CN109652334A

CN109652334A - A kind of complex microbial inoculum and its preparation method and application

Info

Publication number: CN109652334A
Application number: CN201910030845.8A
Authority: CN
Inventors: 谭瑛
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2019-04-19

Abstract

The present invention provides a kind of complex microbial inoculum, including Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis.Complex microbial inoculum of the present invention, survival rate is high after inhibiting pathogen ability strong and being made as freeze-dried powder.Without mutual antagonism between four kinds of bacterial strains of complex microbial inoculum of the present invention, it can be with mutualism, and the bifidobacterium longum, the Bacillus acidi lactici, the streptococcus fecalis have powerful antagonism to pathogen, and then the complex microbial inoculum for forming four kinds of bacterial strains has the function of good recuperating gastrointestinal tract health, improves immunity.

Description

A kind of complex microbial inoculum and its preparation method and application

Technical field

The invention belongs to human body microecology technical field more particularly to a kind of complex microbial inoculum and its preparation sides Method and application.

Background technique

Various microorganisms (bacterium) are often attached to human body from different environment, and can settle down and not medium well at certain position Length raises up seed, this to phenomenon be commonly referred to as " bacteria planting ".The microorganism of field planting must be continuing to supply nutrition by human body Substance could grow and breed, and could and then have an impact to human body.

Typically, human body has following several immunologic mechanisms:

A. structural barriers: " structural barriers " that child resists external cause pathogeny imcrobe infection include body surface barrier and inside Barrier.Body surface barrier is made of the mucous membrane that lining in the cavity for being covered on the skin of body surface and communicating with the outside world, and is body defenses The first line of defence of microorganism.As mucous membrane of small intestine epithelium not only has the function of digesting and assimilating nutriment while and one The important barrier (Kato etc.) of harmful substance (including bacterium, virus, pathogen, allergen macromolecular substances etc.) is resisted, especially It is blood-brain barrier, it can prevent pathogenic microorganism in blood and other macromolecular substances from entering brain tissue and the ventricles of the brain.In addition, The movement of gastral wriggling and respiratory mucosa cilium not only has mechanical scavenging effect, mucous epithelium to microorganism in enteric cavity Periodically fall off replacement reduce the chance that various microorganisms build group.

B. chemical barrier: respiratory tract and the various secretion of each organ secretion of alimentary canal (contain lysozyme, complement, naturally Antibody etc.) to constitute child anti-infectious " chemical barrier ".As the saliva (mainly containing lysozyme) in child oral cavity can kill Multiple pathogenic microorganisms, the gastric acid and pepsin of stomachial secretion can destroy daily ration antigen and can effectively inhibit multiple-microorganism from Oropharynx enters small intestine, and the certain micro-organisms for entering small intestine is also very sensitive to the bile in small intestine；In addition, the egg in pancreatic juice White enzyme can destroy bacterial cell membrane.The mucus of enteron aisle goblet cell secretion can both protect fragile enteric epithelium from mechanical damage, Cultivation antigenic substance is wrapped but also as a kind of adhesive matrix, it is made to be easier to be degraded by the enzyme of pancreas and epithelial cells (Gaskins, 1999)；In addition, mucous membrane can protect the destruction and prevention cause of disease for the digestive ferment that epithelium is secreted from intestinal flora Body invades epithelium (Nevtre etc., 1987)

C. biological barrier: a large amount of normal floras in enteron aisle are otherwise known as " biological barrier ".Hentges etc. (1983) recognizes Main function for normal flora is that invading micro-organism is prevented to build group in enteron aisle.Gaskin etc. (1996) report thinks normal Flora can stimulate the development of animal mechanism to enhance immunity of organism.The various antibacterial materials such as bacteriocin of normal flora secretion It can inhibit certain anaerobic bacteria breedings.In addition, normal flora and pathogenic microorganism are building group point, exist on the life conditions such as nutrition Competitive relation, also inhibit pathogenic microorganism growth and development and breeding.

D. the antibacterial material in phagocyte, natural killer cells and body fluid: the phagocyte (part in child's blood From breast milk) it include neutrophil leucocyte and Monocytes/Macrophages.Neutrophil leucocyte has weight in body defenses bacterium infection It acts on.Sterilization in neutrophil leucocyte/infiltration enhancing albumen (BPl) not only to gram-positive bacteria have wide spectrum inhibit and Lethal effect, and have and neutralize bacterial endotoxin, the effects of inhibiting cytokine release and resist endotoxin lethal effect (Ke Yan etc., 1998).Natural killer cells (NK cell) can produce interferon, plays cytotoxicity and kills infection cell. In addition, the complement, Tuftsin in body fluid all play important nospecific immunity effect.

E. newborn source non-antibody protected protein: human lactoferrin (Loctoferrin, LTF) is that a kind of non-antibody is protected in breast milk Albumen is protected, is that newborn child transports iron ion and as mother's mammary gland and a kind of defensive barrier of ewborn infant enteron aisle, in addition, Also have and adjust nospecific immunity, promotes the functions such as granulocyte and lymphocyte mitosis.

Probiotics is a kind of strain beneficial to human body, has effects that adjust intestinal health, but existing probiotics is general All over having the drawback that 1, inhibit pathogen ability weaker.2, the lyophilized technique of freeze-dried vaccine powder is directly related to bacterium powder after freeze-drying Survival rate, after being made as freeze-dried vaccine powder, number of viable is lower, influences its effectiveness.

Summary of the invention

For this purpose, to be to provide a kind of inhibition pathogen ability strong and be made as first technical problem to be solved by this invention The high complex microbial inoculum of survival rate after freeze-dried powder.Second technical problem to be solved by this invention is in the prior art The low problem of the obtained Viable detection of complex microbial inoculum of freeze-dried vaccine powder technique, and then provide a kind of viable bacteria survival The preparation method of the high long complex microbial inoculum of rate.

Complex microbial inoculum of the invention, including Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis.Institute Lactobacillus brevis is stated in submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) give preservation, deposit number is CGMCC NO. 16125, and Latin is named as Lactobacillus brevis； The bifidobacterium longum is in submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) give preservation, deposit number is CGMCC NO.16126, and Latin is named as Bifldobacterium longum；The saccharomyces bayanus bacterium is in submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms common micro-organisms Center (CGMCC) gives preservation, and deposit number is CGMCC NO. 16127, and Latin is named as Saccharomyces bayanus；The streptococcus fecalis is in the commonly micro- life of submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms Object center (CGMCC) gives preservation, and deposit number is CGMCC NO.16128, and Latin is named as streptococcus faecalis.The address China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), i.e. preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

The 16SrDNA sequence of the Lactobacillus brevis has the sequential structure as shown in SEQ ID NO.1, the SEQ ID The sequential structure of NO.1 is specific as follows:

ggatgaacgctggcggcgtgcctaatacatgcaagtcgaacgagttctcgttgatgatcggtgcttgc ac cgagattcaacatggaacgagtggcggacgggtgagtaacacgtgggtaacctgcccttaagtgggggataac atttggaaacagatgctaataccgcatagatccaagaaccgcatggttcttggctgaaagatggcgtaagctatc gcttttggatggacccgcggcgtattagctagttggtgaggtaatggctcaccaaggcgatgatacgtagccgaa ctgagaggttgatcggccacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaatctt c cacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaaggctttcgggtcgtaaaactctgatgtt g gagaagaatggtcggcagagtaactgttgccggcgtgacggtatccaaccagaaagccacggctaactacgtg cc agcagccgcggtaatacgtaggtggcaagcgttatccagatttattgggcgtaaagcgagcgcaggcggtttt tt aagtctgatgtgaaagccctcggcttaaccgaggaagcgcatcggaaactgggaaacttgagtgcagaagag gac agtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaggcggctgtct ggtc tgtaactgacgctgaggctcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaa acga tgaatgctaggtgttggagggtttccgcccttcagtgccgcagctaacgcattaagcattccgcctggggag tac gaccgcaaggttgaaactcaaaggaattgacgggggcacgcacaagcggtggagcatgtggtttaattcga agca acgcgaagaaccttaccaggtcttgacatcttttgatcacttgagagatcaggtttccccttcgggggcaaa atg acaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctt at gactagttgccagcatttagttgggcactctagtaagactgccggtgacaaaccggaggaaggtggggatga cgt caaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagttgcgagaccg cga ggtcaagctaatctcttaaagccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatc gc tagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgag ag tttgtaacacccgaagccggtggcgtaacccttttagggagcgagccgtct。

The 16SrDNA sequence of the bifidobacterium longum has the sequential structure as shown in SEQ ID NO.2；The SEQ The sequential structure of ID NO.2 is specific as follows:

caggatgaacgctggcggcgtgcttaacacatgcaagtcgaacgggatccctggcagcttgctgtcggg gtgagagtggcgaacgggtgagtaatgcgtgaccaacctgccctgtgcaccggaatagctcctggaaacgggtg g taataccggatgctccgctccatcgcatggtggggtgggaaatgcttttgcggcatgggatggggtcgcgtccta tcagcttgttggcggggtgatggcccaccaaggcgttgacgggtagccggcctgagagggtgaccggccacatt g ggactgagatacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgat gca gcgacgccgcgtgcgggatggaggccttcgggttgtaaaccgcttttgttcaagggcaaggcacggtttcgg ccg tgttgagtggattgttcgaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcgag cgt tatccggatttattgggcgtaaagggctcgtaggcggttcgtcgcgtccggtgtgaaagtccatcgcctaacg gt ggatctgcgccgggtacgggcgggctggagtgcggtaggggagactggaattcccggtgtaacggtggaatg tgt agatatcgggaagaacaccaatggcgaaggcaggtctctgggccgtcactgacgctgaggagcgaaagcg tgggg agcgaacaggattagataccctggtagtccacgccgtaaacggtggatgctggatgtggggccctttcca cgggt cccgtgtcggagccaacgcgttaagcatcccgcctggggagtacggccgcaaggctaaaactcaaaga aattgac gggggcccgcacaagcggcggagcatgcggattaattcgatgcaacgcgaagaaccttacctgggct tgacatgt gccggatcgccgtggagacacggtttcccttcggggccggttcacaggtggtgcatggtcgtcgtcag ctcgtgt cgtgagatgttgggttaagtcccgcaacgagcgcaaccctcgccgcatgttgccagcgggtgatgccg ggaactc atgtgggaccgccggggtcaactcggaggaaggtggggatgacgtcagatcatcatgccccttacgt ccagggct tcacgcatgctacaatggccggtacaacgcggtgcgacacggtgacgtggggcggatcgctgaaa accggtctca gttcggatcgcagtctgcaactcgactgcgtgaaggcggagtcgctagtaatcgcggatcagcaa cgccgcggtg aatgcgttcccgggccttgtacacgccgcccgtcaagtcatgaaagtgggtagcacccgaagcc ggtggcccgac ccttgtggggggagccgtctaaggtgaga。

The 26S rDNA sequence of the saccharomyces bayanus bacterium has the sequential structure as shown in SEQ ID NO.3；The SEQ ID The sequential structure of NO.3 is specific as follows:

atgcttagtacggcgagtgagcggcaaaagctcaaatttgaaatctggtaccttcggtgcccgagttg ta atttggagagggcaactttggggccgttccttgtctatgttccttggaacaggacgtcatagagggtgagaat ccc gtgtggcgaggagtgcggttctttgtaaagtgccttcgaagagtcgagttgtttgggaatgcagctctaagt gggt ggtaaattccatctaaagctaaatattggcgagagaccgatagcgaacaagtacagtgatggaaagatgaa aa gaactttgaaaagagagtgaaaaagtacgtgaaattgttgaaagggaagggcatttgatcagacatggtgttt tg tgccctctgctccttgtgggtaggggaatctcgcatttcactgggccagcatcagttttggtggcaggataaa tcca taggaatgtagcttgcctcggtaagtattatagcctgtgggaatactgccagctgggactgaggactgcga cgta agtcaaggatgctggcataatggttatatgccgcccgtcttgaaacacg。

The 16SrDNA sequence of the streptococcus fecalis has the sequential structure as shown in SEQ ID NO.4.The SEQ ID The sequential structure of NO.4 is specific as follows:

gaacgctggcggcgtgcctaatacatgcaagtagaacgctgaagagaggagcttgctcttcttggatga gttgcgaacgggtgagtaacgcgtaggtaacctgccttgtagcgggggataactattggaaacgatagctaatac cgcataacaatggatgacacatgtcatttatttgaaaggggcaattgctccactacaagatggacctgcgttgtat tagctagtaggtgaggtaatggctcacctaggcgacgatacatagccgacctgagagggtgatcggccacactg g gactgagacacggcccagactcctacgggaggcagcagtagggaatcttcggcaatgggggcaaccctgacc gag caacgccgcgtgagtgaagaaggttttcggatcgtaaagctctgttgtaagtcaagaacgggtgtgagagtg gaa agttcacactgtgacggtagcttaccagaaagggacggctaactacgtgccagcagccgcggtaatacgta ggtc ccgagcgttgtccggatttattgggcgtaaagcgagcgcaggcggtttgataagtctgaagttaaaggctgt ggc tcaaccatagttcgctttggaaactgtcaaacttgagtgcagaaggggagagtggaattccatgtgtagcgg tga aatgcgtagatatatggaggaacaccggtggcgaaagcggctctctggtctgtaactgacgctgaggctcga aag cgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaggtgttggatcct ttc cgggattcagtgccgcagctaacgcattaagcactccgcctggggagtacgaccgcaaggttgaaactcaaa gga attgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggt cttg acatcccgatgctatttctagagatagaaagttacttcggtacatcggtgacaggtggtgcatggttgtcgtc ag ctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccctattgttagttgccatcattcagttggg cactctagcgagactgccggtaataaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacct gggctacacacgtgctacaatggttggtacaacgagttgcgagtcggtgacggcgagctaatctcttaaagccaa tctcagttcggattgtaggctgcaactcgcctacatgaagtcggaatcgctagtaatcgcggatcagcacgccgcg gtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagt。

The complex microbial inoculum is preparing recuperating gastrointestinal tract health and/or is improving the health care product of immunity, cream system Application in product, drug.

Preferably, the complex microbial inoculum is in the health care product of preparation treatment and/or pre- anti-diarrhea, drug Using.

The preparation method of complex microbial inoculum of the invention includes the following steps: to take the Lactobacillus brevis, long pair Discrimination bacillus, saccharomyces bayanus bacterium and streptococcus fecalis are made as freeze-dried powder respectively, remix uniformly to obtain the final product.

Preferably, the preparation method of the complex microbial inoculum specifically comprises the following steps: respectively to the short cream Protective agent is added in bacillus, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis, is uniformly mixed, is set The pre-freeze 1-3h at a temperature of -30~-50 DEG C, distil 10-20h at a temperature of being subsequently placed in -10~-20 DEG C, then is placed in -10 Distil 10-20h at a temperature of~-25 DEG C, then in 20-30 DEG C of at a temperature of secondary distillation 1-5h, places 1-5h under vacuum Afterwards to get the freeze-dried powder of the Lactobacillus brevis, the freeze-dried powder of the bifidobacterium longum, the freeze-dried powder of the saccharomyces bayanus bacterium and institute The freeze-dried powder for stating streptococcus fecalis, by four kinds of freeze-dried powders be uniformly mixed to get.

Preferably, the protective agent be skimmed milk power, maltose, sucrose, soluble starch, mannitol, sodium glutamate, One of L-cysteine, gelatin are a variety of.

Preferably, the preparation method of the complex microbial inoculum, specifically comprises the following steps:

(1) Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis are taken, is fermented respectively Culture, obtains the fermentation liquid of the Lactobacillus brevis, the fermentation liquid of the bifidobacterium longum, the fermentation liquid of the saccharomyces bayanus bacterium, institute State the fermentation liquid of streptococcus fecalis；

(2) four kinds of fermentation liquids that fermentation obtains in step (1) are taken, placing it in revolving speed respectively is 8000-12000 r/min Under, it is centrifuged 10-30min, bacterium mud is collected, obtains four kinds of bacterium muds；

(3) protective agent, the protective agent and the bacterium mud are separately added into bacterium mud obtained in step (2) Weight ratio is 3:1, is uniformly mixed, pre-freeze 3h at a temperature of placing it in -40 DEG C distils at a temperature of being subsequently placed in -15 DEG C 15h, then the 2h that distils at a temperature of being placed in 25 DEG C make the water content of the bacterium mud less than 5%, obtain after placing 2h under vacuum The freeze-dried powder of the Lactobacillus brevis, the freeze-dried powder of the bifidobacterium longum, the saccharomyces bayanus bacterium freeze-dried powder and the excrement hammer The freeze-dried powder of bacterium, by four kinds of freeze-dried powders be uniformly mixed to get.

It is further preferred that in step (1), the Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and institute Streptococcus fecalis is stated, obtains fermentation liquid by three grade fermemtation.

Above-mentioned technical proposal of the invention, has the advantage that compared with prior art

(1) complex microbial inoculum of the present invention separates a large amount of Lactobacillus brevis, long bifid bar from infant faeces Bacterium, saccharomyces bayanus bacterium and streptococcus fecalis, according to the test such as pathogeny bacterium ability, acid producing ability, digestion power is inhibited, filtering out can The preferably bacterial strain of digestible protein, meat slag, corn, starch, leaf, grass, obtains complex microbial inoculum of the present invention, It is used for human body, because homologous strain colonizes well, Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis can inhibit Harmful bacteria, and then safeguard the intestinal flora ecological balance, biological barrier is formed, invasion of the harmful bacteria to enteron aisle is inhibited.In addition, institute Stating complex microbial inoculum can also promote intestines peristalsis and thallus raised growth to change by generating a large amount of short chain fatty acids Osmotic pressure and prevent constipation.In addition, in thallus disintegration product and metabolin containing there are many enzyme, small peptide, short chain fatty acids etc. at Point, the advantages such as supplement the nutrients.

(2) complex microbial inoculum of the present invention, in the enterogastric diseases of people, especially diarrhea has been played very Good preventive and therapeutic action, and the deficiency of antibiotic and vaccine is compensated for through a variety of ways, for the healthy of the mankind and obtain It obtains safe food and opens new approach.

(3) complex microbial inoculum of the present invention, the suppression to Escherichia coli, salmonella, staphylococcus aureus Bacterium is relatively large in diameter, and has stronger inhibition pathogen ability.

(4) complex microbial inoculum of the present invention, acid and bile salt tolerance is good, and by the techniques such as freeze-dried powder behaviour Strain activity is still ideal after work, long shelf-life of seed.And the system of the complex microbial inoculum through the invention Preparation Method preparation, in preparation process, using specific protective agent, the holding for making to be made as each microorganism after freeze-dried powder higher is deposited Motility rate.

Detailed description of the invention

Fig. 1 is the bacterium solution microscopic examination result figure of Lactobacillus brevis of the present invention；

Fig. 2 is the bacterium solution microscopic examination result figure of bifidobacterium longum of the present invention；

Fig. 3 is the bacterium solution microscopic examination result figure of saccharomyces bayanus bacterium of the present invention；

Fig. 4 is the bacterium solution microscopic examination result figure of streptococcus fecalis of the present invention.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its His embodiment, shall fall within the protection scope of the present invention.

The separation screening of 1 bacterial strain of embodiment

The complex microbial inoculum of the present embodiment, including Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis. The Lactobacillus brevis is in submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) give preservation, deposit number is CGMCC NO. 16125；The bifidobacterium longum was submitted on July 17th, 2018 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) gives preservation, deposit number CGMCC NO. 16126；The saccharomyces bayanus bacterium is commonly micro- in submission on July 17th, 2018 China Committee for Culture Collection of Microorganisms Bio-Centers (CGMCC) give preservation, and deposit number is CGMCC NO.16127；The streptococcus fecalis was July 17 in 2018 Day submits China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) to give preservation, and deposit number is CGMCC NO.16128。

The excrement of Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis of the present invention from newborn and infant Just separation in, and by the assay optimizations such as bacteriostatic test, digestion power test, acid and alkali resistance sieve from 84 kinds of beneficial bacteriums therein Wherein four outstanding strains are selected after choosing.It is isolated by the following method:

By 1 gram of human body intestinal canal excrement, loading is put in the sterilizing bottle by 30 glass marbles plus physiological saline makees 100 times of dilutions After sufficiently beating, 10 are remake^-3、10^-4, successively to 10^-8Dilution takes 10^-4、10^-5、 10^-6、10^-7、10^-8Five dilutions Bacterium solution is inoculated in the general agar plate of LYT, Yihong, methylene blue agar, BB (BS) Bifidobacterium selective medium, LBS respectively (LC) Bacillus acidi lactici selective medium, EC enterococcus select base and Sb saccharomycete selects base.Every kind of culture medium connects 6 plates, inoculation Spinning upside down culture dish immediately afterwards spreads inoculation liquid uniformly in dish surface.Wherein make aerobic culture for 3, after 24 hours i.e. Observation.Another 3 are made Anaerobic culturel 48 hours.By visually observing and 45 DEG C of angle refraction light observations of disecting microscope.By every kind Bacterium colony is transplanted to one bacterium colony of one bacterium colony of LYT agar and fluid nutrient medium again, does anaerobism and aerobic respectively, while smear is made Gram stain microscopy isolates 138 bacterial strains, carries out qualification result and belongs to 8 sections, 36 kinds of bacterium of 11 categories, wherein dividing From the strain filtered out, monthly subculture is primary, was lyophilized and saves before 5 generations.

Wherein, the specific method is as follows for the separation and breeding of the saccharomyces bayanus bacterium:

1g sample is taken, is added in 100ml PTYG culture medium, is cultivated in constant-temperature table, 30 DEG C of cultivation temperature, revolving speed 180r/min, incubation time are for 24 hours.

By the sample after enrichment, 1ml is taken to carry out 10 times of gradient dilutions, takes 3 suitable dilutions on improvement SB plate It is coated, 30 DEG C of culture 36h, picking colonies typical is further purified 2~3 times with improvement SB plate streaking.

Wherein, saccharomycete then has no selection characteristic, therefore has no way of preparing selective medium.Because it is to the resistance to of vancomycin By characteristic, and its distinctive colonial morphology is combined, after being cultivated under aerobic conditions, can be distinguished with other bacterium colonies.Therefore, The improvement SB culture medium used includes the following raw material: peptone (Oxoid) 1%, maltose 4%, 10% tartaric acid 1.4%, powdered beef 0.35%, sodium chloride 0.5%, vancomycin 10ug/ml, agar 2%；Its condition of culture is as follows: pH value is 5.8-6.0, temperature are 30 DEG C, cultivate 36h.

The specific method is as follows for the separation and breeding of the streptococcus fecalis:

Excrement 1g is taken, is placed in the triangular flask equipped with 10ml buffered peptone water, shaken well.By 10 times of gradient dilutions, Select each 0.1ml coating KF agar plate of 3 acceptable diluent degree, 37 DEG C of culture 48h.Select white, surface smooth bumps, edge Neatly, the feature bacterium colony of diameter about 1.0~1.5mm.It passes and carries out Gram's staining after PTYG inclined-plane culture, selecting microscopy is Gram-positive, circle form oval, and the bacterial strain of single, pairs of or short catenation purifies culture.By bacterial strain after purification into Row growth characteristics are tested, 6.5% sodium chloride broth culture medium of inoculation and aesculin inclined-plane, after 37 DEG C of culture 48h, select seven leaves The bacterial strain grown in the glycosides hydrolysis positive, 6.5% sodium chloride broth culture medium is as bacterial strain to be sieved.

Strain to be tested is inoculated with PTYG fluid nutrient medium, 37 DEG C of culture 48h, switching plate, picking single colonie connects respectively Kind on the basis PTYG semisolid culturemedium and PTYG solid slope, 38 DEG C of 48 h of culture, and by screen obtain it is acidproof and resistance to Cholate function admirable, and bacterial strain that is well-grown, meeting safety testing.

Wherein, the isolation medium of the streptococcus fecalis is buffered peptone water solution, specifically includes following ingredient: egg White peptone 10g, Na₂HPO₄2g, glucose 5g, K₂HPO₄2g, KCl 5g, distilled water 1000ml.The preparation method is as follows: taking 10ml Buffered peptone water is placed in 50ml triangular flask, is added a small amount of bead, spare after 121 DEG C of sterilizing 15min.Due to streptococcus fecalis There is tolerance to Sodium azide, while there is the characteristic for being resistant to 45 DEG C of high temperature, the streptococcus fecalis selective medium selects city The KF culture medium sold carries out enterococcal separation in conjunction with high temperature culture conditions.By verifying, this method can be efficiently separated Streptococcus fecalis out.

The screening of seed bacterial strain is carried out according to following standard: (1) without interior exotoxin, it is nontoxic, harmless, safe, without secondary work With；(2) may advantageously facilitate internal colony balance or prevention ecological disturbance；(3) high yield antibacterial substance, acid producing ability are strong； (4) just has preferable adhesion property from the probiotics of itself enteron aisle, obtains having inhibition pathogen, adjusting enteron aisle micro- The ecological balance, the bacterial strain for improving immunity of organisms.

The strain of the obtained Lactobacillus brevis is gram-positive bacteria, and characteristic is as follows: the Lactobacillus brevis size is 0.7~1.0 × 2~4 μm, in the arrangement of single or double-strand, bacterium colony is petite, is creamy white, translucent, rough surface, 45 ° of refractive power observations, are evenly distributed, chromatic colour light belt, and microstructure is as shown in Figure 1.

The obtained bifidobacterium longum is Gram-positive bacillus, and characteristic is as follows: size is 0.5-0.8 × 2-8 μ M, in the shape of a rod, rodlike or bifurcated are V, Y type, arrive macrocolony in the bifidobacterium longum, are in canescence, central uplift, surface is put down Sliding or mucus shape.The lower 45 ° of refractivities observation of microscope, colony structure is more careful, and edge is irregular, there is fluorescence rag, side A part of chromatic colour light belt of edge.

The form and dyeing characteristic of the obtained saccharomyces bayanus bacterium: for gram-positive bacteria, 3~6 × 5~14 μ of size M is in oval, sees Fig. 1.Its colony characteristics is medium macrocolony, white, coarse, round or irregular.The saccharomyces bayanus Bacterium be it is obligate aerobic, optimum grow initial pH value be 6.0, optimum cultivation temperature be 30 DEG C, shaking flask culture optimum inoculation Amount is 3%, energy glucose fermentation, maltose, sucrose, fructose and mannitol, azymic galactolipin, gossypose, lactose, nitric acid It is also Antigen positive hybridomas.

The obtained streptococcus fecalis is Gram-positive bacillus, and characteristic is as follows: streptococcus fecalis is Gram-positive ball Bacterium, 0.5-1.0 μm of diameter, thallus is single or exists in pairs, irregular heap is also commonly formed, as shown in Figure 1.The excrement chain The medium bacterium colony bigger than normal of coccus is in canescence, smooth, neat in edge.Its test stone are as follows: should be without miscellaneous bacteria, OD600 value when transferred species For 1.9-2.4.

The Lactobacillus brevis energy glucose fermentation, arabinose, gluconate, maltose, melezitose and ribose.With Under seed is produced to 9084 plants of the Lactobacillus brevis of 1st generation, the 10th generation, the 13rd generation production kind of a 1st generation, the 10th generation, the 13rd generation The bacterium solution viable count of preparation is tested.The metabolite of the bifidobacterium longum is based on acetic acid.Can glucose fermentation, Ah Draw uncle's sugar, xylose, ribose and lactose.1st generation, the 10th generation, the 13rd generation are reached to bifidobacterium longum production seed below, Form, cultural character and the viable count of different generation strains are determined.By the basic bacteria of the saccharomyces bayanus bacterium point It is not seeded to culture medium, stationary culture 18h preparation production seed.Production seed is connected in the medium and passed for 13 generations, observation and survey Form, cultural character and the viable count of fixed different generation strains, to determine the generation of production strain.By the streptococcus fecalis N9024 plants of basic bacteria is inoculated with PTYG fluid nutrient medium, 37 DEG C of stationary culture 18h preparation production seeds of streptococcus fecalis respectively. Production seed is connected in PTYG fluid nutrient medium and passes 13 generations (cultural method is same as above), observes and measure different generation strains Form, cultural character and viable count, to determine the generation of production strain.Its result is as follows:

Through surveying the bifidobacterium longum, the Bacillus acidi lactici, the streptococcus fecalis, the saccharomyces bayanus bacterium 1st generation, the 10th Bacterium solution viable count prepared by generation, the 13rd generation production kind 1st generation, the 10th generation, the 13rd generation production seed, produces seed as the result is shown When reaching for 13 generation, the bifidobacterium longum is not less than 1.0 × 108CFU/mL, and the Bacillus acidi lactici is not less than 1.0 × 108CFU/ ML, shown streptococcus fecalis are not less than 1.0 × 109CFU/mL, and the saccharomyces bayanus bacterium is not less than 1.0 × 108CFU/mL, can Guarantee being normally carried out for biological products production, shows that the production seed of four kinds of bacterial strains is stablized in 1~13 generation vigor.

The preparation of 2 complex microbial inoculum of embodiment

The complex microbial inoculum of the present embodiment, including Lactobacillus brevis described in embodiment 1, bifidobacterium longum, shellfish ferment Female bacterium and streptococcus fecalis.

Preparation method is as follows:

A, Lactobacillus brevis freeze-dried powder is prepared；

(1) Lactobacillus brevis is taken, the 50L of the LYT fermentation medium equipped with 35L is seeded to according to 1% inoculum concentration In fermentor, under conditions of temperature is 37 DEG C, the tank pressure of revolving speed 50rpm, fermentor is 0.05Mpa, ferment 18h, right Number growth period obtains one grade fermemtation liquid；Then, the one grade fermemtation liquid is taken, is seeded to according to 8% inoculum concentration and is sent out equipped with LYT In the fermentor of ferment culture medium, under conditions of temperature is 37 DEG C, the tank pressure of revolving speed 150rpm, fermentor is 0.05Mpa, Anaerobic culturel 20h obtains the second order fermentation liquid of logarithmic phase growth；The second order fermentation liquid is taken again, according to 7.5% inoculum concentration It is seeded in LYT fermentation medium, in the condition that temperature is 37 DEG C, the tank pressure of revolving speed 150rpm, fermentor is 0.05Mpa Under, Anaerobic culturel for 24 hours, obtains the three grade fermemtation liquid of the Lactobacillus brevis of logarithmic phase growth.

Wherein, the pH value of the LYT fermentation medium is 7.0, specifically includes the raw material of following parts by weight: peptone 2.0 Parts by weight；2.0 parts by weight of yeast extract；2.0 parts by weight of glucose；4.0 parts by weight of trace salt, 0.05 weight of L-cysteine salt Measure part；

The trace salt includes the following raw material: calcium chloride 0.2g/L, magnesium sulfate 0.48g/L, sodium bicarbonate 10g/L, phosphoric acid Potassium dihydrogen 1.0g/L, dipotassium hydrogen phosphate 1.0g/L, sodium chloride 2.0g/L.

Since Lactobacillus brevis is facultative anaerobic bacteria, Anaerobic culturel is grown more preferable, therefore, using described in above method culture Lactobacillus brevis, strain activity, flourish situation are more excellent.It further include in preparation institute as the preferred implementation of the present embodiment The step of testing when stating one grade fermemtation liquid, second order fermentation liquid, three grade fermemtation liquid, it is specific as follows: to obtain the level-one hair Zymotic fluid should be pure, and OD₆₀₀Value is 1.8-2.3；The obtained second order fermentation liquid should be pure, and OD₆₀₀Value is 1.8-2.4；? The three grade fermemtation liquid arrived should be pure.

(2) fermentation liquid that fermentation obtains in step (1) is taken, is placed it in centrifuge, revolving speed 8000r/min, It is centrifuged 60min, collects bacterium mud；

(3) be added the protective agent into bacterium mud obtained in step (2), the protective agent be sucrose, skimmed milk power and Gelatin is mixed according to the weight ratio of 5:3:1.The sucrose, the skimmed milk power, the gelatin are uniformly mixed, through 115 DEG C sterilizing 30min, after being cooled to room temperature, aseptically with the bacterium mud according to 3:1 weight ratio mix, bacterium is made Suspension, pre-freeze 3h at a temperature of placing it in -40 DEG C, distil 15h at a temperature of being subsequently placed in -15 DEG C, then is placed in 25 DEG C of temperature The lower 2h that distils of degree makes the water content of the bacterium mud less than 5% to get Lactobacillus brevis freeze-dried powder after placing 2h under vacuum.

B, bifidobacterium longum freeze-dried powder is prepared

(1) take the bifidobacterium longum, temperature be 37 DEG C at, initial pH value be 7.5 solidification cow's milk in culture 36~ 48 hours, as the preferred implementation of the present embodiment, the bifidobacterium longum was seeded to by 3% inoculum concentration as culture On the solidification cow's milk of base, and the culture by the way of shaking flask culture；

(2) fermentation liquid that fermentation obtains in step (1) is taken, is placed it in centrifuge, revolving speed 8000r/min, It is centrifuged 30min, collects bacterium mud；

(3) be added the protective agent into bacterium mud obtained in step (2), the protective agent be sucrose, skimmed milk power and Gelatin is mixed according to the weight ratio of 5:3:1.The sucrose, the skimmed milk power, the gelatin are uniformly mixed, through 115 DEG C sterilizing 30min, after being cooled to room temperature, aseptically with the bacterium mud according to 3:1 weight ratio mix, bacterium is made Suspension, pre-freeze 3h at a temperature of placing it in -40 DEG C, distil 15h at a temperature of being subsequently placed in -15 DEG C, then is placed in 25 DEG C of temperature The lower 2h that distils of degree makes the water content of the bacterium mud less than 5% to get bifidobacterium longum freeze-dried powder after placing 2h under vacuum.

C, saccharomyces bayanus bacterium freeze-dried powder is prepared；

(1) bacterium solution for taking the logarithmic growth phase obtained after saccharomyces bayanus bacterium culture 6h-12h, as primary seed solution, Primary seed solution is inoculated in fermentation medium according to the inoculum concentration of 4-10%, temperature be 28-32 DEG C, revolving speed 150- Under conditions of 200rpm, 24-36h is cultivated, one grade fermemtation liquid is obtained；Then, the one grade fermemtation liquid is taken, according to 4-10%'s Inoculum concentration is seeded in fermentation medium, under conditions of temperature is 28-32 DEG C, revolving speed is 100-180rpm, cultivates 4-6h, Obtain second order fermentation liquid；The second order fermentation liquid is taken again, is seeded in fermentation medium according to the inoculum concentration of 4-10%, in temperature Under conditions of degree is 28-32 DEG C, revolving speed is 150-250rpm, 14-16h is cultivated, three grade fermemtation liquid is obtained.

Wherein, the pH value of the fermentation medium is 6.0-6.5, and including following ingredient: yeast extract, peptone, Portugal Grape sugar and trace salt.As the preferred implementation of the present embodiment, the fermentation medium includes the raw material of following parts by weight: 5 Parts by weight yeast extract, 10 parts by weight peptones, 5 parts by weight glucose, 5 parts by wt NaCl, 1 parts by weight trace salt.It is described Trace salt is in calcium chloride, magnesium sulfate, sodium bicarbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and sodium chloride according to 1:2:6:4:1: 2 weight ratio mixes.

(3) be added the protective agent into bacterium mud obtained in step (2), the protective agent be sucrose, skimmed milk power and Gelatin is mixed according to the weight ratio of 5:3:1.The sucrose, the skimmed milk power, the gelatin are uniformly mixed, through 115 DEG C sterilizing 30min, after being cooled to room temperature, aseptically with the bacterium mud according to 3:1 weight ratio mix, bacterium is made Suspension, pre-freeze 3h at a temperature of placing it in -40 DEG C, distil 15h at a temperature of being subsequently placed in -15 DEG C, then is placed in 25 DEG C of temperature The lower 2h that distils of degree makes the water content of the bacterium mud less than 5% to get saccharomyces bayanus bacterium freeze-dried powder after placing 2h under vacuum.

D, streptococcus fecalis freeze-dried powder is prepared；

(1) streptococcus fecalis is taken, is seeded in one grade fermemtation culture medium and cultivates according to 1% inoculum concentration, in temperature Under conditions of being 100-180rpm for 37 ± 3 DEG C, revolving speed, ferment 12-14h, obtains one grade fermemtation liquid；Then, the level-one is taken Fermentation liquid is seeded in second order fermentation culture medium according to the inoculum concentration of 5-10%, is 37 ± 3 DEG C, revolving speed 100- in temperature Under conditions of 180rpm, 4-6h is cultivated, second order fermentation liquid is obtained；The second order fermentation liquid is taken again, according to the inoculum concentration of 5-12% It is seeded in three grade fermemtation culture medium, under conditions of temperature is 37 ± 3 DEG C, revolving speed is 150-250rpm, cultivates 14-16h, Obtain three grade fermemtation liquid.Wherein, the one grade fermemtation culture medium, the second order fermentation culture medium, the three grade fermemtation culture medium For same culture medium, pH value is 7.0-7.4, and includes following component: 1.5 parts by weight of peptone；Yeast extract powder 0.6 parts by weight；2 parts by weight of glucose；0.05 parts by weight of soluble starch；0.5 parts by weight of sodium chloride；L-cysteine salt 0.05 parts by weight；20 parts by weight of Tomato juice；0.01 parts by weight of Tween-80；0.5 parts by weight of lactoalbumin hydrolysate；2 weight of agar powder Measure part.

(3) be added the protective agent into bacterium mud obtained in step (2), the protective agent be sucrose, skimmed milk power and Gelatin is mixed according to the weight ratio of 5:3:1.The sucrose, the skimmed milk power, the gelatin are uniformly mixed, through 115 DEG C sterilizing 30min, after being cooled to room temperature, aseptically with the bacterium mud according to 3:1 weight ratio mix, bacterium is made Suspension, pre-freeze 3h at a temperature of placing it in -40 DEG C, distil 15h at a temperature of being subsequently placed in -15 DEG C, then is placed in 25 DEG C of temperature The lower 2h that distils of degree makes the water content of the bacterium mud less than 5% to get streptococcus fecalis freeze-dried powder after placing 2h under vacuum.

E, the mixing of four kinds of freeze-dried powders；

By the freeze-drying of the freeze-dried powder, the bifidobacterium longum of the Lactobacillus brevis being prepared in step A, B, C, D The freeze-dried powder of powder, the freeze-dried powder of the saccharomyces bayanus bacterium and the streptococcus fecalis, by four kinds of freeze-dried powders according to the weight of 1:1:1:1 Than be uniformly mixed to get.

The preparation of 3 complex microbial inoculum of embodiment

The complex microbial inoculum of the present embodiment, including Lactobacillus brevis described in embodiment 1, bifidobacterium longum, shellfish ferment Female bacterium and streptococcus fecalis.Preparation method is as follows:

The preparation method of the complex microbial inoculum, specifically comprises the following steps:

(3) protective agent, the protective agent and the bacterium mud are separately added into bacterium mud obtained in step (2) Weight ratio is 3:1, is uniformly mixed, pre-freeze 3h at a temperature of placing it in -40 DEG C distils at a temperature of being subsequently placed in -15 DEG C 15h, then the 2h that distils at a temperature of being placed in 25 DEG C make the water content of the bacterium mud less than 5%, obtain after placing 2h under vacuum The freeze-dried powder of the Lactobacillus brevis, the freeze-dried powder of the bifidobacterium longum, the saccharomyces bayanus bacterium freeze-dried powder and the excrement hammer The freeze-dried powder of bacterium, by four kinds of freeze-dried powders be uniformly mixed to get.Wherein, the protective agent is that sucrose, skimmed milk power and gelatin are pressed It is mixed according to the weight ratio of 5:3:1.

The preparation of 4 bifidobacterium longum freeze-dried powder of embodiment

It is added and protects into the Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis respectively Agent is protected, is uniformly mixed, pre-freeze 1-3h at a temperature of placing it in -30~-50 DEG C is subsequently placed at a temperature of -10~-20 DEG C Distil 10-20h, then the 10-20h that distils at a temperature of being placed in -10~-25 DEG C, then in 20-30 DEG C of at a temperature of secondary distillation 1- 5h, under vacuum to get the freeze-dried powder of the Lactobacillus brevis, the freeze-dried powder of the bifidobacterium longum, the shellfish after placement 1-5h The freeze-dried powder of saccharomycete and the freeze-dried powder of the streptococcus fecalis, by four kinds of freeze-dried powders be uniformly mixed to get.

In the present embodiment, the protective agent is skimmed milk power, be alternative implementation as the present embodiment, the guarantor Shield agent also can be replaced skimmed milk power, maltose, sucrose, soluble starch, mannitol, sodium glutamate, L-cysteine, bright One of glue is a variety of.

Effete test embodiment

Experiment one, the preparation storage life test of the complex microbial inoculum

Take the Lactobacillus brevis that deposit number is CGMCC NO.16125, deposit number be CGMCC NO.16126 The bifidobacterium longum, deposit number be CGMCC NO.16127 the saccharomyces bayanus bacterium, deposit number CGMCC The streptococcus fecalis of NO.16128, basic bacteria freeze-dried powder is saved under the conditions of -80 DEG C, and storage life is 2 years, every two years Detection is primary.

Wherein, the method for count plate is specific as follows:

It is sterile to weigh bifidobacterium longum preparation described in 3g, 27mL fluid nutrient medium or physiological saline is added, sufficiently shakes up (shaken and beaten with several glass marbles) is made 10 times afterwards and is serially diluted, and acceptable diluent degree is selected to be inoculated with.It is selectively cultivated using LBS Each dilution of base, bacterium is inoculated with 3 plates, and rapid pivotal plate by 0.2mL, keeps bacterium solution even in media surface stream. After culture 48 hours, the bacterial growth situation on each plate is observed, and count.When the clump count on each plate is less than 10 Or when being greater than 300, last dilution should be readjusted, is redeterminated.It is calculated according to the following formula according to 3 plate total plate counts Viable count:

Testing result is as follows:

Bacterial strain saves the viable count of different time at -80 DEG C

Bacterial strain saves the viable count of different time at 4 DEG C

Bacterial strain saves the viable count of different time at 25 DEG C

Test the viable count comparative experiments of two kinds of bacterial strains of two, complex microbial inoculum

The Lactobacillus brevis freeze-dried powder being prepared in Example 2, is denoted as CGMCC No.16125 group；In taking Deposit number is the Lactobacillus brevis of CGMCC No.5760 in state patent document CN102660477A, according to the method in the document Freeze-dried powder is prepared, is denoted as CGMCC No.5760 group；It is protected at normal temperature according to the bacterium powder that the method for experiment one detects two groups Deposit the viable count of different time.

Its experimental result is as follows:

The bifidobacterium longum freeze-dried powder being prepared in Example 2, is denoted as CGMCC No.

16126 groups；Take the long bifid that deposit number is CGMCC No.6283 in Chinese patent literature CN104120093A Freeze-dried powder is prepared according to the method in embodiment 2 in bacillus, is denoted as CGMCC No.6283 group；According to the method for experiment one Detect the viable count that two groups of bacterium powder saves different time at normal temperature.

Its experimental result is as follows:

Experiment three, the acid and bile salt tolerance characteristic test of bacterial strain

In order to simulate the intracorporal gastrointestinal tract environment of animal, simulated gastric fluid and simulated intestinal fluid are prepared, is carried out external acidproof resistance to The research of gallbladder performance.Furthermore action time is also one of the major issue be considered as when such test.Due to depositing for cholate In the permeability for changing thallus outer membrane, so generating inhibition, killing effect to probiotics, and then the survival of probiotics is influenced.

(1) Lactobacillus brevis

Stomach juice-resistant test: taking deposit number is the Lactobacillus brevis of CGMCC No.16125, using simulated gastric fluid as base Plinth, has then been activated the liquid culture in two generations by the access of 10% inoculum concentration, and 37 DEG C of stationary cultures take respectively at 0,1,2,4h Sample measures viable count.

The salt test of resistance to cholic acid: taking deposit number is the Lactobacillus brevis of CGMCC No.16125, using simulated intestinal fluid as base 0.3% No. 3 cholate are added in plinth, then activate the Bifidobacterium liquid culture in two generations according to 10% inoculum concentration access, 37 DEG C of stationary cultures are measured by sampling viable count, and calculate survival rate respectively at 0,1,2,4h.

The testing result of stomach juice-resistant is as follows:

The testing result of resistance to cholate is as follows:

(2) bifidobacterium longum

Stomach juice-resistant test: taking deposit number is the bifidobacterium longum of CGMCC No.16126, is placed in simulated gastric fluid In, 37 DEG C of stationary culture 2h are measured by sampling viable count, and calculate survival rate.

The salt test of resistance to cholic acid: taking deposit number is the bifidobacterium longum of CGMCC No.16126, is with simulated intestinal fluid 0.3% cholate is added in basis thereto, and then the bifidobacterium longum is placed in one, in 37 DEG C of stationary culture 2h, sampling Viable count is measured, and calculates survival rate.

The testing result of stomach juice-resistant is survival rate 66.0%；The testing result of resistance to cholate is survival rate 51.1%.

(3) streptococcus fecalis

Stomach juice-resistant test: taking deposit number is the streptococcus fecalis of CGMCC No.16128, is placed in simulated gastric fluid, 37 DEG C of stationary cultures 0h, 1h, 2h, 4h are measured by sampling viable count, and calculate survival rate.

Its experimental result is as follows:

Time	0h	1h	2h	4h
					Viable bacteria rate (%)	100	90.0	87.7	73.8

The salt test of resistance to cholic acid: taking deposit number is the streptococcus fecalis of CGMCC No.16128, using simulated intestinal fluid as base 0.3% cholate is added in plinth thereto, and then the streptococcus fecalis is placed in one, in 37 DEG C of stationary cultures 0h, 1h, 2h, 4h, Viable count is measured by sampling, and calculates survival rate.Its experimental result is as follows:

Time	0h	1h	2h	4h
					Viable bacteria rate (%)	100	83.6	73.6	65.4

Also commercially available three kinds of streptococcus fecalis are tested using the above method in this experiment, commercially available three kinds of excrement hammers Bacterium simulation hydrochloric acid, cholate environment in cultivate 4h after, viable bacteria rate is below 43%.It can be seen that preservation of the invention is compiled Number for CGMCC No.16128 the streptococcus fecalis acidproof and bile tolerance function admirable.

(4) saccharomyces bayanus bacterium

Stomach juice-resistant test: taking deposit number is the saccharomyces bayanus bacterium of CGMCC No.16127, is accessed by 10% inoculum concentration In simulated gastric fluid, 37 DEG C of stationary cultures 0h, 1h, 2h, 4h are measured by sampling viable count, and calculate survival rate.Its experimental result is such as Under:

Time	0h	1h	2h	4h
					Viable bacteria rate (%)	100	100	93	76.7

The salt test of resistance to cholic acid: taking deposit number is the saccharomyces bayanus bacterium of CGMCC No.16127, using simulated intestinal fluid as base Plinth, then 0.3% cholate is added thereto, then the saccharomyces bayanus bacterium is accessed wherein according to 10% inoculum concentration, quiet at 37 DEG C Culture 0h, 1h, 2h, 4h are set, viable count is measured by sampling, and calculate survival rate.Its experimental result is as follows:

Time	0h	1h	2h	4h
					Viable bacteria rate (%)	100	81.4	72.5	57.5

Also commercially available three kinds of saccharomyces bayanus bacterium are tested using the above method in this experiment, commercially available three kinds of saccharomyces bayanus Bacterium simulation hydrochloric acid, cholate environment in cultivate 4h after, viable bacteria rate is below 51%.It can be seen that preservation of the invention is compiled Number for CGMCC No.16127 the saccharomyces bayanus bacterium acidproof and bile tolerance function admirable.

Experiment four inhibits pathogen capacity experimental

(1) Lactobacillus brevis: taking deposit number of the invention is the Lactobacillus brevis of CGMCC No.16125, Yi Jizhong Deposit number is the Lactobacillus brevis of CGMCC No.5760 in state patent document CN102660477A, detects it respectively to virulent big The suppression of enterobacteria EC82-86, virulent salmonella C79-14, staphylococcus saprophyticus SS9084 (being provided by Chinese medicine inspecting institute) Bacterium radius, and record.Its testing result is as follows:

(2) bifidobacterium longum: take deposit number of the invention be CGMCC No.16126 the bifidobacterium longum, in Deposit number is the bifidobacterium longum of CGMCC No.6283 in state patent document CN104120093A, then takes and supervised by Chinese drug Examine provided pathogenic bacteria: pathogenic virulent Escherichia coli 44365, pathogenic virulent salmonella C-79-14, pathogenic virulent Salmonella C-79-20.Each bacterial strain is detected respectively to the antibacterial radius of each pathogenic bacteria, and is recorded.Its testing result is as follows:

(3) streptococcus fecalis: taking deposit number of the invention is the streptococcus fecalis, commercially available of CGMCC No.16128 Streptococcus fecalis, then take and provided pathogenic bacteria: pathogenic virulent Escherichia coli 44365, pathogenic virulent sand are supervised by Chinese drug Door Salmonella C-79-14, pathogenic virulent salmonella C-79-20.Each bacterial strain is detected respectively to the antibacterial radius of each pathogenic bacteria, And it records.Its testing result is as follows:

(4) complex microbial inoculum in embodiment 2: detecting according to the method described above, to the antibacterial radius of each pathogenic bacteria, And it records.Its testing result is as follows: Escherichia coli 38mm, salmonella 32mm, staphylococcus 37mm.It can be seen that of the invention It, can be with mutualism without mutual antagonism between four kinds of bacterial strains of the complex microbial inoculum.

The influence experiment of experiment five, protective agent to freeze-dried powder technique

This experiment is investigated and is acted on protectant frozen-dried protective using survival rate as index.In the way of in embodiment 2 Prepare the complex microbial inoculum, difference is only that: protective agent is replaced with respectively skimmed milk power, maltose, sucrose, can Bifidobacterium longum is prepared in soluble starch, sodium glutamate, L-cysteine, gelatin, mannitol, and that tests respectively is micro- Biological compound fungi agent saves the survival rate after 30 days at normal temperature.

It can be seen that the effect is unsatisfactory as protectant for single ingredient, and protective agent is to the Bifidobacterium Protecting effect differs greatly.In order to verify the stability of the technique, experimental verification three times is carried out to the scheme in embodiment 2, it is living Bacterium rate resultant error rate < 4%, it was demonstrated that result is reliable and stable.

Complex microbial inoculum is prepared in the way of in embodiment 2, difference is only that: protective agent is replaced with respectively Three kinds of skimmed milk power, sucrose, gelatin ingredients are mixed according to the weight ratio of 1:1:1, and are denoted as first group；By defatted milk Three kinds of powder, sucrose, gelatin ingredients mix according to the weight ratio of 1:3:2, and are denoted as second group；By skimmed milk power, sucrose, Three kinds of ingredients of gelatin are mixed according to the weight ratio of 1:5:3, and are denoted as third group；By skimmed milk power, sucrose, three kinds of gelatin Ingredient is mixed according to the weight ratio of 2:1:2, and is denoted as the 4th group；By skimmed milk power, sucrose, three kinds of ingredients of gelatin according to The weight ratio of 2:3:3 mixes, and is denoted as the 5th group；By three kinds of skimmed milk power, sucrose, gelatin ingredients according to 2:5:1's Weight ratio mixing, and it is denoted as the 6th group；Three kinds of skimmed milk power, sucrose, gelatin ingredients are mixed according to the weight ratio of 3:1:3 It closes, and is denoted as the 7th group；By skimmed milk power, sucrose, three kinds of ingredients of gelatin according to 3:3:2 weight ratio mix, and by its It is denoted as the 8th group；The complex microbial inoculum that embodiment 2 is prepared is denoted as the 9th group.Above-mentioned each group is tested respectively to obtain Bifidobacterium longum freeze-dried powder save the survival rate after 30 days at normal temperature.

Its testing result is as follows:

Group	1	2	3	4	5	6	7	8	9
										Viable bacteria rate (%)	53.8	65.9	71.6	64.4	83.0	84.4	64.1	74.7	91.7

Experiment six, the complex microbial inoculum are to small white mouse safety testing

The small white mouse of 20-22g is taken, cleaning grade test uses small white mouse 40, divides 4 groups, every group 10, wherein 1-3 group is given The active bacteria formulation for the complex microbial inoculum being prepared in embodiment 2, dosage are not less than 1.0 × 10⁷CFU gives The medicine period is 3 days, after administration, observes the clinical symptoms of all small white mouses, evaluates test result.

The clinical settings such as spirit, feeding, drinking-water, growth, weight gain of small white mouse after observation medicine feed, and observation experimental period Between animal whether have an adverse reaction relevant to drug, such as breathing, abnormal behavior, spirit inhibits and the abnormal variation feelings of defecation Condition.And the weight for recording every group of small white mouse on the 13rd day that the same day (the 1st day) and experiment terminate before administration, calculate small white mouse Average weight increasing a day.

Its experimental result is as follows:

Each group small white mouse during test is all strong to live, and spirit is good, and honey stomach, the fur colour of skin is normal, defecation urination Color form is normal, no other abnormal clinical symptoms, without sick and dead show.

It weighs within the 13rd day, and calculates to every group of small white mouse after the same day (the 1st day), experiment before administration Each group average daily gain.As a result it see the table below:

Find that the small white mouse Average weight increasing a day of 3 experimental groups is apparently higher than the 4th group of blank control, shows this hair from table The bright complex microbial inoculum has obvious effect of gain to small white mouse, and it is qualified that safety examination is administered.

Experiment seven, the complex microbial inoculum test the improvement of dog intestinal bacilli illness Diarrhea Model

This test, as induced drug, leads to beasle dog enteric flora disturbance, structure by being administered continuously using cefalexin Test dog antibiotic-associated diarrhea is built, specifically comprises the following steps: to take for 3.5~4 monthly ages, 5.0~6.5kg's of weight is common Grade test uses beasle dog 25, wherein 5, as a control group, in addition 20 in such a way that gradient increases induced drug dosage, Continuously give antibiotic induction, building test dog intestinal bacilli illness Diarrhea Model, antibiotic induce 15~20 days, structure mould at Function.Induce through antibiotic 20 beasle dogs are divided into high dose group, middle dose group, low dose group, model group, to high dose Group, middle dose group, low dose group give the active bacteria formulation of the bifidobacterium longum of the present invention of high, medium and low dosage respectively, Model group is not administered, and dosage period is 6~8 days, after administration, observes the clinical symptoms of all dogs only.

Its experimental result is as follows:

Beasle dog weight is an index that is intuitive, easily investigating, and closely related with many factors.To pretherapy and post-treatment ratio The weight of lattice dog is analyzed.Different groups of beasle dog weight is compared two-by-two after modeling, the average body of dog is tested between group Weight P > 0.05 illustrates to influence beasle dog changes of weight using Antibiotics model little.It is compound continuously to give the microorganism After microbial inoculum 7 days, model group, low, middle and high dose groups are compared into P > 0.05 two-by-two, illustrates the microbial inoculum of various dose to test dog Weight influence it is not significant.

Before modeling, each group beasle dog is vivaciously active, and coat is flat and smooth, excrement dry forming, more normally.Modeling knot Shu Shi, control group beasle dog diet is normal, and action is active, defecation frequency rule, excrement dry forming.Through antibiotic induction The dilute wet, agility of stool, which gradually occurs, in test dog to be reduced, occasionally has the symptoms such as anorexia or out of strength, vomiting, is belonged to antibiotic and is excessively caused Classical symptom caused by intestinal bacilli illness.

Preparation is as follows: the influence testing result of test dog excrement state

Wherein, excrement state: -- formed stool；+ forming but moisture it is slightly more；++ sticky half congealed loose stools；+++ watery stool.

After continuously giving active bacteria formulation 5 days, by dog clinical symptoms, excrement situation and bacteria detection as a result, statistics is different Curative effect of the dosage group to puppy intestinal bacilli illness disease.Its testing result is as follows:

Group	Number of animals	It cures	It is effective	Effectively	In vain	It is efficient
							Low dose group	5	2	1	4	1	80%
Middle dose group	5	4	1	5	0	100%
							High dose group	5	4	1	5	0	100%

Complex microbial inoculum 2 times a day, is continuously fed 5 days with 2.0g//times, can be alleviated puppy because of enterobacteriaceae Symptom of diarrhea caused by group's deficiency disorder, promotes dog only to restore colony balance.It can be seen that microbial composite bacteria of the present invention It, can be with mutualism without mutual antagonism between four kinds of bacterial strains of agent.And the bifidobacterium longum, the Bacillus acidi lactici, institute Stating streptococcus fecalis has powerful antagonism to pathogen, and then the complex microbial inoculum for forming four kinds of bacterial strains Have the function of good recuperating gastrointestinal tract health, improve immunity.

Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments. For those of ordinary skill in the art, other various forms of changes can also be made on the basis of the above description Change or changes.There is no necessity and possibility to exhaust all the enbodiments.And obvious change extended from this Change or changes still within the protection scope of the invention.

SEQUENCE LISTING

<110>Tan Ying

<120>a kind of complex microbial inoculum and its preparation method and application

<130> 2018

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 1469

<212> DNA

<213>Lactobacillus brevis

<400> 1

ggatgaacgc tggcggcgtg cctaatacat gcaagtcgaa cgagttctcg ttgatgatcg 60

gtgcttgcac cgagattcaa catggaacga gtggcggacg ggtgagtaac acgtgggtaa 120

cctgccctta agtgggggat aacatttgga aacagatgct aataccgcat agatccaaga 180

accgcatggt tcttggctga aagatggcgt aagctatcgc ttttggatgg acccgcggcg 240

tattagctag ttggtgaggt aatggctcac caaggcgatg atacgtagcc gaactgagag 300

gttgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 360

gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaaggcttt 420

cgggtcgtaa aactctgatg ttggagaaga atggtcggca gagtaactgt tgccggcgtg 480

acggtatcca accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 540

tggcaagcgt tatccagatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg 600

atgtgaaagc cctcggctta accgaggaag cgcatcggaa actgggaaac ttgagtgcag 660

aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720

gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagca tgggtagcga 780

acaggattag ataccctggt agtccatgcc gtaaacgatg aatgctaggt gttggagggt 840

ttccgccctt cagtgccgca gctaacgcat taagcattcc gcctggggag tacgaccgca 900

aggttgaaac tcaaaggaat tgacgggggc acgcacaagc ggtggagcat gtggtttaat 960

tcgaagcaac gcgaagaacc ttaccaggtc ttgacatctt ttgatcactt gagagatcag 1020

gtttcccctt cgggggcaaa atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080

gatgttgggt taagtcccgc aacgagcgca acccttatga ctagttgcca gcatttagtt 1140

gggcactcta gtaagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1200

tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga 1260

gaccgcgagg tcaagctaat ctcttaaagc cattctcagt tcggactgta ggctgcaact 1320

cgcctacacg aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1380

cccgggcctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc gaagccggtg 1440

gcgtaaccct tttagggagc gagccgtct 1469

<210> 2

<211> 1448

<212> DNA

<213>bifidobacterium longum

<400> 2

caggatgaac gctggcggcg tgcttaacac atgcaagtcg aacgggatcc ctggcagctt 60

gctgtcgggg tgagagtggc gaacgggtga gtaatgcgtg accaacctgc cctgtgcacc 120

ggaatagctc ctggaaacgg gtggtaatac cggatgctcc gctccatcgc atggtggggt 180

gggaaatgct tttgcggcat gggatggggt cgcgtcctat cagcttgttg gcggggtgat 240

ggcccaccaa ggcgttgacg ggtagccggc ctgagagggt gaccggccac attgggactg 300

agatacggcc cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag 360

cctgatgcag cgacgccgcg tgcgggatgg aggccttcgg gttgtaaacc gcttttgttc 420

aagggcaagg cacggtttcg gccgtgttga gtggattgtt cgaataagca ccggctaact 480

acgtgccagc agccgcggta atacgtaggg tgcgagcgtt atccggattt attgggcgta 540

aagggctcgt aggcggttcg tcgcgtccgg tgtgaaagtc catcgcctaa cggtggatct 600

gcgccgggta cgggcgggct ggagtgcggt aggggagact ggaattcccg gtgtaacggt 660

ggaatgtgta gatatcggga agaacaccaa tggcgaaggc aggtctctgg gccgtcactg 720

acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780

taaacggtgg atgctggatg tggggccctt tccacgggtc ccgtgtcgga gccaacgcgt 840

taagcatccc gcctggggag tacggccgca aggctaaaac tcaaagaaat tgacgggggc 900

ccgcacaagc ggcggagcat gcggattaat tcgatgcaac gcgaagaacc ttacctgggc 960

ttgacatgtg ccggatcgcc gtggagacac ggtttccctt cggggccggt tcacaggtgg 1020

tgcatggtcg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080

ccctcgccgc atgttgccag cgggtgatgc cgggaactca tgtgggaccg ccggggtcaa 1140

ctcggaggaa ggtggggatg acgtcagatc atcatgcccc ttacgtccag ggcttcacgc 1200

atgctacaat ggccggtaca acgcggtgcg acacggtgac gtggggcgga tcgctgaaaa 1260

ccggtctcag ttcggatcgc agtctgcaac tcgactgcgt gaaggcggag tcgctagtaa 1320

tcgcggatca gcaacgccgc ggtgaatgcg ttcccgggcc ttgtacacgc cgcccgtcaa 1380

gtcatgaaag tgggtagcac ccgaagccgg tggcccgacc cttgtggggg gagccgtcta 1440

aggtgaga 1448

<210> 3

<211> 571

<212> DNA

<213>saccharomyces bayanus bacterium

<400> 3

atgcttagta cggcgagtga gcggcaaaag ctcaaatttg aaatctggta ccttcggtgc 60

ccgagttgta atttggagag ggcaactttg gggccgttcc ttgtctatgt tccttggaac 120

aggacgtcat agagggtgag aatcccgtgt ggcgaggagt gcggttcttt gtaaagtgcc 180

ttcgaagagt cgagttgttt gggaatgcag ctctaagtgg gtggtaaatt ccatctaaag 240

ctaaatattg gcgagagacc gatagcgaac aagtacagtg atggaaagat gaaaagaact 300

ttgaaaagag agtgaaaaag tacgtgaaat tgttgaaagg gaagggcatt tgatcagaca 360

tggtgttttg tgccctctgc tccttgtggg taggggaatc tcgcatttca ctgggccagc 420

atcagttttg gtggcaggat aaatccatag gaatgtagct tgcctcggta agtattatag 480

cctgtgggaa tactgccagc tgggactgag gactgcgacg taagtcaagg atgctggcat 540

aatggttata tgccgcccgt cttgaaacac g 571

<210> 4

<211> 1412

<212> DNA

<213>streptococcus fecalis

<400> 4

gaacgctggc ggcgtgccta atacatgcaa gtagaacgct gaagagagga gcttgctctt 60

cttggatgag ttgcgaacgg gtgagtaacg cgtaggtaac ctgccttgta gcgggggata 120

actattggaa acgatagcta ataccgcata acaatggatg acacatgtca tttatttgaa 180

aggggcaatt gctccactac aagatggacc tgcgttgtat tagctagtag gtgaggtaat 240

ggctcaccta ggcgacgata catagccgac ctgagagggt gatcggccac actgggactg 300

agacacggcc cagactccta cgggaggcag cagtagggaa tcttcggcaa tgggggcaac 360

cctgaccgag caacgccgcg tgagtgaaga aggttttcgg atcgtaaagc tctgttgtaa 420

gtcaagaacg ggtgtgagag tggaaagttc acactgtgac ggtagcttac cagaaaggga 480

cggctaacta cgtgccagca gccgcggtaa tacgtaggtc ccgagcgttg tccggattta 540

ttgggcgtaa agcgagcgca ggcggtttga taagtctgaa gttaaaggct gtggctcaac 600

catagttcgc tttggaaact gtcaaacttg agtgcagaag gggagagtgg aattccatgt 660

gtagcggtga aatgcgtaga tatatggagg aacaccggtg gcgaaagcgg ctctctggtc 720

tgtaactgac gctgaggctc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 780

ccacgccgta aacgatgagt gctaggtgtt ggatcctttc cgggattcag tgccgcagct 840

aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga 900

cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960

ccaggtcttg acatcccgat gctatttcta gagatagaaa gttacttcgg tacatcggtg 1020

acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080

gagcgcaacc cctattgtta gttgccatca ttcagttggg cactctagcg agactgccgg 1140

taataaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200

acacacgtgc tacaatggtt ggtacaacga gttgcgagtc ggtgacggcg agctaatctc 1260

ttaaagccaa tctcagttcg gattgtaggc tgcaactcgc ctacatgaag tcggaatcgc 1320

tagtaatcgc ggatcagcac gccgcggtga atacgttccc gggccttgta cacaccgccc 1380

gtcacaccac gagagtttgt aacacccgaa gt 1412

Claims

1. a kind of complex microbial inoculum, which is characterized in that including Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and excrement hammer Bacterium.

2. complex microbial inoculum according to claim 1, which is characterized in that the deposit number of the Lactobacillus brevis is CGMCC NO.16125；The deposit number of the bifidobacterium longum is CGMCC NO.16126；The preservation of the saccharomyces bayanus bacterium is compiled Number be CGMCC NO.16127；The deposit number of the streptococcus fecalis is CGMCC NO.16128.

3. complex microbial inoculum according to claim 2, which is characterized in that the 16SrDNA sequence of the Lactobacillus brevis With the sequential structure as shown in SEQ ID NO.1；The 16SrDNA sequence of the bifidobacterium longum has such as SEQ ID NO.2 Shown in sequential structure；The 26S rDNA sequence of the saccharomyces bayanus bacterium has the sequential structure as shown in SEQ ID NO.3；It is described The 16SrDNA sequence of streptococcus fecalis has the sequential structure as shown in SEQ ID NO.4.

4. complex microbial inoculum described in any one of claim 1-3 is preparing recuperating gastrointestinal tract health and/or is improving The health care product of immunity, dairy products, the application in drug.

5. application according to claim 4, which is characterized in that the complex microbial inoculum preparation treatment and/or Application in the health care product of pre- anti-diarrhea, drug.

6. a kind of preparation method of complex microbial inoculum, which comprises the steps of: take in claim 1-3 and appoint Lactobacillus brevis, bifidobacterium longum, saccharomyces bayanus bacterium and streptococcus fecalis described in meaning one are made as freeze-dried powder respectively, remix uniformly i.e. ?.

7. the preparation method of complex microbial inoculum according to claim 6, which is characterized in that specifically include following step It is rapid:

Protective agent is added into the Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis respectively, It is uniformly mixed, pre-freeze 1-3h at a temperature of placing it in -30~-50 DEG C, distil 10- at a temperature of being subsequently placed in -10~-20 DEG C 20h, then the 10-20h that distils at a temperature of being placed in -10~-25 DEG C, then in 20-30 DEG C of at a temperature of secondary distillation 1-5h, in vacuum It is lower place 1-5h after to get the freeze-dried powder of the Lactobacillus brevis, the freeze-dried powder of the bifidobacterium longum, the saccharomyces bayanus bacterium jelly Dry powder and the freeze-dried powder of the streptococcus fecalis, by four kinds of freeze-dried powders be uniformly mixed to get.

8. the preparation method of complex microbial inoculum according to claim 7, which is characterized in that the protective agent is degreasing One of milk powder, maltose, sucrose, soluble starch, mannitol, sodium glutamate, L-cysteine, gelatin are a variety of.

9. the preparation method of complex microbial inoculum according to claim 8, which is characterized in that specifically include following step It is rapid:

(1) Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis are taken, respectively fermented and cultured, Obtain the fermentation liquid of the Lactobacillus brevis, the fermentation liquid of the bifidobacterium longum, the fermentation liquid of the saccharomyces bayanus bacterium, the excrement chain The fermentation liquid of coccus；

(2) four kinds of fermentation liquids that fermentation obtains in step (1) are taken, placing it in revolving speed respectively is under 8000-12000r/min, from Heart 10-30min collects bacterium mud, obtains four kinds of bacterium muds；

(3) protective agent, the weight ratio of the protective agent and the bacterium mud are separately added into bacterium mud obtained in step (2) For 3:1, it is uniformly mixed, pre-freeze 3h at a temperature of placing it in -40 DEG C, distil 15h at a temperature of being subsequently placed in -15 DEG C, then sets Distil 2h at a temperature of 25 DEG C, after placing 2h under vacuum, makes the water content of the bacterium mud less than 5%, obtains the short cream The freeze-dried powder of bacillus, the freeze-dried powder of the bifidobacterium longum, the freeze-dried powder of the saccharomyces bayanus bacterium and the freeze-drying of the streptococcus fecalis Powder, by four kinds of freeze-dried powders be uniformly mixed to get.

10. the preparation method of complex microbial inoculum according to claim 9, which is characterized in that described in step (1) Lactobacillus brevis, the bifidobacterium longum, the saccharomyces bayanus bacterium and the streptococcus fecalis obtain fermentation liquid by three grade fermemtation.