Composition combining probiotics and prebiotics, its preparation method and application

Abstract

The present invention provides a composition combining probiotics and prebiotics, its preparation method and application, which relates to the field of biomedical technology. The composition of the present invention comprises components: compound probiotics and compound prebiotics; the compound probiotics are Lactobacillus crispatus and Lactiplantibacillus plantarum with a mass ratio of 0.5-2:3-8; the preservation number of Lactobacillus crispatus is CGMCC No. 26680, and the preservation number of Lactiplantibacillus plantarum is CGMCC No. 26679; the compound prebiotics are galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin, and the mass ratio of galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin is 2.5-5:2-4:2-4:0.5-3:0.5-1.5. The present invention uses prebiotics with specific components and ratios for compounding to protect the activity of compound probiotics and improve the treatment of inflammation and the inhibition of harmful bacteria in vaginitis.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to a composition combining probiotics and prebiotics, and a preparation method and application thereof. Background Art

[0002] Vaginitis is one of the common gynecological diseases. The common types of vaginitis mainly include trichomonal vaginitis, vulvovaginal candidiasis, and bacterial vaginitis. The pathogenic bacteria causing vaginitis mainly include Candida albicans, Staphylococcus aureus, Shigella flexneri, Shigella 301, Salmonella enteritidis, Escherichia coli O157, Salmonella typhimurium, group B hemolytic streptococcus, and Pseudomonas aeruginosa. Therefore, currently, for drugs treating vaginitis, the primary choice is antibacterial drugs aiming at sterilization and anti-inflammation.

[0003] Probiotics are a class of active microorganisms beneficial to the host, and are generally referred to as active beneficial microorganisms that can produce definite health effects, thereby improving the host's microecological balance and exerting beneficial effects. There are various probiotics beneficial to the human body. Chinese invention patent CN115386519A discloses a Lactobacillus composition, a probiotic preparation, and a preparation method and application thereof. In the Lactobacillus composition, through the synergistic cooperation of two Lactobacillus strains, Lactobacillus crispatus DH200 and Lactobacillus plantarum DH230, good improvement effects on vaginitis and urinary tract infections are achieved. However, the activity of probiotics is often affected by the environment, with low survival rate and unstable effects. There are problems of reduced activity and significant decrease in the number of probiotics during storage and use, thus affecting the efficacy of probiotics. Therefore, it is necessary to protect the activity of probiotics.

[0004] Prebiotics are an indigestible food ingredient that selectively stimulates the growth and activity of one or several bacteria, thereby having a beneficial effect on the host and improving the host's health. Chinese invention patent CN107115364A discloses a compound probiotic and a preparation method thereof; the compound probiotic is composed of more than ten types of probiotics and more than two types of prebiotics. The probiotics include Bifidobacterium flora, Lactobacillus casei flora, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus rhamnosus, and Streptococcus thermophilus, and the prebiotics include soybean oligosaccharide and fructooligosaccharide; however, this invention adds more than a dozen types of probiotics, which not only increases the production cost, but also greatly increases the strict control degree of the production environment; even though prebiotics are added, due to the differences in the activity, living environment, etc. of different probiotics, the prebiotics cannot take into account protecting the activity and effective bacterial count of multiple probiotics.

[0005] Chinese Invention Patent CN115531425A discloses a vaginal preparation containing active probiotics and its preparation method. By externally using prebiotics and probiotic components, this invention promotes the restoration of vaginal microecology. The preparation method of the preparation adopts a multi-layer coating technology to store prebiotics and probiotics separately, preventing the problem of prebiotic consumption caused by post-fermentation during product storage, and enabling probiotics to be stored at room temperature without losing their activity. However, the vaginal preparation prepared by this invention is only a suppository, and the preparation method is complex, which is not conducive to industrial production.

[0006] In view of this, it is necessary to find a composition combining probiotics and prebiotics, compounding specific types and dosages of prebiotics to protect the antibacterial activity of probiotics, improve the anti-inflammatory effect, and thus improve the therapeutic effect on vaginitis. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a composition combining probiotics and prebiotics, its preparation method and application. By compounding with prebiotics of specific components and ratios, the activity of composite probiotics is protected, the treatment of inflammation and the inhibition of harmful bacteria in vaginitis are improved, and a significant antibacterial effect is brought.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] First of all, the present invention provides a composition combining probiotics and prebiotics, including components: composite probiotics and composite prebiotics;

[0010] The composite probiotics are a mixture of Lactobacillus crispatus and Lactiplantibacillus plantarum with a mass ratio of 0.5 - 2:3 - 8; the preservation number of Lactobacillus crispatus is CGMCC No. 26680, and the preservation number of Lactiplantibacillus plantarum is CGMCC No.26679;

[0011] The composite prebiotics are galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin, and the mass ratio of galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin is 2.5 - 5:2 - 4:2 - 4:0.5 - 3:0.5 - 1.5.

[0012] Preferably, the mass ratio of galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin is 3 - 4:2.5 - 3.5:2.5 - 3.5:1 - 2:0.8 - 1.2.

[0013] More preferably, the mass ratio of galactooligosaccharides, isomaltooligosaccharides, raffinose, spirulina and inulin is 3.5:3:3:1.5:1.

[0014] Preferably, the composition, by weight, comprises the following components: 0.5 - 2 parts of Lactobacillus crispatus, 3 - 8 parts of Lactiplantibacillus plantarum, 2.5 - 5 parts of galactooligosaccharide, 2 - 4 parts of isomaltooligosaccharide, 2 - 4 parts of raffinose, 0.5 - 3 parts of spirulina, and 0.5 - 1.5 parts of inulin.

[0015] More preferably, the composition, by weight, comprises the following components: 0.8 - 1.2 parts of Lactobacillus crispatus, 4 - 6 parts of Lactiplantibacillus plantarum, 3 - 4 parts of galactooligosaccharide, 2.5 - 3.5 parts of isomaltooligosaccharide, 2.5 - 3.5 parts of raffinose, 1 - 2 parts of spirulina, and 0.8 - 1.2 parts of inulin.

[0016] Even more preferably, the composition, by weight, comprises the following components: 1 part of Lactobacillus crispatus, 5 parts of Lactiplantibacillus plantarum, 3.5 parts of galactooligosaccharide, 3 parts of isomaltooligosaccharide, 3 parts of raffinose, 1.5 parts of spirulina, and 1 part of inulin.

[0017] Then, the present invention provides a method for preparing the above composition, comprising the step of: mixing the compound probiotics and the compound prebiotics to obtain the composition.

[0018] Preferably, the temperature is controlled at 5 - 30 °C during the mixing process.

[0019] Furthermore, the present invention provides a probiotic preparation, comprising the following component: the above composition.

[0020] Preferably, the probiotic preparation further comprises the following component: a pharmaceutical carrier.

[0021] The pharmaceutical carrier used can be solid, liquid or gas. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, and water. Examples of gas carriers include carbon dioxide and nitrogen.

[0022] When preparing the composition in an oral dosage form, any convenient pharmaceutical medium can be used. For example, water, ethanol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations such as suspensions and solutions; while carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrants can be used to form oral solid preparations such as powders, capsules, and tablets. Due to their ease of administration, tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers. Coating of tablets can be optionally carried out using standard aqueous or non-aqueous techniques.

[0023] Tablets containing the Chinese medicine composition or extract of the present invention can be prepared by tabletting or molding, and one or more auxiliary components or adjuvants can be optionally used. Tablets can be prepared by tabletting the active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with binders, lubricants, inert diluents, surface active substances or dispersants. Molded tablets can be molded in a suitable machine, i.e., a mixture of powdered compounds moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of the active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of the active ingredient. For example, a preparation intended for oral administration to humans may contain about 0.5 mg to about 5 g of the active drug, mixed with a suitable and convenient carrier material, which may account for about 5% to 95% of the total composition. The unit dosage form usually contains about 1 mg to about 2 g of the active ingredient, usually 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

[0024] The pharmaceutical composition suitable for parenteral administration in the present invention can be prepared as an aqueous solution or suspension of the active compound. Appropriate surfactants can be included, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures of their oils. In addition, preservatives can be added to prevent the harmful growth of microorganisms.

[0025] The drugs of the present invention can be in a form suitable for topical use, such as aerosols, creams, ointments, lotions, powders or the like. In addition, the composition can be in a suitable form for a transdermal delivery device. The Chinese medicine composition of the present invention can be used to prepare these prescriptions by conventional processing methods. For example, creams or ointments with the desired consistency are prepared by mixing hydrophilic materials and water, and about 5 wt% to about 10 wt% of the compound.

[0026] The drugs of the present invention can be in a form suitable for rectal administration, where the carrier is solid. It is preferably to make the mixture into unit dose suppositories. Suitable carriers include cocoa butter and other commonly used materials in the art. Suppositories can be prepared by first forming a composition containing a softened or melted carrier, and then cooling and shaping in a mold.

[0027] In addition to the above carrier components, the above pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surface active substances, thickening agents, lubricants, preservatives (including antioxidants), etc. In addition, other excipients can be added, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., coloring agents and flavoring agents, etc. Make the preparation isotonic with the blood of the intended recipient. The components containing the Chinese medicine composition of the present invention can also be prepared in powder or concentrated solution form.

[0028] Preferably, the dosage form of the probiotic preparation is not limited to dosage forms for administration such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, ointments, etc., and suitable pharmaceutical carriers in the art can be selected according to different dosage forms.

[0029] Finally, the present invention provides the application of the above composition in the preparation of a drug for treating female reproductive system inflammation.

[0030] Preferably, the application is the application in the preparation of a drug for treating female vaginitis.

[0031] More preferably, the vaginal pathogenic bacteria of the vaginitis are not limited to: Candida albicans, Staphylococcus aureus, Shigella flexneri, Shigella 301, Salmonella enteritidis, Escherichia coli, Salmonella typhimurium, Group B hemolytic streptococcus, and Listeria monocytogenes.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. In the present invention, galactooligosaccharide, isomaltooligosaccharide, raffinose, spirulina, and inulin are selected as composite prebiotics. The components of the composite prebiotics interact with each other, which is beneficial to be decomposed and utilized by probiotics, promotes the antibacterial and anti-inflammatory effects of Lactobacillus crispatus and Lactobacillus plantarum, inhibits the growth of harmful bacteria, simplifies the formula to the greatest extent while improving the improvement effect on vaginitis.

[0034] 2. The composite prebiotic of the present invention is obtained by the mutual combination of specific 5 components. The reasonable ratio of each component can protect the probiotics still having high activity under the conditions of high acid and high bile salts, thereby promoting the growth and activity of probiotics and improving the antibacterial and anti-inflammatory effects.

[0035] 3. The present invention can achieve the protective effect on probiotics by using a small amount of prebiotics. The formula of the composition and the probiotic preparation is simple, and the production conditions are easy to control.

[0036] Deposition description

[0037] Deposition number: CGMCC No. 26680;

[0038] Classification name: Lactobacillus crispatus Lactobacillus crispatus ;

[0039] Deposition time: February 27, 2023;

[0040] Deposition unit: China General Microbiological Culture Collection Center;

[0041] Abbreviation of deposition unit: CGMCC;

[0042] Deposition address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0043] Depositing Description

[0044] Depositing Number: CGMCC No. 26679;

[0045] Taxonomic Nomenclature: Lactiplantibacillus plantarum Lactiplantibacillus plantarum ;

[0046] Depositing Date: February 27, 2023;

[0047] Depositing Institution: China General Microbiological Culture Collection Center;

[0048] Abbreviation of Depositing Institution: CGMCC;

[0049] Depositing Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation Modes

[0050] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary illustration of the scope claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and these should also fall within the scope claimed by the present invention.

[0051] When the examples give a numerical range, it should be understood that unless otherwise specified in the present invention, any one of the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0052] The present invention will be further described below by way of specific examples. All chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified.

[0053] In the following examples, the degree of polymerization of the polygalactose is 2 - 6, and further the degree of polymerization of the galactooligosaccharide is 4; the degree of polymerization of the isomaltooligosaccharide is 2 - 10, and further the degree of polymerization of the isomaltooligosaccharide is 8. For the above components, products from different manufacturers do not have a significant impact on the effect.

[0054] In the present invention, the depositing number of Lactobacillus crispatus is CGMCC No. 26680, and the depositing number of Lactiplantibacillus plantarum is CGMCC No. 26679. The viable count of Lactobacillus crispatus is 1×10 7 CFU / -10×10 7 CFU / g, and the viable count of Lactiplantibacillus plantarum is 1×10 8 CFU / g - 10×108 CFU / g.

[0055] Example 1

[0056] A composition combining probiotics and prebiotics, with the components by weight being: 1 part Lactobacillus crispatus, 5 parts Lactiplantibacillus plantarum, 3.5 parts galactooligosaccharide, 3 parts isomaltooligosaccharide, 3 parts raffinose, 1.5 parts spirulina, and 1 part inulin. Mix the above components evenly under normal temperature conditions to obtain the composition.

[0057] Example 2

[0058] A composition combining probiotics and prebiotics, with the components by weight being: 0.8 part Lactobacillus crispatus, 4 parts Lactiplantibacillus plantarum, 3 parts galactooligosaccharide, 2.5 parts isomaltooligosaccharide, 2.5 parts raffinose, 1 part spirulina, and 0.8 part inulin. Mix the above components evenly under normal temperature conditions to obtain the composition.

[0059] Example 3

[0060] A composition combining probiotics and prebiotics, with the components by weight being: 1.2 parts Lactobacillus crispatus, 6 parts Lactiplantibacillus plantarum, 4 parts galactooligosaccharide, 3.5 parts isomaltooligosaccharide, 3.5 parts raffinose, 2 parts spirulina, and 1.2 part inulin. Mix the above components evenly under normal temperature conditions to obtain the composition.

[0061] Example 4

[0062] A composition combining probiotics and prebiotics, with the components by weight being: 0.5 part Lactobacillus crispatus, 3 parts Lactiplantibacillus plantarum, 2.5 parts galactooligosaccharide, 2 parts isomaltooligosaccharide, 2 parts raffinose, 0.5 part spirulina, and 0.5 part inulin. Mix the above components evenly under normal temperature conditions to obtain the composition.

[0063] Example 5

[0064] A composition combining probiotics and prebiotics, with the components by weight being: 2 parts Lactobacillus crispatus, 8 parts Lactiplantibacillus plantarum, 5 parts galactooligosaccharide, 4 parts isomaltooligosaccharide, 4 parts raffinose, 3 parts spirulina, and 3 parts inulin. Mix the above components evenly under normal temperature conditions to obtain the composition.

[0065] Comparative Example 1

[0066] Differing from Example 1, the ratio of Lactobacillus crispatus to Lactiplantibacillus plantarum is different. Specifically:

[0067] A composition combining probiotics and prebiotics, with the components by weight being: 0.3 parts of Lactobacillus crispatus, 5.7 parts of Lactiplantibacillus plantarum, 3.5 parts of galactooligosaccharide, 3 parts of isomaltooligosaccharide, 3 parts of raffinose, 1.5 parts of spirulina, and 1 part of inulin. Mix the above components evenly at room temperature to obtain the composition.

[0068] Comparative Example 2

[0069] Differing from Example 1, the components of the compound prebiotic are different, with isomaltooligosaccharide replaced by galactose.

[0070] Comparative Example 3

[0071] Differing from Example 1, the components of the compound prebiotic are different, with raffinose replaced by stachyose.

[0072] Comparative Example 4

[0073] Differing from Example 1, the components of the compound prebiotic are galactooligosaccharide, isomaltooligosaccharide, spirulina, and inulin. Specifically:

[0074] A composition combining probiotics and prebiotics, with the components by weight being: 1 part of Lactobacillus crispatus, 5 parts of Lactiplantibacillus plantarum, 3.5 parts of galactooligosaccharide, 3 parts of isomaltooligosaccharide, 1.5 parts of spirulina, and 1 part of inulin. Mix the above components evenly at room temperature to obtain the composition.

[0075] Comparative Example 5

[0076] Differing from Example 1, the component of the compound prebiotic is raffinose. Specifically:

[0077] A composition combining probiotics and prebiotics, with the components by weight being: 1 part of Lactobacillus crispatus, 5 parts of Lactiplantibacillus plantarum, 3 parts of raffinose. Mix the above components evenly at room temperature to obtain the composition.

[0078] Comparative Example 6

[0079] Differing from Example 1, the ratio of each component in the compound prebiotic is different. Specifically:

[0080] A composition combining probiotics and prebiotics, with the components by weight being: 1 part of Lactobacillus crispatus, 5 parts of Lactiplantibacillus plantarum, 1 part of galactooligosaccharide, 1 part of isomaltooligosaccharide, 1 part of raffinose, 4 parts of spirulina, and 5 parts of inulin. Mix the above components evenly at room temperature to obtain the composition.

[0081] Test 1 Detection of acid resistance and bile salt resistance

[0082] 1. Acid resistance experiment

[0083] (1)The compositions of each example and comparative example were cultured in an MRS liquid medium in a constant temperature incubator at 37 °C for 24 - 36 h to prepare a composition bacterial solution; the control group did not add prebiotics.

[0084] (2)Prepare a PBS buffer solution with a pH of 2. Take 1 mL of the bacterial solution cultured for 24 - 36 h, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with PBS, resuspend with 1 mL of PBS with a pH of 2, place it in a constant temperature incubator at 37 °C for 4 h, then dilute and plate, and count the viable bacteria (Lactobacillus plantarum). Three replicates. The results of the viable bacteria count in the acid tolerance experiment are shown in Table 1.

[0085] Table 1

[0086]

[0087] In Table 1, # P <0.05, ## P <0.01, compared with the group of Example 1.

[0088] As can be seen from Table 1, the composite prebiotic of the present invention can protect probiotics, has a high tolerance under strong acidic conditions, and can still grow in a strong acidic environment with a pH of 2. The results of Comparative Example 4 and Comparative Example 5 show that in the composition of the present invention, each prebiotic component interacts with each other, bringing a synergistic effect of protecting the activity of probiotics; the results of Comparative Example 2, Comparative Example 3 and Comparative Example 6 show that the non - specific prebiotic components and ratios of the present invention have a lower protective effect on probiotics.

[0089] 2. Bile salt tolerance experiment

[0090] (1)The compositions of each example and comparative example were cultured in an MRS liquid medium in a constant temperature incubator at 37 °C for 24 - 36 h to prepare a composition bacterial solution;

[0091] (2)Take 10 mL of the bacterial solution cultured for 24 h, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with PBS, and resuspend the bacterial cells with 5 mL of MRS medium. Add bile salts to prepare an MRS culture solution containing 0.2%. Take 200 μL of the resuspended bacterial solution and put it into the prepared 10 mL culture solution and culture at 37 °C for 24 - 36 h, then dilute and plate for counting. Repeat 3 times, and the results of the viable bacteria count (Lactobacillus crispatus) in the bile salt tolerance experiment are shown in Table 2. The control group did not add prebiotics.

[0092] Table 2

[0093]

[0094] In Table 2, #P < 0.05, ## P < 0.01, compared with the group of Example 1.

[0095] As can be seen from Table 2, the compound prebiotic of the present invention can protect probiotics, has a high tolerance at a relatively high bile salt concentration, can still grow in a bile salt environment of 0.2%, and has a significantly increased viable count compared with probiotics without prebiotic compounding (< P < 0.01); the results of Comparative Example 4 and Comparative Example 5 show that in the composition containing the compound prebiotic of the present invention, each prebiotic component interacts with each other, bringing a synergistic effect of protecting the activity of probiotics; the results of Comparative Example 2, Comparative Example 3 and Comparative Example 6 show that the non-specific prebiotic components and ratios of the present invention have a lower protective effect on probiotics.

[0096] Test 2 Antibacterial performance

[0097] (1) Inoculate each composition into MRS liquid medium, culture it in a carbon dioxide incubator at 37 °C for 24 - 36 h, and centrifuge it at 3000 rpm for 10 min to obtain the treated composition;

[0098] (2) Spread 30 μL of Candida albicans, Staphylococcus aureus, and Listeria monocytogenes at 1×10 9 CFU / mL on LB solid medium respectively. Gently place an Oxford cup on the plate, suck 250 μL of the treated composition into the Oxford cup, culture it at a constant temperature of 37 °C, and measure the diameter of the antibacterial zone after 8 h.

[0099] The results of the diameter of the antibacterial zone (mm) of each group are shown in Table 3.

[0100] Table 3

[0101]

[0102] In Table 3, # P < 0.05, ## P < 0.01, compared with the group of Example 1.

[0103] From the results of the diameter of the antibacterial zone in Table 3, it can be seen that the results of the antibacterial performance detection test show that the composition of the combined probiotics and prebiotics of the present invention significantly inhibits the growth of Candida albicans, Staphylococcus aureus, and Listeria monocytogenes, and the antibacterial effect is significantly better than that of the comparative examples. The composition of Comparative Example 1 changed the ratio of Lactobacillus crispatus and Lactiplantibacillus plantarum, which destroyed the synergistic effect between the two probiotics and significantly reduced the antibacterial effect (< P < 0.01, P<0.05); In Comparative Examples 2 - 5, the composition of the prebiotic was changed, resulting in a decrease in the protective effect on the probiotic, a reduction in the efficacy of the probiotic, and a significant impact on the size of the inhibition zone; in Comparative Example 6, the ratio of the prebiotic components was changed, and the antibacterial effect decreased significantly ( P <0.01, P <0.05). Therefore, only by using the composition of combined probiotic and prebiotic described in the present invention can a significantly enhanced antibacterial effect be achieved ( P <0.01, P <0.05).

[0104] Experiment 3 Animal Experiment

[0105] 1. Purchase and living environment of experimental animals

[0106] The animals were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. 104 BALB / c female mice (SPF level, 6 - 8 weeks old, weighing 20 - 22 g). During the experiment, the mice had free access to water and food. The animals were fed with standard pellet feed every day and cleaned regularly. Under specific pathogen - free conditions, the mice were kept in sterile polypropylene cages and maintained in a room with controlled temperature (22°C ± 2°C) and humidity (50% ± 15%) in a light - dark cycle (12 h:12 h), provided with standard diet and water. All mice used in this study were placed in the cages one week before the start of the study to adapt to the housing conditions.

[0107] 2. Establishment of animal model

[0108] The BALB / c female mice were subcutaneously injected with 0.05 mL of 2 mg / mL estradiol phthalate injection, 3 times continuously, once every 2 days. On the 6th day, the vagina of the mice was first rinsed 3 times with sterile PBS solution (pH = 8.5) (interval of 5 min each time). Streptococcus pyogenes, Staphylococcus aureus, and Escherichia coli O157 were mixed in a volume ratio of 1:1:2 to prepare a mixed bacterial solution with a concentration of 1×10 9 CFU / mL; The medical absorbent gelatin sponge was cut into pieces of 0.5 cm × 0.5 cm, 20 μL of the infected bacterial solution was injected into the sponge, and the sponge injected with the bacteria was inserted into the vagina of the mice (inserted about 1 cm - 1.5 cm into the vagina of the mice, and the injection amount of each bacterium was 0.025 mL / 100 g, once a day); when the vagina of the mice was significantly congested, swollen and accompanied by a large amount of purulent secretion, it indicated that the BV model (bacterial vaginitis model) of the mice was successfully prepared; In the same way, estradiol benzoate injection was given, and on the 6th day, a surgical absorbent hemostatic sponge injected with 20 μL of normal saline was inserted into the vagina of the mice as a blank control group.

[0109] 3. Grouping of animal models and treatment with drugs

[0110] (1)Grouping of animal models:

[0111] Using the method of analysis of variance with randomized block design, the mice were grouped according to their body weights. A total of 13 groups were formed, with 8 mice in each group, including: blank control group, model control group, Example 1 - Example 5 groups, Comparative Example 1 - Comparative Example 6 groups.

[0112] (2)Drug treatment:

[0113] Blank control group (C): The vagina was rinsed with 100 μL of normal saline once a day for 14 consecutive days;

[0114] Model control group (M): The vagina was rinsed with 100 μL of normal saline once a day for 14 consecutive days;

[0115] Example groups: The compositions of each example (containing 1 g of probiotics) were taken to rinse the vagina once a day for 14 consecutive days;

[0116] Comparative example groups: The compositions of each comparative example (containing 1 g of probiotics) were taken to rinse the vagina once a day for 14 consecutive days.

[0117] 4. qPCR detection

[0118] Using a reverse transcription and qPCR reaction system kit (purchased from TaKaRa, catalog number RR047A), according to the instructions, the levels of IL-1β, IL-10, and IL-17 in vaginal tissues were detected by qPCR. The detection results are shown in Table 4.

[0119] Table 4

[0120]

[0121] In Table 4, # P <0.05, ## P <0.01, ### P <0.001, compared with Example 1 group; ★ P <0.05, ★★ P <0.01, ★★★ P <0.001, compared with the model control group; △ P <0.05, △△ P <0.01, △△△ P <0.001, compared with the normal control group.

[0122] As can be seen from Table 4, compared with the blank control group, the expression levels of IL-1β and IL-17 in the model control group were significantly increased, and the groups of Examples 1-5 could significantly reduce the expression of these pro-inflammatory factors ( ★★★ P <0.001); although the groups of Comparative Examples 1-6 could also reduce the expression of pro-inflammatory factors, the inhibitory effect was significantly reduced compared with the Example groups ( # P <0.05, ## P <0.01). The expression of the anti-inflammatory factor IL-10 showed the opposite trend.

[0123] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition combining probiotics and prebiotics, characterized in that: Including components: compound probiotics and compound prebiotics; The composite probiotic is a mixture of Lactobacillus crispatus and Lactobacillus plantarum in a mass ratio of 0.5-2:3-8; the Lactobacillus crispatus is deposited as CGMCC No. 26680, and the Lactobacillus plantarum is deposited as CGMCC No. 26679; The composite prebiotics are galacto-oligosaccharide, isomaltooligosaccharide, raffinose, spirulina and inulin, and the mass ratio of galacto-oligosaccharide, isomaltooligosaccharide, raffinose, spirulina and inulin is 2.5-5:2-4:2-4:0.5-3:0.5-1.

5.

2. The composition according to claim 1, characterized in that The mass ratio of the galacto-oligosaccharide, isomaltooligosaccharide, raffinose, spirulina and inulin is 3-4:2.5-3.5:2.5-3.5:1-2:0.8-1.

2.

3. The composition according to claim 2, characterized in that The mass ratio of the galacto-oligosaccharide, isomaltooligosaccharide, raffinose, spirulina and inulin is 3.5:3:3:1.5:

1.

4. The composition according to claim 1, characterized in that The composition comprises the following components in parts by weight: 0.5-2 parts of Lactobacillus crispatus, 3-8 parts of Lactobacillus plantarum, 2.5-5 parts of galacto-oligosaccharide, 2-4 parts of isomaltooligosaccharide, 2-4 parts of raffinose, 0.5-3 parts of spirulina and 0.5-1.5 parts of inulin.

5. The composition according to claim 4, characterized in that The composition comprises the following components in parts by weight: 0.8-1.2 parts of Lactobacillus crispatus, 4-6 parts of Lactobacillus plantarum, 3-4 parts of galacto-oligosaccharide, 2.5-3.5 parts of isomaltooligosaccharide, 2.5-3.5 parts of raffinose, 1-2 parts of spirulina and 0.8-1.2 parts of inulin.

6. The composition according to claim 5, characterized in that The composition comprises the following components in parts by weight: 1 part of Lactobacillus crispatus, 5 parts of Lactobacillus plantarum, 3.5 parts of galacto-oligosaccharides, 3 parts of isomaltooligosaccharides, 3 parts of raffinose, 1.5 parts of spirulina and 1 part of inulin.

7. A method for preparing the composition according to any one of claims 1 to 6, characterized in that: The method comprises the steps of: mixing the compound probiotics and the compound prebiotics to obtain a composition.

8. The preparation method according to claim 7, characterized in that: The temperature of the mixing process is controlled at 5-30°C.

9. A probiotic preparation, characterized in that: The invention comprises the composition according to any one of claims 1 to 6.

10. Use of the composition in the preparation of a drug for treating inflammation of the female reproductive system, characterized in that: The composition is the composition according to any one of claims 1 to 6 or the composition prepared by the preparation method according to any one of claims 7 to 8.

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