CN106957811B - Format lactobacillus and Chinese medicine compound prescription with bacteriostasis and its application in treatment gynaecological imflammation - Google Patents

Abstract

The invention discloses one plant of format lactobacillus with bacteriostasis, it is format lactobacillus (Lactobacillusgasseri) DFNXC2-0121, China typical culture collection center is preserved on March 31st, 2017, address are as follows: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC NO:M 2017162.The invention also discloses the Traditional Chinese medicine probiotic compositions by the format lactobacillus and Chinese medical extract preparation.The present invention is using Tiny ecosystem therapy as guidance, it is separated from normal health vagina, purifying obtains one plant of format lactobacillus, and fermentation is expanded culture together with another lactobacillus plantarum, it is compounded using its metabolite with preferred Chinese herbal medicine extract, so that exploitation obtains the product of nontoxic, without side-effects, safe and efficient treatment gynaecological imflammation.

Description

Format lactobacillus with bacteriostasis is with Chinese medicine compound prescription and its in treatment gynaecological imflammation In application

Technical field

The present invention relates to one plant the format lactobacillus with bacteriostasis and its application in treatment gynaecological imflammation, especially It is the application for being related to the format lactobacillus in the drug of preparation treatment vaginitis, belongs to biomedicine technical field.

Background technique

Gynaecological imflammation is the common disease of Out-patient Clinic of Department of Gynecology, and all age group all has generation, the World Health Organization (WHO) publication Statistics indicate that, the women of 90% or more developing country suffers from gynaecological imflammation, and Chinese women has 70% with different degrees of Reproductive tract infection, due to the imbalance of medical resource, economic condition, cultural knowledge, so that Rural Married Women gynaecological imflammation is sent out Sick rate is more up to 95% or more；85% or more women at least suffers from two or more genital diseases.According to statistics, 2004 it is complete The stream of 14, state, 62, the whole nation in 2005, national 81 gynaecology, hospital in 2006 and more than 50,000 people of family planning outpatient service, which are raised wages, to be expected to show Show, gynecological infection disease (vaginitis, pelvic inflammatory disease, cervicitis) accounts for 55 percent in all diseases of women, thus generates Medical expense every year be up to more than 200 hundred million yuan.It estimates accordingly, annual at least 200,000,000 women of China suffer from reproductive tract infection and correlation Disease, the ten hundreds of women in the whole world are annoying by this vaginopathy always.

Reproductive tract infection has the characteristics that disease incidence height, high recurrence rate, Epidemic Scope are wide, and the body and mind for seriously affecting women is strong Health, shows abnormal leucorrhea yellow green, peculiar smell (fishlike smell, foul smell), itch, burning sensation, lower abdominal pain, intercourse pain, such as Fruit cannot correctly be treated in time, can uplink infection, cause cervicitis, endometritis, pelvic inflammatory disease etc., cause it is infertile not Educate, ectopic pregnancy, pregnant woman leads to miscarriage, stillborn foetus, stillbirth, congenital infection and infection of newborn etc., can make AIDS (AIDS) and The occurrence risk of cervical carcinoma increases, and seriously affects the physical and mental health of women, influences the stable of family's wellbeing and society.

Currently, antibiotic is mainly used in the measure for the treatment of vagina infection, antibiotic is that certain micro-organisms is anti-by enzymatic Metabolite should be changed into structural secondary metabolite.The unreasonable a large amount of uses of antibiotic, abuse, in addition lacking Weary strict supervision, leads to the generation of dead, drug-resistant microorganism and super antibody-resistant bacterium, and the misuse of antibiotic also sets patient in not In necessary resistance risk；Gynemetrics, Peking University First Hospital Liao Qinping professor Chinese women microecology in vaginas study into It is said in Zhan Yiwen, intravaginal normal flora is the core content of microecology in vaginas research, and early in 1892, Dederlein was for the first time The research about human vagina Tiny ecosystem flora is delivered, Dederlein thinks that vagina microorganism only has Gram-positive Bacillus is constituted, i.e. lactobacillus.As that studies vaginal flora deepens continuously, it is found that there is a variety of for intravaginal under normal condition Microorganism, wherein most important, quantity it is most be lactobacillus, separation rate is up to 50%- in the vaginal fluid of healthy women 80%, have determined that now the microorganism being colonized in normal vagina mainly by bacterium, fungi, protozoon, virus, mycoplasma etc., this The balance that the presence of a little floras maintains ecology is not fallen ill.In maintaining the vagina ecological balance, to generate hydrogen peroxide (H₂O₂) Lactobacillus be dominant bacteria, it can generate a variety of antibacterial substances, such as lactic acid, stearic acid, hydrogen peroxide are acidified vaginal environment, Vagina pH, the mass propagation by adherency occupy-place protection, the modes such as immune system being stimulated to inhibit pathogen are reduced, vagina is being maintained The ecological balance in play very important effect.The vagina ecological balance is once broken or foreign pathogens intrusion, can lead Cause inflammation.

During traditional antibiotic treatment, antibiotic also can be the dominant bacteria in vagina while killing pathogenic bacteria It kills, so that vagina is in few bacterium or germ-free condition, and lead to body immunity degradation plus the use of antibiotic, once encounter cause of disease Bacterium is easy to cause to infect, suprainfection.Now, the side effect of antibiotic is cured, medicine circle scientific worker approves, country Gradually limitation uses.

Vagina infection is treated other than oral antibiotic systemic administration, dialectical there are also traditional Chinese medicine treats, and Chinese medicine passes through Benefiting qi and nourishing blood, dispersing stagnated hepatoqi, disinfection carry out systemic administration by the stasis of blood, clearing damp heat, degrading the channel etc., but effect is slow, it is difficult to endure Itch makes one to feel restless, and is difficult to adhere to take Chinese medicine antipruritic.Externally applied drug at present on the market is also dazzling, suppository, glue Capsule, acidification lotion, gelling agent, Chinese herbal fumigation and washing, effervescent tablet etc., many effect of drugs are bad according to investigations, are mainly used for antimicrobial The adjuvant treatment of object cannot eradicate, and be easy repeatedly, especially vulvovaginal candidiasis (VVC), recurrent beads vagina Sick (recurrent vulvovaginal candidiasis, RVVC), treatment course are long easily repeatedly.

To sum up, the effect of antibiotic, traditional Chinese medicine and various externally applied drugs treatment vagina infection is undesirable, has its source in The balance of microecology in vaginas is not restored, but since vaginal flora is a big ecosystem, if the use external source of blindness is micro- Ecological agent is adjusted, it is easy to cause the imbalance of new bacterium germination group.Therefore, how to be treated by adjusting vaginal flora balance Vagina infection is still current difficult point place.

Summary of the invention

For the above-mentioned prior art, the format lactobacillus that the object of the present invention is to provide one plant with bacteriostasis and its Application in gynaecological imflammation.

The present invention separates, purifying obtains one plant of format using Tiny ecosystem therapy as guidance from normal health vagina Lactobacillus, and fermentation is expanded culture together with another lactobacillus plantarum, utilize its metabolite and preferred Chinese herbal medicine Extract is compounded, so that exploitation obtains the product of nontoxic, without side-effects, safe and efficient treatment gynaecological imflammation.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention provides one plant of format lactobacillus with bacteriostasis, is format lactobacillus (Lactobacillusgasseri) DFNXC2-0121 is preserved in China typical culture collection on March 31st, 2017 The heart (abbreviation CCTCC), address are as follows: Wuhan, China, Wuhan University, deposit number are CCTCC NO:M 2017162.

The bacterial strain is isolated from normal health vagina, the physiological and biochemical property of bacterial strain are as follows: colonial morphology For milky, circle, piece shape；Gram-positive, no gemma, nitrate reduction reaction, H₂O₂Enzyme test is negative, does not liquefy Gelatin does not produce indoles.

The second aspect of the present invention, provides a kind of microbial inoculum, and active constituent is the tunning of above-mentioned format lactobacillus.

The present invention also provides the preparation method of above-mentioned microbial inoculum, includes the following steps: the format lactobacillus for fermenting above-mentioned, obtain Tunning.

In above-mentioned preparation method, the fermentation medium for use of fermenting are as follows: MRS culture medium.

In above-mentioned preparation method, the condition of fermentation are as follows: 37 DEG C, stationary culture 18-24h.

The third aspect of the present invention provides above-mentioned format lactobacillus and/or microbial inoculum in the product of preparation treatment gynaecological imflammation In application.

The fourth aspect of the present invention provides a kind of probiotics metabolin composition, the probiotics metabolin composition Include: the fermentation culture medium and deposit number of above-mentioned format lactobacillus are the lactobacillus plantarum of CCTCC NO:M 2010150 Fermentation culture medium.

Viable count in the fermentation culture medium of the format lactobacillus is 1.0 × 10⁸-10.0×10⁸cfu/mL；The guarantor Viable count in the fermentation culture medium for the lactobacillus plantarum that hiding number is CCTCC NO:M 2010150 is 8.0 × 10⁸-20.0× 10⁸cfu/mL。

The plant cream that the fermentation culture medium and the deposit number of the format lactobacillus are CCTCC NO:M 2010150 The fermentation culture medium of bacillus can be with any than mixing；As a preferred option, the fermentation culture medium of the format lactobacillus and The deposit number is that the fermentation culture medium of the lactobacillus plantarum of CCTCC NO:M 2010150 is mixed with volume ratio 1:1.

Further, can also include in above-mentioned probiotics metabolin composition: the groups such as carbomer, glycerol and peppermint oil Point, and the various pharmaceutical dosage forms such as gelling agent is made in conventional manner.

Application of the above-mentioned probiotics metabolin composition in the product of preparation treatment gynaecological imflammation is also guarantor of the invention Protect range.It is found through experiment that probiotics metabolin composition of the invention is apparent for causing the pathogenic bacteria of vaginitis to have Inhibiting effect, especially for causing the staphylococcus aureus of vaginitis, the fungistatic effect of Candida albicans and Escherichia coli It is more significant.

In above-mentioned application, the product includes: the multiple products form such as functional food, health care product, disinfectant and drug.

The fifth aspect of the present invention provides a kind of Traditional Chinese medicine probiotic composition for treating gynaecological imflammation.

The Traditional Chinese medicine probiotic composition for the treatment of gynaecological imflammation of the invention, is made of the component of following parts by weight:

30-40 parts of probiotics metabolin composition, 40-55 parts of Chinese medical extract；

The Chinese medical extract is to be prepared from the following raw materials in parts by weight:

32-40 parts of radix scutellariae, 25-30 parts of kuh-seng, 6-10 parts of Cortex Phellodendri, 4-10 parts of the fruit of summer cypress, 4-10 parts of mulberry leaf.

The sixth aspect of the present invention provides the preparation method of above-mentioned Chinese medicine probiotic composition, and steps are as follows:

(1) preparation of probiotics metabolin composition: being respectively CCTCC by format lactobacillus of the invention and deposit number The lactobacillus plantarum of NO:M 2010150 is activated, and the strain inoculated after activation is cultivated in fermentation medium, until The cell concentration of format lactobacillus ferment culture reaches 1.0 × 10⁸-10.0×10⁸Cfu/mL, deposit number are CCTCC NO: The cell concentration of the lactobacillus plantarum fermentation culture medium of M 2010150 reaches 8.0 × 10⁸-20.0×10⁸Cfu/mL, by format cream The lactobacillus plantarum fermentation culture medium that bacillus fermentation culture and deposit number are CCTCC M 2010150 mixes, spare；

(2) preparation of Chinese medical extract: radix scutellariae, kuh-seng, Cortex Phellodendri, the fruit of summer cypress and mulberry leaf are mixed, are added water to cook, decoction liquor Being concentrated into density is 1.10-1.20g/mL, and centrifugation takes supernatant, spare；

(3) probiotics is metabolized composition and Chinese medical extract is uniformly mixed, that is, Traditional Chinese medicine probiotic composition is prepared.

In step (1), the fermentation medium are as follows: MRS culture medium；Condition of culture are as follows: 37 DEG C, stationary culture.

It in step (2), adds water to cook method particularly includes: radix scutellariae, kuh-seng, Cortex Phellodendri, the fruit of summer cypress and mulberry leaf are mixed, are placed in 40-60min is impregnated in 10 times of water, is decocted, water continues to decoct 50-60min after opening, and the first filtrate is obtained by filtration；8 times of water of the dregs of a decoction Secondary decoction is carried out, water continues to decoct 30-40min after opening, the second filtrate is obtained by filtration and is uniformly mixed with the first filtrate, by true Chinese medicine liquid is concentrated under reduced pressure in sky, until the density of medical fluid is 1.10-1.20g/mL, 3000 turns/min is centrifuged 10min, takes supernatant spare.

Further, the auxiliary material that Traditional Chinese medicine probiotic composition of the invention can be combined with pharmaceutically receiving is prepared into various The product of type, comprising: functional food, health care product, disinfectant and drug (such as: lotion, gelling agent and suppository).

Beneficial effects of the present invention:

(1) present invention is separated from the vagina of healthy women for the first time, purifying obtains one plant of format lactobacillus, which has Acid height is produced, the strong feature of Substance, bacteriostasis is produced.Acidic materials such as lactic acid, formic acid can be generated after strain fermentation And stearic acid etc., these acidic materials can reduce the pH value of genital tract, inhibit the growth of pathogen；Peroxidating can also be generated Hydrogen further inhibits the growth of other bacterium and pathogenic microorganisms.

(2) it is CCTCC that probiotics metabolin composition of the invention, which is by format lactobacillus of the invention and deposit number, The fermentation culture medium of the lactobacillus plantarum of M 2010150 compounds, wherein deposit number is the plant of CCTCC M 2010150 Object lactobacillus is documented in patent " one plant of lactic acid bacteria that can be used for biological preservation and its application " (CN101914475A), should The original effect of lactic acid bacteria is prevention and treatment pericarp browning, reduces fruit disease incidence.The present invention joins the lactic acid bacteria and format lactobacillus It closes, it is found that this two plants of bacterium are used cooperatively with significant synergistic function, it is obvious for causing the pathogenic bacteria of vaginitis to have Inhibiting effect, especially for causing the staphylococcus aureus of vaginitis, the antibacterial effect of Candida albicans and Escherichia coli Fruit is more significant.

(3) Traditional Chinese medicine probiotic preparation of the invention is by probiotics metabolin composition and Chinese medical extract through scientific compatibility It forms, realizes the perfect combination of probiotics and Chinese medical extract, verified by antibacterial experiment in vitro, Chinese medicine of the invention is prebiotic Bacteria preparation is safe and effective, and the probiotic products simple compared with the existing product of existing market or simple tcm product have antimicrobial spectrum Extensively, antipruritic effect of ejecting poison is more obvious.Wherein: Chinese medical extract has apparent inhibition staphylococcus aureus, Candida albicans Bacterium effect and the effect of heat-clearing and damp-drying drug, repellent antipruritic；Probiotics is able to suppress the effect of Escherichia coli, staphylococcus aureus, And it can be acidified the pH of vagina, be conducive to the increment and vaginal mucosa reparation of lactobacillus, the combination of Chinese medicine and probiotics has The therapeutic effect of Chinese medicine has the function of the adjusting microecology in vaginas balance of probiotics again, and expands antimicrobial spectrum, and the two has Synergistic effect.Experiment proves that effect is better than any single raw material.

Detailed description of the invention

Fig. 1: amino decarboxylase testing result；Wherein, Figure 1A is histidine decarboxylase Activity determination as a result, Figure 1B is For tyrosine decarboxylation Enzyme assay as a result, Fig. 1 C is ornithine decarboxylase activity testing result, Fig. 1 D is living for lysine decarboxylase Property testing result.

Fig. 2: the selection result of advantage lactobacillus.

Fig. 3: the phylogenetic tree of bacterial strain C2-0121.

Fig. 4: bacterial strain C2-0121 Fermentative growth curve.

Fig. 5: test group vagina tissue picture and HE stained slice figure；Wherein, Fig. 5 a is negative control group, and Fig. 5 b is contamination Group, Fig. 5 c are positive controls.

Fig. 6: rabbit vagina secretion Flora dynamics during modeling.

Fig. 7: vaginal fluid microscopy picture；Wherein, Fig. 7 a is normal group vaginal fluid microscopy picture, and Fig. 7 b is perfusion Vaginal fluid microscopy picture after antibiotic, Fig. 7 c are the microscopy picture after attacking poison.

Fig. 8: the changes in diet trend of rabbit after treatment 7 days.

Fig. 9: rabbit vagina secretion Flora dynamics during treatment.

Figure 10: each test group vaginal fluid microscopy smear of rabbit；Wherein, Figure 10 a is natural recovering group, and Figure 10 b is mixed Treatment group is closed, Figure 10 c is positive controls, and Figure 10 d is Chinese medicine group, and Figure 10 e is probiotic group.

Figure 11: vagina tissue is sliced during modeling；Wherein, Figure 11 a is Normal group, and Figure 11 b is model group, Figure 11 c For mixing treatment group, Figure 11 d is Chinese medicine group, and Figure 11 e is probiotic group, and Figure 11 f is positive controls.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

As background technique is introduced, it is still current for how treating vagina infection by adjusting vaginal flora balance Where difficult point, it is based on this, present applicant proposes one plant to have the format lactobacillus of bacteriostasis and its in treatment vagina infection Application.

In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool The technical solution of the application is described in detail in the embodiment and comparative example of body.

Embodiment 1: the screening and identification of bacterial strain

1. test material

1.1 separation sources: healthy women vaginal fluid

1.2 culture mediums:

Modified MRS culture medium (calcium carbonate of addition 1% in MRS culture medium when strain isolation), gelatin liquefaction culture medium is (often The commercial product of rule).

H₂O₂Identification of M RS culture medium: including tetramethyl benzidine, the 0.lmg/mL of 0.25mg/mL in MRS agar medium Horseradish peroxidase.

The culture medium of lactobacillus qualification test: the bacterium micro biochemical of Hangzhou day and the production of microorganism reagent Co., Ltd is anti- Ying Guan.

1.3 experimental strains:

Staphylococcus aureus ATCC6538, Escherichia coli 8099 are purchased from Chinese medicine bacterium and contain administrative center, deformation Bacillus and salmonella are purchased from China Agricultural University.

1.4 other

Bacterial genomes extracts kit, drug sensitive test paper, 16S rRNA primer.

2. test method

The acquisition of 2.1 vaginal fluids: doctor assists sampling at the physical examination of 88 gynaecology, hospital of Tai'an, with sterile cotton swab in yin It disposably scrapes and smears clockwise at 1/3 behind road, external genital and introitus must be avoided contact to prevent pollution.Put immediately after taking-up to In MRS fluid nutrient medium, and number record.

2.2 bacterial strains isolating and purifying and identifying

Each separation source is placed in concussion in sterile water (band bead), sample is sufficiently broken up to mixing, takes oscillation liquid successively It is diluted to 10^-2, 10^-3, 10^-4, 10^-5, each dilution take 0.1mL apply plate (MRS+CaCO₃), 2 repetitions, in 37 DEG C of incubators Culture.Bacterium colony on picking culture medium with obvious calcium solusphere is in inclined-plane, Gram's staining, microscopy, until it is determined as pure bacterium, it will After lactobacillus strain after purification is inoculated into MRS culture medium culture, sets in 4 DEG C of refrigerators and save backup.

2.2.1 the measurement of product acid activity and mitotic stability

Isolated strain is inoculated in the MRS culture medium of improvement with 2% inoculum concentration, 37 DEG C of stationary cultures, measurement fermentation The pH value of liquid.

Bacterial strain is continuously transferred 10 days pH value for seeing its stability and fermentation liquid.

2.2.2 the Preliminary Identification of lactobacillus

All Gram-positives, H₂O₂The bacterial strain of enzyme test feminine gender can primarily determine as lactobacillus.

2.2.3 the identification of lactobacillus

(15 DEG C, 45 DEG C), mobility test, nitrate reduction test, gelatin liquefaction test, Yin are tested by growth temperature The identification that diindyl test, hydrogen sulfide production test belong to doubtful bacterial strain.

2.2.4 the identification of lactobacillus species

The identification that acid test is planted is produced by carbohydrate fermentation.The culture medium of various carbohydrate fermentation tests It is all made of bacterium micro biochemical reaction tube.

The biology performance of 2.3 bacterial strains is studied and the screening of advantage lactobacillus

2.3.1 acid resistance test

Hydrochloric acid, which adjusts MRS culture medium, makes its pH value be respectively 1.5,2.5,3.5, respectively the good fermentation liquid of inoculated and cultured, and 1% Inoculum concentration, 37 DEG C culture.Take the bacterium solution plate count of culture 0h and 4h.It calculates survival rate (%).

2.3.2 bile tolerance is tested

Pig gall hydrochlorate is added in MRS culture medium, making its mass fraction is respectively 0.1%, 0.2%, 0.3%, and bacterium solution is pressed 2% inoculum concentration inoculation, 37 DEG C of cultures.The bacterium solution plate count of culture 0h and 4h is taken, is calculated survival rate (%).

2.3.3 extracorporeal bacteria inhibitor test Odontothrips loti

2.3.4 the sensitization test (STT) paper diffusion method of antibiotic

2.3.5 amino decarboxylase detects

After will be for examination actication of culture, it be inoculated in liquid decarboxylase culture and detects, blank cultures check experiment.37 DEG C, after cultivating 4d, observe color change.

2.3.6 the screening of advantage lactobacillus

Using H₂O₂Identification of M RS Screening of Media produces H₂O₂Lactobacillus.37 DEG C of anaerobism after inoculation cultivate 72h, later will Culture medium is exposed in air, produce hydrogen peroxide bacterium colony can in become in 20min blue.

2.4 16S rDNA Molecular Identifications

PCR amplification primer: 1492r 5 '-ggttaccttgttacgactt-3 '；27f 5'- agagttgatcctggctcag-3’。

Bacterium colony PCR reaction condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 1min, 52 DEG C of annealing 1min, 72 DEG C extend 2min, 25 circulations, 72 DEG C of extension 10min.

PCR product after purification through salt-free, completed by Bo Shang Bioisystech Co., Ltd, it is desirable that double by PCR product sequencing It is logical to surveying.Spliced sequence results are inputtedwww.NCBI.nlm.nih.gov/blast, with the sequence in GenBank database Column carry out sequence analysis.

The measurement of 2.5 growth curves

With 2% inoculum concentration by strain inoculated in the MRS culture medium of improvement, 37 DEG C of stationary cultures, in 0h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 20h, for 24 hours, 28h, 30h, 32h, 34h, 36h, 40h, 44h, 48h, 50h OD value and pH is measured by sampling Value, using incubation time as abscissa, corresponding OD value and pH value are ordinate, draw growth curve, find out best incubation time.

3. result and analysis

3.1 isolate and purify the morphological feature of each bacterial strain

Multiple picking single colonie, the bacterial strain isolated and purified are subjected to Gram's staining, the colonial morphology of each strain bacterium is all cream White, round, piece shape, selecting gram-positive, thalli morphology by Gram's staining is rod-shaped bacterial strain, is marked respectively For E-1, G5-7, C2-0109, C2-0121, L-7-3, G5-5, G5-3, H5-2, G5-4, G5-3, H5-3.

3.2 product acid activities, mitotic stability and fermentating liquid PH value (10 generation)

The measurement result (pH value of measurement 20h fermentation liquid) of 1 product acid activity of table

Found out by table 1: C2-0109 and C2-0121 and other Strain comparisons, the pH of fermentation liquid is lower, and both less than 4, it says The bright more other bacterial strains of its acid producing ability are strong.

2 mitotic stability of table and fermentating liquid PH value (10 generation)

Through continuous subculture and comparative test, the physical and chemical shape such as the taxology shape of the bacterial strain, morphological feature is not found Visible change, illustrates the dimensionally stable heredity of the bacterial strain, and the acid producing ability of two bacterial strains is more stable.

The identification of 3.2 lactobacillus

Institute plant division bacterium is Gram-positive, no gemma, nitrate reduction reaction, H₂O₂Enzyme test is negative, does not liquefy Gelatin does not produce indoles, other are shown in Table 3

3 hydrogen sulfide experimental result of table

As can be seen from Table 3: each strain bacterium does not generate hydrogen sulfide, comprehensive each index, bacterial strain C2-0121 and other bacterial strains It is all lactobacillus.

The detection of 3.3 amino decarboxylases

Four kinds of amino acid used in the detection test of amino decarboxylase are tyrosine, histidine, ornithine and lysine. It is respectively formed tyrasamine, histamine, putrescine and cadaverine after deamination, is the primary biological amine substance for causing poisoning symptom.According to Known to amino decarboxylase culture Preliminary detection result: surveyed bacterial strain does not generate ornithine, lysine, histidine and tyrosine Decarboxylase, i.e. not generation amine.Poisoning symptom will not be caused.Histidine decarboxylase Activity determination result is as shown in Figure 1A (in Figure 1A, being from left to right respectively as follows: blank, C2-0109, FNG5-5, FNG5-4, C2-0121, FNG5-3, H5-2), tyrosine Decarboxylase testing result is as shown in Figure 1B (in Figure 1B, from left to right: blank, FNG5-3, FNG5-5, C2-0121, C2- 0109, H5-2, FNG5-4), ornithine decarboxylase activity testing result (is from left to right respectively as shown in Figure 1 C sky in Fig. 1 C White, FNG5-5, C2-0109, FNG5-3, C2-0109/C2-0121, H5-2, FNG5-4), lysine decarboxylase activity detection knot Fruit as shown in figure iD (in Fig. 1 D, from left to right respectively blank, C2-0109, C2-0121, FNG5-4, FNG5-5, C2-0109, FNG5-3)。

The biology performance of 3.4 bacterial strains is studied and the screening of advantage lactobacillus

3.4.1 acid resistance test result

Hydrochloric acid, which adjusts MRS culture medium, makes its pH value be respectively 1.5,2.5,3.5, respectively the good fermentation liquid of inoculated and cultured, and 1% Inoculum concentration, 37 DEG C culture.The bacterium solution plate count of culture 0h and 4h is taken, survival rate (%) is calculated and is shown in Table 4.

Table 4 places the survival rate (%) of 4h in condition of different pH

Bacterial strain/pH degree	1.5	2.5	3.5
				C2-0109	—	—	108%
C2-0121	—	2.1%	93.5%
				C2-0071	—	1.0%	90.5%
G5-7	—	0.8%	88.2%
				G5-5	—	—	91.2%
G5-4	—	—	90.5%
				G5-3	—	1.05%	90.5%
H5-2	—	1.32%	91.8%
				H5-3	—	—	88.6%
L-3-2	—	—	78%

Found out by table 4: each strain bacterium does not all tolerate in 1.5 environment of PH, and bacterial strain C2-0121 is resistant in pH3.5 environment, Survival rate is 93.5%, other than lower than bacterial strain C2-0109, is higher than other bacterial strains.

3.4.2 bile tolerance is tested

Pig gall hydrochlorate is added in MRS culture medium, making its mass fraction is respectively 0.1%, 0.2%, 0.3%, bacterium solution is pressed 2% inoculum concentration inoculation, the bacterium solution plate count of 37 DEG C of culture 0h and 4h calculate survival rate (%), the result is shown in tables 5.

Table 5 places the survival rate (%) of 4h in various concentration cholate condition

Found out by table 5: bacterial strain C2-0109, C2-0121, C2-0071FNG-3 4h in the solution that gallbladder salinity is 0.1% After can survive 85% or more, wherein the ability of C2-0109, C2-0121 bile tolerance is stronger, 37 in 0.1% cholate solution DEG C culture its survival rate of 4h is greater than 95%, and other each Strain survival rates are smaller than 90%.

3.4.3 extracorporeal bacteria inhibitor test

With Odontothrips loti respectively to Escherichia coli 8099, staphylococcus aureus ATCC6538, salmonella and deformed rod The bacteriostasis property of bacterium detects, and is compared with the antibacterial relatively good bacterial strain of format lactobacillus C2-0071 saved in laboratory Compared with the results are shown in Table 6.

The biocidal property result (unit: mm) of 6 each plants of lactobacillus of table

As can be seen from Table 6: each bacterial strain is to pathogenic bacteria staphylococcus aureus, Escherichia coli, salmonella, proteus There is certain biocidal property, and bacterial strain C2-0109, C2-0121, C2-0071 are preferable to the biocidal property of pathogenic bacteria, wherein with bacterial strain C2-0121 is best, all better than the biocidal property of other several plants of bacterium, the inhibition zone than the format lactobacillus C2-0071 that laboratory saves It is all big, big to the biocidal property difference of staphylococcus aureus, Escherichia coli, salmonella, proteus 14.3%, 59.6%, 21.7%, 7.4%.Two plants of bacterium C2-0109, C2-0121 for selecting better performances in the following experiment carry out the reality through a step It tests.

3.4.4 the sensitization test of antibiotic

The sensitive experiment that bacterial strain C2-0109, C2-0121 of better performances are carried out to antibiotic, the results are shown in Table 7.

Sensitization test (antibacterial circle diameter: mm) of the 7 liang of bacterial strains of table to 9 kinds of antibiotic

As can be seen from Table 7: bacterial strain C2-0121 is insensitive to ceftriaxone, cefalexin and the different evil Zo of sulfanilamide (SN), to other Multiple Classes of Antibiotics is sensitive.

3.4.5 biochemical identification test result

The identification that acid test is planted is produced by carbohydrate fermentation.The culture medium of various carbohydrate fermentation tests It is all made of bacterium micro biochemical reaction tube, fermentation results are shown in Table 8.

The sugared fermenting experiment result of table 8

Test result shows that C2-0121 is consistent with the biochemical characteristic of format lactobacillus, according to primary Jie Shi handbook, tentatively It is accredited as format lactobacillus.

3.4.6 the screening of advantage lactobacillus

By the screening of advantage lactobacillus, wherein C2-0121 bacterium colony becomes blue, as a result sees Fig. 2, explanation can produce peroxide Change hydrogen.

In normal vagina flora, lactobacillus is dominant, and lactobacillus is important to maintaining the normal flora of intravaginal to play Effect, on the one hand, lactobacillus can using glycogen generate lactic acid, reduce the pH value of genital tract, inhibit the growth of pathogen；Separately Outside, lactobacillus can also generate hydrogen peroxide, and bacteriocin and class bacteriocin and biosurfactant etc. inhibit other bacterium and cause The growth of sick microorganism, to maintain the balance of microecology in vaginas；Found out by testing above: bacterial strain C2-0121 produces peroxidating Hydrogen, acid producing ability are strong and biocidal property is all better than other bacterial strains, therefore, this plant of bacterium for women genital tract infected with very Good effect has facilitation to maintenance microecology in vaginas balance.

3.5 Molecular Identification results

For determine C2-0121 be lactic acid bacteria type, further done molecular biology identification, by bacterial strain C2-0121 into Row liquid fermentation extracts DNA, and PCR amplification, the sequencing of 16S rDNA is as shown in SEQ ID NO.1, then homology ratio Right, phylogenetic tree construction is shown in Fig. 3.

By the sequencing and sequence analysis of the 16S rDNA to bacterial strain C2-0121, find it with The 16S rDNA of 1902 strain of Lactobacillus hominis CRBIP 24.179FR68 has up to 99% homology, Combining form is accredited as format lactobacillus (Lactobacillusgasseri), is named as format lactobacillus (Lactobacillusgasseri) DFNXC2-0121, and be preserved in China typical culture collection on March 31st, 2017 The heart (abbreviation CCTCC), address are as follows: Wuhan, China, Wuhan University, deposit number are CCTCC NO:M 2017162.

The drafting of 3.6 strain fermentation growth curves

Separately sampled in different time sections after strain inoculated, detection measures its OD value and pH at wavelength 600nm, with culture Time is abscissa, and corresponding OD value and pH value are ordinate, draws growth curve and sees Fig. 4.

Found out by Fig. 4: bacterial strain C2-0121 enters logarithmic growth phase in 8h or so, and strain growth is most fast at this time, at this time under pH Drop also most drops to 5.3 by 6.5 fastly, until OD600 reaches maximum when 16h, pH is 3.99 at this time, learns this by plate count When viable count up to 13.5 × 10⁸Enter stationary phase after cfu/mL, 16h.

Embodiment 2: the preparation of probiotics metabolin composition

(1) preparation of lactobacillus solution: take out culture presevation pipe, with lactic acid bacteria inclined-plane solid medium cross respectively into Row recovery, 37 DEG C of culture 24-36h.Bacterium colony violet staining microscopy on picking slant medium is brevibacterium or long bacillus. Picking colony is inoculated in 100mL lactic acid bacteria culturing medium, is cultivated for 24 hours in 37 DEG C of incubators.Seed uses the inoculation of 2%-5% Amount, is seeded in the big triangular flask of 3L, 37 DEG C of culture -36h for 24 hours, detects its bacterial concentration, and (deposit number is lactobacillus plantarum CCTCC M 2010150) bacterial content be more than the bacterial content of 1,000,000,000/milliliter and bacterial strain C2-0121 be more than 200,000,000/milliliter, Ji Kezuo For the inoculation of lactobacillus liquid spawn.

(2) strain inoculated after activation is cultivated in MRS culture medium, fermented and cultured to format lactobacillus ferment is trained The cell concentration for supporting object reaches 1.0 × 10⁸-10.0×10⁸Cfu/mL, deposit number are the plant cream bar of CCTCC M 2010150 The cell concentration of bacterium fermentation culture medium reaches 8.0 × 10⁸-20.0×10⁸cfu/mL。

The MRS culture medium: glucose 2.0%, ammonium citrate 0.2%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.5%, manganese sulfate 0.02%, magnesium sulfate 0.05%, peptone 1.0%, beef extract 1.0%, yeast extract 0.5%, Tween 80 0.1%, adjust pH to 6.0；Solid medium adds 1.5% agar powder.Medium sterilization condition are as follows: 0.10-0.15MPa, 121 DEG C Sterilize 20min.

(3) count respectively, by the fermentation culture medium of step (2) with normal saline dilution to its viable count be all 1 × 10⁷cfu/mL；Take the bacterium solution mixing for standby use of same volume.

Embodiment 3: the preparation of Chinese medical extract

By 35 parts of radix scutellariae, 30 parts of kuh-seng, 8 parts of Cortex Phellodendri, 6 parts of the fruit of summer cypress, 8 parts of mulberry leaf mixing, mixture is placed in 10 times of water Middle immersion 40-60min is decocted, and water continues to decoct 50-60min after opening, and the first filtrate is obtained by filtration；The dregs of a decoction carry out two with 8 times of water Secondary decoction, water continue 30-40min of decoction after opening, the second filtrate is obtained by filtration and is uniformly mixed with the first filtrate, is subtracted by vacuum Rotary Evaporators concentrated traditional Chinese medicine liquid is pressed, until the density of Chinese medicine liquid is 1.1g/mL-1.2g/mL, left undisturbed overnight, 3000 turns of centrifugations 10min collects supernatant, spare.

Embodiment 4: the preparation of Traditional Chinese medicine probiotic preparation

Prepared by 40 parts of probiotics metabolin composition prepared by embodiment 2 and embodiment 3 45 parts of Chinese medical extract are mixed It closes uniformly, that is, Traditional Chinese medicine probiotic preparation is prepared.

Embodiment 5: the preparation of the gelling agent of vaginitis is treated

942 2.5g of carbomer is weighed, Traditional Chinese medicine probiotic preparation 850mL prepared by embodiment 4 is measured, is added to and fills card In the container of wave nurse, overnight, stir evenly, until it is fine and smooth uniformly without particle, pH value is adjusted with the sodium hydroxide that mass concentration is 10% To 5.0, glycerol 5g and peppermint oil 0.2g is added, add water to 1000g stir evenly to get.

Application examples 1: the antibacterial experiment in vitro of probiotics

1. test material:

1.1 strains testeds: lactobacillus plantarum BLCC2-0001 (deposit number is CCTCC M 2010150), format cream bar Bacterium DFNXC2-0121 (screening of embodiment 1 obtains).

1.2 indicator bacterias: Candida albicans, staphylococcus aureus, Escherichia coli, Lactobacillus crispatus BLCC2-0109, it is thermophilic Lactobacillus lactis 1.2467, Lactobacillus casei 1.0062, each indicator bacteria are commercial product.

2. method and result:

Odontothrips loti

Take the fermentation liquid 10000rpm centrifugation of bacterial strain lactobacillus plantarum BLCC2-0001, format lactobacillus DFNXC2-0121 10min takes supernatant, carries out biocidal property test；The indicator bacteria of selection is respectively Candida albicans, staphylococcus aureus, big Enterobacteria, Lactobacillus crispatus BLCC2-0109, lactobacillus acidophilus 1.2467, Lactobacillus casei 1.0062.Test result is shown in Table 9.

Diameter (unit: mm) of 9 bacterial strain of table to the inhibition zone of each indicator bacteria

Note :-indicate no apparent inhibition zone.

Test result shows: bacterial strain BLCC2-0001, DFNXC2-0121 are to Escherichia coli, staphylococcus aureus, sramana Salmonella has stronger biocidal property, to Lactobacillus casei 1.0062, lactobacillus acidophilus, Lactobacillus crispatus BLCC2-0109 without antibacterial Property.Therefore, this bacterial strain is selective depression pathogenic bacteria Escherichia coli, salmonella and the stronger bacterial strain of staphylococcus aureus.

Application examples 2: the antibacterial experiment in vitro of Chinese medical extract

1. test material:

1.1 for trying Chinese medicine: Chinese medical extract prepared by embodiment 3

1.2 indicator bacterias: Candida albicans, staphylococcus aureus, Escherichia coli, Lactobacillus crispatus BLCC2-0109, it is thermophilic Lactobacillus lactis 1.2467, Lactobacillus casei 1.0062, each indicator bacteria are commercial product.

2. method and result

2.1 methods: Odontothrips loti

2.2 results: Chinese medical extract is to the biocidal property of each indicator bacteria as a result, being shown in Table 10.

Table 10: biocidal property result of the Chinese medical extract to each indicator bacteria

Found out by experimental result: Chinese medicine has very strong biocidal property to staphylococcus aureus and Candida albicans, and right Escherichia coli and sramana's sterilization are then without biocidal property, to lactic acid bacteria equally also without biocidal property.

Application examples 3: the antibacterial experiment in vitro of Traditional Chinese medicine probiotic preparation

1. test material:

1.1 for trying Chinese medicine: Traditional Chinese medicine probiotic preparation prepared by embodiment 4

1.2 indicator bacterias: Candida albicans, staphylococcus aureus, Escherichia coli, Lactobacillus crispatus BLCC2-0109, it is thermophilic Lactobacillus lactis 1.2467, Lactobacillus casei 1.0062, each indicator bacteria are commercial product.

2. method and result

2.1 methods: Odontothrips loti

2.2 results: Traditional Chinese medicine probiotic preparation is to the biocidal property of each indicator bacteria as a result, being shown in Table 11.

Antibacterial result of the 11 Traditional Chinese medicine probiotic preparation of table to each strain bacterium

As can be seen from Table 11: to Escherichia coli, staphylococcus aureus and white after Chinese medicine and the mixing of lactobacillus ferment liquid Color candida albicans, salmonella have very strong biocidal property, but expand after illustrating mixing to lactobacillus still without biocidal property Antimicrobial spectrum, the two have concertedness, fungistatic effect more wide spectrum.

Application examples 4: rabbit vagina irritant test is (according to 2002 sterile products inspection technology specifications-vaginal mucosa stimulation examination It tests)

1. experimental animal: selecting the Female white rabbit of health, the non-estrus just grown up, Belgian rabbit, weight 2.5kg.Whether there is or not secretion, hyperemia, oedema and other degree of impairment for pretest inspection animal introitus.If any inflammation or (and) damage Wound, should abandon.

2. test grouping: it is divided into contamination group, negative control group and positive controls, every group 3.Wherein, contamination group uses Gelling agent prepared by the embodiment of the present invention 5, negative control group pour into physiological saline, and positive controls pour into commercial product Jilin one The bacteriostatic gel of Biotechnology Co., Ltd, positive medicine company group

3. experimental working technique

3.1 connect the syringe of blunt nosed hose and 5mL that length is 8cm or so.Syringe and conduit are filled by test solution It is spare.Every animal prepares a set of.

The contamination method of 3.2 mucosa irritation tests: animal is faced upward fixation, by conduit with by test solution or comparison liquid it is wet after It gently is inserted into vagina (4cm-5cm), and is slowly injected into 2mL by test solution with syringe, withdrawal conduit completes contamination.It is negative right Make same processing according to group animal physiological saline.

The contamination method of more than 3.3 Tests For Irritating Effects: pressing above-mentioned 3.2 contamination method, every repeatedly contamination one for 24 hours It is secondary, continuous 5d.Negative control group animal physiological saline makees same processing, the positive control positive medicine company collection in commercial product Jilin one The bacteriostatic gel of Biotechnology Co., Ltd, group

3.4 injected by test solution after may have spilling, can be wiped away with aseptic cotton or soft paper.

After the contamination of 3.5 lasts for 24 hours, animal is put to death using aeroembolism method, cuts open the belly and takes out complete vagina, longitudinally slit, naked eyes See whether that hyperemia, oedema etc. show, for referring to when pathologic sampling.Then vagina is put into 10% formalin solution solid Determine more than for 24 hours, the tissue film-making for choosing the both ends and central 3 positions of vagina carries out histopathological examination after HE dyeing.

4 evaluation of result

Histopathological examination is as a result, score to the stimulate the reaction of vaginal mucosa by table 12 is fixed.It is dynamic by test group 3 After the stimulate the reaction integral at 3 positions of object is added, then divided by total (number of animals × 3) are observed, show that test group vaginal mucosa pierces Swash the average integral of reaction, maximum is scored as 16 (being shown in Table 12).Control group methods of marking is same as above.Test group average integral is subtracted After control group average integral obtains stimulus index, stimulus intensity classification is carried out by table 13.

4.1 vaginal mucosa stimulate the reaction standards of grading are shown in Table 12.

12 vaginal mucosa stimulate the reaction standards of grading of table

Stimulate the reaction integral=A+B+C+D.

The classification of 13 vaginal mucosa stimulus intensity of table

4.2 each test group vagina tissue pictures and HE stained slice figure

Negative control group vagina epithelium is complete, and cell-free necrosis falls off and inflammatory cell infiltration, contamination group and positive control Group is also likewise, result is shown in Fig. 5 a- Fig. 5 c.

4.3 each test group vaginal mucosa stimulation scores are shown in Table 14.

Each test group vaginal mucosa stimulus index of table 14

Test result shows: testing the vaginal mucosa stimulus index < 1 of intervention group, is classified according to vaginal mucosa stimulus intensity Standard is it is found that test group is nonirritant to rabbit vagina mucous membrane, by vagina tissue picture and HE stained slice it can also be seen that yin Road is without congested, oedema and inflammatory cell, and vaginal mucosa is complete, cell-free necrosis, obscission.

Application examples 5: the inhibiting effect research of probiotics and Chinese medicine composition to rabbit vagina inflammation

1. materials and methods

1.1 experimental material

1.1.1 experimental animal Healthy female rabbit, weight 3.0kg, with production 2 times or more, by Tran town fishpond village Pang Property raiser provide, totally 30.

1.1.2 drug and reagent hydrochloric acid lincomycin (being purchased from the big pharmacy of Tai'an Tongrentang), Candida albicans CMCC (F) 98001 (being purchased from Chinese pharmaceutical biological product identification institute), positive bacteriostatic gel (being purchased from the big pharmacy in a Tai'an Wanning) Wuhan is new The production of ecologic biological Engineering Co., Ltd

1.1.3 culture medium selective medium: EMB, LBS, TSA, CQ, MRS, being purchased from Qingdao hypo biotechnology has Limit company.

1.2 experimental method

Rabbit is first divided into two groups, A group Normal group 5, B group model group 25 takes vagina after pre- feeding 3 days at random Secretion microscopy simultaneously counts Escherichia coli, staphylococcus aureus, enterococcus, lactic acid bacteria number.With No. 18 after the disinfection of A group external genital Rat oral gavage device saline injection 1mL, after perfusion 11 days, gas fastens method and kills rabbit, takes the fixed system of 10% formaldehyde of vagina tissue Piece after HE dyeing, carries out pathological examination scoring, classification.Lincomycin hydrochloride injection 1mL is perfused in B group, 1 time a day, perfusion 5 days, restores 1 day, then use Candida albicans viable count 1.5 × 10⁷0.5mL is perfused in cfu/mL, 1 time a day, after 4 days, at random It takes vaginal fluid microscopy and counts Escherichia coli, staphylococcus aureus, enterococcus, Candida albicans and lactic acid bacteria number, really After determining modeling success, model group rabbit gas fastens method and kills rabbit 5, takes the fixed film-making of 10% formaldehyde of vagina tissue, after HE dyeing, It carries out pathological examination and scores referring to table.Then rabbit is grouped again, is divided into 5 groups, B1 group is natural recovering group, and B2 group is mixed (Chinese medicine and probiotics) treatment group is closed, B3 group is Chinese medicine group, and B4 group is probiotic group, B5 group positive controls, every group 5, is opened Begin to treat.Only, the gelling agent 1.5g/ of embodiment 5 is perfused only in B2 group to Blank gel 1.5g/ of the B1 group perfusion without any drug, B3 group is the prebiotic bacterium solution 1.5mL of Chinese medicine liquid 1.5mL, the B4 embodiment 2 of embodiment 3, B5 group one positive bacteriostatic gel 1.5g/, Feed relative is weighed during treatment daily all to take vaginal fluid microscopy after treatment 6 days and count Escherichia coli, golden yellow Staphylococcus, enterococcus, Candida albicans and lactic acid bacteria number, gas fastens method and kills rabbit after 7 days, takes 10% formaldehyde of vagina tissue solid Piece is customized, after HE dyeing, carries out pathological examination and referring to table 4, the scoring of table 5, classification.

2 results and analysis

The sign variation of each test group rabbit during 2.1 tests

2.1.1 the sign of rabbit changes during modeling

After pre- feeding 5 days, model group rabbit coat is pure white smooth, and diet is normal, and spirit is good.After antibiotic, spirit Depressed, appetite decline, shape of crawling slightly is had loose bowels, 3 death, and after Candida albicans attacks poison, spirit is depressed, and do not feel like eating is crawled Shape is had loose bowels, and has 1 death.

2.1.2 rabbit vagina secretion Flora dynamics result is shown in Fig. 6 during modeling.As seen from Figure 6: rabbit is normal In the case of, there is a large amount of bacterial strain in vagina, wherein quantity is more with Bacillus acidi lactici to be main excellent；After using antibiotic, respectively Kind bacterium all largely reduces, and attacks after poison, and the breeding of the pathogenic bacteria such as staphylococcus aureus, Escherichia coli and Candida albicans increases It is more, it is main bacterial strain, and Bacillus acidi lactici is reduced accordingly, this illustrates being successfully established for the model of rabbit vagina inflammation.

Fig. 7 a is normal group vaginal fluid microscopy picture, it is seen that has a large amount of bacillus, Fig. 7 b is yin after perfusion antibiotic Road secretion microscopy picture, Microflora are seldom；Fig. 7 c is the microscopy picture after attacking poison, there is a large amount of Candida albicans, drop The pathogenic bacteria such as worm, coccus, this also illustrates model foundation success.

Indices after 2.2 treatments

2.2.1 15 the sign variation of each group rabbit during treating after modeling: be the results are shown in Table.

The sign variation of each group rabbit during being treated after 15 modeling of table

2.2.2 the changes in diet trend result of rabbit is shown in Fig. 8 after treating 7 days.

Test result shows: first day treatment group's dietary amount is most, natural recovering group is minimum, still, with prolonging for time Long natural recovering group dietary amount gradually increases, and mixing treatment group, Chinese medicine group, probiotic group and control group dietary amount are located always In higher level, it is minimum to illustrate that mixing treatment group stimulates the stomach of rabbit for mixing treatment group highest.

2.2.3 rabbit vagina secretion Flora dynamics result is shown in Fig. 9 during treating, and is found out by Fig. 9: the lactic acid normally organized Bacterium highest is dominant bacteria；It is dominant bacteria that lactic acid bacteria, which significantly increases, after the treatment of mixing treatment group, with normally organizing compared to without aobvious Sex differernce is write, probiotic group, Chinese medicine group, positive controls its lactobacillus quantity compared with natural recovering group are increased, still The lactobacillus quantity of mixing group is less than compared with mixing group, and natural recovering group lactic acid bacterium number is minimum, still in weak tendency, because This, inflammation does not disappear, it is therefore seen that, after mixing treatment group (Chinese medicine and probiotics) treatment, lactic acid bacterium number is greatly improved, It has the advantage status, restores to illustrate that composition has controlling for highly significant to the experimental rabbit with vaginitis to normal level Treatment effect.

Each test group vaginal fluid microscopy smear results of 2.3 rabbit are shown in Figure 10 a- Figure 10 e.

Natural recovering group (Figure 10 a) is visible minute quantity bacillus, and mixing treatment group (Figure 10 b) has a large amount of bacillus, sun Property control group (Figure 10 c) has a large amount of bacillus but also has Candida albicans, and Chinese medicine group (Figure 10 d) Microflora is less, probiotics Still pathogenic bacteria are also more though there is lactobacillus for group (Figure 10 e), and especially Candida albicans, each group compare, and illustrate prepared by embodiment 4 Traditional Chinese medicine probiotic preparation cause vaginitis treatment to have mixed infection to inhibit pathogen effect well.

Vagina tissue is sliced during 2.4 modelings, sees Figure 11 a to Figure 11 f.

The Normal group lyra of vagina complete display it can be seen from Figure 11 a, the epithelial cell of mucous membrane is without lesion and necrosis Phenomenon；Model group (Figure 11 b) lyra of vagina is shallow, and stratum supravasculare tissue is imperfect, and muscle layer is festered, atrophy, cell detachment, necrosis； Mixing treatment group (Figure 11 c) lyra of vagina is deep and more, and the upper layer mucosa cells of mucous membrane are more clear than more complete, and nothing falls off, necrosis is existing As.Chinese medicine group (Figure 11 d), probiotic group (Figure 11 e), positive controls (Figure 11 f) have high-visible lyra of vagina, mucous membrane Upper layer is complete, no ulcer surface, exuviation phenomenon, illustrates mixing treatment group, Chinese medicine group, probiotic group and positive controls to yin The experimental rabbit of road inflammation has a therapeutic effect, and mixing treatment group is compared with Chinese medicine group, probiotic group, vaginal wall gauffer it is more and Deep, mucous membrane upper layer is complete, illustrates the experimentalist of probiotics and Chinese medicine composition than single Chinese medicine or probiotics to vaginitis Rabbit therapeutic effect is significant.Mixing treatment group had both included the clearing heat and detoxicating, antibacterial of Chinese medicine, to staphylococcus aureus and Candida albicans Bacterium inhibit, and adds probiotics, can supplement lactobacillus definite value in vagina and generate ablastins, organic acid and hydrogen peroxide etc. Inhibit pathogen growth, the balance of microecology in vaginas can be promoted, quickly repair vagina mucous membrane, therefore, Chinese medicine with it is prebiotic Bacteria composition has good therapeutic effect to the experimental rabbit of vaginitis.

3 conclusions

3.1 use lincomycin hydrochloride injection 1mL, and perfusion 1 time daily totally 5 days, restores 1 day, then uses Candida albicans Viable count 1.5 × 10⁷0.5mL is perfused in cfu/mL, 1 time a day, is perfused 4 days, is stimulated by microscopy vaginal fluid, vaginal mucosa Index can be seen that model is set up.

The 3.2 Traditional Chinese medicine probiotic preparation perfusions prepared with embodiment 4,1.5g/ only, after treatment 6 days, pass through microscopy vagina point Flora in secretion, check vagina lesion situation and vagina tissue slice it can be seen that: model group vagina is seriously congested, Epithelial cell is imperfect, and phenomena such as lesion, fall off occurs in mucous layer, and is perfused with Traditional Chinese medicine probiotic preparation prepared by embodiment 4 Treatment group, positive controls it is complete with the vaginal epithelial tissue normally organized, phenomena such as mucous membrane is smooth, no lesion；It is more than summary Description of test: probiotics metabolin and Chinese medicine composition are safe and non-stimulating, do not influence its diet；To the pathogenic bacteria for causing vaginitis There is good inhibiting effect, promotes the flora in the experimental rabbit vagina with vaginitis to restore, and can quickly repair Impaired vaginal mucosa.

Application examples 6: clinical trial

(1) vaginitis and cervicitis

1 material

1.1 given the test agent: gelling agent prepared by embodiment 5 is sepia or auburn gel.Recommend dosage 3g/ People, day, inject, and 6 days are a small course for the treatment of.

1.2 tested crowds

Subject is included in standard: vaginitis (bacterial vaginitis, colpomycosis and trichomonas vaginitis) 160, Cervicitis 160, the age 18 years old~65 years old, equal sexual intercourse history.The non-menstrual period；Clinical symptoms meet clinically bacterial vaginosis Scorching diagnostic criteria, patient exclude the patient for having great mental disease, prohibit sexual life in 1W；Be not used in 1W before testing its medicine and Vaginal cleaning agent, or it is invalid using other drugs.

1.3 treatment method

80 patients of control group take metronidazole 400mg, 2 times a day, take orally 7 days, and 80 patients of observation group are using implementation The gelling agent of example, 1 time a day, each 3g, after patient cleans vulva, every night is pushed into 3g gel, two groups of patient's continuous uses 1 Clinical effectiveness is observed after week.

1.4 observation index

Curative effect is divided into three indexs, and A is cured: the clinical symptoms of patient disappear, and testing result is feminine gender；B is effective: patient's Clinical symptoms are substantially achieved improvement, and testing result is feminine gender；C is invalid: the clinical symptoms of patient do not improve or aggravation, There is the phenomenon that recurrence after treatment 3 months.Total effective rate=(healing+effective)/total case × 100%

1.5 statistical method

Statistical software SPSS16.0 handles data, and the explanation of p < 0.05 has significant difference.

2 results

2.1 two groups of patient outcomes are comprehensively compared

Observe 80 patients after the treatment, treatment total effective rate is apparently higher than control group, and the treatment for comparing two groups of patients is total Effective percentage has significant difference, and P < 0.05 is statistically significant, and bacterial vaginitis is detailed in the following table 16, and cervicitis is shown in Table 17

16 vaginitis of table, two groups of patient medication's effect comparative analysis (%)

Two groups efficient (healing+improvement) are compared through statistics.P < 0.05, difference are statistically significant

17 cervicitis of table, two groups of patient medication's effect comparative analysis (%)

Group	Number of cases	It cures	Effectively	In vain	Total effective rate
						Observation group	80	60 (75%)	14 (17.5%)	6 (7.5%)	92.5%
Control group	80	52 (65%)	16 (20%)	12 (15%)	88%
						P	-	-	<0.05	<0.05

The recurrence rate of 2.2 two groups of patients compares

For 80 patients of observation group after treatment, clinical recurrence rate is substantially less than control group, two groups of Patients on Recurrence rate differences Significant P < 0.05, it is statistically significant.

18 two groups of vaginitis Patients on Recurrence rates of table compare (%)

Group	Number of cases	Recurrence number	Recurrence rate
				Observation group	80	2	2.5%
Control group	80	12	15%
				P	-	<0.05	<0.05

19 two groups of virus in patients with cervicitis recurrence rates of table compare (%)

Group	Number of cases	Recurrence number	Recurrence rate
				Observation group	80	4	5%
Control group	80	15	18.8%
				P	-	<0.05	<0.05

By table 18, table 19 it can be seen that probiotics and Chinese medicine composition in the microbial vaginitis for the treatment of Different Kinds of Pathogens and In terms of cervicitis compared with metronidazole, cure rate is high, and recurrence rate is low.

(2) adnexitis, pelvic inflammatory disease and pelvic hydrops

Adnexitis 8: underbelly falling inflation pain, tenderness is pressed in left side, with diseases such as leucorrhea increasing peculiar smell, Low Back Pain, menstrual disorder Shape.After one course for the treatment of of gel made of embodiment 5, many canescence and purulent secretion, pain relief, peculiar smell are excluded It disappears；After product 2 weeks of invention, discharge has dark brown clot, and pain disappears.Recheck 6 recoveries from illness.

Pelvic inflammatory disease simultaneously has pelvic hydrops 12: hospital checks with pelvic inflammatory disease and with pelvic hydrops, lower abdominal pain And have a fever, abdominal compression pain, lumbosacral region is ached, behind made of embodiment 5 gel 2 days, start be discharged purulent secretion and Water sample object, after 6 days, secretion increases, and no longer has a fever, and after 12 days, pain in the loins mitigates, after secondary month reuses 12 days, pain It disappears, makes a definite diagnosis recovery from illness 9.

3. conclusion

In this experiment, with Chinese medicine come heat-clearing toxin expelling, the effects of anti-inflammatory clearing damp, antipruritic dispelling abnormal flavor, and probiotics is used While inhibiting pathogenic bacteria, and it can be colonized in flourish in genital tract, the objects such as metabolite lactic acid and hydrogen peroxide Mass-energy keeps genital tract normal acidic environment, inhibits and eliminate the growth of pathogenic bacteria.To adjust and restore micro- life of genital tract State balance.Therefore, there is good therapeutic effect to the disease in terms of gynaecological imflammation, and recurrence rate is low.

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

SEQUENCE LISTING

<110>Tai'an legendary god of farming pharmaceutical Co. Ltd generally

<120>the format lactobacillus with bacteriostasis and Chinese medicine compound prescription and its application in treatment gynaecological imflammation

<130> 2017

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1442

<212> DNA

<213>bacterial strain C2-0121

<400> 1

gcaagtcgag cgagcttgcc tagatgaatt tggtgcttgc accaaatgaa actagataca 60

agcgagcggc ggacgggtga gtaacacgtg ggtaacctgc ccaagagact gggataacac 120

ctggaaacag atgctaatac cggataacaa cactagacgc atgtctagag tttaaaagat 180

ggttctgcta tcactcttgg atggacctgc ggtgcattag ctagttggta aggtaacggc 240

ttaccaaggc aatgatgcat agccgagttg agagactgat cggccacatt gggactgaga 300

cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg acgcaagtct 360

gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc gtaaagctct gttggtagtg 420

aagaaagata gaggtagtaa ctggccttta tttgacggta attacttaga aagtcacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttattgg 540

gcgtaaagcg agtgcaggcg gttcaataag tctgatgtga aagccttcgg ctcaaccgga 600

gaattgcatc agaaactgtt gaacttgagt gcagaagagg agagtggaac tccatgtgta 660

gcggtggaat gcgtagatat atggaagaac accagtggcg aaggcggctc tctggtctgc 720

aactgacgct gaggctcgaa agcatgggta gcgaacagga ttagataccc tggtagtcca 780

tgccgtaaac gatgagtgct aagtgttggg aggtttccgc ctctcagtgc tgcagctaac 840

gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tccagtgcaa acctaagaga ttaggtgttc ccttcgggga cgctgagaca 1020

ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gtcattagtt gccatcatta agttgggcac tctaatgaga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa gtcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatggacggt acaacgagaa gcgaacctgc gaaggcaagc ggatctctga 1260

aagccgttct cagttcggac tgtaggctgc aactcgccta cacgaagctg gaatcgctag 1320

taatcgcgga tcagcacgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccatgag agtctgtaac acccaaagcc ggtgggataa cctttatagg agtcagccgt 1440

ct 1442

Claims (7)

1. one plant of format lactobacillus with bacteriostasis, is format lactobacillus (Lactobacillusgasseri) DFNXC2-0121 is preserved in China typical culture collection center, address on March 31st, 2017 are as follows: Wuhan, China, it is military Chinese university, deposit number are CCTCC NO:M 2017162.

2. a kind of microbial inoculum, active constituent is the tunning of format lactobacillus described in claim 1；

The preparation method of the microbial inoculum, which comprises the steps of: ferment format cream bar described in claim 1 Bacterium obtains tunning, the condition of fermentation are as follows: and 37 DEG C, stationary culture 18-24h.

3. the production that format lactobacillus described in claim 1 and/or microbial inoculum as claimed in claim 2 treat gynaecological imflammation in preparation Application in product.

4. a kind of probiotics metabolin composition, which is characterized in that the probiotics metabolin composition includes: claim The fermentation culture medium of format lactobacillus and deposit number described in 1 are the fermentation of the lactobacillus plantarum of CCTCC NO:M 2010150 Culture.

5. probiotics metabolin composition as claimed in claim 4, which is characterized in that the fermented and cultured of the format lactobacillus Viable count in object is 1.0 × 10⁸-10.0×10⁸cfu/mL；The deposit number is the plant of CCTCC NO:M 2010150 Viable count in the fermentation culture medium of lactobacillus is 8.0 × 10⁸-20.0×10⁸cfu/mL。

6. application of the probiotics metabolin composition described in claim 4 or 5 in the product of preparation treatment gynaecological imflammation.

7. a kind of Traditional Chinese medicine probiotic composition for treating gynaecological imflammation, which is characterized in that be made of the component of following parts by weight: benefit 30-40 parts of raw bacterium metabolin composition, 40-55 parts of Chinese medical extract；The Chinese medical extract is by the raw material of following parts by weight Medicine is made: 32-40 parts of radix scutellariae, 25-30 parts of kuh-seng, 6-10 parts of Cortex Phellodendri, 4-10 parts of the fruit of summer cypress, 4-10 parts of mulberry leaf；

The preparation method of the Traditional Chinese medicine probiotic composition, which is characterized in that steps are as follows:

(1) preparation of probiotics metabolin composition: it is by format lactobacillus described in claim 1 and deposit number respectively The lactobacillus plantarum of CCTCC M 2010150 is activated, and the strain inoculated after activation is cultivated in fermentation medium, Cell concentration to format lactobacillus ferment culture reaches 1.0 × 10⁸-10.0×10⁸Cfu/mL, deposit number CCTCC The cell concentration of the lactobacillus plantarum fermentation culture medium of NO:M2010150 reaches 8.0 × 10⁸-20.0×10⁸Cfu/mL, by format The lactobacillus plantarum fermentation culture medium that lactobacillus ferment culture and deposit number are CCTCC NO:M 2010150 mixes, standby With；

(2) preparation of Chinese medical extract: radix scutellariae, kuh-seng, Cortex Phellodendri, the fruit of summer cypress and mulberry leaf being mixed, are added water to cook, decoction liquor concentration It is 1.10-1.20g/mL to density, centrifugation takes supernatant, spare；

(3) probiotics is metabolized composition and Chinese medical extract is uniformly mixed, that is, Traditional Chinese medicine probiotic composition is prepared.