A collagen-probiotic microsphere for repairing cells of a female reproductive system and increasing elasticity of a vagina and a preparation method thereof

Abstract

The present application provides a kind of collagen-probiotic microspheres for female reproductive system cell repair, increase the elasticity of vagina collagen and preparation method thereof, it relates to the field of biological medicine.The collagen-probiotic microspheres of the present application can effectively protect the biological activity of collagen and probiotics, synergistically and stably exist in the microspheres, realize the sustained release of active ingredients in the local environment of vagina, and can be stored stably for a long time, can realize the colonization of beneficial bacteria, is not easy to inactivate, promote the migration and proliferation of vaginal epithelial cells, realize the repair of damaged tissue.The collagen-probiotic microspheres of the present application are colonized synergistically in vaginal mucosa and metabolize acid efficiently, restore the pH value of the disordered vagina to the normal physiological range, exclude the colonization of pathogenic bacteria such as gardnerella and candida albicans, comprehensively repair the damaged vaginal microecological barrier, and have good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a collagen-probiotic microsphere for repairing female reproductive system cells and increasing vaginal elasticity, and its preparation method. Background Technology

[0002] The health of the female reproductive system is a crucial factor affecting women's quality of life. With age, childbirth, hormonal changes (especially the decline in estrogen levels during and after menopause), and certain pathological conditions, the female reproductive system often undergoes a series of degenerative changes, primarily including vaginal mucosal atrophy, decreased vaginal wall elasticity, vaginal dryness, and reduced lubrication. These symptoms not only lead to painful intercourse and a decline in the quality of sexual life but can also trigger complications such as recurrent vaginitis and urinary tract infections, seriously impacting women's physical and mental health.

[0003] Currently, the commonly used interventions for the above problems in clinical practice mainly include the following categories: (1) local estrogen preparations, such as estradiol ointment or vaginal tablets, which promote vaginal epithelial proliferation and angiogenesis by supplementing exogenous estrogen, but long-term use carries the risk of endocrine disruption; (2) vaginal lubricants, such as hyaluronic acid or glycerin matrix preparations, which only provide temporary lubrication and cannot fundamentally improve tissue elasticity and repair damaged mucosa; (3) pelvic floor physical therapy, such as laser or radiofrequency therapy, which can stimulate collagen regeneration, but are expensive and require multiple treatments, resulting in limited patient compliance. None of the above methods can simultaneously meet the three major needs of tissue repair, elasticity restoration, and microecological balance regulation.

[0004] Collagen is a key extracellular matrix component for maintaining the structure and elasticity of the vaginal wall, primarily consisting of type I and type III collagen. Studies have shown that a decrease in collagen content and cross-linking in the vaginal wall is a significant molecular basis for vaginal laxity and reduced elasticity. While orally administered collagen hydrolysates can provide amino acid substrates to support collagen synthesis, the proportion that reaches target tissues after absorption through the digestive tract is limited. Topical application of exogenous collagen faces challenges in maintaining its activity and insufficient mucosal permeability.

[0005] Furthermore, maintaining a balanced vaginal microecology is crucial. In healthy women, the vaginal flora, dominated by Lactobacillus, maintains an acidic environment (pH 3.8-4.5) by producing lactic acid, inhibiting the colonization of pathogenic microorganisms. Simultaneously, their metabolites play an important role in maintaining mucosal immune function. When the number of Lactobacillus decreases or the flora structure becomes imbalanced, pathogenic bacteria proliferate more easily, leading to infectious diseases such as bacterial vaginosis and aerobic vaginitis. Currently, commercially available probiotic vaginal preparations are mostly in suppository or capsule form, which have shortcomings such as short activity retention time, low colonization efficiency, and poor release controllability.

[0006] Therefore, developing a microsphere formulation that can simultaneously deliver collagen and probiotics, and has good mucosal adhesion and sustained-release properties, is of great clinical significance and application value for the repair of female reproductive system cells and the restoration of vaginal elasticity. Summary of the Invention

[0007] Therefore, this invention proposes a collagen-probiotic microsphere for repairing female reproductive system cells and increasing vaginal elasticity, and its preparation method.

[0008] The technical solution of this invention is implemented as follows: A method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity, comprising the following steps: S1. Preparation of probiotic powder (1) Preparation of strain activation and seed culture: Inoculate probiotics into modified MRS liquid medium and incubate under anaerobic conditions at 36-38℃ for 16-20h to obtain primary seed culture. Transfer the primary seed culture to fresh modified MRS liquid medium at an inoculation rate of 3%-5% and incubate under the same conditions for 16-20h to obtain secondary seed culture. (2) High-density fermentation: Inoculate the secondary seed liquid into the fermentation medium at an inoculation rate of 5%-7%, ferment at 35-38℃, pH=6.0-6.5, and 100-200rpm for 24-28h, and collect the fermentation liquid; (3) Cell wall breaking separation: Centrifuge the fermentation broth, collect the fermentation supernatant and bacterial sludge, mix the bacterial sludge with physiological saline and resuspend, homogenize under high pressure to obtain cell wall breaking bacterial suspension, mix the fermentation supernatant and cell wall breaking bacterial suspension to obtain fermentation cell wall breaking liquid, and store at 3-5℃ for later use. (4) First, add 2%-3% trehalose to the fermentation broth and stir. Then add 3%-5% skim milk powder and stir. Next, add 0.8%-1% sodium glutamate and stir. Then add 1%-1.5% glycerol and stir. Finally, add 0.3%-0.5% L-cysteine ​​and stir. Pre-freeze at -40~-50℃ for 2-4 hours and freeze-dry under vacuum for 15-20 hours to obtain probiotic powder. S2. Preparation of Collagen-Probiotic Microspheres (1) Add mannitol, serine and trehalose to an aqueous acetic acid solution to obtain an aqueous phase. Heat and mix palmitic acid and Tween 80 to obtain an oil phase. Add the oil phase to the aqueous phase, shear at high speed, and cool to obtain an emulsion. Add collagen to an aqueous acetic acid solution to prepare a collagen solution with a concentration of 7-25 g / L. Add the collagen solution to the emulsion and stir to obtain a modified collagen solution. (2) Using coaxial electrostatic spraying, the inner axis is a modified collagen solution and the outer axis is sodium alginate solution and probiotic powder. The formed droplets are collected and cross-linked in calcium chloride solution to obtain collagen-probiotic microspheres.

[0009] Furthermore, the probiotics are Lactobacillus rhamnosus YSs069, Lactobacillus reuteri YSs207, Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and Lactobacillus reuteri HA188 in a mass ratio of 1:(1-2):(0.8-1.5):(2-3):(1.5-2).

[0010] Furthermore, in the preparation step (1) of the probiotic powder, the modified MRS liquid culture medium contains the following per 1L of purified water: 10g rapeseed peptone, 10g corn peptone, 8g yeast fluid extract, 30g glucose, 5g potassium sulfate, 2g dipotassium hydrogen phosphate, 1g magnesium sulfate, and 3mL Tween-80.

[0011] Furthermore, in step (2) of preparing the probiotic powder, the fermentation culture medium includes: rapeseed peptone 15-20 g / L, corn peptone 5-7 g / L, oligosaccharide 1-2 g / L, L-cysteine ​​hydrochloride 2-3 g / L, yeast extract 5-10 g / L, manganese sulfate monohydrate 0.2-0.4 g / L, magnesium chloride hexahydrate 0.3-0.5 g / L, sodium chloride 0.2-0.3 g / L, calcium carbonate 0.1-0.2 g / L, dipotassium hydrogen phosphate 0.2-0.5 g / L, and Tween-80 1-2 mL / L.

[0012] Furthermore, in step (2) of preparing the probiotic powder, the number of live bacteria in the fermentation broth is ≥2.8×10⁻⁶. 10 CFU / mL.

[0013] Furthermore, in the preparation step (3) of the probiotic powder, the centrifugation is carried out at 3-5℃ and 7000-9000r / min for 10-20min; the mass ratio of the bacterial sludge to physiological saline is 1:5-7; and the high-pressure homogenization is carried out by 2-4 cycles at 700-800bar.

[0014] Furthermore, in the preparation step (4) of the probiotic powder, the stirring speed is 200-300 rpm, the temperature is 4-10℃, and the time is 10-20 min.

[0015] Furthermore, in step (1) of the preparation of collagen-probiotic microspheres, the solid-liquid ratio of mannitol, serine, and trehalose to acetic acid aqueous solution is 1g:(0.5-0.7)g:(3-5)g:(10-15)mL; the mass concentration of the acetic acid aqueous solution is 5%-8%; the mass ratio of palmitic acid to Tween 80 is 1:3-5; the heating and mixing is carried out at 60-70℃ for 30-40min; the volume ratio of the oil phase to the water phase is 1:2-3; the high-speed shearing is carried out at 6000-7000rpm for 3-5min; and the volume ratio of collagen solution to emulsion is 2-3:1.

[0016] Furthermore, in step (2) of preparing collagen-probiotic microspheres, the coaxial electrostatic spray has an inner diameter of 0.8-1.0 mm, an outer diameter of 1.4-1.6 mm, a flow rate ratio of 1:3-4 between the inner and outer axes, an applied voltage of 10-15 kV, and a receiving distance of 8-10 cm; the volume ratio of sodium alginate solution to calcium chloride solution and modified collagen solution is 5-7:0.5-0.8:1, the concentration of calcium chloride solution is 0.1-0.3 mol / L, and the mass concentration of sodium alginate solution is 3-5%; the particle size of the collagen-probiotic microspheres is 30-100 μm.

[0017] A collagen-probiotic microsphere for repairing female reproductive system cells and increasing vaginal elasticity is prepared by any of the above-described preparation methods.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. The collagen-probiotic microspheres of the present invention can effectively protect the bioactivity of collagen and probiotics, which exist synergistically and stably in the microspheres, realize the sustained release of active ingredients in the local vaginal environment, and can be stored stably for a long time. They can achieve the colonization of beneficial bacteria, are not easily inactivated, promote the migration and proliferation of vaginal epithelial cells, and achieve the repair of damaged tissues.

[0019] 2. The collagen-probiotic microspheres of the present invention synergistically colonize the vaginal mucosa and efficiently metabolize and produce acid, restoring the imbalanced vaginal pH value to the normal physiological range, rejecting the colonization of pathogenic bacteria such as Gardnerella vaginalis and Candida albicans, and comprehensively repairing the damaged vaginal microecological barrier.

[0020] 3. The improved MRS liquid culture medium and fermentation culture medium of the present invention can obtain high viable bacterial counts and maintain high activity. The addition of trehalose, skim milk powder, monosodium glutamate, glycerol and L-cysteine ​​in batches in sequence can maintain a high survival rate of probiotics throughout the entire process. Detailed Implementation

[0021] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0022] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0024] The probiotics of the present invention are Lactobacillus rhamnosus YSs069, Lactobacillus reuteri YSs207, Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and Lactobacillus reuteri HA188 in a mass ratio of 1:1:1:2.5:1.8.

[0025] Lactobacillus rhamnosus YSs069 and Lactobacillus rhamnosus R0011 were both derived from the China Center for Type Culture and Preservation.

[0026] Both Lactobacillus reuteri YSs207 and Lactobacillus reuteri HA188 were obtained from the Shanghai Center for Microbial Preservation.

[0027] Lactobacillus helveticus R0052 was obtained from the China Center for Type Culture and Preservation.

[0028] The collagen of this invention is recombinant type III human collagen.

[0029] Preparation Example Preparation of probiotic powder (1) Preparation of strain activation and seed culture: Each probiotic was inoculated into a modified MRS liquid medium and cultured statically at 37°C under anaerobic conditions for 18 hours to obtain a primary seed culture. The primary seed culture was then transferred to a fresh modified MRS liquid medium at an inoculation rate of 4% and cultured under the same conditions for 18 hours to obtain a secondary seed culture. The modified MRS liquid culture medium contains the following per 1L of purified water: 10g rapeseed peptone, 10g corn peptone, 8g yeast fluid extract, 30g glucose, 5g potassium sulfate, 2g dipotassium hydrogen phosphate, 1g magnesium sulfate, and 3mL Tween-80. (2) High-density fermentation: The secondary seed culture was inoculated into the fermentation medium at an inoculation rate of 6%, and fermented at 37℃, pH=6.0-6.5, and 150 rpm for 24 h. The fermentation broth was collected, and the viable count in the fermentation broth was ≥2.8×10⁻⁶. 10 CFU / mL; The fermentation medium consisted of: 18 g / L rapeseed peptone, 6 g / L corn peptone, 1.5 g / L oligosaccharide, 2.5 g / L L-cysteine ​​hydrochloride, 8 g / L yeast extract, 0.3 g / L manganese sulfate monohydrate, 0.4 g / L magnesium chloride hexahydrate, 0.25 g / L sodium chloride, 0.15 g / L calcium carbonate, 0.3 g / L dipotassium hydrogen phosphate, and 1.5 mL / L Tween-80. (3) Cell wall breaking separation: The fermentation broth was centrifuged at 4℃ and 8000r / min for 15min, and the fermentation supernatant and bacterial sludge were collected. The bacterial sludge with a mass ratio of 1:6 was mixed with physiological saline and resuspended. The mixture was circulated 3 times at 750bar to obtain the cell wall broken bacterial suspension. The fermentation supernatant and the cell wall broken bacterial suspension were mixed to obtain the fermentation cell wall broken liquid, which was stored at 4℃ for later use. (4) First, add 2.5% trehalose to the fermentation broth and stir at 250 rpm and 8℃ for 15 min. Then add 4% skim milk powder and stir at 250 rpm and 8℃ for 15 min. Next, add 0.9% sodium glutamate and stir at 250 rpm and 8℃ for 15 min. Then add 1.3% glycerol and stir at 250 rpm and 8℃ for 15 min. Finally, add 0.4% L-cysteine ​​and stir at 250 rpm and 8℃ for 15 min. Pre-freeze at -50℃ for 3 h and freeze-dry under vacuum for 15 h to obtain the probiotic powders.

[0030] Example 1 A method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity, comprising the following steps: (1) Add mannitol, serine and trehalose to a 7wt% acetic acid aqueous solution. The solid-liquid ratio of mannitol, serine and trehalose to the acetic acid aqueous solution is 1g:0.6g:4g:15mL to obtain an aqueous phase. Mix palmitic acid and Tween 80 at 65℃ for 35min with a mass ratio of 1:4 to obtain an oil phase. Add the oil phase to the aqueous phase. The volume ratio of the oil phase to the aqueous phase is 1:2.5. Shear at 6500rpm for 4min and cool to obtain an emulsion. Add collagen to a 7wt% acetic acid aqueous solution to prepare a collagen solution with a concentration of 15g / L. Add the collagen solution to the emulsion. The volume ratio of the collagen solution to the emulsion is 2.5:1. Stir for 1h to obtain a modified collagen solution. (2) The coaxial electrostatic spraying method was adopted. The inner axis was a modified collagen solution, and the outer axis was a 4wt% sodium alginate solution and probiotic powder. The inner diameter of the inner axis was 0.9 mm, the inner diameter of the outer axis was 1.5 mm, the flow rate ratio of the inner axis to the outer axis was 1:3.5, the applied voltage was 13 kV, the receiving distance was 9 cm, and the formed droplets were collected in a 0.2 mol / L calcium chloride solution for cross-linking. The volume ratio of sodium alginate solution to calcium chloride solution and modified collagen solution was 6:0.7:1 to obtain collagen-probiotic microspheres with a particle size of 50±2 μm.

[0031] Example 2 A method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity, comprising the following steps: (1) Add mannitol, serine and trehalose to 5wt% acetic acid aqueous solution. The solid-liquid ratio of mannitol, serine and trehalose to acetic acid aqueous solution is 1g:0.5g:3g:10mL to obtain an aqueous phase. Mix palmitic acid and Tween 80 at 60℃ for 30min with a mass ratio of 1:3 to obtain an oil phase. Add the oil phase to the aqueous phase. The volume ratio of oil phase to water phase is 1:2. Shear at 6000rpm for 3min and cool to obtain an emulsion. Add collagen to 5wt% acetic acid aqueous solution to prepare a collagen solution with a concentration of 7g / L. Add the collagen solution to the emulsion. The volume ratio of collagen solution to emulsion is 2:1. Stir for 1h to obtain a modified collagen solution. (2) The coaxial electrostatic spraying method was adopted. The inner axis was a modified collagen solution, and the outer axis was a 3wt% sodium alginate solution and probiotic powder. The inner diameter of the inner axis was 0.8 mm, the inner diameter of the outer axis was 1.4 mm, the flow rate ratio of the inner axis to the outer axis was 1:3, the applied voltage was 10 kV, the receiving distance was 8 cm, and the formed droplets were collected in a 0.1 mol / L calcium chloride solution for cross-linking. The volume ratio of sodium alginate solution to calcium chloride solution and modified collagen solution was 5:0.5:1 to obtain collagen-probiotic microspheres with a particle size of 32±2 μm.

[0032] Example 3 A method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity, comprising the following steps: (1) Add mannitol, serine and trehalose to an 8wt% acetic acid aqueous solution. The solid-liquid ratio of mannitol, serine and trehalose to the acetic acid aqueous solution is 1g:0.7g:5g:15mL to obtain an aqueous phase. Mix palmitic acid and Tween 80 at 70℃ for 40min with a mass ratio of 1:5 to obtain an oil phase. Add the oil phase to the aqueous phase. The volume ratio of the oil phase to the aqueous phase is 1:3. Shear at 7000rpm for 5min and cool to obtain an emulsion. Add collagen to an 8wt% acetic acid aqueous solution to prepare a collagen solution with a concentration of 25g / L. Add the collagen solution to the emulsion. The volume ratio of the collagen solution to the emulsion is 3:1. Stir for 1h to obtain a modified collagen solution. (2) The coaxial electrostatic spraying method was adopted. The inner axis was a modified collagen solution, and the outer axis was a 5wt% sodium alginate solution and probiotic powder. The inner diameter of the inner axis was 1.0 mm, the inner diameter of the outer axis was 1.6 mm, the flow rate ratio of the inner axis to the outer axis was 1:4, the applied voltage was 15 kV, the receiving distance was 10 cm, and the formed droplets were collected in a 0.3 mol / L calcium chloride solution for cross-linking. The volume ratio of sodium alginate solution to calcium chloride solution and modified collagen solution was 7:0.8:1 to obtain collagen-probiotic microspheres with a particle size of 98±2 μm.

[0033] Comparative Example 1 The difference from Comparative Example 1 is that the modified MRS liquid medium lacks Tween-80 and rapeseed peptone, otherwise it is the same as Example 1.

[0034] Comparative Example 2 The difference from Comparative Example 1 is that the fermentation medium lacks Tween-80 and corn peptone, otherwise it is the same as Example 1.

[0035] Comparative Example 3 The difference from Comparative Example 1 is that: the cell wall breaking separation was not performed. Instead, 2.5% trehalose was added directly to the fermentation broth and stirred at 250 rpm and 8°C for 15 min. Then, 4% skim milk powder was added and stirred at 250 rpm and 8°C for 15 min. Next, 0.9% monosodium glutamate was added and stirred at 250 rpm and 8°C for 15 min. Then, 1.3% glycerol was added and stirred at 250 rpm and 8°C for 15 min. Finally, 0.4% L-cysteine ​​was added and stirred at 250 rpm and 8°C for 15 min. The mixture was then pre-frozen at -50°C for 3 h and vacuum freeze-dried for 15 h to obtain the various probiotic powders. The rest was the same as in Example 1.

[0036] Comparative Example 4 The difference from Comparative Example 1 is that trehalose, skim milk powder, monosodium glutamate, glycerol and L-cysteine ​​are mixed and added to the fermentation cell wall breaking liquid all at once, while the rest is the same as in Example 1.

[0037] Comparative Example 5 The difference from Comparative Example 1 is that collagen was directly added to a 7wt% acetic acid aqueous solution to prepare a collagen solution with a concentration of 15g / L. The coaxial electrostatic spraying method was used, with the collagen solution as the inner axis. Everything else was the same as in Example 1.

[0038] Comparative Example 6 The difference from Comparative Example 1 is that: instead of using the coaxial electrostatic spraying method, the modified collagen solution, probiotic powder and sodium alginate solution were directly mixed and cross-linked in calcium chloride solution to obtain collagen-probiotic microspheres. The rest is the same as in Example 1.

[0039] Test Example 1 The collagen-probiotic microspheres prepared in Examples 1-3 and Comparative Examples 1-6 were tested for their antibacterial effects against vaginal pathogens: Gardnerella vaginalis, Candida albicans, and Escherichia coli. Experimental method: The Oxford cup method was used to adjust the concentration of each pathogenic bacterium to 1×10⁻⁶. 6 Take 0.1 mL of the bacterial suspension (CFU / mL) and spread it evenly on the surface of the corresponding solid culture medium. Place it in an Oxford cup and add 200 μL of the test sample solution (10 mg / mL) to each cup. Incubate at 37℃ for 24 h, measure the diameter of the inhibition zone, and calculate the inhibition rate.

[0040] Antibacterial rate (%) = (D 样品 D 空白 / D 阳性对照 D 空白 )×100%.

[0041] D 样品 The average diameter (mm) of the inhibition zone in the sample group is the arithmetic mean of measurements taken in two perpendicular directions; D 空白 : The average diameter (mm) of the inhibition zone in the blank control group, i.e., the diameter of the inhibition zone around the Oxford cup with an equal volume of physiological saline added; D 阳性对照 The average diameter (mm) of the inhibition zone in the positive control group was measured using an antibiotic (metronidazole 50 μg / mL) as the positive control.

[0042] The results are shown in Table 1.

[0043] Table 1

[0044] As can be seen from Table 1, the collagen-probiotic microspheres prepared in Examples 1-3 of this invention can significantly inhibit pathogenic bacteria.

[0045] Test Example 2 The collagen-probiotic microspheres prepared in Examples 1-3 and Comparative Examples 1-6 were tested for their effects on improving vaginal laxity and increasing elasticity.

[0046] Experimental Methods: 110 female SD rats, weighing 200-250g, were randomly divided into a blank control group (n=10) and an experimental group (n=100). The experimental group was anesthetized by injecting 2% lidocaine with a syringe. Under sterile conditions, an incision was made inside the vagina of each rat to create a vaginal defect. Exogenous relaxin was then administered to promote vaginal tissue proliferation and relaxation for 7 consecutive days. After 7 days, the skin, tissues, and muscles near the vagina were observed to be relaxed. Due to the relaxation of vaginal muscles, the vulva was somewhat open, slightly red and congested, and the vulva was moist with discharge. All rats in the experimental group successfully developed the model. Eighty successfully modeled rats were randomly divided into 10 groups of 10 each: the model group, Examples 1-3, and Comparative Examples 1-6. In Examples 1-3 and Comparative Examples 1-6, the corresponding collagen-probiotic microspheres were administered to the vaginal opening at a dose of 2 mg each time. The model group received no treatment. The above procedure was repeated after 24 hours. The efficacy was evaluated after 7 days of treatment.

[0047] The evaluation indicators include: 1. TNF-α (tumor necrosis factor-α) affects the structure and function of vaginal tissue by regulating inflammatory responses and apoptosis; 2. VEGF (vascular endothelial growth factor) is a protein that promotes angiogenesis and repair, playing a crucial role in maintaining normal tissue blood supply and repairing damaged tissue. In the vagina, VEGF can promote vascular endothelial cell proliferation, stimulate the formation of new blood vessels, thereby increasing local blood flow, improving tissue nutrition, and helping to restore tissue elasticity and function; 3. Collagen I (COL-1) is the most abundant connective tissue protein in vaginal tissue, forming a fibrous structural support network that maintains the overall structure and morphology of vaginal tissue. The presence of COL-1 gives vaginal tissue a certain degree of tension and elasticity, contributing to vaginal tightness and strength; 4. Elastin is an elastic protein that helps maintain vaginal elasticity and tightness.

[0048] After homogenizing vaginal tissue, the expression of the above indicators was detected using an ELISA kit.

[0049] The results are shown in Table 2.

[0050] Table 2

[0051] Note: a) Compared with the control group, P<0.05; b) Compared with the model group, P<0.05.

[0052] As can be seen from Table 2, compared with the control group, the TNF-α content in the model group was significantly increased, while the VEGF, COL-1 and Elastin content was significantly decreased. After administering the collagen-probiotic microspheres prepared in Examples 1-3 of this invention, the TNF-α content in rats decreased, while the VEGF, COL-1 and Elastin content increased.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity, characterized in that, The specific steps include: S1. Preparation of probiotic powder (1) Preparation of strain activation and seed culture: Inoculate probiotics into modified MRS liquid medium and incubate under anaerobic conditions at 36-38℃ for 16-20h to obtain primary seed culture. Transfer the primary seed culture to fresh modified MRS liquid medium at an inoculation rate of 3%-5% and incubate under the same conditions for 16-20h to obtain secondary seed culture. (2) High-density fermentation: Inoculate the secondary seed liquid into the fermentation medium at an inoculation rate of 5%-7%, ferment at 35-38℃, pH=6.0-6.5, and 100-200rpm for 24-28h, and collect the fermentation liquid; (3) Cell wall breaking separation: Centrifuge the fermentation broth, collect the fermentation supernatant and bacterial sludge, mix the bacterial sludge with physiological saline and resuspend, homogenize under high pressure to obtain cell wall breaking bacterial suspension, mix the fermentation supernatant and cell wall breaking bacterial suspension to obtain fermentation cell wall breaking liquid, and store at 3-5℃ for later use. (4) First, add 2%-3% trehalose to the fermentation broth and stir. Then add 3%-5% skim milk powder and stir. Next, add 0.8%-1% sodium glutamate and stir. Then add 1%-1.5% glycerol and stir. Finally, add 0.3%-0.5% L-cysteine ​​and stir. Pre-freeze at -40~-50℃ for 2-4 hours and freeze-dry under vacuum for 15-20 hours to obtain probiotic powder. S2. Preparation of Collagen-Probiotic Microspheres (1) Add mannitol, serine and trehalose to an aqueous acetic acid solution to obtain an aqueous phase. Heat and mix palmitic acid and Tween 80 to obtain an oil phase. Add the oil phase to the aqueous phase, shear at high speed, and cool to obtain an emulsion. Add collagen to an aqueous acetic acid solution to prepare a collagen solution with a concentration of 7-25 g / L. Add the collagen solution to the emulsion and stir to obtain a modified collagen solution. (2) Using coaxial electrostatic spraying, the inner axis is a modified collagen solution and the outer axis is sodium alginate solution and probiotic powder. The formed droplets are collected and cross-linked in calcium chloride solution to obtain collagen-probiotic microspheres.

2. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, The probiotics are Lactobacillus rhamnosus YSs069, Lactobacillus reuteri YSs207, Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and Lactobacillus reuteri HA188 in a mass ratio of 1:(1-2):(0.8-1.5):(2-3):(1.5-2).

3. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In the preparation step (1) of the probiotic powder, the modified MRS liquid culture medium contains the following per 1L of purified water: 10g rapeseed peptone, 10g corn peptone, 8g yeast fluid extract, 30g glucose, 5g potassium sulfate, 2g dipotassium hydrogen phosphate, 1g magnesium sulfate, and 3mL Tween-80.

4. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In step (2) of preparing probiotic powder, the fermentation culture medium includes: rapeseed peptone 15-20 g / L, corn peptone 5-7 g / L, oligopolysaccharide 1-2 g / L, L-cysteine ​​hydrochloride 2-3 g / L, yeast extract 5-10 g / L, manganese sulfate monohydrate 0.2-0.4 g / L, magnesium chloride hexahydrate 0.3-0.5 g / L, sodium chloride 0.2-0.3 g / L, calcium carbonate 0.1-0.2 g / L, dipotassium hydrogen phosphate 0.2-0.5 g / L, and Tween-80 1-2 mL / L.

5. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In step (2) of the preparation of probiotic powder, the number of live bacteria in the fermentation broth is ≥2.8×10⁻⁶. 10 CFU / mL.

6. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In the preparation step (3) of probiotic powder, the centrifugation is carried out at 3-5℃ and 7000-9000r / min for 10-20min; the mass ratio of bacterial sludge to physiological saline is 1:5-7; and the high-pressure homogenization is carried out by 2-4 cycles at 700-800bar.

7. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In step (4) of preparing probiotic powder, the stirring speed is 200-300 rpm, the temperature is 4-10℃, and the time is 10-20 min.

8. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In step (1) of the preparation of collagen-probiotic microspheres, the solid-liquid ratio of mannitol, serine, and trehalose to acetic acid aqueous solution is 1g:(0.5-0.7)g:(3-5)g:(10-15)mL; the mass concentration of acetic acid aqueous solution is 5%-8%; the mass ratio of palmitic acid to Tween 80 is 1:3-5; the heating and mixing is carried out at 60-70℃ for 30-40min; the volume ratio of oil phase to water phase is 1:2-3; the high-speed shearing is carried out at 6000-7000rpm for 3-5min; and the volume ratio of collagen solution to emulsion is 2-3:

1.

9. The method for preparing collagen-probiotic microspheres for repairing female reproductive system cells and increasing vaginal elasticity as described in claim 1, characterized in that, In step (2) of preparing collagen-probiotic microspheres, the coaxial electrostatic spray has an inner diameter of 0.8-1.0 mm, an outer diameter of 1.4-1.6 mm, a flow rate ratio of 1:3-4 between the inner and outer axes, an applied voltage of 10-15 kV, and a receiving distance of 8-10 cm; the volume ratio of sodium alginate solution to calcium chloride solution and modified collagen solution is 5-7:0.5-0.8:1, the concentration of calcium chloride solution is 0.1-0.3 mol / L, and the mass concentration of sodium alginate solution is 3-5%; the particle size of the collagen-probiotic microspheres is 30-100 μm.

10. A collagen-probiotic microsphere for repairing female reproductive system cells and increasing vaginal elasticity, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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