lactobacillus rhamnosus, was deposited in the General Microbiology Center of China Culture Collection Administration on August 1, 2022, with the deposit number CGMCC No. 25449, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. DETAILED DESCRIPTION

[0050] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0051] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0052] Matrine, CAS: 519-02-8, commercially available product, purity ≥ 98%;

[0053] Cnidium monnieri, a Chinese medicine name, is the dried mature fruit of the Apiaceae plant Cnidium monnieri (L.) Cuss., and is a commercially available product;

[0054] Lactoferrin, commercially available product, purity ≥95%;

[0055] Hyaluronic acid, commercially available product, purity ≥96%;

[0056] Borneol is natural borneol, a commercially available product with a purity of 96% to 98%;

[0057] Aloe vera (L.) Burm. f., which is commercially available, is a herbaceous plant of the family Asphodeloides in the order Liliales.

[0058] Bio-lysozyme was purchased from Yuanye Biotechnology Co., Ltd.;

[0059] Licorice extract was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd.;

[0060] Dandelion extract was purchased from Baichuan Kangze Biotechnology Co., Ltd.;

[0061] Honeysuckle extract was purchased from Xi'an Changyue Biotechnology Co., Ltd.;

[0062] Nanosilver was purchased from Nanjing Dongna Biotechnology Co., Ltd.

[0063] Improved BHI liquid medium: 10.0 g of tryptone, 17.5 g of beef heart extract powder, 5.0 g of sodium chloride, 2.5 g of disodium hydrogen phosphate, 2.0 g of glucose, 1000 ml of distilled water, and then additionally add yeast extract with a final concentration of 10% by mass, 0.1% by mass maltose, and 0.1% by mass glucose, and adjust the pH value to 7.4. Sterilize at 121°C for 15 min. Before use, add sterile fetal bovine serum with a final concentration of 10% by mass to the culture medium.

[0064] Example 1: Acquisition of Lactobacillus paracasei ProSci-92

[0065] Using naturally fermented camel milk from Alxa as the sample, 0.5 mL of the sample was added to 5 mL of MRS liquid culture medium (purchased from Beijing Road & Bridge Technology Co., Ltd.), and anaerobically cultured at 37 °C for 24 h to obtain the enriched sample; 0.5 mL of the enriched sample was added to 4.5 mL of sterile saline to obtain 10 -1 Repeat this process for 10 -1 The diluent was diluted in series to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilution; 100 μL of the gradient dilution was applied to MRS solid culture medium, and cultured anaerobically at 37°C for 48 hours to obtain colonies; colonies with typical characteristics of Lactobacillus paracasei on the MRS solid culture medium were selected, and colonies were streaked on the MRS solid culture medium with an inoculation loop, and cultured anaerobically at 37°C for 48 hours to obtain purified single colonies; the purified single colonies were picked and inoculated into 5 mL of MRS liquid culture medium, and cultured anaerobically at 37°C for 24 hours to obtain bacterial liquid; each strain corresponding to each bacterial liquid was numbered, and Gram staining, strain identification, physiological and biochemical experiments and genome identification analysis were performed to select rod-shaped Gram-positive bacteria with typical characteristics of Lactobacillus paracasei to obtain strain ProSci-92; strain ProSci-92 The 16S rDNA sequence was used for identification, and the identification result showed that it was Lactobacillus paracasei. It was named Lactobacillus paracasei ProSci-92 and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.26526.

[0066] Example 2: Postbiotic Composition

[0067] This embodiment provides a postbiotic composition, wherein the components of the postbiotic composition include inactivated bacteria and metabolites of Lactobacillus paracasei ProSci-92, and inactivated bacteria and metabolites of Lactobacillus rhamnosus ProSci-109; and the preparation method of the postbiotic composition is as follows:

[0068] Seed culture: 0.1 mL of the bacterial solution of Lactobacillus paracasei ProSci-92 (deposit number CGMCC No. 26526) was inoculated into MRS liquid medium (purchased from Beijing Luqiao Technology Co., Ltd.), and cultured at 30° C. for 30 h to obtain ProSci-92 seed solution; 0.1 mL of the bacterial solution of Lactobacillus rhamnosus ProSci-109 (deposit number CGMCC No. 25449) was inoculated into MRS liquid medium, and cultured at 30° C. for 30 h to obtain ProSci-109 seed solution;

[0069] Fermentation: ProSci-92 seed solution and ProSci-109 seed solution were inoculated into the fermentation medium. The total viable count of Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109 was 2×10 6 CFU / mL, and then cultured at 30°C for 30 h to obtain a fermentation broth; during inoculation, the ratio of the number of viable bacteria of Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109 was 1:1;

[0070] Post-treatment: sterilize the fermentation broth at 85°C for 15 min to obtain a sterilized fermentation broth; spray dry the sterilized fermentation broth to obtain a postbiotic composition (the total number of cells of Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109 in the postbiotic composition is ≥2.0×10 11 pcs / g);

[0071] Wherein, the preparation method of fermentation medium is as follows:

[0072] The fermentation medium consists of 1% defatted soy flour, 8% defatted milk powder and the balance water in percentage by mass; the components of the fermentation medium are mixed in proportion, and the mixture is stirred at 58° C. for 15 minutes (i.e., stirred at 150 rpm) to obtain a liquid; the liquid is homogenized once at 58° C. at a primary pressure of 19 MPa and a secondary pressure of 5.0 MPa to obtain a homogenized liquid; the homogenized liquid is sterilized at 93° C. for 30 minutes to obtain a sterilized liquid; the sterilized liquid is cooled to 35° C. to obtain a fermentation medium.

[0073] Embodiment 3: Gynecological antibacterial gel

[0074] This embodiment provides a gynecological antibacterial gel, including the following raw materials:

[0075] Matrine 110g, Cnidium monnieri 100g, bio-lysozyme 3.5g, lactoferrin 2.9g, the postbiotic composition in Example 2 115g, licorice extract 7g, dandelion extract 7g, honeysuckle extract 4.3g, aloe 65g, borneol 2.8g, hyaluronic acid 3.5g, nanosilver 0.0008g, and carbomer 2g.

[0076] Preparation method:

[0077] (1) According to the formula, Cnidium monnieri, Licorice extract, Taraxacum extract and Honeysuckle extract were mixed, and water was added and decocted for 3 times. The amount of water added was 6 times the total weight of the above raw materials. The decocting temperature was 100°C, and each time was 3 hours. The filtrate was combined, filtered, and concentrated to obtain a concentrated solution for standby use.

[0078] (2) Place carbomer in distilled water, soak and dissolve, and then filter to obtain a first filtrate for later use.

[0079] (3) Mix matrine, borneol, the postbiotic composition, biological lysozyme, lactoferrin, and hyaluronic acid, dissolve in distilled water, and filter to obtain a second filtrate for later use.

[0080] (4) Mix the nanosilver, the liquid after crushing and filtering of aloe vera (leaves), the first filtrate, the second filtrate and the concentrated solution, mix thoroughly and add water to 1000 g, adjust the pH to 6.0±0.2, and can.

[0081] Embodiment 4: Gynecological antibacterial gel

[0082] This embodiment provides a gynecological antibacterial gel, including the following raw materials:

[0083] Matrine 50g, Cnidium monnieri 150g, bio-lysozyme 0.7g, lactoferrin 4.2g, the postbiotic composition in Example 2 57g, licorice extract 10.1g, dandelion extract 2.6g, honeysuckle extract 6.7g, aloe 29g, borneol 1.1g, hyaluronic acid 5.2g, nanosilver 0.0003g, and carbomer 3.88g.

[0084] Preparation method:

[0085] (1) According to the formula, Cnidium monnieri, Licorice extract, Taraxacum extract and Honeysuckle extract were mixed, and water was added and decocted once. The amount of water added was 10 times the total weight of the above raw materials. The decocting temperature was 80°C and the decocting time was 5 hours. The filtrate was combined, filtered and concentrated to obtain a concentrated solution for standby use.

[0086] (2) Place carbomer in distilled water, soak and dissolve, and then filter to obtain a first filtrate for later use.

[0087] (3) Mix matrine, borneol, the postbiotic composition, biological lysozyme, lactoferrin, and hyaluronic acid, dissolve in distilled water, and filter to obtain a second filtrate for later use.

[0088] (4) Mix the nanosilver, the liquid after crushing and filtering of aloe vera (leaves), the first filtrate, the second filtrate and the concentrated solution, mix them thoroughly and add water to 1000 g, adjust the pH to 7.0±0.2, and can the mixture.

[0089] Embodiment 5: Gynecological antibacterial gel

[0090] This embodiment provides a gynecological antibacterial gel, including the following raw materials:

[0091] Matrine 170g, Cnidium monnieri 60g, bio-lysozyme 6.3g, lactoferrin 0.5g, the postbiotic composition in Example 2 139g, licorice extract 3.8g, dandelion extract 7.4g, honeysuckle extract 1.9g, aloe 73g, borneol 4.5g, hyaluronic acid 1.7g, nanosilver 0.001g, and carbomer 0.17g.

[0092] Preparation method:

[0093] (1) According to the formula, Cnidium monnieri, Licorice extract, Taraxacum extract and Honeysuckle extract were mixed, and water was added and decocted for 4 times. The amount of water added was 5 times the total weight of the above raw materials. The decocting temperature was 90°C, and each time was 1 hour. The filtrate was combined, filtered, and concentrated to obtain a concentrated solution for standby use.

[0094] (2) Place carbomer in distilled water, soak and dissolve, and then filter to obtain a first filtrate for later use.

[0095] (3) Mix matrine, borneol, the postbiotic composition, biological lysozyme, lactoferrin, and hyaluronic acid, dissolve in distilled water, and filter to obtain a second filtrate for later use.

[0096] (4) Mix the nanosilver, the liquid after crushing and filtering of aloe vera (leaves), the first filtrate, the second filtrate and the concentrated solution, mix thoroughly and add water to 1000 g, adjust the pH to 5.0±0.2, and can.

[0097] Experimental Example 1: Determination of the Acid Production Capacity of Lactobacillus

[0098] Lactobacillus rhamnosus R9639, Lactobacillus rhamnosus LGG, and Lactobacillus rhamnosus GR-1 are commercially available, and the remaining 6 strains of Lactobacillus were obtained from Beijing Ketuo Hengtong Biotechnology Co., Ltd. The specific strain information is shown in Table 1.

[0099] The experimental strains were inoculated into MRS liquid medium with an inoculation loop, cultured anaerobically at 37°C for 24 hours, and activated for three generations. The culture solution of the third generation of strains was centrifuged at 8000g for 10 minutes at 4°C, and filtered through a 0.22μm sterile filter membrane to obtain a cell-free supernatant. The pH value measured by the uninoculated MRS medium was used as a control, and the pH value of the lactobacillus supernatant after 24 hours of culture was measured to determine the acid production ability of the lactobacillus.

[0100] Table 1, determination of lactobacillus acid production capacity

[0101] Lactobacillus strains pH Uninoculated MRS medium 6.20±0.20 Lactobacillus rhamnosus R9639 3.91±0.01 Lactobacillus rhamnosus ProSci-109 3.67±0.02 Lactobacillus rhamnosus LR-82 4.02±0.00 Lactobacillus paracasei ProSci-92 3.86±0.01 Lactobacillus paracasei PC-101 4.16±0.03 Probiosci-101 3.97±0.00 Lactobacillus paracasei LC-01 4.02±0.01 Lactobacillus rhamnosus LGG 3.88±0.00 Lactobacillus rhamnosus GR-1 3.90±0.01

[0102] The pH value of the vaginal environment of healthy women is between 3.8 and 4.5, which is mainly attributed to the fact that lactobacilli colonized in the vagina use glucose in vaginal epithelial cells to produce a large amount of organic acid. A large number of in vitro studies have confirmed that the acidic environment of the vagina is not conducive to the colonization and growth of pathogenic bacteria related to vaginal diseases. In this experiment, the acid production capacity of the above 9 strains of bacteria was measured, and the results are shown in Table 1. The results showed that there was no significant difference in the acid production capacity among the lactobacilli, and the pH value was between 3.60-4.20, which is within the pH range of the vaginal environment of healthy women, indicating that the 9 lactobacilli used in the experiment have the potential to improve the pH of the vaginal environment of women with bacterial vaginitis.

[0103] Experimental Example 2: Effect of inactivated lactobacillus on the proliferation activity of Gardnerella

[0104] The lactobacillus strains in Experimental Example 1 were inoculated into MRS liquid culture medium, cultured anaerobically at 37°C for 24 h, and the fermentation broth was sterilized and inactivated at 85°C for 15 min to obtain the sterilized and inactivated fermentation broth. The bacterial count was adjusted to 1.0×10 8 The Gardnerella strain was inoculated into the modified BHI liquid medium, cultured anaerobically at 37°C for 24 h, centrifuged at 4°C (4000 r / min) for 5 min, the supernatant was discarded, the bacteria were washed three times with PBS solution and then added back into the modified BHI liquid medium, and the bacterial concentration was adjusted to 1.0×10 8 CFU / mL is reserved.

[0105] The bacterial count was set to 1.0×10 8 0.5 mL of inactivated Lactobacillus solution with a concentration of 100 / mL and 0.5 mL of Gardnerella solution were mixed, and a control group (without Lactobacillus solution) was set up. The mixture was cultured anaerobically at 37°C. After 4 h of culture, 0.2 mL of the mixed solution was taken and the concentration was adjusted to 0.5 McFarland turbidity standard. The mixture was then graded and diluted (the dilution factor was 10 -9 ), the diluted bacterial solution was inoculated onto a modified BHI agar plate and cultured anaerobically at 37°C for 24 h, and the number of viable bacteria was calculated based on the number of Gardnerella colonies.

[0106] Table 2. Effect of inactivated lactobacilli on the proliferation activity of Gardnerella

[0107]

[0108] A total of 9 inactivated lactobacillus strains were tested for their effects on the proliferation activity of vaginal Gardnerella. The activity of vaginal Gardnerella was inhibited to varying degrees, among which Lactobacillus rhamnosus ProSci-109 and Lactobacillus paracasei ProSci-92 had better inhibitory effects. This further shows that the inactivated bacterial liquids of these strains have good antagonistic effects on vaginal Gardnerella and can be used as potential probiotics for the treatment of vaginitis.

[0109] Experimental Example 3 Effect of inactivated lactobacillus combination on the proliferation activity of vaginal Gardnerella

[0110] The experiment on the effect of different inactivated lactobacillus combinations on the proliferation activity of vaginal Gardnerella was prepared according to the method of Experimental Example 2, except that in the lactobacillus combination, the inoculation ratio of each lactobacillus strain was 1:1, and the fermentation liquid after sterilization and inactivation was finally obtained, and the total number of bacteria was adjusted to 1.0×10 8 The results are shown in Table 3.

[0111] Table 3. Effect of inactivated lactobacilli on the proliferation activity of Gardnerella

[0112]

[0113]

[0114] As can be seen from the above table, a total of 31 groups of inactivated lactobacillus compositions including Lactobacillus rhamnosus + Lactobacillus rhamnosus, Lactobacillus rhamnosus + Lactobacillus paracasei and Lactobacillus paracasei + Lactobacillus paracasei on the proliferation activity of vaginal Gardnerella were detected, and the activity of vaginal Gardnerella was inhibited to varying degrees, among which the most significant inhibitory effect was the inactivated lactobacillus composition of the combination of Lactobacillus rhamnosus ProSci-109 + Lactobacillus paracasei ProSci-92, further illustrating that the inactivated bacteria of the combination of Lactobacillus rhamnosus ProSci-109 + Lactobacillus paracasei ProSci-92 of the present invention and its metabolites have good antagonistic effect on vaginal Gardnerella.

[0115] Experimental Example 4: Adding postbiotic composition Effects of gynecological antibacterial gel on inflammatory response and vaginal environment of vaginitis

[0116] 1 Experimental methods

[0117] A total of 120 patients with vaginitis aged 25 to 40 years who were admitted to the Affiliated Hospital of Inner Mongolia Medical University from February 2023 to May 2023 were selected and randomly divided into three groups:

[0118] Postbiotic group A: 40 cases, Gynecological antibacterial gel; the postbiotic composition is the combination of Lactobacillus rhamnosus ProSci-109 + Lactobacillus paracasei PC-101 in Experimental Example 3, prepared according to Example 2; The preparation method of gynecological antibacterial gel is the same as that of Example 3;

[0119] Postbiotic group B: 40 cases, Gynecological antibacterial gel; the postbiotic composition is the postbiotic composition of Lactobacillus rhamnosus ProSci-109 + Lactobacillus paracasei ProSci-92 prepared in Example 2; The preparation method of gynecological antibacterial gel is the same as that of Example 3;

[0120] Control group: 40 cases, without postbiotic composition The preparation method of the gynecological antibacterial gel is the same as that of Example 3, except that the postbiotic composition is not added.

[0121] The above groups Gynecological antibacterial gel, specification: 3g×6 pieces / box.

[0122] Among the cases in the above groups, the average age of the postbiotic group A was (31.90±1.07) years old; the average age of the postbiotic group B was (30.70±1.16) years old; and the average age of the control group was (31.05±2.15) years old. There was no significant difference in the age of the cases among the three groups (P>0.05). The independent ethics committee (IEC) reviewed and approved the test. The subjects cleaned their hands and vulva with warm water every night before use, avoiding the menstrual period. One gynecological antibacterial gel product, lying on your back with your buttocks slightly raised, slowly insert the gel catheter containing the gel solution deep into the vagina, push it into the vulva with a push rod, and keep the position for about 15 minutes. Use it continuously for 28 days, one bottle every night. During this period, family members were advised not to have sexual intercourse or other vaginal invasive operations, and not to use other antibiotics, probiotics and other drugs during the treatment. The difference is that the three groups used Whether the postbiotic composition or the postbiotic composition is added to the gynecological antibacterial gel is different.

[0123] There was no significant difference in the general data of the three groups (P>0.05). Exclusion criteria: ① Vaginitis induced by infection; ② Taking related hormone drugs 6 months before treatment; ③ Allergic to the drugs used in this treatment.

[0124] Observation indicators: ①Efficacy observation, comparing the effects before treatment (day 0) and after treatment (day 28). The clinical treatment effect was analyzed in combination with the patient's symptoms and vaginal secretions, and divided into three situations: marked effect (itching and other discomfort symptoms completely disappeared, the vaginal mucosa was light pink, and the cleanliness of the secretions was I-II), effective (itching and other discomfort symptoms were significantly alleviated, the congestion of the vaginal mucosa was improved, and the cleanliness of the secretions was II-III), and ineffective (the discomfort symptoms did not change significantly or even worsened). The total effective rate = marked effective rate + effective rate, marked effective rate = number of marked effective cases / total number of cases × 100%, effective rate = number of effective cases / total number of cases × 100%. ②Duration of symptoms and signs: Symptoms and signs observation indicators include vaginal itching, vaginal pain, and abnormal secretions. The changes in disease symptoms of the three groups of patients before and after treatment (day 0 and day 28) were observed, and the score was set from 0 to 10 points, where 0 was asymptomatic and 10 was severe symptoms. The score was positively correlated with the symptom performance. ③pH value detection. The changes in vaginal pH values ​​of the three groups of patients before and after treatment (day 0 and day 28) were measured using test strips (Merck SuPelco pH test strips), and the measurement method followed the instructions for use of the test strips. ④ Detection of inflammatory factors. 3 mL of fasting venous blood was drawn from the three groups of patients before and after treatment (day 0 and day 28), and the levels of inflammatory factors were detected using enzyme-linked immunosorbent assay. The indicators included: tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and high-sensitivity C-reactive protein (hs-CRP). ⑤ Recurrence rate: Follow-up for 6 months, record the recurrence of vaginitis symptoms in patients (at least one of clinical symptoms, pH value, and inflammatory factors returned to the pre-treatment level), recurrence rate = number of relapses / total number of people × 100%.

[0125] Data processing method: SPSS25.0 was used for statistical analysis.

[0126] 2 Experimental results

[0127] After treatment, the results are shown in Table 4 below. The total effective rate of the postbiotic group was above 95%, which was significantly higher than that of the control group, indicating that the use of the postbiotic combination was effective. Gynecological antibacterial gel can effectively enhance the overall treatment effect and accelerate the recovery of physical signs. Compared with the postbiotic group A, the postbiotic group B has a higher total effective rate and a higher significant effective rate, indicating that the therapeutic effect of the postbiotic combination group B is significantly higher than that of the postbiotic group A.

[0128] Table 4 Comparison of the effects of patients [number of cases (%)]

[0129]

[0130] After treatment, the symptom scores of the three groups of patients were lower than before treatment, and the postbiotic group was lower than the control group, and the duration of symptoms was shortened, as shown in Table 5. Gynecological antibacterial gel can treat or relieve symptoms such as vaginal itching, vaginal pain, and abnormal vaginal discharge, and shorten the duration of symptoms. Compared with the postbiotic A group, the postbiotic B group had lower symptom scores and shorter duration after treatment, and the difference was statistically significant, indicating that the postbiotic B group was significantly better than the postbiotic A group in treating symptoms such as vaginal itching, vaginal pain, and abnormal vaginal discharge, as well as shortening the duration of symptoms.

[0131] Table 5 Comparison of patient symptom assessment

[0132]

[0133] Note: Compared with the control group, *, **, *** respectively indicate p < 0.05, p < 0.01, p < 0.001, and the difference is statistically significant. Among the indicators, #, ##, ### respectively indicate p < 0.05, p < 0.01, p < 0.001, and the difference is statistically significant after treatment compared with before treatment.

[0134] After treatment, the vaginal pH values ​​of the three groups of patients were lower than before treatment, and the pH value of the postbiotic group was lower than that of the control group, as shown in Table 6. This therapeutic effect may be caused by the fact that the postbiotic composition contains bioactive substances such as organic acids, antimicrobial peptides, and short-chain fatty acids, which inhibit the growth and proliferation of harmful bacteria in the vagina, promote the decomposition and conversion of glycogen in the vagina to lactic acid, provide favorable conditions for the growth of lactic acid bacteria, maintain the stability of the acidic environment in the vagina, and enhance antibacterial ability.

[0135] Table 6 Comparison of vaginal pH values ​​in patients

[0136]

[0137] Note: Compared with the control group, *, **, *** respectively indicate p < 0.05, p < 0.01, p < 0.001, and the difference is statistically significant. Among the indicators, #, ##, ### respectively indicate p < 0.05, p < 0.01, p < 0.001, and the difference is statistically significant after treatment compared with before treatment.

[0138] After treatment, TNF-α, IL-6 and hs-CRP in the three groups were lower than before treatment, and the inflammatory indicators in the postbiotic group were lower than those in the control group, as shown in Table 7. Gynecological antibacterial gel has the effects of sterilization and anti-inflammatory, and improves vaginitis inflammation. At the same time, adding the postbiotic composition has a synergistic effect. Compared with the postbiotic A group, the TNF-α level of the postbiotic B group after treatment was lower, and the difference was statistically significant, indicating that the postbiotic B group has significantly better effects of sterilization and anti-inflammatory, and improving vaginitis inflammation than the postbiotic A group.

[0139] Table 7 Comparison of inflammation indicators in patients

[0140]

[0141]

[0142] Note: Compared with the control group, *, **, *** respectively indicate p < 0.05, p < 0.01, p < 0.001, and the difference is statistically significant. # , ## , ### It means p<0.05, p<0.01, and p<0.001 respectively, and the difference is statistically significant.

[0143] In the comparison of relapse rates, the relapse rate of the postbiotic group was less than 10% at 6 months of follow-up, while the relapse rate of the control group was more than 50%. The overall relapse rate of the postbiotic group was lower than that of the control group, which further demonstrated the effect of adding the postbiotic combination. Gynecological antibacterial gel is effective and lasting in improving the inflammatory response of vaginitis and the vaginal environment.

[0144] Table 8 Comparison of recurrence rate among patients [number of cases (%)]

[0145]

[0146] In summary, the #imgpt41# gynecological antibacterial gel with the addition of a postbiotic composition is effective in improving the symptoms of vaginitis. It can treat or relieve symptoms such as vaginal itching, vaginal pain, and abnormal vaginal discharge. At the same time, it can shorten the duration of symptoms, effectively regulate the patient's vaginal microenvironment, and reduce inflammation, with significant results.

[0147] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A postbiotic composition, characterized in that The postbiotic composition comprises inactivated bacteria of the probiotic composition and metabolites thereof; The probiotic composition comprises Lactobacillus paracasei ProSci-92 and Lactobacillus rhamnosus ProSci-109; the preservation number of the Lactobacillus paracasei ProSci-92 is CGMCC No. 26526; the preservation number of the Lactobacillus rhamnosus ProSci-109 is CGMCC No. 25449.

2. The postbiotic composition according to claim 1, characterized in that The total number of bacteria in the postbiotic composition is ≥1.0×10 6 pcs / g; Optionally, the total number of bacteria in the postbiotic composition is ≥ 2.0×10 11 Pieces / g.

3. The postbiotic composition according to claim 1 or 2, characterized in that When the probiotic composition is inoculated, the ratio of the number of live bacteria of Lactobacillus paracasei to that of Lactobacillus rhamnosus is 1:

1.

4. A method for preparing the postbiotic composition according to any one of claims 1 to 3, characterized in that: The live bacteria of the probiotic composition are inoculated into a fermentation medium for cultivation to obtain a fermentation liquid; and the fermentation liquid is inactivated to obtain a postbiotic composition.

5. The postbiotic composition of any one of claims 1 to 3 comprises the following uses: (1) Use in the preparation of products for improving or treating vaginitis; (2) Use in the preparation of products for improving vaginal environment; (3) Use in the preparation of products for relieving or treating vaginal itching; (4) Use in the preparation of products for relieving or treating vaginal pain; (5) Use in the preparation of products for relieving or treating abnormal vaginal discharge; (6) Use in inhibiting vaginitis pathogenic bacteria or in preparing products for inhibiting vaginitis pathogenic bacteria.

6. The use according to claim 5, characterized in that The vaginitis is mixed vaginitis; And / or, the product includes cosmetics, medicines or sanitary disinfection products.

7. The use according to claim 6, characterized in that The sanitary disinfection products include private parts care liquid, private parts care wet wipes, private parts care gel, private parts care suppository, private parts care sanitary napkin, or private parts care spray; Or, the medicine includes external medicine or oral medicine; the external medicine includes: cream, ointment, lotion, suppository, gel or skin patch; the oral medicine includes: injection, tablet, capsule, granule or powder.

8. A method for improving the inflammatory response of vaginitis and the vaginal environment Gynecological antibacterial gel, characterized in that: A postbiotic composition comprising any one of claims 1 to 3.

9. The method according to claim 8 Gynecological antibacterial gel, characterized in that: In the product, the addition amount of the postbiotic composition is ≥0.01% by mass; Optionally, the postbiotic composition is added in an amount of 5 to 70% by mass; Optionally, the The gynecological antibacterial gel also includes acceptable excipients, which may be excipients acceptable in the fields of medicines, cosmetics or sanitary disinfection products; Preferably, the The gynecological antibacterial gel includes the following raw materials in parts by weight: 50-170 parts of matrine, 60-150 parts of cnidium monnieri, 0.7-6.3 parts of biological lysozyme, 0.5-4.2 parts of lactoferrin, 57-139 parts of postbiotic composition, 3.8-10.1 parts of licorice extract, 2.6-7.4 parts of dandelion extract, 1.9-6.7 parts of honeysuckle extract, 29-73 parts of aloe, 1.1-4.5 parts of borneol, 1.7-5.2 parts of hyaluronic acid, 0.0003-0.001 parts of nanosilver, and 0.17-3.88 parts of carbomer; Optionally, the The gynecological antibacterial gel also includes water, which is supplemented to 1000 parts by weight.

10. A method for improving the inflammatory response of vaginitis and the vaginal environment as claimed in any one of claims 8 to 9 The preparation method of gynecological antibacterial gel is characterized in that: The steps include: Weigh according to the formula, add or not add auxiliary materials, and prepare Gynecological antibacterial gel; Optionally, the following steps are included: According to the formula, add Cnidium monnieri, licorice extract, dandelion extract, and honeysuckle extract, and boil them in water for 1-4 times, the amount of water added is 5-10 times the total weight of the raw materials, the boiling temperature is 80-100° C., each time for 1-5 hours, the filtrate is combined, filtered, and concentrated to obtain a concentrated solution; According to the formula, carbomer is added into water, soaked and dissolved, and filtered to obtain a first filtrate; According to the formula, matrine, borneol, postbiotic composition, biological lysozyme, lactoferrin, and hyaluronic acid are mixed, dissolved in water, and filtered to obtain a second filtrate; According to the formula, the nano silver, the liquid after the aloe vera is crushed and filtered, the first filtrate, the second filtrate and the concentrated liquid are mixed, mixed thoroughly, water is added to 1000 parts by weight, and the pH is adjusted to 5-7.

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