CN106957811A - Application of the form lactobacillus with bacteriostasis with Chinese medicine compound prescription and its in treatment gynaecological imflammation - Google Patents

Abstract

The invention discloses the form lactobacillus that one plant has bacteriostasis, it is form lactobacillus (Lactobacillusgasseri) DFNXC2 0121, is preserved in China typical culture collection center on March 31st, 2017, address is：Wuchang, wuhan Luo Jia Shan, its deposit number is CCTCC NO:M 2017162.The invention also discloses the Traditional Chinese medicine probiotic composition by the form lactobacillus and Chinese medical extract preparation.The present invention is used as guidance using Tiny ecosystem therapy, separated from normal health vagina, purifying obtains one plant of form lactobacillus, and it is enlarged cultivation and fermentation together with another lactobacillus plantarum, compounded using its metabolite with preferred Chinese herbal medicine extract, product that is nontoxic, having no side effect, safely and efficiently treat gynaecological imflammation is obtained so as to develop.

Description

Form 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 have bacteriostasis form lactobacillus and its treatment gynaecological imflammation in application, especially It is the application for being related to the form lactobacillus in the medicine for preparing treatment vaginitis, belongs to biomedicine technical field.

Background technology

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) issue As shown by data, the women of developing country more than 90% suffers from gynaecological imflammation, and Chinese women has 70% with different degrees of RTI, due to medical resource, economic condition, cultural knowledge imbalance so that Rural Married Women gynaecological imflammation send out Sick rate is more up to more than 95%；More than 85% women at least suffers from two or more genital diseases.According to statistics, 2004 it is complete 14, state, 62, the whole nation in 2005, the stream of national 81 gynaecology of hospital and family planning outpatient service people more than 50,000 in 2006, which is raised wages, expects aobvious Show, gynecological infection disease (vaginitis, pelvic infecton, cervicitis) accounts for 55 percent in all diseases of women, thus produces Medical expense every year up to more than 200 hundred million yuan.Estimate accordingly, annual at least 200,000,000 women of China suffer from RTI and correlation Disease, the whole world ten hundreds of women annoying by this vaginopathy always.

The characteristics of RTI has incidence of disease height, high recurrence rate, wide Epidemic Scope, the body and mind for having a strong impact on women is good for 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 up infection, cause cervicitis, endometritis, pelvic infecton etc., cause it is infertile not Educate, ectopic pregnancy, pregnant woman causes miscarriage, stillborn foetus, stillbirth, congenital infection and infection of newborn etc., can make AIDS (AIDS) and The occurrence risk increase of cervical carcinoma, has a strong impact on the physical and mental health of women, influence family's wellbeing and society it is stable.

At present, the measure for the treatment of vagina infection, which is mainly, uses antibiotic, and antibiotic is that certain micro-organisms is anti-by enzymatic Metabolite should be changed into structural secondary metabolite.The irrational a large amount of uses of antibiotic, abuse, are added scarce Weary strict supervision, causes the generation of dead, drug-resistant microorganism and super antibody-resistant bacterium, and the misuse of antibiotic also puts patient in not In necessary resistance risk；Gynemetrics of Peking University First Hospital Liao Qinping professor Chinese women microecology in vaginas study into Said in Zhan Yiwen, intravaginal normal flora is the core content of microecology in vaginas research, and early in 1892, Dederlein was first The research on human vagina Tiny ecosystem flora is delivered, Dederlein thinks vagina microorganism only Gram-positive Bacillus is constituted, i.e. lactobacillus.With deepening continuously for studying vaginal flora, intravaginal exists a variety of under discovery normal condition Microorganism, wherein, most important, quantity at most be lactobacillus, separation rate is up to 50%- in the vaginal fluid of healthy women 80%, have determined that the microorganism that is colonized in normal vagina is main by bacterium, fungi, protozoon, virus, mycoplasma etc. now, this The balance that the presence of a little floras maintains ecology is not fallen ill.In the vagina ecological balance is maintained, to produce hydrogen peroxide (H₂O₂) Lactobacillus be dominant bacteria, it can produce a variety of antibacterial substances, such as lactic acid, stearic acid, hydrogen peroxide acidifying vaginal environment, Reduce vagina pH, suppress the amount reproduction of pathogen by adhering to the modes such as occupy-place protection, stimulating immune system, maintain vagina The ecological balance in play very important effect.The vagina ecological balance is once broken or foreign pathogens intrusion, you can led Cause inflammation.

During traditional antibiotic therapy, antibiotic also can be the dominant bacteria in vagina while pathogenic bacteria are killed Kill, vagina is in few bacterium or germ-free condition, the use plus antibiotic causes body immunity degradation again, once run into cause of disease Bacterium is easy to cause infection, suprainfection.Now, the side effect of antibiotic is cured, medicine circle scientific worker approves, country Progressively limitation is used.

Vagina infection is treated in addition to oral antibiotic systemic administration, also the dialectical of traditional Chinese medicine treats, and the traditional Chinese medical science passes through Benefiting qi and nourishing blood, dispersing stagnated hepatoqi, sterilization carry out systemic administration by the stasis of blood, clearing damp heat, degrading the channel etc., but effect is slow, it is difficult to stand Itch makes one to feel restless, it is difficult to adhere to taking Chinese medicine antipruritic.Externally applied drug at present on the market is also a feast for the eyes, suppository, glue Capsule, acidifying lotion, gel, Chinese herbal fumigation and washing, effervescent tablet etc., many effect of drugs are not good according to investigations, are mainly used for antimicrobial The auxiliary treatment of thing, it is impossible to effect a radical cure, easily repeatedly, particularly VVC (VVC), recurrent beads vagina Sick (recurrent vulvovaginal candidiasis, RVVC), treatment course length is easily repeatedly.

To sum up, the effect of antibiotic, traditional Chinese medicine and various externally applied drugs treatment vagina infection is undesirable, and it has its source in Do not recover the balance of microecology in vaginas, but because 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.

The content of the invention

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

The present invention is separated, purifying obtains one plant of form using Tiny ecosystem therapy as guidance from normal health vagina Lactobacillus, and cultivation and fermentation is enlarged together with another lactobacillus plantarum, utilize its metabolite and preferred Chinese herbal medicine Extract is compounded, and product that is nontoxic, having no side effect, safely and efficiently treat gynaecological imflammation is obtained so as to develop.

To achieve the above object, the present invention uses following technical proposals：

The first aspect of the present invention is there is provided the form lactobacillus that one plant has bacteriostasis, and it is form lactobacillus (Lactobacillusgasseri) DFNXC2-0121, is preserved in China typical culture collection on March 31st, 2017 The heart (abbreviation CCTCC), address is：Wuhan, China, Wuhan University, its deposit number is CCTCC NO:M 2017162.

The bacterial strain is isolated from normal health vagina, and the physiological and biochemical property of bacterial strain is：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 is there is provided a kind of microbial inoculum, and its active component is the tunning of above-mentioned form lactobacillus.

The present invention also provides the preparation method of above-mentioned microbial inoculum, comprises the following steps：The above-mentioned form lactobacillus of fermentation, is obtained Tunning.

In above-mentioned preparation method, ferment the fermentation medium that uses for：MRS culture mediums.

In above-mentioned preparation method, the condition of fermentation is：37 DEG C, quiescent culture 18-24h.

The third aspect of the present invention is preparing the product for the treatment of gynaecological imflammation there is provided above-mentioned form lactobacillus and/or microbial inoculum In application.

There is provided a kind of probiotics metabolome compound, described probiotics metabolome compound for the fourth aspect of the present invention Comprising：The fermentation culture medium and deposit number of above-mentioned form lactobacillus are CCTCC NO:M 2010150 Lactobacillus plantarum Fermentation culture medium.

Viable count in the fermentation culture medium of the form lactobacillus is 1.0 × 10⁸-10.0×10⁸cfu/mL；It is described to protect It is CCTCC NO to hide numbering:Viable count in the fermentation culture medium of M 2010150 Lactobacillus plantarum is 8.0 × 10⁸-20.0× 10⁸cfu/mL。

The fermentation culture medium and the deposit number of the form lactobacillus are CCTCC NO:M 2010150 plant breast The fermentation culture medium of bacillus can be with any than mixing；As preferred scheme, the fermentation culture medium of the form lactobacillus and The deposit number is CCTCC NO:The fermentation culture medium of M 2010150 Lactobacillus plantarum is with volume ratio 1：1 is mixed.

Further, it can also be included in above-mentioned probiotics metabolome compound：The groups such as Carbomer, glycerine and peppermint oil Point, and the various pharmaceutical dosage forms such as gel are made in conventional manner.

Application of the above-mentioned probiotics metabolome compound in the product for preparing treatment gynaecological imflammation is also the guarantor of the present invention Protect scope.Found through experiment, probiotics metabolome compound of the invention is obvious for causing the pathogenic bacteria of vaginitis to have Inhibitory action, especially for the fungistatic effect for staphylococcus aureus, Candida albicans and the Escherichia coli for causing vaginitis It is more notable.

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

There is provided a kind of Traditional Chinese medicine probiotic composition for treating gynaecological imflammation for the fifth aspect of the present invention.

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

30-40 parts of probiotics metabolome compound, 40-55 parts of Chinese medical extract；

The Chinese medical extract is made up of the bulk drug of following parts by weight：

32-40 parts of the root of large-flowered skullcap, 25-30 parts of kuh-seng, 6-10 parts of golden cypress, 4-10 parts of the fruit of summer cypress, 4-10 parts of mulberry leaf.

The sixth aspect of the present invention is there is provided the preparation method of above-mentioned Traditional Chinese medicine probiotic composition, and step is as follows：

(1) preparation of probiotics metabolome compound：It is respectively CCTCC by the form lactobacillus and deposit number of the present invention NO:M 2010150 Lactobacillus plantarum is activated, and the inoculation after activation is cultivated in fermentation medium, extremely The cell concentration of form lactobacillus ferment culture reaches 1.0 × 10⁸-10.0×10⁸Cfu/mL, deposit number is CCTCC NO: The cell concentration of M 2010150 Lactobacillus plantarum fermentation culture medium reaches 8.0 × 10⁸-20.0×10⁸Cfu/mL, by form breast Bacillus fermentation culture and deposit number mix for CCTCC M 2010150 Lactobacillus plantarum fermentation culture medium, standby；

(2) preparation of Chinese medical extract：The root of large-flowered skullcap, kuh-seng, golden cypress, the fruit of summer cypress and mulberry leaf are mixed, added water to cook, decoction liquor Density is concentrated into for 1.10-1.20g/mL, centrifugation takes supernatant, standby；

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

In step (1), the fermentation medium is：MRS culture mediums；Condition of culture is：37 DEG C, quiescent culture.

In step (2), the specific method added water to cook is：The root of large-flowered skullcap, kuh-seng, golden cypress, the fruit of summer cypress and mulberry leaf are mixed, are placed in 40-60min is soaked in 10 times of water, is decocted, water continues to decoct 50-60min after opening, and is filtrated to get the first filtrate；The dregs of a decoction are with 8 times of water Secondary decoction is carried out, water continues to decoct 30-40min after opening, is filtrated to get the second filtrate and is well mixed with the first filtrate, by true Sky is concentrated under reduced pressure herb liquid, and the density to decoction is 1.10-1.20g/mL, 3000 turns/min centrifugation 10min, takes supernatant standby.

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, including：Functional food, health products, disinfectant and medicine are (such as：Lotion, gel and suppository etc.).

Beneficial effects of the present invention：

(1) present invention is separated from the vagina of healthy women first, purifying obtains one plant of form lactobacillus, and the bacterial strain has Production acid is high, production Substance, the characteristics of bacteriostasis is strong.Acidic materials such as lactic acid, formic acid can be produced after strain fermentation And stearic acid etc., these acidic materials can reduce the pH value of genital tract, suppress the growth of pathogen；Peroxidating can also be produced Hydrogen, further suppresses the growth of other bacterium and pathogenic microorganisms.

(2) it by the form lactobacillus and deposit number of the present invention is CCTCC that probiotics metabolome compound of the invention, which is, The fermentation culture medium compounding of M 2010150 Lactobacillus plantarum is formed, wherein, deposit number is CCTCC M 2010150 plant Thing lactobacillus is documented in patent " one plant of lactic acid bacteria and its application available for biological preservation " (CN101914475A), should The original effect of lactic acid bacteria is preventing and treating pericarp browning, reduces the fruit incidence of disease.The present invention joins the lactic acid bacteria and form lactobacillus Close, it is found that this two plants of bacterium are used cooperatively with significant synergistic function, have for the pathogenic bacteria for causing vaginitis obvious Inhibitory action, especially for the antibacterial effect for staphylococcus aureus, Candida albicans and the Escherichia coli for causing vaginitis Fruit is more notable.

(3) Traditional Chinese medicine probiotic preparation of the invention is through scientific compatibility by probiotics metabolome compound and Chinese medical extract Form, realize the perfect adaptation of probiotics and Chinese medical extract, verified by antibacterial experiment in vitro, Chinese medicine of the invention is prebiotic Safely and effectively, the probiotic products simple compared with the existing product of existing market or simple tcm product have antimicrobial spectrum to bacteria preparation Extensively, antipruritic effect of ejecting poison is more obvious.Wherein：Chinese medical extract has obvious suppression staphylococcus aureus, Candida albicans Bacterium act on, and heat-clearing and damp-drying drug, repellent antipruritic effect；Probiotics can suppress the effect of Escherichia coli, staphylococcus aureus, And the pH of vagina can be acidified, be conducive to the increment and vaginal mucosa reparation of lactobacillus, the combination of Chinese medicine and probiotics has The therapeutic action of the Chinese medicine effect that the regulation microecology in vaginas with probiotics is balanced again, and antimicrobial spectrum is expanded, the two has Synergy.Experiment proves that, effect is better than any single raw material.

Brief description of the drawings

Fig. 1：Amino decarboxylase testing result；Wherein, Figure 1A is histidine decarboxylase Activity determination result, Tu1BWei Tyrosine decarboxylation Enzyme assay result, Fig. 1 C are ornithine decarboxylase activity testing result, and Fig. 1 D live for lysine decarboxylase Property testing result.

Fig. 2：The selection result of advantage lactobacillus.

Fig. 3：Bacterial strain C2-0121 phylogenetic tree.

Fig. 4：Bacterial strain C2-0121 Fermentative growth curves.

Fig. 5：Test group vagina tissue picture and HE stained slice figures；Wherein, Fig. 5 a are negative control group, and Fig. 5 b are 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 are normal group vaginal fluid microscopy picture, and Fig. 7 b are perfusion Vaginal fluid microscopy picture after antibiotic, Fig. 7 c are the microscopy picture attacked after poison.

Fig. 8：The changes in diet trend of rabbit after treating 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 are natural recovering group, and Figure 10 b are mixed Treatment group is closed, Figure 10 c are positive controls, and Figure 10 d are Chinese medicine group, and Figure 10 e are probiotic group.

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

Embodiment

It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

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

In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool The embodiment of body describes the technical scheme of the application in detail with comparative example.

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 mediums during strain isolation), gelatin liquefaction culture medium is (often The commercially available prod of rule).

H₂O₂Identification of M RS culture mediums：Tetramethyl benzidine comprising 0.25mg/mL, 0.lmg/mL in MRS agar mediums Horseradish peroxidase.

The culture medium of lactobacillus qualification test：The bacterium micro biochemical that Hangzhou day and microorganism reagent Co., Ltd produce 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 primers.

2. test method

The collection of 2.1 vaginal fluids：Doctor assists sampling at the physical examination of 88 gynaecology of hospital of Tai'an, with sterile cotton swab in the moon Disposably scrape and smear 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 mediums, and number record.

2.2 bacterial strains isolating and purifying and identify

Each separation source is placed in sterilized water (band bead) and shaken, sample is fully broken up to mixing, vibration liquid is taken successively It is diluted to 10^-2, 10^-3, 10^-4, 10^-5, each dilution factor takes 0.1mL to apply flat board (MRS+CaCO₃), 2 repetitions, in 37 DEG C of incubators Culture.The bacterium colony with obvious calcium solusphere is in inclined-plane, Gram's staining, microscopy on picking culture medium, until it is defined as pure bacterium, will Lactobacillus strain after purification is inoculated into after MRS medium cultures, is put in 4 DEG C of refrigerators and is saved backup.

2.2.1 the measure and mitotic stability of product acid activity

The strain of separation is inoculated in the MRS culture mediums of improvement with 2% inoculum concentration, 37 DEG C of quiescent cultures, determines fermentation The pH value of liquid.

Continuously its stability and the pH value of zymotic fluid are seen in switching for 10 days to bacterial strain.

2.2.2 the Preliminary Identification of lactobacillus

Every Gram-positive, H₂O₂The negative bacterial strain of enzyme test can be primarily determined that 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 experiment, hydrogen sulfide production test are belonged to doubtful bacterial strain.

2.2.4 the identification of lactobacillus species

The identification that acid experiment is planted is produced by carbohydrate fermentation.The culture medium of various carbohydrate fermentation experiments Use bacterium micro biochemical reaction tube.

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

2.3.1 acid resistance test

Hydrochloric acid regulation MRS culture mediums make its pH value be respectively 1.5,2.5,3.5, respectively the good zymotic fluid of inoculated and cultured, 1% Inoculum concentration, 37 DEG C culture.Take culture 0h and 4h bacterium solution plate count.Calculate survival rate (%).

2.3.2 bile tolerance is tested

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

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 is detected

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 culture 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, afterwards will Culture medium be exposed to air in, produce hydrogen peroxide bacterium colony can in be changed into 20min blueness.

2.4 16S rDNA Molecular Identifications

Pcr amplification primer thing：1492r 5’-ggttaccttgttacgactt-3’；27f 5’- agagttgatcctggctcag-3’。

Bacterium colony PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 1min, 52 DEG C of annealing 1min, 72 DEG C of extensions 2min, 25 circulations, 72 DEG C of extension 10min.

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

The measure of 2.5 growth curves

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

3. result and analysis

3.1 isolate and purify the morphological feature of each bacterial strain

Multiple picking single bacterium colony, the bacterial strain isolated and purified are subjected to Gram's staining, the colonial morphology of each strain bacterium is all breast White, circular, piece shape, select gram-positive, thalli morphology by Gram's staining and are shaft-like bacterial strain, mark 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 for determining 20h zymotic fluids) of the product acid activity of table 1

Found out by table 1：C2-0109 and C2-0121 and other Strain comparisons, the pH of its zymotic fluid are relatively low, and both less than 4, say The bright more other bacterial strains of its acid producing ability are strong.

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

Through continuous subculture and contrast test, the physics and chemistry shape such as taxology shape, morphological feature of the bacterial strain 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, and other are shown in Table 3

The hydrogen sulfide experimental result of table 3

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

3.3 amino decarboxylases are detected

Detect that the four kinds of amino acid used in experiment are tyrosine, histidine, ornithine and lysine in amino decarboxylase. Form tyrasamine, histamine, putrescine and cadaverine respectively after deamination, be the primary biological amine substance for causing poisoning symptom.According to Amino decarboxylase culture Preliminary detection result is understood：Surveyed bacterial strain does not produce 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, it is respectively from left to right：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- 0109th, H5-2, FNG5-4), ornithine decarboxylase activity testing result (is respectively as shown in Figure 1 C from left to right sky in Fig. 1 C In vain, 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 research and the screening of advantage lactobacillus of 3.4 bacterial strains

3.4.1 acid resistance test result

Hydrochloric acid regulation MRS culture mediums make its pH value be respectively 1.5,2.5,3.5, respectively the good zymotic fluid of inoculated and cultured, 1% Inoculum concentration, 37 DEG C culture.Culture 0h and 4h bacterium solution plate count is taken, survival rate (%) is calculated and is shown in Table 4.

Table 4 places 4h survival rate (%) 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 is not all tolerated in the environment of PH 1.5, and bacterial strain C2-0121 is resistant in pH3.5 environment, Survival rate is 93.5%, in addition to less than bacterial strain C2-0109, higher than other bacterial strains.

3.4.2 bile tolerance is tested

Pig gall hydrochlorate is added in MRS culture mediums, it is respectively 0.1%, 0.2%, 0.3% to make its mass fraction, bacterium solution is pressed 2% inoculum concentration inoculation, 37 DEG C of culture 0h and 4h bacterium solution plate count calculates survival rate (%), it the results are shown in Table 5.

Table 5 places 4h survival rate (%) in various concentrations cholate condition

Found out by table 5：Bacterial strain C2-0109, C2-0121, C2-0071FNG-3 4h in gallbladder salinity is 0.1% solution After can survive more than 85%, the ability of wherein C2-0109, C2-0121 bile tolerance is stronger, 37 in 0.1% cholate solution DEG C culture 4h its survival rate is more 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 detection of bacterium, and compared with the antibacterial relatively good bacterial strains of form lactobacillus C2-0071 that are preserved in laboratory Compared with the results are shown in Table 6.

Biocidal property result (the unit of 6 each plants of lactobacillus of table：mm)

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 Preferably, the biocidal property than other several plants of bacterium is all good by C2-0121, the form lactobacillus C2-0071 preserved than laboratory inhibition zone It is all big, it is big by 14.3% to the biocidal property difference of staphylococcus aureus, Escherichia coli, salmonella, proteus, 59.6%, 21.7%th, 7.4%.Two plants of bacterium C2-0109, C2-0121 that better performances are selected in following experiment enter the reality for a step of passing through Test.

3.4.4 the sensitization test of antibiotic

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

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

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 result of the test

The identification that acid experiment is planted is produced by carbohydrate fermentation.The culture medium of various carbohydrate fermentation experiments Bacterium micro biochemical reaction tube is used, fermentation results are shown in Table 8.

The sugared fermenting experiment result of table 8

Result of the test shows that C2-0121 is consistent with the biochemical characteristic of form lactobacillus, the primary Jie Shi handbooks of foundation, tentatively It is accredited as form lactobacillus.

3.4.6 the screening of advantage lactobacillus

By the screening of advantage lactobacillus, wherein C2-0121 bacterium colonies are changed into blueness, as a result see 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 produce lactic acid, reduce genital tract pH value, suppress pathogen growth；Separately Outside, lactobacillus can also produce hydrogen peroxide, and bacteriocin and class bacteriocin and biosurfactant etc. suppress other bacterium and cause The growth of sick microorganism, so as to maintain the balance of microecology in vaginas；Experiment is found out more than：Bacterial strain C2-0121 produces peroxidating Hydrogen, acid producing ability are strong and biocidal property is all better than other bacterial strains, therefore, and this plant of bacterium is for the genital tract of women infected with very Good effect, to safeguarding that microecology in vaginas balance has facilitation.

3.5 Molecular Identification results

To determine that C2-0121 is the species of lactic acid bacteria, molecular biology identification is further done, bacterial strain C2-0121 has been entered Row liquid fermentation, extracts DNA, PCR amplifications, and 16S rDNA sequencing 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 the strains of Lactobacillus hominis CRBIP 24.179FR68 1902 have up to 99% homology, Combining form, is accredited as form lactobacillus (Lactobacillusgasseri), is named as form lactobacillus (Lactobacillusgasseri) DFNXC2-0121, and be preserved on March 31st, 2017 in China typical culture collection The heart (abbreviation CCTCC), address is：Wuhan, China, Wuhan University, its deposit number is CCTCC NO:M 2017162.

The drafting of 3.6 strain fermentation growth curves

Separately sampled in different time sections after inoculation, detection determines its OD value and pH at wavelength 600nm, to cultivate Time is abscissa, and corresponding OD values are ordinate with pH value, draws growth curve and sees Fig. 4.

Found out by Fig. 4：Bacterial strain C2-0121 enters exponential phase in 8h or so, and now strain growth is most fast, now under pH Drop also most drops to 5.3 by 6.5 soon, and during to 16h, OD600 reaches maximum, and now pH is 3.99, and this is learnt by plate count When viable count up to 13.5 × 10⁸Enter stationary phase after cfu/mL, 16h.

Embodiment 2：The preparation of probiotics metabolome compound

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

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

Described MRS culture mediums：Glucose 2.0%, ammonium citrate 0.2%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.5%th, 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 is：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 the root of large-flowered skullcap, 30 parts of kuh-seng, 8 parts of golden cypress, 6 parts of the fruit of summer cypress, 8 parts of mixing of mulberry leaf, mixture is placed in 10 times of water Middle immersion 40-60min, is decocted, and water continues to decoct 50-60min after opening, and is filtrated to get the first filtrate；The dregs of a decoction carry out two with 8 times of water Secondary to decoct, water continues to decoct 30-40min after opening, and is filtrated to get the second filtrate and is well mixed with the first filtrate, is subtracted by vacuum Press Rotary Evaporators concentrated traditional Chinese medicine liquid, to herb liquid density be 1.1g/mL-1.2g/mL, left undisturbed overnight, 3000 leave the heart 10min, collects supernatant, standby.

Embodiment 4：The preparation of Traditional Chinese medicine probiotic preparation

45 parts of Chinese medical extract prepared by 40 parts of probiotics metabolome compound and embodiment 3 prepared by embodiment 2 is mixed Close uniform, that is, prepare Traditional Chinese medicine probiotic preparation.

Embodiment 5：Treat the preparation of the gel of vaginitis

The 2.5g of Carbomer 942 is weighed, the Traditional Chinese medicine probiotic preparation 850mL of the preparation of embodiment 4 is measured, is added to and fills card In the container of ripple nurse, overnight, stir evenly, until it is fine and smooth uniform without particle, adjust pH value with mass concentration for 10% NaOH To 5.0, glycerine 5g and peppermint oil 0.2g is added, 1000g is added water to and stirs evenly, produce.

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), the newborn bar of form Bacterium DFNXC2-0121 (screening of embodiment 1 is obtained).

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 commercially available prod.

2. method and result：

Odontothrips loti

Take bacterial strain Lactobacillus plantarum BLCC2-0001, form lactobacillus DFNXC2-0121 zymotic fluid 10000rpm centrifugations 10min, takes supernatant, carries out biocidal property experiment；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.Result of the test is shown in Table 9.

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

Note：- represent without obvious inhibition zone.

Result of the test 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 examination 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 commercially available prod.

2. method and result

2.1 methods：Odontothrips loti

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

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 are sterilized 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 examination 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 commercially available prod.

2. method and result

2.1 methods：Odontothrips loti

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

Antibacterial result of the Traditional Chinese medicine probiotic preparation of table 11 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 lactobacillus is expanded still without biocidal property after illustrating mixing Antimicrobial spectrum, the two has concertedness, fungistatic effect more wide spectrum.

Application examples 4：Rabbit vagina irritant test (stimulates examination according to 2002 sterile products inspection technology specifications-vaginal mucosa Test)

1. experimental animal：From health, the Female white rabbit for the non-estrus just grown up, Belgian rabbit, body weight 2.5kg.Pretest inspection animal introitus whether there is secretion, hyperemia, oedema and other degree of impairments.If any inflammation or (and) damage Wound, should be abandoned.

2. experiment packet：It is divided into contamination group, negative control group and positive controls, every group 3.Wherein, contamination group is used Gel prepared by the embodiment of the present invention 5, negative control group pours into physiological saline, and positive controls pour into commercially available prod Jilin one The bacteriostatic gel of bio tech ltd of positive medicine company group

3. experimental working technique

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

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

The contamination method of more than 3.3 Tests For Irritating Effects：By above-mentioned 3.2 contamination method, contamination one is repeated every 24h It is secondary, continuous 5d.Negative control group animal physiological saline makees same processing, the positive control positive medicine company collection in commercially available prod Jilin one The bacteriostatic gel of bio tech ltd of group

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

24h after the contamination of 3.5 lasts, animal is put to death using aeroembolism method, is cut open the belly and is taken out complete vagina, longitudinally slit, naked eyes See whether that hyperemia, oedema etc. are showed, referred to during for pathologic sampling.Then vagina is put into 10% formalin solution solid Determine more than 24h, choose the tissue film-making at the two ends and central 3 positions of vagina, after HE dyeing, carry out histopathological examination.

4 evaluation of result

Histopathological examination result, scores by the fixed stimulate the reaction to vaginal mucosa of table 12.By test group, 3 are moved After the stimulate the reaction integration at 3 positions of thing is added, then divided by observation it is total (number of animals × 3), show that test group vaginal mucosa is pierced Swash the average integral of reaction, maximum is scored as 16 (being shown in Table 12).Control group methods of marking is ibid.Test group average integral is subtracted Control group average integral is drawn after stimulus index, and stimulus intensity classification is carried out by table 13.

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

The vaginal mucosa stimulate the reaction standards of grading of table 12

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

The vaginal mucosa stimulus intensity of table 13 is classified

4.2 each test group vagina tissue picture and HE stained slice figures

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

4.3 each test group vaginal mucosas stimulate scores to be shown in Table 14.

Each test group vaginal mucosa stimulus index of table 14

Result of the test shows：The vaginal mucosa stimulus index ＜ 1 of intervention group is tested, is classified according to vaginal mucosa stimulus intensity Standard understands that test group is nonirritant to rabbit vagina mucous membrane, by vagina tissue picture and HE stained slices it can also be seen that the moon Road is without congested, oedema and inflammatory cell, and vaginal mucosa is complete, acellular downright bad, obscission.

Application examples 5：Probiotics and Chinese medicine composition are to the scorching inhibitory action research of rabbit vagina

1. materials and methods

1.1 experiment materials

1.1.1 experimental animal Healthy female rabbit, body weight is 3.0kg, to produce more than 2 times, huge by Tran town fishpond village Property raiser provide, totally 30.

1.1.2 medicine 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 Ecologic biological Engineering Co., Ltd produces

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

1.2 experimental methods

Rabbit is first divided into two groups, A groups Normal group 5, B group models group 25 after raising 3 days in advance, takes vagina at random Secretion microscopy simultaneously counts Escherichia coli, staphylococcus aureus, enterococcus, lactic acid bacteria number.With No. 18 after the sterilization of A groups external genital Rat oral gavage device saline injection 1mL, after irrigating 11 days, gas fastens method and kills rabbit, takes the formaldehyde of vagina tissue 10% to fix system Piece, after HE dyeing, carries out pathological examination scoring, classification.B groups irrigate lincomycin hydrochloride injection 1mL, 1 time a day, perfusion 5 days, recover 1 day, then with Candida albicans viable count 1.5 × 10⁷Cfu/mL irrigates 0.5mL, 1 time a day, after 4 days, at random Take vaginal fluid microscopy and count Escherichia coli, staphylococcus aureus, enterococcus, Candida albicans and lactic acid bacteria number, really Determine after modeling success, model group rabbit gas fastens method and kills rabbit 5, take the formaldehyde of vagina tissue 10% to fix film-making, after HE dyeing, Carry out pathological examination and reference table scoring.Then rabbit is grouped again, is divided into 5 groups, B1 groups are natural recovering group, and B2 groups are mixed (Chinese medicine and probiotics) treatment group is closed, B3 groups are Chinese medicine group, and B4 groups are probiotic group, B5 group positive controls, every group 5, are opened Begin to treat.Only, B2 groups irrigate the gel 1.5g/ of embodiment 5 only to Blank gel 1.5g/ of the B1 groups perfusion without any medicine, B3 groups for herb liquid 1.5mL, the B4 embodiment 2 of embodiment 3 prebiotic bacterium solution 1.5mL, B5 groups one positive bacteriostatic gel 1.5g/, Feed relative is weighed during treatment daily, after treating 6 days, vaginal fluid microscopy is all taken and counts Escherichia coli, golden yellow Staphylococcus, enterococcus, Candida albicans and lactic acid bacteria number, gas fastens method and kills rabbit after 7 days, takes the formaldehyde of vagina tissue 10% to consolidate Piece is customized, after HE dyeing, pathological examination is carried out and scores, is classified with reference to table 4, table 5.

2 results and analysis

The sign change of each test group rabbit during 2.1 experiments

2.1.1 the sign of rabbit changes during modeling

After pre- feeding 5 days, model group rabbit is pure white smooth by hair, and diet is normal, and spirit is good.After antibiotic, spirit Depressed, appetite declines, and shape of crawling slightly is had loose bowels, and 3 death, Candida albicans is attacked after poison, spiritual depressed, do not feel like eating, crawls Shape, has 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 substantial amounts of bacterial strain in vagina, wherein using Bacillus acidi lactici to be main excellent, quantity is more；After using antibiotic, respectively Plant bacterium all largely to reduce, and attack after poison, the breeding of the pathogenic bacteria such as staphylococcus aureus, Escherichia coli and Candida albicans increases It is many, it is main bacterial strain, and Bacillus acidi lactici is reduced accordingly, this illustrates being successfully established for the scorching model of rabbit vagina.

Fig. 7 a are normal group vaginal fluid microscopy picture, it is seen that have substantial amounts of bacillus, and Fig. 7 b are cloudy after perfusion antibiotic Road secretion microscopy picture, Microflora is seldom；Fig. 7 c are the microscopy picture attacked after poison, there is substantial amounts of Candida albicans, drop The pathogenic bacteria such as worm, coccus, this also illustrates that model is successfully established.

Indices after 2.2 treatments

2.2.1 the sign change of each group rabbit during being treated after modeling：It the results are shown in Table 15.

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

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

Result of the test shows：First day treatment group's dietary amount is most, natural recovering group is minimum, still, prolonging over 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, mixing treatment group highest illustrates that mixing treatment group stimulates minimum to the stomach of rabbit.

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 of normal group Bacterium highest, is dominant bacteria；Lactic acid bacteria is significantly raised as dominant bacteria after the treatment of mixing treatment group, and the nothing of comparing with normal group shows Sex differernce is write, probiotic group, Chinese medicine group, positive controls are compared its lactobacillus quantity with natural recovering group and increased, still Compared the lactobacillus quantity of less than mixing group 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, Have the advantage status, recover to normal level, illustrate that composition has controlling for highly significant to the experimental rabbit with vaginitis Treatment is acted on.

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 very small amount bacillus, and mixing treatment group (Figure 10 b) has substantial amounts of bacillus, sun Property control group (Figure 10 c) has substantial amounts 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), particularly Candida albicans, and each group compares, and illustrates prepared by embodiment 4 Traditional Chinese medicine probiotic preparation mixed infection is caused vaginitis treatment have well suppress pathogen effect.

Vagina tissue is cut into slices 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 strata mucosa cells of mucous membrane are more clear than more complete, and nothing comes 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 strata is complete, and no ulcer surface, exuviation phenomenon illustrate mixing treatment group, Chinese medicine group, probiotic group and positive controls to the moon The scorching experimental rabbit in road has therapeutic action, and mixing treatment group is compared with Chinese medicine group, probiotic group, more than vaginal wall gauffer and Deep, mucous membrane upper strata is complete, illustrate probiotics and Chinese medicine composition than the experimentalist of single Chinese medicine or probiotics to vaginitis Rabbit therapeutic effect is notable.Mixing treatment group was both clearing heat and detoxicating, antibacterial including Chinese medicine, to staphylococcus aureus and Candida albicans The progress of bacterium suppresses, and adds probiotics, can supplement lactobacillus definite value and produce ablastins, organic acid and hydrogen peroxide etc. in vagina Suppress the growth of pathogen, the balance of microecology in vaginas can be promoted, the quick mucous membrane for repairing vagina, therefore, Chinese medicine with it is prebiotic Bacteria composition has good therapeutic action to the experimental rabbit of vaginitis.

3 conclusions

3.1 use lincomycin hydrochloride injection 1mL, daily perfusion 1 time, totally 5 days, recover 1 day, then use Candida albicans Viable count 1.5 × 10⁷Cfu/mL irrigates 0.5mL, 1 time a day, irrigates 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 preparations prepared with embodiment 4 are irrigated, and 1.5g/ only, after treating 6 days, passes through microscopy vagina point Flora in secretion, check vagina lesion situation and vagina tissue section it can be seen that：Model group vagina is seriously congested, Epithelial cell is imperfect, the phenomenon such as mucous layer lesion occurs, come off, and the Traditional Chinese medicine probiotic preparation prepared with embodiment 4 is irrigated Treatment group, positive controls and normal group vaginal epithelial tissue it is complete, mucous membrane is smooth, the phenomenon such as no lesion；It is more than summary Description of test：Probiotics metabolin and Chinese medicine composition are safe and non-stimulating, and its diet is not influenceed；Pathogenic bacteria to causing vaginitis There is good inhibiting effect, promote the flora in the experimental rabbit vagina with vaginitis to recover, and can quickly repair Impaired vaginal mucosa.

Application examples 6：Clinical trial

(1) vaginitis and cervicitis

1 material

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

1.2 tested crowds

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

1.3 treatment methods

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

1.4 observation index

Curative effect is divided into three indexs, and A is cured：The clinical symptom disappearance of patient, 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 of recurrence after treating 3 months.Total effective rate=(healing+effective)/total case × 100%

1.5 statistical methods

Statistical software SPSS16.0 processing datas, p<0.05 explanation has significant difference.

2 results

2.1 two groups of patient outcomes' Integrated comparatives

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

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

Two groups efficient (healings+improvement) compare through statistics.P<0.05, difference is statistically significant

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

Group	Number of cases	Cure	Effectively	It is invalid	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

80 patients of observation group are after treatment, and clinical recurrence rate is substantially less than control group, two groups of Patients on Recurrence rate differences Notable 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

It can be seen that by table 18, table 19：Probiotics and Chinese medicine composition in the treatment microbial vaginitis of Different Kinds of Pathogens and Compared in terms of cervicitis with metronidazole, cure rate is high, recurrence rate is low.

(2) adnexitis, pelvic infecton and pelvic hydrops

Adnexitis 8：Tenderness is pressed in underbelly falling inflation pain, 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 being made using embodiment 5, many canescence of exclusion and purulent secretion, pain relief, peculiar smell Disappear；After the product 2 weeks of invention, discharge carries dark brown clot, and pain disappears.Recheck 6 recoveries from illness.

Pelvic infecton simultaneously carries pelvic hydrops 12：Examination in hospital is with pelvic infecton and with pelvic hydrops, lower abdominal pain And have a fever, abdominal compression bitterly, lumbosacral region is ached, and the gel being made using embodiment 5 is after 2 days, start discharge purulent secretion and Water sample thing, after 6 days, secretion increase is no longer had a fever, and after 12 days, pain in the back mitigates, and reuses after 12 days within secondary month, pain Disappear, make a definite diagnosis recovery from illness 9.

3. conclusion

In this experiment, with Chinese medicine come heat-clearing toxin expelling, anti-inflammatory clearing damp, the effect such as antipruritic dispelling abnormal flavor, and use probiotics While pathogenic bacteria are suppressed, and flourish in genital tract, the thing such as its metabolite lactic acid and hydrogen peroxide can be colonized in Mass-energy keeps genital tract normal acidic environment, suppresses and eliminate the growth of pathogenic bacteria.So as to adjust and recover micro- life of genital tract State is balanced.Therefore, there is good therapeutic effect to the disease in terms of gynaecological imflammation, and recurrence rate is low.

The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair Change, equivalent, improvement etc., should be included within the protection domain of the application.

SEQUENCE LISTING

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

<120>Application of the form lactobacillus with bacteriostasis with Chinese medicine compound prescription and its 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 (10)

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

2. a kind of microbial inoculum, its active component is the tunning of the form lactobacillus described in claim 1.

3. the preparation method of the microbial inoculum described in claim 2, it is characterised in that comprise the following steps：Described in claim 1 of fermenting Form lactobacillus, obtain tunning.

4. preparation method as claimed in claim 3, it is characterised in that the condition of fermentation is：37 DEG C, quiescent culture 18-24h.

5. the microbial inoculum described in form lactobacillus and/or claim 2 described in claim 1 is preparing the production for the treatment of gynaecological imflammation Application in product.

6. a kind of probiotics metabolome compound, it is characterised in that described probiotics metabolome compound is included：Claim The fermentation culture medium and deposit number of form lactobacillus described in 1 are CCTCC NO:The fermentation of M 2010150 Lactobacillus plantarum Culture.

7. probiotics metabolome compound as claimed in claim 6, it is characterised in that the fermented and cultured of the form lactobacillus Viable count in thing is 1.0 × 10⁸-10.0×10⁸cfu/mL；The deposit number is CCTCC NO:M 2010150 plant Viable count in the fermentation culture medium of lactobacillus is 8.0 × 10⁸-20.0×10⁸cfu/mL。

8. application of the probiotics metabolome compound in the product for preparing treatment gynaecological imflammation described in claim 6 or 7.

9. a kind of Traditional Chinese medicine probiotic composition for treating gynaecological imflammation, it is characterised in that be made up of the component of following parts by weight：

30-40 parts of probiotics metabolome compound, 40-55 parts of Chinese medical extract；

The Chinese medical extract is made up of the bulk drug of following parts by weight：

32-40 parts of the root of large-flowered skullcap, 25-30 parts of kuh-seng, 6-10 parts of golden cypress, 4-10 parts of the fruit of summer cypress, 4-10 parts of mulberry leaf.

10. the preparation method of the Traditional Chinese medicine probiotic composition described in claim 9, it is characterised in that step is as follows：

(1) preparation of probiotics metabolome compound：It is by the form lactobacillus and deposit number described in claim 1 respectively CCTCC M 2010150 Lactobacillus plantarum is activated, and the inoculation after activation is cultivated in fermentation medium, Cell concentration to form lactobacillus ferment culture reaches 1.0 × 10⁸-10.0×10⁸Cfu/mL, deposit number is CCTCC NO:The cell concentration of M 2010150 Lactobacillus plantarum fermentation culture medium reaches 8.0 × 10⁸-20.0×10⁸Cfu/mL, by lattice Formula lactobacillus ferment culture and deposit number are CCTCC NO:M 2010150 Lactobacillus plantarum fermentation culture medium mixing, it is standby With；

(2) preparation of Chinese medical extract：The root of large-flowered skullcap, kuh-seng, golden cypress, the fruit of summer cypress and mulberry leaf are mixed, added water to cook, decoction liquor concentration It is 1.10-1.20g/mL to density, centrifugation takes supernatant, standby；

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