A probiotic for regulating vaginal microflora and a method for preparing the same

Abstract

The present application relates to the technical field of probiotic composition, and discloses a bacterial agent for regulating vaginal microbial flora and a preparation method thereof, which comprises the following steps: preparing Lactobacillus paracasei KFY202407 powder into a seed solution; performing segmented fermentation culture on the seed solution; obtaining bacterial slurry after centrifugal enrichment of the fermentation product; mixing the bacterial slurry with a composite freeze-drying protective agent and then performing freeze-drying to obtain freeze-dried bacterial powder; and performing gelatinization treatment on the freeze-dried bacterial powder to obtain the bacterial agent. The present application realizes rapid proliferation of bacterial cells and promotes accumulation of active metabolites by adopting a two-stage fermentation process of liquid-state fermentation followed by solid-state fermentation, thereby solving the technical problems of low active bacterial proliferation efficiency and insufficient metabolites in the prior art. Meanwhile, the present application adopts a freeze-drying process of two-stage pre-freezing and drying sublimation, so that ice crystals are removed in the form of sublimation instead of melting, thereby solving the technical problem of bacterial cell damage and uncontrollable inactivation due to ice crystals in the traditional freeze-drying technology.

Description

Technical Field

[0001] This invention relates to the field of probiotic preparation technology, and more specifically, to a probiotic preparation for regulating vaginal microbiota and its preparation method. Background Technology

[0002] Vaginitis is a common reproductive tract infection in women, with candidiasis (yeast infection) causing a persistently high incidence rate. Approximately 75% of women have experienced this condition, and the recurrence rate is as high as 45%, causing significant psychological stress and disruption to daily life. Conventional clinical treatment relies on antifungal drugs such as fluconazole and clotrimazole; however, long-term use can lead to increased drug resistance in Candida albicans, resulting in treatment delays or recurrence. This has led to a year-on-year increase in the incidence of vaginitis, posing a serious challenge to current treatment options.

[0003] Lactobacillus paracasei KFY202407 is a strain with potential probiotic functions isolated from fermented yogurt in Korla, Xinjiang. Experiments have shown that it can effectively alleviate Candida albicans-induced vaginal inflammation by regulating serum inflammatory factor secretion, reducing vaginal myeloperoxidase (MPO) activity, and modulating the expression of inflammation-related genes, providing a new probiotic resource for the treatment of vaginitis. However, existing preparation technologies for converting this strain into a practical bacterial agent have several key shortcomings: First, the proliferation efficiency of live bacteria is limited during the fermentation process. The ratio of substrate to fermentation broth and the culture conditions during solid-state fermentation lack targeted optimization, making it difficult to obtain highly active fermented products. Second, the freeze-drying process is unreasonable. Traditional freeze-drying technology is prone to bacterial inactivation due to ice crystal damage and uncontrolled heating rate, resulting in low survival rate of live bacteria. Third, the preparation of bacterial agents lacks process design adapted to the vaginal microenvironment. The finished product has weak colonization ability in the vagina, is easily washed away by secretions, and is difficult to maintain the dominant bacterial population status for a long time.

[0004] Therefore, the present invention proposes a bacterial agent for regulating vaginal microbiota and its preparation method, which has important practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes a bacterial agent for regulating vaginal microbiota and a method for preparing the same, aiming to solve at least one of the problems in the background art mentioned above.

[0006] This invention proposes a bacterial agent for regulating vaginal microbiota and its preparation method, comprising the following preparation steps: Lactobacillus paracasei KFY202407 bacterial powder was inoculated into an activation medium for bacterial activation culture to obtain an activated bacterial solution; The activated bacterial solution was inoculated into a seed culture medium for seed culture to obtain a seed culture. The seed liquid was subjected to segmented fermentation culture to obtain the fermentation product; The fermented material was enriched to obtain a microbial sludge; After mixing the bacterial sludge with a composite freeze-drying protectant, a bacterial suspension is obtained; The bacterial suspension was freeze-dried to obtain freeze-dried bacterial powder; After sterilizing the gel matrix, sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder are added and stirred evenly. The pH is adjusted to between 4.0 and 4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0007] Furthermore, when inoculating the Lactobacillus paracasei KFY202407 bacterial powder into the activation medium, the inoculation was carried out at a volume ratio of Lactobacillus paracasei KFY202407 bacterial powder to activation medium of 1:30; the activation culture temperature of the strain was 36℃ and the culture time was 22h.

[0008] Furthermore, when the activated bacterial solution is inoculated into the seed culture medium, the inoculation is carried out at a volume ratio of 1:70 for the activated bacterial solution to the seed culture medium; the seed culture is carried out at a temperature of 36°C, a shaking speed of 130 r / min, and a culture time of 18 h.

[0009] Furthermore, the segmented fermentation culture specifically includes: The seed culture was inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture was carried out at 35°C for 18 hours to obtain the fermentation broth. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermented product.

[0010] Furthermore, the enrichment process is as follows: The fermentation product was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 15 min, and the precipitate was collected to obtain bacterial sludge.

[0011] Furthermore, it is characterized by, When mixing the bacterial sludge with the composite freeze-drying protectant, the bacterial sludge is added to the composite freeze-drying protectant at a mass ratio of (3-8):1, and stirred at 4°C for 30 minutes.

[0012] Furthermore, the freeze-drying process specifically includes: The bacterial suspension was pre-frozen in liquid nitrogen for 1.5 min, and then frozen at -45℃ for 3 h to obtain the pre-frozen material; The pre-frozen material was placed in an environment with a temperature of -20°C and a vacuum of 5Pa. The ambient temperature was increased from -20°C to 0°C at a rate of 0.7°C / min and maintained for 28 hours. Then the temperature was increased to 28°C and maintained for 12 hours. After the process, the material was crushed and passed through an 80-mesh sieve to obtain freeze-dried bacterial powder.

[0013] Furthermore, the composite lyophilization protectant comprises the following components in parts by weight: 8-12 parts trehalose, 5-8 parts lactose, 2-3 parts monosodium glutamate, and 1-2 parts tremella polysaccharide.

[0014] Furthermore, the gel matrix comprises the following components in parts by weight: 60-70 parts of polyethylene glycol 400 and 15-20 parts of polyethylene glycol 4000.

[0015] The present invention also provides a method for preparing a bacterial agent for regulating vaginal microbiota, which is used to prepare a bacterial agent for regulating vaginal microbiota.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention adopts a two-stage fermentation process of liquid fermentation followed by solid fermentation. First, liquid fermentation provides sufficient nutrition and an aerobic environment for Lactobacillus paracasei KFY202407 to achieve rapid cell proliferation. Then, solid fermentation is carried out using the loose and porous structure of the soybean protein isolate-konjac flour composite matrix as a carrier to provide an anaerobic microenvironment suitable for the cells, promote the accumulation of active metabolites, and solve the technical problems of low live cell proliferation efficiency and insufficient metabolites in existing fermentation technologies.

[0017] 2. This invention employs a two-stage freeze-drying process involving pre-freezing and sublimation. First, liquid nitrogen is used for rapid freezing to quickly freeze the surface moisture of the bacteria and prevent ice crystal growth. Then, deep freezing at -45°C is used to achieve uniform vitrification of the bacteria's interior. Finally, in the sublimation drying stage, the temperature is slowly raised to 0°C and then to 28°C. Without exceeding the eutectic point of the bacteria (-25~-30°C), the latent heat of sublimation is continuously provided to ensure that the ice crystals are removed through sublimation rather than melting. This solves the technical problems of bacterial cell inactivation due to ice crystal damage and uncontrolled temperature rise in traditional freeze-drying technologies. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0019] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention provides a bacterial agent for regulating vaginal microbiota and a method for preparing the same, comprising the following preparation steps: Lactobacillus paracasei KFY202407 bacterial powder was inoculated into an activation medium for bacterial activation culture to obtain an activated bacterial solution; The activated bacterial solution was inoculated into a seed culture medium for seed culture to obtain a seed culture. The seed liquid was subjected to segmented fermentation culture to obtain the fermentation product; The fermented material was enriched to obtain a microbial sludge; After mixing the bacterial sludge with a composite freeze-drying protectant, a bacterial suspension is obtained; The bacterial suspension was freeze-dried to obtain freeze-dried bacterial powder; After sterilizing the gel matrix, sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder are added and stirred evenly. The pH is adjusted to between 4.0 and 4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0024] Specifically, the Lactobacillus paracasei KFY202407 has the accession number CGMCC NO.30736 and is deposited at the China General Microbiological Culture Collection Center on May 22, 2024.

[0025] Specifically, the gel matrix is ​​heated to 115°C and kept at that temperature for 3 hours for sterilization. After cooling to 30°C, sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder are added and stirred evenly. The pH is then adjusted to 4.0-4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0026] Specifically, the mass ratio of the gel matrix, sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder is 83:4:0.8:0.4:11.8.

[0027] Specifically, the preparation method of the activation culture medium is as follows: Formula (based on 1L of culture medium): 10g peptone, 8g beef extract, 5g yeast extract, 20g glucose, 3g fructooligosaccharides, 2g triammonium citrate, 5g sodium acetate, 2g potassium dihydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1mL Tween-80, 0.01g vitamin B1, with the remainder being deionized water; Preparation steps: Weigh out peptone, beef extract, yeast extract, glucose, fructooligosaccharides, triammonium citrate, sodium acetate, potassium dihydrogen phosphate, magnesium sulfate, and manganese sulfate according to the proportions, place them in a 1L beaker, add 800mL of deionized water, stir until completely dissolved, add Tween-80 and vitamin B1, continue stirring until homogeneous, adjust the pH of the system to 6.3 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide, add deionized water to make up to 1L, and sterilize at 21℃ and 0.1MPa for 20min.

[0028] Specifically, the method for preparing the seed culture medium is as follows: Formula (based on 1L of culture medium): 15g peptone, 10g yeast extract, 20mL corn steep liquor, 25g glucose, 5g sucrose, 2.5g potassium dihydrogen phosphate, 3g sodium acetate, 0.6g magnesium sulfate, 0.3g manganese sulfate, 0.5g L-cysteine ​​hydrochloride, with the remainder being deionized water; Preparation steps: Weigh peptone, yeast extract, glucose, sucrose, potassium dihydrogen phosphate, sodium acetate, magnesium sulfate, manganese sulfate, and L-cysteine ​​hydrochloride according to the proportions, put them into a 1L beaker, add 850mL of deionized water, stir until the solids are completely dissolved, slowly add corn steep liquor while stirring, adjust the pH of the culture medium to 6.4 with 1mol / L sodium hydroxide solution, add deionized water to 1L, stir thoroughly, and sterilize at 21℃ and 0.1MPa for 18min.

[0029] It is understood that this invention adopts a two-stage fermentation process of liquid fermentation followed by solid fermentation. First, liquid fermentation provides sufficient nutrition and an aerobic environment for Lactobacillus paracasei KFY202407 to achieve rapid cell proliferation. Then, solid fermentation is carried out using the loose and porous structure of the soybean protein isolate-konjac flour composite matrix as a carrier to provide an anaerobic microenvironment suitable for the cells, promote the accumulation of active metabolites, and solve the technical problems of low live cell proliferation efficiency and insufficient metabolites in existing fermentation technologies.

[0030] Understandably, this invention employs a two-stage freeze-drying process involving pre-freezing and sublimation. First, liquid nitrogen is used for rapid freezing to quickly freeze the surface moisture of the bacteria and prevent ice crystal growth. Then, deep freezing at -45°C is used to achieve uniform vitrification of the bacteria's interior. Finally, in the sublimation drying stage, the temperature is first slowly raised to 0°C and then to 28°C. Under the premise of not exceeding the eutectic point of the bacteria (-25~-30°C), the latent heat of sublimation is continuously provided to ensure that the ice crystals are removed in the form of sublimation rather than melting. This solves the technical problems of bacterial cell inactivation due to ice crystal damage and uncontrolled temperature rise in traditional freeze-drying technology.

[0031] In this invention, when inoculating Lactobacillus paracasei KFY202407 bacterial powder into the activation medium, the inoculation is carried out at a volume ratio of Lactobacillus paracasei KFY202407 bacterial powder to activation medium of 1:30; the activation culture temperature of the strain is 36℃ and the culture time is 22h.

[0032] When the activated bacterial solution is inoculated into the seed culture medium, the inoculation is carried out at a volume ratio of 1:70 for the activated bacterial solution to the seed culture medium; the seed culture temperature is 36℃, the shaking speed is 130r / min, and the culture time is 18h.

[0033] Understandably, by inoculating Lactobacillus paracasei KFY202407 bacterial powder at a volume ratio of 1:30 into an activation medium suitable for the strain's nutritional needs, and culturing it at 36℃ under sealed static conditions for 20-22 hours, the strain's dormant metabolic system can be rapidly activated, restoring its proliferative capacity. At the same time, components such as oligofructose and vitamin B1 in the culture medium can specifically enhance the strain's activation efficiency. Subsequently, the activated bacterial solution is transferred to a more nutrient-rich seed culture medium at a volume ratio of 1:70, and cultured continuously at 36℃ and 130 r / min for 18 hours. This not only increases the dissolved oxygen content and nutrient contact efficiency of the bacterial solution through shaking, but also meets the strain's rapid proliferation needs with the help of components such as corn steep liquor and L-cysteine ​​hydrochloride in the seed culture medium, ultimately obtaining a highly active seed solution.

[0034] In this invention, the segmented fermentation culture specifically refers to: The seed culture was inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture was carried out at 35°C for 18 hours to obtain the fermentation broth. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermented product.

[0035] Specifically, during fermentation in a liquid fermentation medium, the fermentation pH is controlled between 6.2 and 6.5 by adding 10% by volume of sterile calcium carbonate suspension or 5% by volume of sterile lactic acid solution.

[0036] Specifically, the preparation method for liquid fermentation medium is as follows: Formula (based on 1L of culture medium): 12g soybean peptone, 7g yeast powder, 30g maltodextrin, 8g lactose, 1.5g triammonium citrate, 3g dipotassium hydrogen phosphate, 4g sodium acetate, 0.65g magnesium sulfate, 0.3g manganese sulfate, 1.5mL Tween-80, 5mL compound trace element solution, with the remainder being deionized water; The compound trace element solution formula (100mL) is as follows: zinc sulfate 0.1g, copper sulfate 0.05g, ferric chloride 0.03g, and deionized water 100mL.

[0037] Preparation steps: Weigh zinc sulfate, copper sulfate, and ferric chloride according to the proportion, add 100mL of deionized water, stir to dissolve, and seal to obtain a compound trace element solution; Weigh soybean peptone, yeast powder, maltodextrin, lactose, triammonium citrate, dipotassium hydrogen phosphate, sodium acetate, magnesium sulfate, and manganese sulfate according to the proportion, place them in a 1L beaker, add 800mL of deionized water, heat to 40℃ and stir continuously until all solid components are completely dissolved, add Tween-80 and 5mL of compound trace element solution, stir evenly, adjust the pH to 6.2 with 1mol / L hydrochloric acid, add deionized water to 1L, stir to mix evenly, and sterilize at 21℃ and 0.1MPa for 25min.

[0038] Specifically, the preparation method of the soy protein isolate-konjac flour composite matrix is ​​as follows: Formula (based on the preparation of 1kg composite matrix): 200g soy protein isolate, 80g konjac flour, 150g corn flour, 500g wheat bran, 50g glucose, 10g ammonium dihydrogen phosphate, 10g calcium carbonate; Preparation steps: Weigh out soy protein isolate, konjac flour, corn flour, wheat bran, glucose, ammonium dihydrogen phosphate and calcium carbonate according to the formula ratio and stir at low speed for 15 minutes. Then add deionized water until the water content is between 55% and 60% (mass fraction) and continue stirring for 20 minutes. Then sterilize at 21℃ and 0.1MPa for 30 minutes.

[0039] Understandably, in the liquid fermentation stage: the highly active seed liquid is inoculated at an 8% inoculation rate into a liquid fermentation medium with optimized nutrient ratio, and cultured for 18 hours in an anaerobic environment at 35℃ and under suitable acid-base conditions of pH 6.2-6.5. The uniformity of the liquid environment can ensure that the cells are fully in contact with nutrients, rapidly achieve proliferation, and provide a sufficient source of cells for subsequent solid-state fermentation. Solid-state fermentation stage: Solid-state fermentation uses a loose and porous soybean protein isolate-konjac flour composite substrate as a carrier and is cultured at 37℃ under anaerobic static conditions for 30 hours. This provides a suitable anaerobic microenvironment and continuous nutrient supply for the bacteria, promoting further proliferation and stable colonization of the bacteria in the substrate. It also promotes the synthesis and accumulation of a large number of active metabolites related to the relief of vaginitis.

[0040] In this invention, the enrichment process is as follows: The fermentation product was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 15 min, and the precipitate was collected to obtain bacterial sludge.

[0041] Understandably, washing twice with sterile saline can effectively remove residual liquid / solid culture medium components, bacterial metabolic byproducts, a small amount of miscellaneous bacteria, and fragments of the soy protein isolate-konjac flour composite matrix from the fermentation product. This avoids impurities interfering with the full binding of the subsequent compound freeze-drying protectant to the bacteria, while also reducing the impact of miscellaneous bacteria on the safety of the final inoculum. Centrifugation at 4000 rpm for 15 minutes can quickly separate the bacteria from the washing liquid, efficiently enriching Lactobacillus paracasei KFY202407 dispersed in the fermentation system, significantly increasing the bacterial concentration, and reducing bacterial loss during subsequent freeze-drying and formulation processes.

[0042] In this invention, when mixing the bacterial sludge with the composite freeze-drying protectant, the bacterial sludge is added to the composite freeze-drying protectant at a mass ratio of (3-8):1, and stirred at 4°C for 30 minutes; the bacterial sludge:composite freeze-drying protectant ratio is preferably 5:1. The freeze-drying process specifically involves: The bacterial suspension was pre-frozen in liquid nitrogen for 1.5 min, and then frozen at -45℃ for 3 h to obtain the pre-frozen material; The pre-frozen material was placed in an environment with a temperature of -20°C and a vacuum of 5Pa. The ambient temperature was increased from -20°C to 0°C at a rate of 0.7°C / min and maintained for 28 hours. Then the temperature was increased to 28°C and maintained for 12 hours. After the process, the material was crushed and passed through an 80-mesh sieve to obtain freeze-dried bacterial powder.

[0043] Specifically, the bacterial suspension was pre-frozen in liquid nitrogen for 1.5 minutes, and then transferred to a -45°C freezer for 3 hours to obtain pre-frozen material. The pre-frozen material was placed in a freeze dryer with a vacuum of 5 Pa, and the temperature of the plate was increased from -20°C to 0°C at a rate of 0.7°C / min for 28 hours. Then the temperature was increased to 28°C and maintained for 12 hours. After the process, the material was pulverized and passed through an 80-mesh sieve to obtain freeze-dried bacterial powder.

[0044] Understandably, the two-stage pre-freezing process involves first rapidly freezing the surface water of the bacteria in liquid nitrogen for 1.5 minutes to form a dense "protective shell," preventing ice crystals from growing and damaging the cell membrane during subsequent freezing. Then, the bacteria are transferred to a -45°C freezer for 3 hours to uniformly freeze the internal water, forming a stable glassy structure and completely locking in their active state. During the sublimation drying stage, the temperature of the plate is raised from -20°C to 0°C at a slow rate of 0.7°C / h under low pressure. This provides continuous latent heat for ice crystal sublimation without exceeding the eutectic point of the bacteria, allowing the frozen water to be removed in gaseous form, preventing melting and bacterial inactivation. Finally, the desorption drying stage raises the temperature to 28°C and maintains it for 12 hours to deeply remove residual bound water from the bacteria, significantly improving storage stability.

[0045] In this invention, the composite freeze-drying protectant comprises the following components in parts by weight: 8-12 parts trehalose, 5-8 parts lactose, 2-3 parts monosodium glutamate, and 1-2 parts tremella polysaccharide; preferably, each component comprises 10 parts trehalose, 6 parts lactose, 2.5 parts monosodium glutamate, and 1.5 parts tremella polysaccharide.

[0046] Specifically, the preparation method of the composite lyophilization protectant is as follows: Formula: 8-12 parts trehalose, 5-8 parts lactose, 2-3 parts monosodium glutamate, 1-2 parts tremella polysaccharide, solvent is deionized water, and the amount used is 6 times the mass of the total solid components.

[0047] Preparation method: Weigh trehalose, lactose, monosodium glutamate, and tremella polysaccharide according to the proportions, place them in a sterile beaker, add 6 times the mass of deionized water, and stir at 300 r / min for 15 min at room temperature to completely dissolve trehalose, lactose, and monosodium glutamate. Then place the beaker in a constant temperature water bath, heat to 45℃, and continue stirring at 300 r / min for 30 min to promote the full dissolution of tremella polysaccharide. Adjust the pH of the protective agent system to 6.0-6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and sterilize at 121℃ and 0.1 MPa for 15 min.

[0048] It is understandable that trehalose and tremella polysaccharide can form a dense and stable glassy structure, which tightly wraps the cell membrane of the bacteria during the two-stage pre-freezing, sublimation, and desorption drying process, avoiding cell membrane shrinkage and rupture caused by ice crystal damage and rapid water loss. Lactose and monosodium glutamate synergistically regulate the osmotic pressure inside the bacteria, maintain the activity of metabolic enzymes, reduce protein denaturation during freeze-drying and storage, and provide rapidly available nutrients for the revival of the bacteria.

[0049] In this invention, the gel matrix comprises the following components in parts by weight: 60-70 parts of polyethylene glycol 400 and 15-20 parts of polyethylene glycol 4000; preferably, the parts by weight of each component of the gel matrix are 65 parts of polyethylene glycol 400 and 18 parts of polyethylene glycol 4000.

[0050] Example 1 Reagent preparation: The activation culture medium, seed culture medium, liquid fermentation culture medium, and soybean protein isolate-konjac flour composite matrix were prepared according to the preparation methods disclosed above. Composite freeze-drying protectant: Take 8 parts trehalose, 5 parts lactose, 2 parts monosodium glutamate, and 1 part Tremella polysaccharide, place them in a sterile beaker, add 6 times the mass of deionized water, and stir at 300 r / min for 15 min at room temperature to completely dissolve the trehalose, lactose, and monosodium glutamate. Then place the beaker in a constant temperature water bath, heat to 45℃, and continue stirring at 300 r / min for 30 min to fully dissolve the Tremella polysaccharide. Adjust the pH of the protectant system to 6.0-6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and sterilize at 121℃ and 0.1 MPa for 15 min.

[0051] Gel matrix: Mix 60 parts of polyethylene glycol 400 and 15 parts of polyethylene glycol 4000.

[0052] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 bacterial powder into activation medium at a volume ratio of 1:30, and incubate in a sealed container at 36℃ for 22 hours to obtain activated bacterial solution. S2. Inoculate the activated bacterial solution into the seed culture medium at a volume ratio of 1:70, and culture at 36℃ with shaking at 130 r / min for 18 h to obtain the seed solution. S3. The seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture is carried out at 35°C for 18 hours to obtain the fermentation broth. During the fermentation, the pH is controlled between 6.2 and 6.5 by adding 10% by volume of sterile calcium carbonate suspension or 5% by volume of sterile lactic acid solution. S4. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermentation product. S5. Wash the fermentation material twice with sterile physiological saline, centrifuge at 4000 rpm for 15 min, collect the precipitate to obtain bacterial sludge, add the bacterial sludge to the compound freeze-drying protectant at a mass ratio of bacterial sludge:compound freeze-drying protectant of 3:1, stir at 4℃ for 30 min to obtain bacterial suspension. S6. Pre-freeze the bacterial suspension in liquid nitrogen for 1.5 min, then transfer it to a -45℃ low-temperature freezer for 3 h to obtain pre-frozen material; place the pre-frozen material in a freeze dryer and control the vacuum degree to 5 Pa, raise the temperature of the plate from -20℃ to 0℃ at a rate of 0.7℃ / min for 28 h, then raise the temperature to 28℃ and maintain it for 12 h, after which pulverize the material and pass it through an 80-mesh sieve to obtain freeze-dried bacterial powder; S7. Sterilize the gel matrix by heating it to 115℃ and holding it at that temperature for 3 hours. After cooling it to 30℃, add sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder to the gel matrix according to the mass ratio of gel matrix: sodium carboxymethyl cellulose: sodium ascorbate: disodium hydrogen phosphate: lyophilized bacterial powder of 83:4:0.8:0.4:11.8. Stir well and then adjust the pH to 4.0-4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0053] Example 2 Reagent preparation: The activation culture medium, seed culture medium, liquid fermentation culture medium, and soybean protein isolate-konjac flour composite matrix were prepared according to the preparation methods disclosed above. Composite freeze-drying protectant: Take 10 parts trehalose, 6 parts lactose, 2.5 parts monosodium glutamate, and 1.5 parts tremella polysaccharide, place them in a sterile beaker, add 6 times the mass of deionized water, and stir at 300 r / min for 15 min at room temperature to completely dissolve the trehalose, lactose, and monosodium glutamate. Then place the beaker in a constant temperature water bath, heat to 45℃, and continue stirring at 300 r / min for 30 min to promote the full dissolution of tremella polysaccharide. Adjust the pH of the protectant system to 6.0-6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and sterilize at 121℃ and 0.1 MPa for 15 min.

[0054] Gel matrix: Mix 65 parts of polyethylene glycol 400 and 18 parts of polyethylene glycol 4000.

[0055] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 bacterial powder into activation medium at a volume ratio of 1:30, and incubate in a sealed container at 36℃ for 22 hours to obtain activated bacterial solution. S2. Inoculate the activated bacterial solution into the seed culture medium at a volume ratio of 1:70, and culture at 36℃ with shaking at 130 r / min for 18 h to obtain the seed solution. S3. The seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture is carried out at 35°C for 18 hours to obtain the fermentation broth. During the fermentation, the pH is controlled between 6.2 and 6.5 by adding 10% by volume of sterile calcium carbonate suspension or 5% by volume of sterile lactic acid solution. S4. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermentation product. S5. Wash the fermentation material twice with sterile physiological saline, centrifuge at 4000 rpm for 15 min, collect the precipitate to obtain bacterial sludge, add the bacterial sludge to the compound freeze-drying protectant at a mass ratio of bacterial sludge:compound freeze-drying protectant of 5:1, stir at 4℃ for 30 min to obtain bacterial suspension. S6. Pre-freeze the bacterial suspension in liquid nitrogen for 1.5 min, then transfer it to a -45℃ low-temperature freezer for 3 h to obtain pre-frozen material; place the pre-frozen material in a freeze dryer and control the vacuum degree to 5 Pa, raise the temperature of the plate from -20℃ to 0℃ at a rate of 0.7℃ / min for 28 h, then raise the temperature to 28℃ and maintain it for 12 h, after which pulverize the material and pass it through an 80-mesh sieve to obtain freeze-dried bacterial powder; S7. Sterilize the gel matrix by heating it to 115℃ and holding it at that temperature for 3 hours. After cooling it to 30℃, add sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder to the gel matrix according to the mass ratio of gel matrix: sodium carboxymethyl cellulose: sodium ascorbate: disodium hydrogen phosphate: lyophilized bacterial powder of 83:4:0.8:0.4:11.8. Stir well and then adjust the pH to 4.0-4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0056] Example 3 Reagent preparation: The activation culture medium, seed culture medium, liquid fermentation culture medium, and soybean protein isolate-konjac flour composite matrix were prepared according to the preparation methods disclosed above. Composite freeze-drying protectant: Take 12 parts trehalose, 8 parts lactose, 3 parts monosodium glutamate, and 2 parts tremella polysaccharide, place them in a sterile beaker, add 6 times the mass of deionized water, and stir at 300 r / min for 15 min at room temperature to completely dissolve the trehalose, lactose, and monosodium glutamate. Then place the beaker in a constant temperature water bath, heat to 45℃, and continue stirring at 300 r / min for 30 min to promote the full dissolution of tremella polysaccharide. Adjust the pH of the protectant system to 6.0-6.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and sterilize at 121℃ and 0.1 MPa for 15 min.

[0057] Gel matrix: Mix 70 parts of polyethylene glycol 400 and 20 parts of polyethylene glycol 4000.

[0058] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 bacterial powder into activation medium at a volume ratio of 1:30, and incubate in a sealed container at 36℃ for 22 hours to obtain activated bacterial solution. S2. Inoculate the activated bacterial solution into the seed culture medium at a volume ratio of 1:70, and culture at 36℃ with shaking at 130 r / min for 18 h to obtain the seed solution. S3. The seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture is carried out at 35°C for 18 hours to obtain the fermentation broth. During the fermentation, the pH is controlled between 6.2 and 6.5 by adding 10% by volume of sterile calcium carbonate suspension or 5% by volume of sterile lactic acid solution. S4. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermentation product. S5. Wash the fermentation material twice with sterile physiological saline, centrifuge at 4000 rpm for 15 min, collect the precipitate to obtain bacterial sludge, add the bacterial sludge to the compound freeze-drying protectant at a mass ratio of bacterial sludge to compound freeze-drying protectant of 8:1, stir at 4℃ for 30 min to obtain bacterial suspension. S6. Pre-freeze the bacterial suspension in liquid nitrogen for 1.5 min, then transfer it to a -45℃ low-temperature freezer for 3 h to obtain pre-frozen material; place the pre-frozen material in a freeze dryer and control the vacuum degree to 5 Pa, raise the temperature of the plate from -20℃ to 0℃ at a rate of 0.7℃ / min for 28 h, then raise the temperature to 28℃ and maintain it for 12 h, after which pulverize the material and pass it through an 80-mesh sieve to obtain freeze-dried bacterial powder; S7. Sterilize the gel matrix by heating it to 115℃ and holding it at that temperature for 3 hours. After cooling it to 30℃, add sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder to the gel matrix according to the mass ratio of gel matrix: sodium carboxymethyl cellulose: sodium ascorbate: disodium hydrogen phosphate: lyophilized bacterial powder of 83:4:0.8:0.4:11.8. Stir well and then adjust the pH to 4.0-4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0059] Comparative Example 1 Reagent preparation: The activation culture medium, seed culture medium, liquid fermentation culture medium, and soybean protein isolate-konjac flour composite matrix were prepared according to the preparation methods disclosed above. Compound freeze-drying protectant (by mass): 10% skim milk, 5% trehalose, 3% glycerin, with the remainder being deionized water.

[0060] Gel matrix: Mix 65 parts of polyethylene glycol 400 and 18 parts of polyethylene glycol 4000.

[0061] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 bacterial powder into activation medium at a volume ratio of 1:30, and incubate in a sealed container at 36℃ for 22 hours to obtain activated bacterial solution. S2. Inoculate the activated bacterial solution into the seed culture medium at a volume ratio of 1:70, and culture at 36℃ with shaking at 130 r / min for 18 h to obtain the seed solution. S3. The seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture is carried out at 35°C for 18 hours to obtain the fermentation broth. During the fermentation, the pH is controlled between 6.2 and 6.5 by adding 10% by volume of sterile calcium carbonate suspension or 5% by volume of sterile lactic acid solution. S4. Centrifuge the fermentation broth at 4000 rpm for 15 min, collect the precipitate, wash it twice with sterile physiological saline to obtain bacterial sludge, add the bacterial sludge to the compound freeze-drying protectant at a mass ratio of bacterial sludge:compound freeze-drying protectant of 5:1, stir at 4℃ for 30 min to obtain bacterial suspension. S6. Transfer the bacterial suspension to a -45℃ low temperature freezer and freeze for 3 hours to obtain pre-frozen material; put the pre-frozen material into a freeze dryer and control the vacuum degree to 5Pa, set the plate temperature to -20℃ for 28 hours, then raise the temperature to 28℃ and maintain it for 12 hours. After the end, crush the material and pass it through an 80-mesh sieve to obtain freeze-dried bacterial powder. S7. Sterilize the gel matrix by heating it to 115℃ and holding it at that temperature for 3 hours. After cooling it to 30℃, add sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder to the gel matrix according to the mass ratio of gel matrix: sodium carboxymethyl cellulose: sodium ascorbate: disodium hydrogen phosphate: lyophilized bacterial powder of 83:4:0.8:0.4:11.8. Stir well and then adjust the pH to 4.0-4.5 to obtain a bacterial agent for regulating vaginal microbiota.

[0062] Test Example 1 Sample preparation: Three batches of freeze-dried bacterial powder were prepared according to the preparation methods of Example 2 and Comparative Example 1. 1g of freeze-dried bacterial powder was taken from each batch to prepare bacterial agents according to the preparation methods of Example 2 and Comparative Example 1.

[0063] Test method: Samples were taken at key points in the sample preparation and storage process, and the number of viable bacteria at each point was calculated; Sampling nodes: T0: The bacterial sludge collected in step S5 of Example 2 and the bacterial sludge collected in step S5 of Comparative Example 1; T1: Freeze-dried bacterial powder prepared in Example 2 and Comparative Example 1; T2: Seal the bacterial powder, place it in a 4℃ refrigerator, and take a sample after one month of storage; T3: Seal the bacterial powder, place it in a 4℃ refrigerator, and take a sample after 3 months of storage; T4: Seal the bacterial powder, place it in a 4℃ refrigerator, and take samples after 6 months of storage; T5: Final inoculum.

[0064] Viable cell count: 1g of each sample was taken, serially diluted, and then anaerobically cultured on MRS agar using the pour plate method (37℃, 48h). Each dilution was performed in triplicate, and the average value was taken.

[0065] The test results are shown in Table 1: Table 1: Changes in viable bacterial counts before and after freeze-drying and during storage (CFU / g, ×10⁻⁶) 10 )

[0066] As can be seen, the freeze-dried survival rate of Example 2 of the present invention is as high as 92.8%, which is significantly better than that of Comparative Example 1 (83.3%). After being stored at 4°C for 6 months, the viable bacteria retention rate of Example 2 of the present invention remained at 95.7% (i.e., a decrease of only 4.3%), while that of Comparative Example 1 decreased by 20.0% and 30.0%. Moreover, after further formulating the bacterial powder into a gel form, the viable bacteria retention rate of Example 2 of the present invention was still as high as 90.5%, while that of Comparative Example 1 was only 80.0%.

[0067] Test Example 2 Cell culture: Human vaginal epithelial cell line VK2 / E6E7 was seeded into 24-well plates and cultured until a complete monolayer was formed.

[0068] Bacterial preparation: Take the lyophilized bacterial powders from Example 2 and Comparative Example 1 at time point T1 in Test Example 1, revive them with sterile PBS, and wash by centrifugation. Resuspend them in antibiotic-free cell maintenance medium, and adjust the concentration of all bacterial suspensions to 1.0 × 10⁻⁶. 8 CFU / mL (initial adjustment by McFarland turbidimetric method, accurate quantification by plate count method).

[0069] Co-incubation and washing: Aspirate the culture medium from the cell wells, add 1 mL of bacterial suspension to each well, and co-incubate at 37°C and 5% CO2 for 2 hours. After incubation, gently wash 5 times with pre-warmed PBS to thoroughly remove any unattached bacteria.

[0070] Cell lysis and counting: Add 1 mL of PBS solution containing 0.1% Triton X-100 to each well, and pipette to lyse cells and adhering bacteria. Collect the lysate, perform serial dilutions, and pour plates for counting; this represents the number of viable adhering bacteria.

[0071] The test results are shown in Table 2: Table 2: Results of in vitro cell adhesion test

[0072] As can be seen, the in vitro cell adhesion rate of the bacteria in Example 2 of the present invention reached 5.40%, which is significantly improved by 35.0% compared with the comparative example 1 (4.00%) of the traditional process.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a bacterial agent for regulating vaginal microbiota, characterized in that, The preparation steps include the following: Lactobacillus paracasei KFY202407 bacterial powder was inoculated into an activation medium for bacterial activation culture to obtain an activated bacterial solution; The activated bacterial solution was inoculated into a seed culture medium for seed culture to obtain a seed culture. The seed liquid was subjected to segmented fermentation culture to obtain the fermentation product; The fermented material was enriched to obtain a microbial sludge; After mixing the bacterial sludge with a composite freeze-drying protectant, a bacterial suspension is obtained; The bacterial suspension was freeze-dried to obtain freeze-dried bacterial powder; After sterilizing the gel matrix, sodium carboxymethyl cellulose, sodium ascorbate, disodium hydrogen phosphate, and lyophilized bacterial powder are added and stirred evenly. The pH is adjusted to between 4.0 and 4.5 to obtain a bacterial agent for regulating vaginal microbiota.

2. The method for preparing a bacterial agent for regulating vaginal microbiota according to claim 1, characterized in that, When inoculating Lactobacillus paracasei KFY202407 bacterial powder into the activation medium, the inoculation was carried out at a volume ratio of Lactobacillus paracasei KFY202407 bacterial powder to activation medium of 1:30; the activation culture temperature of the strain was 36℃ and the culture time was 22h.

3. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 2, characterized in that, When the activated bacterial solution is inoculated into the seed culture medium, the inoculation is carried out at a volume ratio of 1:70 for the activated bacterial solution to the seed culture medium; the seed culture temperature is 36℃, the shaking speed is 130r / min, and the culture time is 18h.

4. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 3, characterized in that, The segmented fermentation culture specifically refers to: The seed culture was inoculated into the liquid fermentation medium at a volume ratio of 1:25, and anaerobic culture was carried out at 35°C for 18 hours to obtain the fermentation broth. The fermentation broth was inoculated into the soybean protein isolate-konjac flour composite matrix at a ratio of 1.6 ml: 1 g, and anaerobic culture was carried out at 37°C for 30 h to obtain the fermented product.

5. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 4, characterized in that, The enrichment process is as follows: The fermentation product was washed twice with sterile physiological saline, centrifuged at 4000 rpm for 15 min, and the precipitate was collected to obtain bacterial sludge.

6. The preparation method of the bacterial agent for regulating vaginal microbiota according to claim 5, characterized in that, When mixing the bacterial sludge with the composite freeze-drying protectant, the bacterial sludge is added to the composite freeze-drying protectant at a mass ratio of (3-8):1, and stirred at 4°C for 30 minutes.

7. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 6, characterized in that, The freeze-drying process specifically involves: The bacterial suspension was pre-frozen in liquid nitrogen for 1.5 min, and then frozen at -45℃ for 3 h to obtain the pre-frozen material; The pre-frozen material was placed in an environment with a temperature of -20°C and a vacuum of 5Pa. The ambient temperature was increased from -20°C to 0°C at a rate of 0.7°C / min and maintained for 28 hours. Then the temperature was increased to 28°C and maintained for 12 hours. After the process, the material was crushed and passed through an 80-mesh sieve to obtain freeze-dried bacterial powder.

8. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 7, characterized in that, The composite lyophilization protectant comprises the following components in parts by weight: 8-12 parts trehalose, 5-8 parts lactose, 2-3 parts monosodium glutamate, and 1-2 parts tremella polysaccharide.

9. The method for preparing the bacterial agent for regulating vaginal microbiota according to claim 8, characterized in that, The gel matrix comprises the following components in parts by weight: 60-70 parts of polyethylene glycol 400 and 15-20 parts of polyethylene glycol 4000.

10. A bacterial agent for regulating vaginal microbiota prepared by the method for preparing a bacterial agent for regulating vaginal microbiota according to any one of claims 1-9.

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