Preparation method, product and application of composite wormwood probiotic postbiotics

Abstract

The invention relates to a preparation method, a product and application of a composite wormwood probiotic metagen, and the preparation method of the metagen comprises the following steps: inoculating lactobacillus gasseri TM13 and lactobacillus crispatus LG55 into a culture medium containing a wormwood extract, and fermenting to obtain fermentation liquor; and inactivating the fermentation liquor to obtain a finished product. The lactobacillus gasseri TM13 and the lactobacillus crispatus LG55 can mutually cooperate and cooperatively grow, and the wormwood extract is added in the fermentation process, so that the fermentation biomass can be remarkably increased, the pH value can be reduced, the bacteriostatic ability of a fermentation product on vaginal harmful bacteria can be improved, particularly, the candida albicans can be inhibited, and the synergistic interaction effect of the plant extract and the strain can be exerted. The form of a metagen product can break through the limitation of application of viable bacteria, external intervention can directly act on an affected part to regulate the micro-ecological environment of the vagina, loss caused by similar extreme environments of gastrointestinal tracts is avoided, and the effect is more prominent.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology and relates to a preparation method of a composite wormwood probiotic postbiotic and its product and application. Background Art

[0002] The female reproductive tract is home to a diverse population of microorganisms, forming a unique microecological environment. In the reproductive tracts of healthy women of childbearing age, Lactobacillus species, which can ferment and produce large amounts of lactic acid and hydrogen peroxide, predominate. The most common species are Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners. When present in large numbers in the female reproductive tract, these species can inhibit the growth of pathogenic and opportunistic microorganisms through mechanisms such as lactic acid, hydrogen peroxide, antimicrobial peptides, and immune regulation, thereby protecting female reproductive health. If the number of Lactobacilli decreases, their inhibitory effect on other microorganisms will also decrease, allowing these previously harmful microorganisms to proliferate, potentially leading to reproductive tract infections and adverse pregnancy outcomes.

[0003] Vaginal infections are common reproductive tract infections in women. Bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and aerobic vaginitis (AV) are relatively common. Reproductive tract infections are closely associated with an imbalance in the vaginal microbiome. BV is the most common type of vaginitis, causing inflammation and redness in the female reproductive tract, primarily due to a decrease in Lactobacilli and an increase in facultative and anaerobic bacteria. VVC refers to vaginal inflammation caused by Candida species, primarily Candida albicans, with Candida glabrata as a minority. AV is caused by a decrease or absence of Lactobacilli in the vagina and an increase in aerobic bacteria. Its pathogens are relatively complex, with increases in various aerobic and facultative anaerobic bacteria, such as Group B Streptococcus, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis. The massive proliferation of these pathogens often increases the pH of the reproductive tract environment, causing an imbalance in the vaginal microbiome and leading to a severe inflammatory response, often accompanied by imbalances in sex hormone levels. Reproductive tract infections generally cause both physical and psychological harm to patients, seriously affecting their normal lives. Therefore, there is an urgent need to develop effective treatments.

[0004] Antibiotics are the most common treatment for genital tract infections, typically administered orally, systemically, or topically, or by selecting appropriate antibiotics and routes of administration for specific pathogens. In recent years, probiotics and postbiotics have been introduced clinically as adjunctive treatments and preventive measures. In combination with medication, oral or topical probiotic / postbiotic preparations increase the rate of lactic acid bacteria colonization in the female genital tract, inhibiting the growth of harmful bacteria and partially regulating the homeostasis of the genital tract microenvironment.

[0005] However, with the increasing use of antimicrobial drugs, pathogen resistance continues to rise. Furthermore, while antibiotics eliminate infectious pathogens, they also affect other healthy genital microorganisms, potentially further disrupting the vaginal microbiome and increasing the risk of recurrence. In fact, standard antibiotic treatment often only provides temporary relief. Furthermore, due to the high rate of natural resistance among Candida species, vaginal infections such as vaginal genital warts and vaginal genital cirrhosis (VVC) are likely to recur within a few months after initial cure. This is due to the fact that efforts to address vaginal microbiome imbalance and achieve long-term cure have largely been unsuccessful. Furthermore, the use of probiotics and postbiotics has largely remained limited to suppressing infection with a subset of genital pathogens and lacks broad efficacy against genital pathogens, particularly Candida albicans. Furthermore, most probiotics used are not the most common bacteria in the healthy female genital tract, and their targeted effects and restorative effects on the vaginal microbiome may not be as strong as those found in high abundance in the vagina, such as Lactobacillus crispatus and Lactobacillus gasseri. In addition, the results of studies on the efficacy of a single strain are unsatisfactory, and research on multi-strain composite and plant-based composite probiotic preparations is still insufficient. It is necessary to continue to develop composite preparations that are beneficial for the prevention and treatment of female reproductive tract infections and to improve their efficacy. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a preparation method of a composite wormwood probiotic postbiotic and its product and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a composite wormwood probiotic postbiotic, the method comprising:

[0009] (1) inoculating Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 into a culture medium containing an wormwood extract, and fermenting to obtain a fermentation liquid;

[0010] (2) Inactivate the fermentation liquid to obtain the product.

[0011] The technical problem to be solved by the present invention is to overcome the shortcomings of the probiotic postbiotics disclosed in the prior art, such as the lack of extensive and prominent activity in inhibiting genital pathogens, the unclear composition and process, and the insufficient effective ingredients. A composite wormwood probiotic postbiotic product is provided, which is mainly composed of Lactobacillus crispatus and Lactobacillus gestatus, supplemented by wormwood extract, and has extensive and effective antibacterial ability, and the postbiotic product has rich antibacterial ingredients. The composite wormwood probiotic postbiotic product provided by the present invention is obtained by inactivating wormwood after fermentation of probiotics. The probiotics refer to Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55. Lactobacillus gasseri TM13 is isolated from healthy human intestinal samples, has the effect of producing a variety of bioactive substances, effectively inhibiting the growth of genital pathogens, and improving genital tract infections. Lactobacillus crispatus LG55 is isolated from healthy human intestinal samples, has the effect of producing a large amount of lactic acid and hydrogen peroxide, effectively inhibiting the growth of genital pathogens, and regulating vaginal flora. Mugwort extract is the dried powder after processing mugwort. Mugwort has the effect of removing dampness and relieving itching, and has inhibitory effects on a variety of bacteria and fungi.

[0012] When Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 are co-fermented with mugwort, the resulting product has stronger antibacterial activity. The lack of any one of them will affect the effect of postbiotics, and the effect of mixed fermentation of Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 is better.

[0013] Preferably, the viable count of Lactobacillus gasseri TM13 in the culture medium is 1×10 7 -3×10 7 CFU / mL, for example 1×10 7 CFU / mL, 1.5×10 7 CFU / mL, 2×10 7 CFU / mL, 2.5×10 7 CFU / mL, 3×10 7 CFU / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0014] Preferably, the viable count of Lactobacillus crispatus LG55 in the culture medium is 1×10 7 -3×10 7 CFU / mL, for example 1×10 7 CFU / mL, 1.5×10 7 CFU / mL, 2×10 7 CFU / mL, 2.5×10 7 CFU / mL, 3×10 7 CFU / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0015] Preferably, the ratio of the viable cell counts of Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 is (1-2):(1-2).

[0016] The specific point values ​​in (1-2) can be selected as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc. Other specific point values ​​within the above numerical range can be selected and will not be listed here one by one.

[0017] Preferably, the added amount of the mugwort extract is 0.5-1.5wt%, for example, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0018] Preferably, the culture medium comprises a carbon source, a nitrogen source, inorganic salts, growth factors and water.

[0019] Preferably, the culture medium comprises MRS medium.

[0020] Preferably, the fermentation temperature is 35-39° C. and the fermentation time is 24-120 h.

[0021] The temperature can be selected as 35℃, 36℃, 37℃, 38℃, 39℃, etc. The time can be selected as 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, 72h, 78h, 84h, 90h, 96h, 102h, 108h, 114h, 120h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here.

[0022] Preferably, the fermentation method is static fermentation.

[0023] Preferably, the inactivation method includes heat treatment inactivation, high pressure treatment inactivation or radiation treatment inactivation.

[0024] In a second aspect, the present invention provides a composite wormwood probiotic postbiotic prepared according to the preparation method of the composite wormwood probiotic postbiotic described in the first aspect.

[0025] A total of 13 metabolites with antibacterial activity that increased significantly after fermentation were identified in the composite wormwood probiotic postbiotics prepared by the present invention, including 3-hydroxypropionic acid, succinic acid, 2-hydroxyisocaproic acid, gentisic acid, dihydrocaffeic acid, quebrachol, 4-hydroxyphenyllactic acid, catechin, syringic acid, lactic acid, 4-hydroxyphenyl alcohol, 7-hydroxycoumarin and cornflower flavonoids.

[0026] In a third aspect, the present invention provides a use of the composite wormwood probiotic postbiotic according to the second aspect in the preparation of a product for preventing or treating female reproductive tract infections.

[0027] Preferably, the product is a female vaginal external preparation.

[0028] The product prepared by the present invention can directly enter the vagina to exert an antibacterial effect.

[0029] Preferably, the female reproductive tract infection includes bacterial vaginosis, vulvovaginal candidiasis or aerobic vaginitis.

[0030] Preferably, the pathogenic bacteria of bacterial vaginosis include the facultative anaerobic bacterium Gardnerella vaginalis.

[0031] Preferably, the pathogenic bacteria of vulvovaginal candidiasis include Candida albicans and / or Candida glabrata.

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

[0033] The present invention develops a novel topical composite wormwood probiotic postbiotic. The Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 used in the fermentation process are both human symbiotic bacteria. Adding wormwood extract during the fermentation process significantly increases the growth biomass of the strains, lowers the pH, and enhances their antibacterial ability, demonstrating that wormwood is a good fermentation substrate for both strains. The two strains can cooperate and grow synergistically, further increasing their biomass and enhancing the antibacterial ability of the wormwood probiotic postbiotic against reproductive tract pathogens, particularly Candida albicans, thereby exerting a synergistic effect between the plant extract and the strains. The mixed fermentation of mugwort with Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 produces a richer array of antibacterial substances, including 3-hydroxypropionic acid, succinic acid, 2-hydroxyisocaproic acid, gentisic acid, dihydrocaffeic acid, quebrachol, 4-hydroxyphenyllactic acid, catechin, syringic acid, lactic acid, 4-hydroxyphenyl alcohol, 7-hydroxycoumarin, and cornflower flavonoids. These postbiotics exhibit effective antibacterial properties even at relatively low concentrations. Postbiotic formulations transcend the limitations of live bacteria. Topical interventions can directly affect the affected area, regulating the vaginal microecological environment without the damage often associated with the extreme gastrointestinal environment, resulting in even more pronounced results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the result of the changes in biomass and pH of the compound mugwort probiotic postbiotics at different fermentation times.

[0035] Figure 2This is the inhibition rate of compound wormwood probiotic postbiotics with different fermentation times on five common reproductive tract pathogens, among which Figure A is Candida albicans, Figure B is Candida glabrata, Figure C is Gardnerella vaginalis, Figure D is Escherichia coli, and Figure E is Staphylococcus aureus. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0037] The sources of the functional ingredients contained in the products involved in the following examples and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0038] The mugwort extract is a water-extracted mugwort extract powder product purchased from Shanghai Jianyi Supply Chain Management Co., Ltd.

[0039] MRS medium: Add 10 g of casein digest, 10 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, and 1 g of Tween 80 to each liter of deionized water. Sterilize at 121°C for 15 min.

[0040] Artemisia annua-MRS medium: Add 10 g of artemisia annua extract, 10 g of casein digest, 10 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, and 1 g of Tween 80 to each liter of deionized water. Sterilize at 121°C for 15 min.

[0041] Sabouraud dextrose broth medium: Add 10 g of a mixture of equal amounts of animal tissue pepsin hydrolysate and trypticase and 20 g of glucose to each liter of deionized water and sterilize at 115°C for 30 min.

[0042] Brain heart infusion broth (BHI): Add 10 g of tryptone, 17.5 g of beef heart extract powder, 5 g of sodium chloride, 2 g of glucose, and 2.5 g of disodium hydrogen phosphate dodecahydrate to each liter of deionized water and sterilize at 121°C for 15 min.

[0043] The Lactobacillus gasseri TM13 strain involved in the following content is classified as Lactobacillus gasseri, with a deposit number of GDMCC No: 60405, a deposit date of August 24, 2018, and a depository unit of Guangdong Provincial Microbiological Culture Collection, with a deposit address of 5th Floor, No. 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0044] The Lactobacillus crispatus LG55 strain involved in the following content is classified as Lactobacillus crispatus, with a deposit number of GDMCC No: 60407, a deposit date of August 24, 2018, and a depository unit of Guangdong Provincial Microbiological Culture Collection, with a deposit address of 5th Floor, No. 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0045] Example 1

[0046] This embodiment provides a preparation method of a composite wormwood probiotic postbiotic, the preparation method comprising:

[0047] (1) Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 were inoculated into mugwort-MRS medium. The viable count of Lactobacillus gasseri TM13 was 1.0×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 was 1.0×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0048] (2) Inactivate the fermentation broth at 65°C for 30 minutes.

[0049] Example 2

[0050] This embodiment provides a preparation method of a composite wormwood probiotic postbiotic, the preparation method comprising:

[0051] (1) Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 were inoculated into mugwort-MRS medium. The viable count of Lactobacillus gasseri TM13 was 1.5×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 was 1.5×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0052] (2) Inactivate the fermentation broth at 65°C for 30 minutes.

[0053] Example 3

[0054] This embodiment provides a preparation method of a composite wormwood probiotic postbiotic, the preparation method comprising:

[0055] (1) Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 were inoculated into mugwort-MRS medium. The viable cell count of Lactobacillus gasseri TM13 was 1×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 was 2×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0056] (2) Inactivate the fermentation broth at 65°C for 30 minutes.

[0057] Example 4

[0058] This embodiment provides a preparation method of a composite wormwood probiotic postbiotic, the preparation method comprising:

[0059] (1) Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 were inoculated into mugwort-MRS medium. The viable count of Lactobacillus gasseri TM13 was 2×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 is 1×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0060] (2) Inactivate the fermentation broth at 65°C for 30 minutes.

[0061] Comparative Example 1

[0062] This comparative example provides a preparation method of a composite wormwood probiotic postbiotic, which differs from Example 1 only in that step (1) is "inoculating Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 into MRS culture medium, with the viable count of Lactobacillus gasseri TM13 being 1.0×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 was 1.0×10 7 CFU / mL, ferment at 37 °C for 24 h to obtain the fermentation broth”, and other operations remained unchanged.

[0063] Comparative Example 2

[0064] This comparative example provides a preparation method of a composite wormwood probiotic postbiotic, which differs from Example 2 only in that step (1) is "inoculating Lactobacillus gasseri TM13 into wormwood-MRS culture medium, and the number of viable bacteria of Lactobacillus gasseri TM13 is 3×10 7 CFU / mL, ferment at 37 °C for 24 h to obtain the fermentation broth”, and other operations remained unchanged.

[0065] Comparative Example 3

[0066] This comparative example provides a preparation method of a composite wormwood probiotic postbiotic, which differs from Example 2 only in that step (1) is "inoculating Lactobacillus crispatus LG55 into wormwood-MRS culture medium, and the number of viable Lactobacillus crispatus LG55 is 3×10 7 CFU / mL, ferment at 37 °C for 24 h to obtain the fermentation broth”, and other operations remained unchanged.

[0067] Comparative Example 4

[0068] This comparative example provides a method for preparing a composite wormwood probiotic postbiotic, the method comprising:

[0069] (1) Lactobacillus gasseri TM13 was inoculated into mugwort-MRS medium. The viable count of Lactobacillus gasseri TM13 was 3×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0070] Lactobacillus crispatus LG55 was inoculated into mugwort-MRS medium. The number of viable bacteria of Lactobacillus crispatus LG55 fermented was 3×10 7 CFU / mL, fermented at 37 °C for 24 h to obtain the fermentation broth;

[0071] (2) Take an equal volume of fermentation broth and inactivate it at 65℃ for 30 minutes.

[0072] Comparative Example 5

[0073] This comparative example provides a preparation method of a composite wormwood probiotic postbiotic, which differs from comparative example 4 only in that step (2) is "mixing Lactobacillus gasseri fermentation broth and Lactobacillus crispatus fermentation broth in a volume ratio of 1:2, and inactivating them at 65°C for 30 minutes to obtain", and other operations remain unchanged.

[0074] Comparative Example 6

[0075] This comparative example provides a preparation method of a composite wormwood probiotic postbiotic, which differs from comparative example 4 only in that step (2) is "mixing Lactobacillus gasseri fermentation broth and Lactobacillus crispatus fermentation broth in a volume ratio of 2:1, and inactivating them at 65°C for 30 minutes to obtain", and other operations remain unchanged.

[0076] Test Example 1

[0077] Effects of Artemisia argyi extract on the biomass and antibacterial ability of probiotic postbiotics

[0078] Samples to be tested: Example 1, Comparative Example 1.

[0079] Sample pretreatment: The samples obtained in Example 1 and Comparative Example 1 were centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm sterile filter membrane.

[0080] Test method:

[0081] The cells were activated in Sabouraud dextrose broth and prepared at a concentration of 2 × 10 6 CFU / mL of Candida albicans suspension was mixed and diluted 10 times with Sabouraud medium, and then diluted 1:100 to obtain a concentration of 2×10 3CFU / mL (2 times the inoculum concentration). In a 96-well microplate, add 100 μL of the pre-treated sample to be tested, then add 100 μL of the 2 times concentration of the bacterial suspension, mix well, and then measure the OD at 0 hours. 600 The value was recorded as OD experiment 1; MRS medium and mugwort-MRS medium were used as blank controls, recorded as OD blank 1; the microplate was placed at 35℃ for 24h and the OD was measured again. 600 The values ​​were recorded as OD experiment 2 and OD blank 2, and three parallel experiments were performed.

[0082]

[0083] Table 1

[0084] <![CDATA[Biomass (OD 600 )]]> Candida albicans inhibition rate (%) Example 1 <![CDATA[1.86±0.03 b ]]> <![CDATA[91.60±0.84 b ]]> Comparative Example 1 <![CDATA[1.33±0.01 a ]]> <![CDATA[85.70±0.79 a ]]>

[0085] The results are shown in Table 1. Different letters in the same column a and b indicate significant differences. Adding 1% wormwood extract significantly increased the biomass of the strain and improved the inhibitory effect of the fermentation broth on Candida albicans, indicating that wormwood extract can enhance the reproduction and metabolic capacity of Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55.

[0086] Test Example 2

[0087] Effects of different inoculation ratios on the growth and antibacterial ability of compound wormwood probiotics

[0088] Samples to be tested: Examples 2-4 and Comparative Examples 2-6.

[0089] The sample pretreatment and testing methods were the same as those in Test Example 1, and the differences in antibacterial ability of different composite wormwood probiotic postbiotic concentrations (25%, 50%, 100%) were detected. The results are shown in Table 2.

[0090] Table 2

[0091]

[0092] The results are shown in Table 2. Compared with the single strain inoculation, the biomass (OD 600 ) increased significantly, and the inhibitory effect on Candida albicans was enhanced. Among them, when the inoculation ratio was Lactobacillus gasseri TM13: Lactobacillus crispatus LG55 = 2:1 (Example 4), the antibacterial effect was the best, indicating that the mixed inoculation of the two strains had a synergistic effect and could jointly enhance the reproduction and metabolic capacity.

[0093] Test Example 3

[0094] Effects of different fermentation times on the growth and antibacterial ability of compound wormwood probiotics

[0095] Test method:

[0096] (1) Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 were inoculated into mugwort-MRS medium. The viable count of Lactobacillus gasseri TM13 was 2×10 7 CFU / mL, the number of viable bacteria in the fermentation of Lactobacillus crispatus LG55 is 1×10 7 CFU / mL, ferment at 37℃, and take samples at 0h, 24h, 48h, 72h, 96h, and 120h. After the fermentation liquid is thoroughly mixed, the absorbance at 600nm and pH value are measured.

[0097] Using Candida albicans ATCC10231, Candida glabrata ATCC2001, Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213 and Gardnerella vaginalis ATCC14018 as indicator bacteria, the antibacterial ability of the compound wormwood probiotic postbiotics at three concentrations of 100%, 50% and 25% was tested. The concentration of the bacterial suspension of Candida albicans and Candida glabrata was 2×10 3 CFU / mL (2 times the inoculum concentration), the culture medium is Sabouraud medium; the concentration of the bacterial suspension of Escherichia coli and Staphylococcus aureus is 1×10 6 CFU / mL (2 times the inoculum concentration), the culture medium is BHI medium; the concentration of the vaginal Gardnerella suspension is 1×10 6 CFU / mL (2 times the inoculum concentration), the culture medium is MRS medium; except for Gardnerella vaginalis, which is cultured anaerobically at 35°C for 48 hours, all other bacteria are cultured aerobically at 35°C for 24 hours. The rest of the operations are the same as those in Test Example 1.

[0098] The results are as follows Figure 1 As shown in Figure 2, after one day of fermentation, the pH of the postbiotics decreased significantly to 3.84, and the biomass (OD 600 ) increased significantly to 1.87; pH value decreased slightly during the 1-3 days of fermentation and remained unchanged after 3 days; biomass (OD 600 The above results show that the effect of mixed fermentation of Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 gradually increases with the increase of fermentation time, and basically reaches stability after 3 days of fermentation.

[0099] The antibacterial ability test results are as follows Figure 2As shown in the results, the compound wormwood probiotic postbiotic had the best inhibitory effect on Candida glabrata and Escherichia coli, and could basically completely inhibit the two pathogens at a low concentration of 25%; followed by Staphylococcus aureus, with the inhibition rates of 25% postbiotics after fermentation for 1 day and 2 days were 20.62% and 71.77%, respectively. The inhibition rates of postbiotics at other fermentation times and concentrations were all over 98%; at concentrations of 25% and 50%, the inhibitory effect of postbiotics fermented for 3 days on Candida albicans was The results were stronger than those of postbiotics fermented for 1-2 days. After three days of fermentation, the 50% concentration of postbiotics had an inhibition rate of over 92% against Candida albicans, and the 100% concentration had an inhibition rate of over 99% against Candida albicans. With increasing concentration and fermentation time, the inhibition rate of postbiotics against Gardnerella vaginalis gradually increased. The 50% concentration of postbiotics fermented for 3-5 days had a similar inhibitory effect, but was higher than that of postbiotics fermented for 1-2 days. After 2 days of fermentation, the 100% concentration of postbiotics had an inhibition rate of over 95% against Gardnerella vaginalis. These results indicate that the compound wormwood probiotic postbiotic has excellent inhibitory effects against Candida albicans, Candida glabrata, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus, pathogens that cause female reproductive tract infections. Considering the antibacterial effect and fermentation time cost, a 3-day fermentation period is optimal.

[0100] Test Example 4

[0101] Determination of active components of postbiotics from compound mugwort probiotics

[0102] Take 100 μL of 0-5 day fermentation wormwood probiotics after centrifugation and filtration, fermentation method reference example 1, add 1mL extract (methanol: acetonitrile: water = 2: 2: 1, v / v), shake and mix, ice water bath ultrasonic treatment 10min, liquid nitrogen quick freezing 1min, repeated three times. After standing at -20 ° C for 1h, 13000r / min, 4 ° C centrifugation 15min. Take the supernatant, blow dry with a nitrogen blower, add 100 μL acetonitrile: water = 1: 1 (v / v) to redissolve, shake 30s, ice water bath ultrasonic treatment 10min, 13000r / min at 4 ° C centrifugation 15min. Take the supernatant, and the machine performs LC / MS detection of fermentation liquid components.

[0103] Table 3

[0104]

[0105]

[0106] The results are shown in Table 3. A total of 13 metabolites with antibacterial activity that increased significantly after fermentation were identified, including 3-hydroxypropionic acid, succinic acid, 2-hydroxyisocaproic acid, gentisic acid, dihydrocaffeic acid, quebrachol, 4-hydroxyphenyllactic acid, catechin, syringic acid, lactic acid, 4-hydroxyphenyl alcohol, 7-hydroxycoumarin and cornflower flavonoids. The contents of these antibacterial metabolites increased significantly during fermentation days 0-3 and remained basically stable during days 3-5, which was consistent with the results of the in vitro antibacterial experiment.

[0107] The applicant declares that the present invention illustrates the preparation method of a composite wormwood probiotic postbiotic and its products and applications through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the raw materials of the product of the present invention, the addition of auxiliary ingredients, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

[0108] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0109] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a composite wormwood probiotic postbiotic, characterized in that: The preparation method of the composite wormwood probiotic postbiotic comprises: (1) inoculating Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 into a culture medium containing an wormwood extract, and fermenting to obtain a fermentation liquid; (2) Inactivate the fermentation liquid to obtain the product.

2. The preparation method of the composite wormwood probiotic postbiotic according to claim 1, characterized in that, The viable bacterial count of Lactobacillus gasseri TM13 in the culture medium was 1×10 7 -3×10 7 CFU / mL; Preferably, the viable count of Lactobacillus crispatus LG55 in the culture medium is 1×10 7 -3×10 7 CFU / mL.

3. The method for preparing the composite wormwood probiotic postbiotic according to claim 1 or 2, characterized in that: The ratio of the viable counts of Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 is (1-2):(1-2).

4. The method for preparing the composite wormwood probiotic postbiotic according to any one of claims 1 to 3, characterized in that: The added amount of the wormwood extract is 0.5-1.5 wt %.

5. The method for preparing the composite wormwood probiotic postbiotic according to any one of claims 1 to 4, characterized in that: The culture medium comprises a carbon source, a nitrogen source, inorganic salts, growth factors and water; Preferably, the culture medium comprises MRS medium.

6. The method for preparing the composite wormwood probiotic postbiotic according to any one of claims 1 to 5, characterized in that: The fermentation temperature is 35-39°C and the fermentation time is 24-120h; Preferably, the fermentation method is static fermentation.

7. The method for preparing the composite wormwood probiotic postbiotic according to any one of claims 1 to 6, characterized in that: The inactivation method includes heat treatment inactivation, high pressure treatment inactivation or radiation treatment inactivation.

8. A composite wormwood probiotic postbiotic prepared by the method for preparing a composite wormwood probiotic postbiotic according to any one of claims 1 to 7.

9. Use of the compound wormwood probiotic postbiotic according to claim 8 in the preparation of a product for preventing or treating female reproductive tract infections.

10. The use according to claim 9, characterized in that The female reproductive tract infection includes bacterial vaginosis, vulvovaginal candidiasis or aerobic bacterial vaginitis.

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