CN106176821A - A kind of Tiny ecosystem vagina microbial inoculum and its preparation method and application - Google Patents
A kind of Tiny ecosystem vagina microbial inoculum and its preparation method and application Download PDFInfo
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- CN106176821A CN106176821A CN201610799951.9A CN201610799951A CN106176821A CN 106176821 A CN106176821 A CN 106176821A CN 201610799951 A CN201610799951 A CN 201610799951A CN 106176821 A CN106176821 A CN 106176821A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The present invention provides a kind of Tiny ecosystem vagina microbial inoculum, the fermentation liquid obtained including curling lactobacillus and the fermentation of Jansen lactobacillus compatibility.Present invention also offers the preparation method of Tiny ecosystem vagina microbial inoculum, curling lactobacillus and Jansen lactobacillus are mixed strain compatibility, mixed strain bacterium solution is seeded to enrichment culture in enrichment culture, obtains enrichment culture liquid；Enrichment culture liquid is seeded to amplification culture on amplification culture base, obtains scale-up medium；Described scale-up medium being inoculated into fermentation medium top fermentation cultivate, the fermentation liquid obtained is Tiny ecosystem vagina microbial inoculum.The described Tiny ecosystem vagina microbial inoculum that the present invention provides application in the medicine of preparation prevention and treatment recurrent vaginal inflammation or secretions exception gynaecopathia.Described Tiny ecosystem vagina microbial inoculum cure rate is 88%～98%, and effective percentage is 92%～100%, and in 3 months, relapse rate is respectively 0%～6%, and adverse reaction rate is low, illustrates that complex microbial inoculum treats various recurrent vaginal inflammation the most effective.
Description
Technical field
The present invention relates to prevention and the microbial inoculum technical field for the treatment of vagina, be specifically related to a kind of Tiny ecosystem vagina microbial inoculum
And its preparation method and application.
Background technology
Adding up according to authoritative department, the prevalence of China's adult female's gynaecopathia is up to 87.5%.Elderly woman, because of ovary
Deterioration, estrogen level reduces, vaginal wall atrophy, and mucosa is thinning, and in epithelial cell, glycogen content reduces, intravaginal pH value
Rising, locally resistance reduces, and pathogenic bacterium are easily invaded breeding and cause inflammation.Gynecological inflammation includes female vulva inflammation, vaginitis, palace
Neck inflammation, pelvic inflammatory disease etc., if preventing not in time and treat to cause other pathological changes, such as, can affect the up of sperm, harm
Hinder the combination of sperm and ovum, or hinder the implantation of germ cell to grow, or cause not ovulation etc., and then just easily cause women not
Pregnant.If gestation pregnant female infects gynecological inflammation, the most also gestation can be adversely affected, it may occur that intrauterine infection,
And miscarriage, premature labor, the low inferior problem of fetal intelligence, it is unfavorable for prenatal and postnatal care.
Commonly used gynaecologic washing lotion suffers from woman with the women of antibiotic compared with the women not using gynaecologic washing lotion and antibiotic
The probability of section's inflammation is low by 73%, but women uses no matter one week antibiotic is oral or for Other diseases continuously, and 50%
Vaginitis is suffered from by above women's federation.Therefore, using gynaecologic washing lotion is prevention and the effective ways for the treatment of gynecological inflammation.Treatment at present
The washing liquid of gynecological inflammation is of a great variety, and it is public that the effect having the yellow Pu clean skin microbial inoculum of pure Chinese medicine to sterilize comes from Cortex Phellodendri, Rhizoma Coptidis and Pu
The Chinese medicine medicines compositions such as English；Also has the microbial inoculum that Chinese medicine and chemical composition combine, such as: JIEERYIN, woman's inflammation are clean, gynecological's a thousand pieces of gold washing liquid
Etc., utilize the bactericidal action of chemical composition to take stopgap measures, utilize Chinese medicine ingredients to effect a permanent cure.But the purest Chinese medicine or Chinese medicine and change
Study point microbial inoculum combined, all can directly result in vagina bacterium owing to abusing these Fungicidal substance heavy damage vagina self-cleaning systems
Group is disorderly, makes the easy recurrent exerbation of gynaecopathia, and therapeutic effect is undesirable.
Summary of the invention
In view of this, it is an object of the invention to a kind of Tiny ecosystem vagina microbial inoculum and preparation method thereof, by regulation women
Vaginal microbial flora, forms the microecosystem that a uniqueness is stable and harmless, reaches effectively prevent and treat gynaecopathia
Purpose.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of Tiny ecosystem vagina microbial inoculum, ferment including curling lactobacillus and Jansen lactobacillus compatibility
The fermentation liquid arrived.
Preferably, described fermentation liquid crimps viable count 1～1.5 hundred million CFU/ml of lactobacillus；Described Jansen lactobacillus
Viable count 0.5～100,000,000 CFU/ml.
Present invention also offers the preparation method of described Tiny ecosystem vagina microbial inoculum, comprise the following steps:
1) curling lactobacillus and Jansen lactobacillus are mixed strain compatibility, mixed strain bacterium solution is seeded on enrichment medium enrichment
Cultivate, obtain enrichment culture liquid；
2) by described step 1) the enrichment culture liquid that obtains is seeded to amplification culture on amplification culture base, obtains amplification culture
Liquid；
3) by described step 2) scale-up medium that obtains is inoculated into fermentation medium top fermentation and cultivates, the fermentation liquid obtained
For Tiny ecosystem vagina microbial inoculum.
Preferably, described step 1) crimp lactobacillus and Jansen lactobacillus according to quantity than for (1～2): (2～1) is entered
The mixed strain of row.
Preferably, described step 1) inoculum concentration of mixed strain bacterium solution is 5～10%.
Preferably, described step 1) enrichment medium includes following components: MgSO4·7H2O1.0～1.5g, KH2PO4
0.5～1.0g, K2HPO43.0～6g, NaCl 1.0～2g, CaCl215～25mg, FeSO43～8mg, MnCl24H2O 3～
8mg, CuCl20.3～0.6mg, liquid microelement 5～8ml, concentration is the MCs of the MC-LR of MC-RR and 13mg/L of 15mg/L
Crude extract 200～300ml；PH value≤4.5 of described enrichment medium；Described liquid microelement contains the group of following mass content
Point: ZnSO40.29g/L, CaCl20.24g/L, CuSO40.25g/L, MgSO40.17g/L and FeSO40.28g/L.Preferably, institute
State step 1) in time of enrichment culture be 1～2 day；Described step 1) in the temperature of enrichment culture be 28～30 DEG C；Described step
Rapid 1) in, enrichment culture is carried out under the speed conditions of 100rpm～140rpm.
Preferably, described step 2) in amplification culture base include following components content: peptone 8～15g, yeast extract 4～
10g, NaCl3～8g, distilled water supplements surplus to 1L；
Preferably, pH value≤4.5 of described amplification culture base；
Preferably, described step 2) in time of amplification culture be 3～5 days；Described step 2) in the temperature of amplification culture
It it is 28～30 DEG C；
Preferably, described step 2) in amplification culture carry out under the speed conditions of 100rpm～140rpm；
Preferably, described step 2) in the inoculum concentration of amplification culture be 5%～10%.
Preferably, described step 3) in fermentation medium include the component of following weight portion: the useful micro-life of EM of 2～3 parts
Thing flora, the brown sugar of 2～3 parts, the L-AA of 0.2～0.3 part, the potassium dihydrogen phosphate of 0.05～0.2 part, 0.2～0.5 part
Earthworm antimicrobial peptide solution, the Mel of 0.3～0.8 part and the water of 48～52 parts；
Preferably, described step 3) in fermentation inoculum concentration be 1%～3%；
Preferably, described step 3) in fermentation condition be under normal temperature environment；
Preferably, described step 3) in fermentation time be 12～15 days.
Preferably, described step 3) in the preparation method of earthworm antimicrobial peptide solution, comprise the following steps:
The Lumbricus that lives is (4～6) with brown sugar according to quality by A: the ratio of (2～3.5) mixes, and obtains Lumbricus after Lumbricus dissolving
Liquid；
The earthworm liquid that described step A is obtained by B separates, and collects supernatant；
The supernatant that described step B is obtained by C carries out boiling water bath 5～10min, separates, and collects and takes supernatant；
The supernatant liquid filtering that described step C is obtained by D collects filtrate, obtains earthworm antimicrobial peptide solution.
Tiny ecosystem vagina microbial inoculum prepared by the method that the present invention provides described Tiny ecosystem vagina microbial inoculum or the present invention to provide
Application in the medicine of preparation prevention and treatment recurrent vaginal inflammation or secretions exception gynaecopathia.
Technique effect
A kind of Tiny ecosystem vagina microbial inoculum that the present invention provides, obtains for curling lactobacillus and the fermentation of Jansen lactobacillus compatibility
Fermentation liquid, curling lactobacillus and Jansen lactobacillus can produce H2O2, for beneficial microbe, scientific combination, mixed strain compatibility, utilize
Symbiosis between these microorganisms, forms a complexity and stable microecosystem, acts on the intravaginal of patient, make
The probioticss such as lactic acid bacteria are strong Tiny ecosystem group along with the function bacterium formation in woman vagina, occupies firmly in vagina
The Ecological niche, while suppressing other various pathogenic bacterium, retains the probiotics in woman vagina, makes intravaginal Tiny ecosystem arrive flat
Between weighing apparatus pH value 4～4.5, enhancing human body immunity power and vagina self-cleaning function, reach effect of clear-cutting forestland.The present invention provides
Tiny ecosystem vagina microbial inoculum cure rate is more than 90%, and effective percentage is more than 92%, and in 3 months, relapse rate is respectively less than 3%, no
Good reaction incidence rate is relatively low.It is the most effective that complex microbial inoculum treats various recurrent vaginal inflammation.
Meanwhile, a kind of Tiny ecosystem vaginal lotion that the present invention provides is regenerative microorganism, and pH value, between 3～4.5, is
The opponent that pathogen is feared most, its therapeutical effect is not breed by controlling harmful bacteria, does not has antibiotics to annihilate the character gone out,
Because of without produce there is variant as resistant bacterium, also would not make body produce drug resistance, thus safety without bad instead
Should.
Present invention also offers the preparation method of Tiny ecosystem vagina microbial inoculum, be curling lactobacillus and Jansen lactobacillus useful
Microorganism is probiotics, by antibacterial to EM beneficial microbe colony, brown sugar, L-AA, the potassium dihydrogen phosphate of 0.1 part, Lumbricus
Peptide solution and Mel chelating fermentation, preparation method is simple, with low cost, reproducible.
Detailed description of the invention
The invention provides a kind of Tiny ecosystem vagina microbial inoculum, ferment including curling lactobacillus and Jansen lactobacillus compatibility
The fermentation liquid arrived.
A kind of Tiny ecosystem vagina microbial inoculum that the present invention provides, ties with medical science microecology mutually by advanced biotechnology
Close, curling lactobacillus and Jansen lactobacillus as beneficial microbe, scientific combination, mixed strain compatibility, utilize these microorganisms it
Between symbiosis, form a complexity and stable microecosystem, while suppressing other various pathogenic bacterium, retain woman
Probiotics in cysthus road, makes intravaginal Tiny ecosystem arrive between equilibrium ph 4～4.5, and enhancing human body immunity power and vagina are certainly
Clean function, reaches effect of clear-cutting forestland.
In the present invention, described fermentation liquid crimps viable count 1～1.5 hundred million CFU/ml of lactobacillus；Described Jansen breast bar
Viable count 0.5～100,000,000 CFU/ml of bacterium.
In the present invention, described Tiny ecosystem vagina microbial inoculum further preferably includes the adjuvant medically accepted.In the present invention, described
Adjuvant preferably include porous-starch.The mass volume ratio of described porous-starch and fermentation liquid be preferably 1.5～2.5:3 (mg:
ML), more preferably 2:3 (mg:L).
Present invention also offers the preparation method of described Tiny ecosystem vagina microbial inoculum, comprise the following steps:
1) it is seeded to enrichment culture on enrichment medium after curling lactobacillus and Jansen lactobacillus being mixed strain compatibility, obtains
Enrichment culture liquid；
2) by described step 1) the enrichment culture liquid that obtains is seeded to amplification culture on amplification culture base, obtains amplification culture
Liquid；
3) by described step 2) scale-up medium that obtains is inoculated into fermentation medium top fermentation and cultivates, the fermentation liquid obtained
For Tiny ecosystem vagina microbial inoculum.
The preparation method of the described Tiny ecosystem vagina microbial inoculum that the present invention provides, method is simple, reproducible, low cost
Honest and clean.
The present invention is seeded to enrichment culture on enrichment medium after curling lactobacillus and Jansen lactobacillus are mixed strain compatibility,
Obtain enrichment culture liquid.
In the present invention, the kind of described curling lactobacillus and Jansen lactobacillus is not particularly limited, and uses this area skill
The curling lactobacillus preparing vagina microbial inoculum known to art personnel and the kind of Jansen lactobacillus.
In the present invention, the source of described curling lactobacillus and Jansen lactobacillus be healthy women vaginal secretions in sieve
Choosing obtains.The method of described screening includes isolating lactobacillus according to bacteriology's separation method, then tests (15 by growth temperature
DEG C～45 DEG C), mobility test, nitrate reduction test, gelatin liquefaction test, indole (indole) test, hydrogen sulfide production test
It is Lactobacillus by the doubtful strain dientification of bacteria；Use carbohydrate fermentation to produce the qualification that lactobacillus is carried out planting by acid test again, enter
One step filters out the dominant strain producing hydrogen peroxide, and uses 16S rRNA sequence analysis method to carry out genotype identification.This
In invention, described 16S rRNA sequence analysis method is not particularly limited, and uses 16S well-known to those skilled in the art
RRNA sequence analysis method.The primer sequence used in described 16S rRNA sequence analysis method is preferably those skilled in the art
Known bacteria molecule identifies 16S rRNA primer forward sequence 5'-AGAGTTTGATCATGGCTCAG-3' used, 16S
RRNA primer reverse sequence 5'-TAGGGTTACCTTGTTACGACTT-3'.Described 16S rRNA primer is by Shanghai raw work synthesis.
In the present invention, described curling lactobacillus and Jansen lactobacillus are preferably (1～2) according to quantity ratio: (2～1) is entered
Row mixed strain compatibility, more preferably 1:1.
The mixed strain of described curling lactobacillus and Jansen lactobacillus is preferably 5～10% to inoculate, more according to inoculum concentration
It is preferably 8%.
In the present invention, described enrichment medium preferably includes following components: MgSO4·7H2O 1.0～1.5g,
KH2PO40.5～1.0g, K2HPO43.0～6g, NaCl 1.0～2g, CaCl215～25mg, FeSO43～8mg, MnCl2·4H2O
3～8mg, CuCl20.3～0.6mg, liquid microelement 5～8ml, concentration is the MC-RR (Algae toxins RR) and 13mg/L of 15mg/L
The MCs crude extract 200～300ml of MC-LR (Algae toxins LR), distilled water supplements surplus；Described enrichment medium pH value≤
4.5；More preferably MgSO4·7H2O 1.2g, KH2PO40.8g, K2HPO44g, NaCl 1.5g, CaCl220mg, FeSO45mg,
MnCl2·4H2O 5mg, CuCl20.5mg.Liquid microelement 6ml, concentration is the MC-LR's of MC-RR and 13mg/L of 15mg/L
MCs crude extract 200～300ml, distilled water is supplemented to 1000ml.The pH value of described enrichment medium is more preferably 4.0～4.4.
The method that the pH value of described enrichment medium adjusts uses the HCl solution of volumetric concentration 0.1% and mass concentration to be 0.1%
NaOH solution is adjusted.
In the present invention, described liquid microelement preferably includes the component of following mass content: ZnSO40.29g/L,
CaCl20.24g/L, CuSO40.25g/L, MgSO40.17g/L and FeSO40.28g/L；Solvent in described liquid microelement is
Distilled water.
It is well known to those skilled in the art that the present invention is not particularly limited employing to the compound method of described enrichment medium
Culture medium compound method.
In the present invention, the preparation method of described MCs crude extract preferably includes following steps:
Lyophilizing chlorella powder volumetric concentration be 5%～10% acetic acid extraction 3～4 times, the suspension centrifuging and taking obtained
Supernatant, and being adsorbed by the active carbon adsorption column of pretreatment, cleaning active charcoal adsorption column subsequently, with volumetric concentration be 15%～
20% methanol is washed once, then is 100% methanol-eluted fractions toxin by volumetric concentration；It is that 100% meoh eluate exists by volumetric concentration
Being evaporated on Rotary Evaporators, the dry powder obtained is thick Microcystin.Described preprocess method is preferably by volumetric concentration 100%
Methanol rinse, after use deionized water rinsing.
In the present invention, the time of described enrichment culture is preferably 1～2 day, more preferably 1.5 days.Described enrichment culture
Temperature is preferably 28～30 DEG C, more preferably 29 DEG C.Described enrichment culture is preferably in the condition that rotating speed is 100rpm～140rpm
Under carry out, more preferably 120rpm.
After obtaining enrichment culture liquid, described enrichment culture liquid is seeded to amplification culture on amplification culture base by the present invention,
To scale-up medium；
In the present invention, described amplification culture base preferably includes the component of following mass content: peptone 8～15g/L, yeast
Cream 4～10g/L, NaCl 3～8g/L；Solvent in described amplification culture base is distilled water；More preferably peptone 10g/L, ferment
Female cream 46g/L, NaCl 5g/L；Described amplification culture base pH≤4.5, more preferably 4.0～4.4.The control method of described pH value
Preferably employ the HCl solution that volumetric concentration is 0.1% and the NaOH solution that mass concentration is 0.1% regulation.
In the present invention, the time of described amplification culture is 3～5 days, more preferably 4 days.The temperature of described amplification culture is
28～30 DEG C, more preferably 29 DEG C.Carry out under described amplification culture preferred earthquake condition of culture, the rotating speed that described concussion is cultivated
For 100rpm～140rpm；More preferably 120rpm.The inoculum concentration of described amplification culture is 5%～10%, more preferably 8%.
After obtaining scale-up medium, described scale-up medium is inoculated into fermentation medium top fermentation and cultivates by the present invention,
To fermentation liquid be Tiny ecosystem vagina microbial inoculum.
In the present invention, described fermentation medium includes the component of following weight portion: the EM beneficial microbe colony of 2～3 parts,
The brown sugar of 2～3 parts, the L-AA of 0.2～0.3 part, the potassium dihydrogen phosphate of 0.05～0.2 part, the Lumbricus of 0.2～0.5 part
Antibacterial peptide solution, the Mel of 0.3～0.8 part and the water of 48～52 parts, the EM beneficial microbe colony of more preferably 2.5 parts, 2.5
The brown sugar of part, the L-AA of 0.25 part, the potassium dihydrogen phosphate of 0.1 part, the earthworm antimicrobial peptide solution of 0.3 part, the honeybee of 0.5 part
Honey and the water of 50 parts.The present invention does not has special restriction to the kind of described water, uses cultivation well known to those skilled in the art
Base use water, concrete, described water is cold water.
In the present invention, the inoculum concentration of described fermentation culture is preferably 1%～3%, more preferably 2%；Described fermentation training
Support and preferably carry out under normal temperature environment；The time of described fermentation culture is 12～15 days, more preferably 13～14 days.
In the present invention, the source of described EM beneficial microbe colony is not particularly limited, and uses those skilled in the art institute
Known to the source of EM beneficial microbe colony.The source of EM beneficial microbe colony described in the embodiment of the present invention be from
Jiangxi providence biology company limited is commercially available.
In the present invention, the preparation method of described earthworm antimicrobial peptide solution preferably includes following steps:
The Lumbricus that lives is (4～6) with brown sugar according to quality by A: the ratio of (2～3.5) mixes, and obtains Lumbricus after Lumbricus dissolving
Liquid；
The earthworm liquid that described step A is obtained by B separates, and collects supernatant；
The supernatant that described step B is obtained by C carries out boiling water bath 5～10min, separates, and collects and takes supernatant；
The supernatant liquid filtering that described step C is obtained by D collects filtrate, obtains earthworm antimicrobial peptide solution.
The Lumbricus that lives be (4～6) with brown sugar according to quality by the present invention: the ratio of (2～3.5) mixes, must after Lumbricus dissolving
To earthworm liquid；The ratio mixing of more preferably 5:3.
In the present invention, described mixing is not particularly limited the technical scheme using mixing well-known to those skilled in the art
?.
In the present invention, described Lumbricus alive was preferably carried out totally before mixing with brown sugar.In the present invention, described earthworm alive
The source of earthworm and brown sugar is not particularly limited, and uses Lumbricus alive well-known to those skilled in the art and the source of brown sugar.
In the present invention, lived Lumbricus can be dissolved under the effect of brown sugar, obtains earthworm liquid.
After obtaining earthworm liquid, described earthworm liquid is separated by the present invention, collects supernatant.
In the present invention, the method for described separation is not particularly limited, use described in those skilled in the art separation
Technical scheme.In the embodiment of the present invention, the scheme of described separation is centrifugal.Described centrifugal rotating speed be preferably 100rpm～
140rpm；More preferably 120rpm.The described centrifugal time is preferably 5～8min；More preferably 6～7min.Described centrifugal
Temperature is preferably 18 DEG C-35 DEG C；More preferably 30 DEG C.
After obtaining supernatant, described supernatant is carried out boiling water bath 5～10min by the present invention, separates, and collects and takes supernatant.
In the present invention, supernatant carries out the time 5～10min of boiling water bath, more preferably 8min.Described supernatant boils
Preferably dilute with acetate buffer before water-bath.The concentration of described acetate buffer is preferably 30%～40%；More preferably
36%.The multiple of described dilution is preferably 80～120 times, more preferably 100 times.
In the present invention, described boiling water bath is preferably carried out under conditions of stirring.The speed of described stirring is not had by the present invention
Particular restriction, uses boiling water bath mixing speed well-known to those skilled in the art.
After obtaining supernatant, described supernatant liquid filtering is collected filtrate by the present invention, obtains earthworm antimicrobial peptide solution.
In the present invention, described filtration is preferably employed and can be carried out by the ultrafiltration system of molecular weight 10000Da specification.
In the present invention, the process of described fermentation culture specifically preferably includes following steps:
I, brown sugar, potassium dihydrogen phosphate are mixed with cold water, be stirred well to all dissolve；
II, adding EM beneficial microbe colony, described scale-up medium and earthworm antimicrobial peptide solution, stirring, in semitight
Under state, 25～35 DEG C, intensity of illumination ferment in the environment of 100～550Lex, every three days stirring once, stir 1 every time
～2min；
III, when fermentation proceeds to the 3rd day, the water surface there will be foam, and addition Mel, to accelerate fermenting speed, promotes useful micro-
Biological generation；When the 8th day, foam slowly disappears, and the water surface floats the float of a piece of white, adds L-AA, to increase
Strong anti-oxidation ability, during by the 13rd day, float forms precipitate, and pH value is below 4.5, and solid-liquid separation, in the fermentation obtained
Clear liquid is Tiny ecosystem vagina microbial inoculum.
In the present invention, the state that under described semitight state, preferred round closes the lid.
In the present invention, described fermentation liquid preferably employs porous-starch absorption, obtains the composite bacteria agent capable of porous-starch absorption, spray
The dried product of mist is distributed into capsule, prepares Tiny ecosystem vagina bacteria capsule.The inlet temperature of described spray drying is excellent
Elect 150～180 DEG C as；More preferably 170 DEG C.The leaving air temp of described spray drying is preferably 60～70 DEG C；More preferably 65
℃。
In the present invention, described porous-starch is preferably 1.5～2.5:3 (mg:mL) with the mass volume ratio of fermented supernatant fluid,
More preferably 2:3 (mg:L).The source of described porous-starch is not particularly limited, and uses well-known to those skilled in the art many
Hole starch.
Feature based on above-mentioned Tiny ecosystem vagina microbial inoculum, the Tiny ecosystem vagina microbial inoculum that the present invention provides is in preparation prevention and controls
Treat the application in the medicine of recurrent vaginal inflammation or secretions exception gynaecopathia.
In the present invention, described Tiny ecosystem vagina microbial inoculum use during, preferably continuous use 5d～6d, every day 1 time,
Each 1 seed lac capsule.The viable count containing described Tiny ecosystem vagina microbial inoculum in described capsule is preferably 8～1,500,000,000 CFU/g.
Carry out detailed below in conjunction with a kind of Tiny ecosystem vagina microbial inoculum that the present invention is provided by embodiment and preparation method thereof
Illustrate, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
Work is washed Lumbricus, with Lumbricus with the ratio of brown sugar 5:3, adds brown sugar (having another name called cane suger), wait Lumbricus molten
Xie Hou, by earthworm liquid centrifugation, takes supernatant, after diluting with acetate buffer, enters boiling water bath, and limit heating edge stirs 5min,
It is then centrifuged for separating, takes supernatant, after diluting with acetate buffer, with the ultrafiltration system that can pass through molecular weight 10000Da specification
Filter or high speed centrifuge isolated earthworm antimicrobial peptide solution.
Embodiment 2
Work is washed Lumbricus, with Lumbricus with the ratio of brown sugar 4:3.5, adds brown sugar (having another name called cane suger), wait Lumbricus
After dissolving, by earthworm liquid centrifugation, taking supernatant, after diluting with acetate buffer, enter boiling water bath, limit heating edge stirs
8min, is then centrifuged for separating, takes supernatant, after diluting with acetate buffer, with the ultrafiltration that can pass through molecular weight 10000Da specification
System filters or high speed centrifuge isolated earthworm antimicrobial peptide solution.
Embodiment 3
Work is washed Lumbricus, with Lumbricus with the ratio of brown sugar 6:2, adds brown sugar (having another name called cane suger), wait Lumbricus molten
Xie Hou, by earthworm liquid centrifugation, takes supernatant, after diluting with acetate buffer, enters boiling water bath, and limit heating edge stirs 10min,
It is then centrifuged for separating, takes supernatant, after diluting with acetate buffer, with the ultrafiltration system that can pass through molecular weight 10000Da specification
Filter or high speed centrifuge isolated earthworm antimicrobial peptide solution.
Embodiment 4
Filtering out and crimp lactobacillus and Jansen lactobacillus probiotics in healthy women vagina, mixed strain compatibility, according to 5%
Inoculum concentration is seeded in enrichment medium, and with 120 turns/min, at 30 DEG C, concussion is cultivated 1 day, obtains probiotics culture fluid, enrichment
Culture medium culturing base consist of MgSO4·7H2O 1.0g, KH2PO41.0g, K2HPO43.0g, NaCl 2g, CaCl215mg,
FeSO48mg, MnCl2·4H2O 3mg, CuCl20.6mg, liquid microelement 5ml, concentration is MC-RR and 13mg/L of 15mg/L
The MCs crude extract 200ml of MC-LR, distilled water is supplemented to 1000ml, and regulation pH is 4.5, and wherein liquid microelement is 1L distillation
Containing ZnSO in water40.29g, CaCl20.24g, CuSO40.25g, MgSO40.17g and FeSO40.28g.By culture fluid by 5%
Inoculum concentration is inoculated on amplification culture base, with 120rpm, continues to cultivate 5 days, obtain fermentation liquid, screen excellent species at 30 DEG C,
Described amplification culture base consist of peptone 8g, yeast extract 10g, NaCl 3g, distilled water is supplemented to 1000ml, regulation pH and is
4.5.The amplification culture fluid strain obtained is accessed the EM strain brown sugar Han 2kg, 0.05kg phosphoric acid according to the amount that inoculum concentration is 1%
Prepare 0.2 part of potassium dihydrogen and embodiment 1 the aqueous solution of 50kg of solution of earthworm antimicrobial peptide, stirring, close half
Under envelope state, room temperature 35 DEG C, intensity of illumination ferment in the environment of 100Lex, every three days stirring once, stir 2min every time；
Fermentation is when proceeding to the 3rd day, and the water surface there will be foam, need to add 0.3kg Mel to accelerate fermenting speed, promotion beneficial microbe
Generation；When the 8th day, foam slowly disappears, and the water surface floats the float of a piece of white, need to add 0.2kg L-AA,
To strengthen oxidation resistance, during by the l3 days, float formed precipitate, pH value 4.5, solid-liquid separation, collect fermentation supernatant
Liquid, sprays after adding porous-starch according to the ratio of porous-starch with the mass volume ratio 1.5:3 (mg:mL) of fermented supernatant fluid
Mist is dried, and obtains Tiny ecosystem vagina microbial inoculum dry powder.By in dry powder subpackage to capsule, it is ensured that the Tiny ecosystem vagina contained in every capsule
The viable count of microbial inoculum is 800,000,000 CFU/g, i.e. obtains Tiny ecosystem vagina microbial inoculum product.
Embodiment 5
Filtering out and crimp lactobacillus and Jansen lactobacillus probiotics in healthy women vagina, mixed strain compatibility, according to 10%
Inoculum concentration be seeded in enrichment medium, with 100 turns/min, at 28 DEG C, concussion is cultivated 3 days, obtains probiotics culture fluid, rich
Collect culture medium culturing base consists of MgSO4·7H2O 1.5g, KH2PO40.5g, K2HPO46g, NaCl 1.0g, CaCl225mg,
FeSO43mg, MnCl2·4H2O3mg, CuCl20.3mg, liquid microelement 8ml, concentration is MC-RR's and 13mg/L of 15mg/L
The MCs crude extract 200～300ml of MC-LR, distilled water 800～1000ml, regulation pH is 4, and wherein liquid microelement is 1L distillation
Containing ZnSO in water40.29g, CaCl20.24g, CuSO40.25g, MgSO40.17g and FeSO40.28g.By culture fluid by 8%
Inoculum concentration is inoculated on amplification culture base, with 140rpm, continues to cultivate 3 days, obtain fermentation liquid, screen excellent species at 30 DEG C,
Described amplification culture base consist of peptone 15g, yeast extract 4g, NaCl 8g, distilled water supplements 1L, and regulation pH is 4.Will
To amplification culture fluid strain access the EM strain brown sugar Han 3kg, 0.2kg potassium dihydrogen phosphate and reality according to amount that inoculum concentration is 1%
Execute the aqueous solution of the 52kg of the solution of the earthworm antimicrobial peptide of 0.5 part that example 2 prepares, stirring, under semitight state, room temperature
25 DEG C, intensity of illumination ferment in the environment of 550Lex, every three days stirring once, stir 1min every time；Fermentation proceeds to the 3rd
It time, the water surface there will be foam, need to add 0.8kg Mel to accelerate fermenting speed, promotes that beneficial microorganism generates；8th day
Time, foam slowly disappears, and the water surface floats the float of a piece of white, need to add 0.3kg L-AA, to strengthen antioxidation
Ability, during by the l3 days, float formed precipitate, pH value 4.2, solid-liquid separation, fermented supernatant fluid, according to porous-starch with
It is spray-dried after the ratio porous-starch of the mass volume ratio 2.5:3 (mg:mL) of fermented supernatant fluid, obtains Tiny ecosystem vagina microbial inoculum
Dry powder.By in dry powder subpackage to capsule, it is ensured that the viable count of the Tiny ecosystem vagina microbial inoculum contained in every capsule is 1,500,000,000 CFU/g,
I.e. obtain Tiny ecosystem vagina microbial inoculum product.
Embodiment 6
Filtering out and crimp lactobacillus and Jansen lactobacillus probiotics in healthy women vagina, mixed strain compatibility, according to 8%
Inoculum concentration is seeded in enrichment medium, and with 140 turns/min, at 29 DEG C, concussion is cultivated 2 days, obtains probiotics culture fluid, enrichment
Culture medium culturing base consist of MgSO4·7H2O 1.2g, KH2PO40.8g, K2HPO44g, NaCl 1.5g, CaCl220mg,
FeSO45mg, MnCl2·4H2O 5mg, CuCl20.5mg, liquid microelement 6ml, concentration is MC-RR and 13mg/L of 15mg/L
The MCs crude extract 200～300ml of MC-LR, distilled water is supplemented to 1000ml, and regulation pH is 4.2, and wherein liquid microelement is
Containing ZnSO in 1L distilled water40.29g, CaCl20.24g, CuSO40.25g, MgSO40.17g and FeSO40.28g.By culture fluid
It is inoculated on amplification culture base by the inoculum concentration of 10%, with 100rpm, continues at 30 DEG C to cultivate 4 days, obtain fermentation liquid, screen excellent
Good strain, described amplification culture base consist of peptone 10g, yeast extract 46g, NaCl 5g, distilled water supplements 1L, regulates pH
It is 4.2.The amplification culture fluid strain obtained is accessed the EM strain brown sugar Han 2.5kg, 0.1kg phosphorus according to the amount that inoculum concentration is 1%
The aqueous solution of the 48kg of the solution of the earthworm antimicrobial peptide of prepare 0.3 part of acid dihydride potassium and embodiment 3, stirring, close half
Under envelope state, room temperature 30 DEG C, intensity of illumination ferment in the environment of 300Lex, every three days stirring once, stir every time
1.5min；Fermentation is when proceeding to the 3rd day, and the water surface there will be foam, need to add 0.5kg Mel to accelerate fermenting speed, promote have
The generation of beneficial microorganism；When the 8th day, foam slowly disappears, and the water surface floats the float of a piece of white, need to add 0.25kg L-
Ascorbic acid, to strengthen oxidation resistance, during by the l3 days, float formed precipitate, pH value 4.0, solid-liquid separation, fermentation
Supernatant, according to spray dried after the porous-starch ratio porous-starch with the mass volume ratio 2:3 (mg:mL) of fermented supernatant fluid
Dry, obtain Tiny ecosystem vagina microbial inoculum dry powder.By in dry powder subpackage to capsule, it is ensured that the Tiny ecosystem vagina microbial inoculum contained in every capsule
Viable count be 1,000,000,000 CFU/g, i.e. obtain Tiny ecosystem vagina microbial inoculum product.
Embodiment 7～10
By outpatient service 200 example recurrent vaginal inflammation patient according to dominant bacteria and flora closeness, multifarious bacterium different, special
Detection, body inflammatory reaction and the clinical manifestation of group are divided into 4 groups, often organize 50 examples.
A group: still have symptom after vaginitis treatment, microecology in vaginas performance without dominant bacteria, closeness+, multiformity+, give
Emphasis recovers acid vagina environment, supplements lactobacillus, vagina complex microbial inoculum 5d, 1 time every night.
B group: still having symptom after vaginitis treatment, microecology in vaginas performance gram positive coccus or gram negative bacilli are excellent
Gesture bacterium, closeness and multiformity areGive to recover acid vagina environment, apply respectively for gram positive coccus or
It is main antibiotic therapy 1 week for gram negative bacilli, vagina complex microbial inoculum 5d, 1 time every night.
C group: trichomonacide, is shown in infusorian under mirror, gives after oral nitre azole treats 1 week, to use complex microbial inoculum 5d, often
Evening 1 time.
D group: candidiasis vaginitis recurs, and sees mycelia or spore, dysbacteriosis under mirror, and clinic has pruritus vulvae
Symptom, leucorrhea is typical or is not true to type, oral fluconazol 150mg (the 1st day) vagina daktarin 400mg simultaneously, 1 time every night, even
With 3d, vagina complex microbial inoculum 5d after drug withdrawal, 1 time every night.
Evaluation curative effect.Result: 4 groups of cure rates of A, B, C, D are respectively 98%, 96%, 96%, 88%, effective percentage is respectively
100%, 98%, 98%, 92%, in 3 months, relapse rate is respectively 0%, and 2%, 2%, 6%, adverse reaction rate is low.Micro-life
It is the most effective that thing composite bacteria agent capable treats various recurrent vaginal inflammation.
Embodiment 11～14
It is divided into 4 groups according to secretions color, abnormal smells from the patient, pH value and microecology in vaginas diagnosis, clues cell, specific disease substance.
A group: have a clinical symptoms, Tiny ecosystem checks that all antibacterials are the fewest, is presented without dominant bacteria, closeness+, multiformity
+, treatment is it is important that recover acid vagina environment, and 3% boric acid demibain, every day 2 times, it is deep that Lactobacillus 1 piece puts vagina
Place, 1 time every night, 5d altogether.
B group: having clinical symptoms, microecology in vaginas performance gram negative bacilli is that dominant bacteria, closeness and multiformity areApplying for gram negative bacilli is main antibiotic ciprofloxacin 500mg, every day 2 times, treats 7d altogether, as found
Having clues cell, ammonia test is positive, vagina pH > 4.5, and the secretions diagnosing bacterial vagina disease of vagina homogeneous lean gives first
Nitre azoles 400mg is administered orally, every day 2 times, altogether 7d.If still there being symptom after vaginitis treatment, microecology in vaginas performance gram positive coccus,
Apply the antibiotic cefradine 0.5g for gram positive coccus, every day 4 times, altogether 7d.
C group: typical yellow green foam sample leucorrhea, pruritus, scorching hot, pain, infusorian seen from secretions sessile drop method, treatment is adopted
By metronidazole or tinidazole 2g single oral, locally rinsing by 3% boric acid solution, sexual partner rules together, live lactobacillus capsule after 1 week
Put vagina depths, 1 time every night, be total to 5d for 1 piece.
D group: clinical symptoms and sign typical case or be not true to type, it is seen that spore or mycelia, oral fluconazol 150mg (the 1st day)
Vagina daktarin 400mg simultaneously, 1 time every night, is used in conjunction 3d, uses Lactobacillus 5d after drug withdrawal, 1 time every night.
Evaluation curative effect.Result: 4 groups of cure rates of A, B, C, D are respectively 97%, 95%, 98%, 90%, effective percentage is respectively
100%, 98%, 98%, 92%, in 3 months, relapse rate is respectively 1%, and 1%, 0%, 3%, adverse reaction rate is low.Micro-life
It is the most effective that thing composite bacteria agent capable treats various recurrent vaginal inflammation.
As seen from the above embodiment, a kind of Tiny ecosystem vagina microbial inoculum that the present invention provides, by advanced biotechnology
Combining with medical science microecology, curling lactobacillus and Jansen lactobacillus are joined as beneficial microbe, scientific combination, mixed strain
5, application for the treatment of gynaecopathia, the Tiny ecosystem vagina microbial inoculum that the result display present invention provides can effectively prevent and treat multiple woman
Section's disease, and use safety to have no adverse reaction.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a Tiny ecosystem vagina microbial inoculum, it is characterised in that include crimping lactobacillus and the fermentation of Jansen lactobacillus compatibility obtains
Fermentation liquid.
Tiny ecosystem vagina microbial inoculum the most according to claim 1, it is characterised in that crimp lactobacillus in described fermentation liquid
Viable count 1～1.5 hundred million CFU/ml；Viable count 0.5～100,000,000 CFU/ml of described Jansen lactobacillus.
3. the preparation method of the Tiny ecosystem vagina microbial inoculum described in claim 1 or 2, it is characterised in that comprise the following steps:
1) curling lactobacillus and Jansen lactobacillus are mixed strain compatibility, mixed strain bacterium solution is seeded on enrichment medium enrichment training
Support, obtain enrichment culture liquid；
2) by described step 1) the enrichment culture liquid that obtains is seeded to amplification culture on amplification culture base, obtains scale-up medium；
3) by described step 2) scale-up medium that obtains is inoculated into fermentation medium top fermentation and cultivates, and the fermentation liquid obtained is micro-
Ecological vagina microbial inoculum.
Preparation method the most according to claim 3, it is characterised in that described step 1) crimp lactobacillus and Jansen breast bar
Bacterium is (1～2) according to quantity ratio: (2～1) carries out mixed strain compatibility.
5. according to the preparation method described in claim 3 or 4, it is characterised in that described step 1) inoculum concentration of mixed strain bacterium solution is 5
～10%.
Preparation method the most according to claim 5, it is characterised in that described step 1) enrichment medium includes following group
Point: MgSO4·7H2O1.0～1.5g, KH2PO40.5～1.0g, K2HPO43.0～6g, NaCl 1.0～2g, CaCl215～
25mg, FeSO43～8mg, MnCl2·4H2O 3～8mg, CuCl20.3～0.6mg, liquid microelement 5～8mL, concentration is
The MCs crude extract 200～300mL of the MC-LR of MC-RR and 13mg/L of 15mg/L, distilled water supplements surplus to 1L；
PH value≤4.5 of described enrichment medium；
Described liquid microelement contains the component of following mass content: ZnSO40.29g/L, CaCl20.24g/L, CuSO4
0.25g/L, MgSO40.17g/L and FeSO40.28g/L；
Described step 1) in time of enrichment culture be 1～2 day；Described step 1) in the temperature of enrichment culture be 28～30 DEG C；
Described step 1) in enrichment culture carry out under the speed conditions of 100rpm～140rpm.
Preparation method the most according to claim 5, it is characterised in that described step 2) in amplification culture base include following matter
The component of amount content: peptone 8～15g/L, yeast extract 4～10g/L, NaCl 3～8g/L, the pH value of described amplification culture base
≤4.5；
Described step 2) in the inoculum concentration of amplification culture be 5%～10%；
Described step 2) in time of amplification culture be 3～5 days；Described step 2) described in the temperature of amplification culture be 28～30
℃；Described step 2) in amplification culture carry out under conditions of rotating speed is 100rpm～140rpm.
Preparation method the most according to claim 5, it is characterised in that described step 3) in fermentation medium include following heavy
Amount part component: the EM beneficial microbe colony of 2～3 parts, the brown sugar of 2～3 parts, the L-AA of 0.2～0.3 part, 0.05
～the potassium dihydrogen phosphate of 0.2 part, the earthworm antimicrobial peptide solution of 0.2～0.5 part, the Mel of 0.3～0.8 part and 48～52 parts
Water；
Described step 3) in the inoculum concentration of fermentation culture be 1%～3%；
Described step 3) in fermentation time be 12～15 days.
Preparation method the most according to claim 9, it is characterised in that described step 3) in the preparation of earthworm antimicrobial peptide solution
Method, comprises the following steps:
The Lumbricus that lives is (4～6) with brown sugar according to quality by A: the ratio of (2～3.5) mixes, and obtains earthworm liquid after Lumbricus dissolving；
The earthworm liquid that described step A is obtained by B separates, and collects supernatant；
The supernatant that described step B is obtained by C carries out boiling water bath 5～10min, separates, and collects and takes supernatant；
The supernatant liquid filtering that described step C is obtained by D, the filtrate obtained is earthworm antimicrobial peptide solution.
10. prepared by the Tiny ecosystem vagina microbial inoculum described in claim 1 or 2 or the method described in claim 3～9 any one
The application in the medicine of preparation prevention and treatment recurrent vaginal inflammation or secretions exception gynaecopathia of the Tiny ecosystem vagina microbial inoculum.
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| CN106754585A (en) * | 2017-03-23 | 2017-05-31 | 北京农学院 | A kind of Bacillus foecalis alkaligenes YS302 and its application |
| CN106822318A (en) * | 2016-12-31 | 2017-06-13 | 健务（上海）生物科技有限公司 | Treat topical agent of senile vahinitis and preparation method thereof |
| CN110320085A (en) * | 2019-06-30 | 2019-10-11 | 江西业力医疗器械有限公司 | A kind of gynecological disease detection preparation and treatment preparation |
| CN113274415A (en) * | 2021-06-29 | 2021-08-20 | 镇江远森医药科技有限公司 | Probiotics compound for treating inflammatory gynecological diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106822318A (en) * | 2016-12-31 | 2017-06-13 | 健务（上海）生物科技有限公司 | Treat topical agent of senile vahinitis and preparation method thereof |
| CN106754585A (en) * | 2017-03-23 | 2017-05-31 | 北京农学院 | A kind of Bacillus foecalis alkaligenes YS302 and its application |
| CN106754585B (en) * | 2017-03-23 | 2020-04-24 | 北京农学院 | Alcaligenes faecalis YS302 and application thereof |
| CN110320085A (en) * | 2019-06-30 | 2019-10-11 | 江西业力医疗器械有限公司 | A kind of gynecological disease detection preparation and treatment preparation |
| CN113274415A (en) * | 2021-06-29 | 2021-08-20 | 镇江远森医药科技有限公司 | Probiotics compound for treating inflammatory gynecological diseases |
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