A synbiotic composition capable of improving bacterial vaginitis and its application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a synbiotic composition that can improve bacterial vaginitis and its application. After extensive research, the present invention found that the extract of the medicine-food combination can replace glucose as the carbon source required for the growth of Lactobacillus reuteri; and the extract of the medicine-food combination has high polysaccharides, quercetin and other effective ingredients, which can promote the proliferation of Lactobacillus reuteri; the flavonoids, alkaloids and other ingredients contained therein can also effectively inhibit the proliferation of pathogenic bacteria in the private parts. The synbiotic composition obtained by compounding Lactobacillus reuteri and the extract of the medicine-food combination has a synergistic effect in antibacterial and anti-inflammatory effects, and can effectively improve the symptoms of bacterial vaginitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a synbiotic composition capable of improving bacterial vaginitis and application thereof. Background Art

[0002] Bacterial vaginosis (BV) is a common vaginal infection in women of childbearing age, which can lead to premature birth, infertility or other vaginal diseases. The specific cause of BV has not yet been elucidated, but most scholars believe that the occurrence of the disease is closely related to the structure of the vaginal microbiome. There are a variety of microbial communities distributed in the female vagina, and different microorganisms antagonize and coexist with each other to maintain the physiological function of the female reproductive tract. The flora of a healthy female vagina is mainly lactobacillus, while this type of flora is reduced in the vagina of BV patients, resulting in excessive growth of other pathogenic bacteria, such as Gardnerella vaginalis, which changes the vaginal environment and causes inflammation.

[0003] Currently, the treatments for bacterial vaginitis mainly include: oral antibiotics, such as metronidazole, tinidazole, etc., which can effectively inhibit the growth and reproduction of bacteria; the use of vaginal washes and vaginal suppositories, such as metronidazole suppositories, tinidazole suppositories, etc.; or the use of Chinese herbal prescriptions or Chinese patent medicines, such as Chinese herbal decoctions that clear away heat and toxic substances, and reduce inflammation and kill bacteria, for external use as auxiliary treatments for bacterial vaginitis.

[0004] Compared with drug treatment, probiotics have less toxic and side effects, can selectively stimulate the proliferation of specific natural bacterial strains in the vagina, restore the balance of vaginal flora, and thus play a therapeutic role. At present, a large number of studies on probiotics are focused on topical probiotics that directly act on the vagina. There are mainly two vaginal probiotics: lactobacillus vaginal capsules and Dingjunsheng, with active ingredients of Streptococcus faecalis and Lactobacillus delbrueckii, respectively. However, there are relatively few studies on improving bacterial vaginitis through oral probiotics. Currently, there are UREX and ASTARTE developed by Chr. Hansen on the market, which are used in various oral products for female reproductive tract health. CN108576802B discloses a composition for maintaining women's health, including cranberry powder, yeast β-glucan, Lactobacillus acidophilus, Lactobacillus rosenbergii and Lactobacillus rhamnosus. CN106511976A provides a composition for alleviating gynecological inflammation and improving immunity, including stachyose, inulin, cranberry extract, isomaltooligosaccharide, collagen, plant lactobacillus, Lactobacillus reuteri TR02, Lactobacillus rhamnosus TR08, Lactobacillus acidophilus. The prebiotics added in the above invention are mostly traditional prebiotics, such as carbohydrate compounds such as inulin, stachyose, and beta-glucan, but they can only be used as nutrients for probiotics, and they have no obvious preventive and therapeutic effects on bacterial vaginitis, etc. Although cranberry extract can play a certain role in female private protection, it has not been confirmed that it has a proliferative effect on probiotics. Therefore, it is a very meaningful research direction to find a new type of prebiotic that has both female private protection and probiotic nutrients, and is used for the preparation of female private protection synbiotics. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a synbiotic composition that can improve the symptoms of bacterial vaginitis.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a synbiotic composition, comprising the following components in parts by weight: 5-15 parts of Lactobacillus reuteri powder and 15-50 parts of a medicinal and edible combination extract; the medicinal and edible combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials, and the extraction solvent used is a water-soluble substance of betaine, fructose, xylitol, lactic acid and glycine.

[0008] The present invention selects two herbs, dandelion and cornus officinalis, as raw materials for extraction to obtain a medicinal and edible composite extract. Dandelion has the effects of clearing away heat and detoxification, removing dampness and clearing heat, and anti-inflammatory and sterilization, and can effectively eliminate pathogens. At the same time, removing dampness and clearing heat helps to improve the damp and hot environment in the vagina, thereby alleviating inflammatory symptoms; Cornus officinalis has the characteristics of nourishing the liver and kidney, astringing and consolidating, and has a good conditioning effect on women's menstrual disorders, metrorrhagia and leukorrhea. The composite use of these two medicinal and edible raw materials can comprehensively exert multiple effects such as clearing away heat and detoxification, antibacterial and anti-inflammatory, and immunomodulation, thereby effectively treating bacterial vaginitis.

[0009] The present invention has found through extensive research that the extract of the medicine-food combination can replace glucose as the carbon source required for the growth of Lactobacillus reuteri; and the extract of the medicine-food combination has high polysaccharides, quercetin and other effective ingredients, which can promote the proliferation of Lactobacillus reuteri; the flavonoids, alkaloids and other ingredients contained therein can also effectively inhibit the proliferation of pathogenic bacteria in the private parts. The present invention has proved in vitro that the extract of the medicine-food combination has the effect of probiotic proliferation, and the synbiotic composition obtained by compounding Lactobacillus reuteri and the extract of the medicine-food combination is a complementary synbiotic, which has a synergistic effect in antibacterial and anti-inflammatory effects, and can effectively improve the symptoms of bacterial vaginitis.

[0010] In the extraction solvent used in the present invention, betaine, lactic acid, and glycine serve as hydrogen bond acceptors, while fructose and xylitol have the characteristics of hydrogen bond donors. The hydrogen bond acceptors and hydrogen bond donors interact with each other, so that the mixed extract has good permeability, which plays an effect similar to supramolecular extraction. The active ingredients in the plants will further form hydrogen bonds and van der Waals forces with the components of the natural deep eutectic solvent, and more binding forces are introduced into the system. These interaction forces help to extract the active ingredients more effectively and targetedly, thereby more efficiently extracting the effective ingredients in the mixed raw materials; at the same time, the present invention uses ultrasound-assisted extraction to improve the extraction efficiency of the raw materials and protect the biological activity of the functional substances in the raw materials.

[0011] Preferably, the synbiotic composition comprises the following components in parts by weight: 15 parts of Lactobacillus reuteri powder and 50 parts of the food-drug combination extract.

[0012] Preferably, in the extraction solvent, the weight ratio of betaine, fructose, xylitol, lactic acid and glycine is 5:5:5:30:30:25.

[0013] Preferably, the preparation method of the food-drug combination extract comprises the following steps:

[0014] (1) adding an extraction solvent to dandelion powder, performing ultrasonic extraction, centrifuging, and collecting a filtrate;

[0015] (2) Adding cornus officinalis powder to the filtrate obtained in step (1), performing ultrasonic extraction, centrifugation, concentration, and drying to obtain the medicine-food combination extract.

[0016] The present invention has found through a large number of experimental explorations that the various plant raw materials in the medicinal and edible composition have different ingredients, such as dandelion, which is rich in flavonoids and can be well extracted in natural low eutectic solvents. At the same time, flavonoids are rich in hydroxyl groups, which are very good hydrogen bond donors and can further enhance the permeability of natural low eutectic solvents. Cornus officinalis is rich in sugars and organic acids, and has a relatively large viscosity. Therefore, the post-extraction of Cornus officinalis can ensure the fluidity of the solvent. The present invention can promote the connection of related substances through hydrogen bonds through step-by-step extraction and the properties of the extracted substances, and fully extract the effective components of each raw material.

[0017] Preferably, in steps (1) and (2), the conditions for ultrasonic extraction are: ultrasonic extraction at 30°C for 30 min, and the ultrasonic power is 250w.

[0018] Preferably, the Lactobacillus reuteri is Lactobacillus reuteri HCS02-001.

[0019] Preferably, in the synbiotic composition, the number of viable bacteria in the Lactobacillus reuteri powder is at least 10 10 CFU.

[0020] In a second aspect, the present invention provides use of the above-mentioned synbiotic composition in the preparation of a preparation or medicine for improving or treating vaginitis.

[0021] Preferably, the vaginitis comprises bacterial vaginitis.

[0022] In a third aspect, the present invention provides a preparation or medicine for improving or treating vaginitis, comprising the above-mentioned synbiotic composition.

[0023] The beneficial effects of the present invention are:

[0024] The present invention has found through extensive research that the extract of the medicine-food combination can replace glucose as the carbon source required for the growth of Lactobacillus reuteri; and the extract of the medicine-food combination has high polysaccharides, quercetin and other effective ingredients, which can promote the proliferation of Lactobacillus reuteri; the flavonoids, alkaloids and other ingredients contained in it can also effectively inhibit the proliferation of pathogenic bacteria in the private parts. The synbiotic composition obtained by compounding Lactobacillus reuteri and the extract of the medicine-food combination has a synergistic effect in antibacterial and anti-inflammatory effects, and can effectively improve the symptoms of bacterial vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A typical graph of the fluorescence intensity of Gardnerella vaginalis in the zebrafish urogenital tract after sample treatment.

[0026] Figure 2 A typical graph of the fluorescence intensity of Staphylococcus aureus in the zebrafish urogenital tract after sample treatment. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] Unless otherwise specified, the experimental methods used in the embodiments and / or comparative examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0029] Carbon-deficient MRS broth liquid culture medium: peptone 10g, yeast extract 5g, beef extract powder 10g, K2HPO4 2g, CH3COONa·3H2O 5g, ammonium citrate tribasic 2g, MgSO4·7H2O 0.1g, MnSO4·4H2O 0.05g, Tween 80 1mL, dilute to 1L deionized water, sterilize at 121℃ for 15min;

[0030] MRS broth liquid culture medium: peptone 10g, yeast extract 5g, beef extract powder 10g, glucose 20g, K2HPO42g, CH3COONa·3H2O 5g, triammonium citrate 2g, MgSO4·7H2O 0.1g, MnSO4·4H2O 0.05g, Tween 801mL, dilute to 1L deionized water, sterilize at 121℃ for 15min;

[0031] The raw materials used in the present invention include: MRS solid culture medium purchased from Beijing Luqiao Technology Co., Ltd.; Lactobacillus reuteri HCS02-001 powder purchased from Jiangxi Renren Health Microecology Technology Co., Ltd., with a specification of 10 11 CFU / g (1g of bacterial powder contains 10 11 CFU Lactobacillus reuteri HCS02-001).

[0032] Example 1

[0033] An embodiment of the synbiotic composition of the present invention comprises the following components in parts by weight: 5 parts of Lactobacillus reuteri HCS02-001 powder and 15 parts of a medicinal and edible combination extract; the medicinal and edible combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials.

[0034] The preparation method of the medicine-food combination extract comprises the following steps:

[0035] S1, raw material pretreatment; the dried dandelion and cornus raw materials are washed and dusted, dried, crushed and sieved to obtain dandelion powder and cornus powder;

[0036] S2. Prepare extraction solvent: the extraction solvent comprises the following components in weight percentage: 5% betaine, 5% fructose, 5% xylitol, 30% lactic acid, 30% glycine, and 25% water.

[0037] S3, weigh 100g of dandelion powder, add 2000g of extraction solvent, the extraction temperature is 30°C, the extraction time is 30min, and the ultrasonic power is 250w;

[0038] S4, after the extraction is completed, centrifuge to obtain a filtrate, add 100g of Cornus officinalis powder to the filtrate, and perform ultrasonic extraction at 30°C for 30min with an ultrasonic power of 250w;

[0039] S5. After the extraction is completed, the filtrate is centrifuged and concentrated at low temperature to Brix = 20-25%;

[0040] S6, spray drying, the air inlet temperature is controlled at 170-180° C., and the air outlet temperature is 70-80° C.; the medicinal and edible combined extract (brown solid powder, weighed to be 26.5 g) is obtained.

[0041] Example 2

[0042] An embodiment of the synbiotic composition of the present invention comprises the following components in parts by weight: 10 parts of Lactobacillus reuteri HCS02-001 powder and 35 parts of a medicinal and edible combination extract; the medicinal and edible combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials.

[0043] The preparation method of the medicine-food combination extract is the same as that in Example 1.

[0044] Example 3

[0045] An embodiment of the synbiotic composition of the present invention comprises the following components in parts by weight: 15 parts of Lactobacillus reuteri HCS02-001 powder and 50 parts of a medicine-food combination extract; the medicine-food combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials.

[0046] The preparation method of the medicine-food combination extract is the same as that in Example 1.

[0047] Comparative Example 1

[0048] A comparative example of the synbiotic composition of the present invention, the difference between this comparative example and Example 3 is that this comparative example contains only 15 parts of Lactobacillus reuteri HCS02-001 powder.

[0049] Comparative Example 2

[0050] A comparative example of the synbiotic composition of the present invention, the difference between this comparative example and Example 3 is that this comparative example only contains 50 portions of the medicinal and edible combination extract.

[0051] Comparative Example 3

[0052] A comparative example of the synbiotic composition of the present invention, the synbiotic composition comprises the following components in parts by weight: 15 parts of Lactobacillus plantarum HCS03-001 powder and 50 parts of a medicine-food combination extract; the medicine-food combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials.

[0053] The preparation method of the medicine-food combination extract is the same as that in Example 1.

[0054] The Lactobacillus plantarum HCS03-001 powder was purchased from Jiangxi Renren Healthy Microecology Technology Co., Ltd.

[0055] Comparative Example 4

[0056] A comparative example of the synbiotic composition of the present invention, the synbiotic composition comprises the following components in parts by weight: 15 parts of Lactobacillus reuteri HCS02-001 powder and 50 parts of oligofructose.

[0057] The oligofructose was purchased from Quantum Hi-Tech (Guangdong) Biotechnology Co., Ltd.

[0058] Comparative Example 5

[0059] A comparative example of the synbiotic composition of the present invention comprises the following components in parts by weight: 15 parts of Lactobacillus plantarum HCS03-001 powder and 50 parts of oligofructose.

[0060] The Lactobacillus plantarum HCS03-001 powder was purchased from Jiangxi Renren Healthy Microecology Technology Co., Ltd.

[0061] The oligofructose was purchased from Quantum Hi-Tech (Guangdong) Biotechnology Co., Ltd.

[0062] Comparative Example 6

[0063] A comparative example of the synbiotic composition of the present invention, the difference between this comparative example and Example 1 is that the composition and weight percentage ratio of the extraction solvent in this comparative example are: 5% fructose, 5% xylitol, 30% lactic acid, 30% glycine, and 25% water. The rest is the same as Example 1.

[0064] Comparative Example 7

[0065] A comparative example of the synbiotic composition of the present invention is different from Example 1 only in that the composition and weight percentage ratio of the extraction solvent in this comparative example are: 15% betaine, 30% lactic acid, 30% glycine, and 25% water. The rest is the same as Example 1.

[0066] Comparative Example 8

[0067] A comparative example of the synbiotic composition of the present invention, the difference between this comparative example and Example 1 is only that the composition of the extraction solvent of this comparative example and its weight percentage ratio are: 25% betaine, 10% fructose, 10% xylitol, 12% lactic acid, 12% glycine, and 31% water.

[0068] Comparative Example 9

[0069] A comparative example of the synbiotic composition of the present invention, the difference between this comparative example and Example 1 is only that the composition of the extraction solvent in this comparative example and its weight percentage ratio are: 25% glucose, 10% fructose, 10% xylitol, 12% lactic acid, 12% glycine, and 31% water.

[0070] Test Example 1: Probiotic Proliferation Experiment

[0071] 1. Proliferation culture of Lactobacillus reuteri HCS02-001

[0072] Lactobacillus reuteri HCS02-001 powder was dissolved with as little MRS culture medium as possible, inoculated into 10 mL MRS liquid culture medium, and cultured statically. After culturing at 37°C for 24 hours, the plate was streaked, and a single colony on the plate was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours. After microscopic examination, it was determined that a pure bacterial solution of Lactobacillus reuteri HCS02-001 (10 8 CFU / mL) and then stored in a -80°C refrigerator with 30% glycerol.

[0073] 2. The food and medicine extract prepared in Example 1 was dissolved in sterile water to prepare a 10% (m / v) food and medicine extract solution. After filtering through a 0.22 μm aqueous filter, 1 mL was added to 9 mL of carbon-deficient MRS liquid culture medium to make the final concentration of the food and medicine extract in the mixed solution 1% (m / v) as the test group.

[0074] 1 mL of 10% oligofructose solution was added into 9 mL of MRS liquid culture medium as the oligofructose group, wherein the oligofructose was purchased from Quantum Hi-Tech (Guangdong) Biotechnology Co., Ltd.

[0075] 1 mL of sterile water was added to 9 mL of carbon-deficient MRS liquid culture medium as a blank control group.

[0076] 1 mL of 10% glucose was added to 9 mL of MRS liquid culture medium as a positive control group.

[0077] 3. The Lactobacillus reuteri HCS02-001 bacterial solution (10 8 CFU / mL) was inoculated into the liquid culture medium of each group obtained in step (2) at an inoculum size of 2%, and the culture was placed at 37°C for 48 hours. After 48 hours, samples were taken and the number of viable bacteria in the fermentation broth was determined by the plate count method to observe the proliferation effect of the medicine-food combination extract on Lactobacillus reuteri HCS02-001. The results are shown in Table 1.

[0078] Table 1 Effect of the combined extracts of medicine and food on the viable count of Lactobacillus reuteri HCS02-001

[0079]

[0080]

[0081] Note: Values ​​are expressed as mean ± SD. Different letters in the same column indicate significant differences.

[0082] As shown in Table 1, glucose, oligofructose and the extract of the medicine-food combination can significantly promote the growth of Lactobacillus reuteri HCS02-001 (p < 0.0001); there is no significant difference in the number of live bacteria of Lactobacillus reuteri HCS02-001 between the positive control group and the oligofructose group.

[0083] The number of live bacteria of Lactobacillus reuteri HCS02-001 in the test group was significantly higher than that in the positive control group (p < 0.0001), which indicates that the medicine-food combination extract can replace glucose as the carbon source required for the growth of Lactobacillus reuteri HCS02-001 and its effect is better.

[0084] The number of live bacteria of Lactobacillus reuteri HCS02-001 in the test group was significantly higher than that in the oligofructose group (p < 0.0001), which indicates that compared with traditional prebiotics on the market, the medicine-food combination extract provided by the present invention can more significantly promote the growth of Lactobacillus reuteri HCS02-001. This is because after optimizing the extraction method of the present invention, the medicine-food combination extract prepared has higher polysaccharides, quercetin and other effective ingredients that can promote the proliferation of probiotics.

[0085] Test Example 2: Functional in vitro experiment

[0086] 1. Preparation of experimental solution

[0087] 1 g of synbiotic composition powder (Example 1-3, Comparative Example 1-5) was dissolved in 10 mL of sterile water, centrifuged at 4°C, 4500×g for 10 min, the supernatant was concentrated 3 times, and stored at 4°C for later use.

[0088] 2. Activation of vaginal Gardnerella

[0089] 1 mL of the bacterial liquid of Gardnerella vaginalis was taken from a cryopreservation tube and inoculated into 100 mL of TSB medium + 5% defibrinated sheep blood, and cultured in a micro-anaerobic gas production bag (5% CO2) environment at 37°C for 24 hours.

[0090] 3. Antibacterial experiment

[0091] Take vaginal Gardnerella liquid (100 μL, concentration is about 1×10 8 CFU / mL) was spread on the agar plate, and the sterilized filter paper was placed in the plate, four of them were placed in parallel. The prepared experimental solution was taken, 20 μL was absorbed and placed on the filter paper, and sterile water was used as a blank control. After the plate was left to stand for 20 minutes, it was slowly placed in a 37°C micro-anaerobic gas production bag (5% CO2) and cultured for 48 hours. After the culture, the inhibition zone was observed and the diameter of the inhibition zone was measured. The results are shown in Table 2.

[0092] Table 2 Antibacterial test results of synbiotic composition on vaginal Gardnerella

[0093]

[0094]

[0095] As can be seen from the above table, after being treated with the synbiotic composition of each example group and the comparative example group, an antibacterial zone appeared, indicating that the synbiotic composition provided by each example group and the comparative example group has an antibacterial effect on vaginal Gardnerella.

[0096] Compared with the blank control group and the comparative example 1-5 groups, the diameter of the inhibition zone of the embodiment 1-3 groups is larger, and the difference is significant (p < 0.05), indicating that in the synbiotic composition provided by the present invention, the Lactobacillus reuteri HCS02-001 powder and the medicine-food homologous combination extract produce a synergistic effect with each other; in the synbiotic composition provided by the comparative example, any component is missing, or the Lactobacillus reuteri HCS02-001 powder in the synbiotic composition is replaced with Lactobacillus plantarum HCS03-001 powder, or the medicine-food homologous combination extract is replaced with oligofructose, and the final effect is significantly worse than the synbiotic composition prepared in the embodiment of the present invention.

[0097] In addition, the inventors also found that the homemade food-medicine combination extract of the present invention can significantly promote the proliferation of Lactobacillus reuteri HCS02-001, and the proliferation degree is higher than that of Lactobacillus plantarum HCS03-001, so Lactobacillus reuteri HCS02-001 and the food-medicine combination extract produce a synergistic effect with each other. Compared with oligofructose, which only has the effect of proliferating Lactobacillus reuteri HCS02-001, the flavonoids, alkaloids and other components in the food-medicine combination extract prepared by the present invention also have the effect of effectively inhibiting the proliferation of private pathogens, so the antibacterial effect of the synbiotic composition containing the food-medicine combination extract prepared by the present invention is better than that of the synbiotic composition containing oligofructose.

[0098] Compared with the comparative examples 6-9, the diameter of the antibacterial zone of the embodiment 1 group is larger, and the difference is significant (p < 0.05). Although the comparative examples 6-9 groups also have certain antibacterial effects, their effects are not as good as those of the embodiment groups of the present invention, indicating that the composition of the extraction solvent and the changes in its ratio will also significantly affect the effect of the synbiotic composition. The lack of any component in the extraction solvent, or the ratio of each solvent exceeds the scope of the present invention, or the replacement of any component will result in a final effect that is significantly worse than the synbiotic composition prepared by the embodiment of the present invention. The present invention has obtained a specific combination of extraction solvents and their ratios after a lot of research, so that the raw materials are fully extracted, so that the Lactobacillus reuteri powder and the medicinal and edible homologous combination extract can better exert synergistic effects.

[0099] Among Examples 1-3, the diameter of the antibacterial zone of the synbiotic composition of Example 3 is the largest, and the difference is significant (p < 0.05), indicating that this example is the optimal example of the present invention and has the best antibacterial effect. This example was selected for the subsequent animal experiments.

[0100] Test Example 3: Functional Animal Experiment

[0101] 1. Experiment on the anti-infection of zebrafish by synbiotic combination

[0102] According to the results of the previous concentration exploration, the No Observed Adverse Effect Level (NOAEL) of the synbiotic combination on normal zebrafish is 1000 μg·mL -1 The low, medium and high dose groups were set at 1 / 4, 1 / 2 and 1 of the NOAEL dose, that is, the mass concentration of the synbiotic composition was 250 μg mL -1 , 500μg·mL -1 and 1000 μg mL -1 .

[0103] The single extract of the combination of medicine and food was used as the prebiotic group, and the mass concentration of the extract of the combination of medicine and food was 1000 μg·mL-1 .

[0104] Lactobacillus reuteri HCS02-001 powder was used as the probiotic group, and the mass concentration of Lactobacillus reuteri HCS02-001 powder was 1000 μg mL -1 .

[0105] (1) Anti-Staphylococcus aureus infection experiment

[0106] Wild-type AB zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each well (experimental group). The synbiotic composition was dissolved in fish water according to the preset concentration, and the zebrafish in the model control group were treated with fish water only, with a volume of 3 mL per well. After continuous treatment at 28°C for 18 hours, Staphylococcus aureus labeled with red fluorescent CM-Dil was injected into the intestinal cavity of zebrafish in each experimental group, and the medium was changed and continued to be treated at 28°C for 3 hours to establish a zebrafish Staphylococcus aureus infection model. Whether the model was successful was determined by whether there was red fluorescence in the intestinal cavity of zebrafish.

[0107] (2) Anti-Gardnerella vaginalis infection experiment

[0108] Wild-type AB zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each well (experimental group). The synbiotic composition was dissolved in fish water according to the preset concentration, and the zebrafish in the model control group were treated with fish water only, with a volume of 3 mL per well. After continuous treatment at 28°C for 18 hours, each experimental group was injected with vaginal Gardnerella labeled with green fluorescent CM-DiI into the intestinal cavity of zebrafish, and the medium was changed and continued to be treated at 28°C for 3 hours to establish a zebrafish vaginal Gardnerella infection model. Whether the modeling was successful was determined by whether there was green fluorescence in the intestinal cavity.

[0109] After the above experiments, 10 zebrafish were randomly selected from each group and photographed using a fluorescence microscope. The data were analyzed and collected using NIS-ElementsD 3.20 advanced image processing software to analyze the fluorescence intensity and antibacterial rate of pathogenic bacteria in the intestinal cavity of zebrafish to evaluate the anti-infection efficacy of the synbiotic composition. The antibacterial rate was calculated according to formula (1).

[0110] Bacterial inhibition rate (%) = 100% × (F 模型 -F 实验 ) / F 模型 ——Formula (1)

[0111] Table 3 Fluorescence intensity of pathogenic bacteria in the intestinal cavity of zebrafish and the antibacterial rate of the synbiotic composition on pathogenic bacteria

[0112]

[0113] Note: Values ​​are expressed as mean ± SD. Different letters in the same column indicate significant differences.

[0114] The results are shown in Table 3. Figure 1 , Figure 2 As shown. Compared with the model control group, the fluorescence intensity of pathogenic bacteria in zebrafish was reduced after 24 hours of water-soluble administration of the synbiotic composition. The extract of the medicine-food combination, Lactobacillus reuteri HCS02-001, and the synbiotic composition all had antibacterial ability against Staphylococcus aureus and Gardnerella vaginalis; among them, the synbiotic composition had a better antibacterial effect, with a low concentration (250μg·mL -1 ) showed antibacterial effect.

[0115] With a mass concentration of 1000 μg mL -1 Compared with the medicinal and edible combined extract and Lactobacillus reuteri HCS02-001, 500 μg mL -1 The effect of the synbiotic combination was more significant. The inhibition rate of the synbiotic combination on Staphylococcus aureus gradually increased with the increase of concentration, with the highest being 54.32% (P < 0.0001); the inhibition rate of the synbiotic combination on Gardnerella vaginalis gradually increased with the increase of concentration, with the highest being 38.32% (P < 0.0001).

[0116] 2. Experiment on improving inflammation in zebrafish using synbiotic combination

[0117] Transgenic neutrophil green fluorescent MPX strain zebrafish 4 days after fertilization were placed in a 6-well plate, and 30 fish were randomly placed in each group (normal control group, model control group, and low, medium, and high dose groups), for a total of 150 fish. The synbiotic composition was dissolved in fish culture water, and the concentration was the same as the above anti-zebrafish infection experiment. The zebrafish in the normal control group and the model control group were only treated with fish culture water, and the volume of each well was 3 mL. After continuous treatment at 28°C for 18 hours, Staphylococcus aureus was injected into the intestinal cavity of zebrafish except the normal control group to establish a zebrafish inflammatory infection model, and the medium was changed and continued to be treated at 28°C for 3 hours. After the experiment, 10 zebrafish were randomly selected from each group and photographed with a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils (N) in the intestinal cavity of zebrafish and the inflammation inhibition rate, which were used to evaluate the inflammation inhibition rate of the synbiotic composition. The inflammation inhibition rate was calculated according to formula (2).

[0118] Inflammation inhibition rate (%) = 100% × (N 模型对照组平均值 -N 实验组 ) / (N 模型对照组平均值 -N 正常对照组平均值 )

[0119] ——Formula (2)

[0120] Table 4 The number of neutrophils in the intestinal cavity of zebrafish and the inhibition rate of zebrafish intestinal inflammation by the synbiotic composition

[0121]

[0122] Note: Values ​​are expressed as mean ± SD. Different letters in the same column indicate significant differences.

[0123] The results are shown in Table 4. The number of neutrophils in the intestinal cavity of the normal control group was significantly lower than that of the model control group (P < 0.0001), indicating that the modeling was successful. The extract of the medicine-food combination, Lactobacillus reuteri HCS02-001 and the synbiotic combination all have anti-inflammatory ability; among them, the synbiotic combination has a better anti-inflammatory effect, and at a low concentration (250 μg mL -1 ) has been shown to have anti-inflammatory effects.

[0124] With a mass concentration of 1000 μg mL -1 Compared with the medicinal and edible combined extract and Lactobacillus reuteri HCS02-001, 500 μg mL -1 The effect of the synbiotic combination was more significant. The inflammation inhibition rate of the synbiotic combination gradually increased with the increase of concentration, with the highest being 97.61% (P < 0.0001), which was consistent with the anti-Staphylococcus aureus infection effect of the synbiotic combination. It is speculated that it may achieve anti-inflammatory effect by resisting Staphylococcus aureus infection.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A synbiotic composition, characterized in that: The invention comprises the following components in parts by weight: 5-15 parts of Lactobacillus reuteri powder and 15-50 parts of a medicine-food combination extract; the medicine-food combination extract is obtained by ultrasonic extraction using dandelion powder and cornus officinalis powder as raw materials, and the extraction solvent used is a water-soluble substance of betaine, fructose, xylitol, lactic acid and glycine.

2. The synbiotic composition according to claim 1, characterized in that The invention comprises the following components in parts by weight: 15 parts of Lactobacillus reuteri powder and 50 parts of the medicine-food homologous combination extract.

3. The synbiotic composition according to claim 1, characterized in that In the extraction solvent, the weight ratio of betaine, fructose, xylitol, lactic acid and glycine is 5:5:5:30:30:

25.

4. The synbiotic composition according to claim 1, characterized in that The preparation method of the medicine-food combination extract comprises the following steps: (1) adding an extraction solvent to dandelion powder, ultrasonically extracting, centrifuging, and collecting a filtrate; (2) Adding cornus officinalis powder to the filtrate obtained in step (1), performing ultrasonic extraction, centrifugation, concentration, and drying to obtain the medicine-food combination extract.

5. The synbiotic composition according to claim 4, characterized in that In the steps (1) and (2), the conditions for ultrasonic extraction are: ultrasonic extraction at 30° C. for 30 min and an ultrasonic power of 250 W.

6. The synbiotic composition according to claim 1, characterized in that The Lactobacillus reuteri is Lactobacillus reuteri HCS02-001.

7. The synbiotic composition according to claim 1, characterized in that In the synbiotic composition, the number of viable bacteria in the Lactobacillus reuteri powder is at least 10 10 CFU.

8. Use of the synbiotic composition according to any one of claims 1 to 7 in the preparation of a preparation or medicine for improving or treating vaginitis.

9. The use according to claim 8, characterized in that The vaginitis includes bacterial vaginitis.

10. A preparation or medicine for improving or treating vaginitis, characterized in that: The invention relates to a synbiotic composition comprising the synbiotic composition according to any one of claims 1 to 7.

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