CN105567598A

CN105567598A - Tibetan pig origin bacillus amyloliquefaciens and application thereof

Info

Publication number: CN105567598A
Application number: CN201610027456.6A
Authority: CN
Inventors: 曹斌云; 杨伟平; 王建刚; 王方圆; 赵越; 徐然; 于占涛; 程颍州
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2016-01-15
Filing date: 2016-01-15
Publication date: 2016-05-11
Anticipated expiration: 2036-01-15
Also published as: CN105567598B

Abstract

The invention discloses Tibetan pig origin bacillus amyloliquefaciens and application thereof. The disclosed Tibetan pig origin bacillus is named as bacillus amyloliquefaciens BY-5 through morphological identification, biochemical identification and molecular identification, and the preservation number is CCTCC NO: M 2013739. The bacillus amyloliquefaciens is separated from Tibetan pig cecum contents, can effectively produce cellulose clastic enzyme and has the capability of degrading sodium carboxymethyl cellulose. In addition, the in-vitro characteristic evaluation on the strain shows that the strain has a strong adverse-resistant characteristic, can be resistant to heat, acid and bile salt and also has a certain capability of inhibiting escherichia coli, salmonella and staphylococcus aureus. Furthermore, the Tibetan pig origin bacillus amyloliquefaciens also has a certain capability of inhibiting adhesion of three types of pathogenic bacteria to the mucous membranes of small intestines of pigs.

Description

A kind of Tibetan pig source bacillus amyloliquefaciens and application thereof

Technical field

The invention belongs to microorganism field, be specifically related to a kind of Tibetan pig source bacillus amyloliquefaciens and application thereof.

Background technology

1, the source of cellulose-decomposing bacterium and separation:

In livestock industry and fodder industry, in order to alleviate the situation of current energy feed shortage, the separation screening of the cellulose-decomposing bacterium of application of cellulase and cellulase-producing is taken seriously always.From the source of cellulase, microorganism is the topmost source of cellulase.At present, occurring in nature can comprise fungi, bacterium, actinomycetes to the microorganism that baroque Mierocrystalline cellulose carries out effectively degrading, wherein the degradation capability of fungi is the strongest, mainly comprises the bacterial strain of Trichoderma, Aspergillus, Penicillium and the mould genus of branch top spore, the mould genus of paint spot etc.In these fungies, fungus Trichoderma because of produce institute and formed and eccrine fiber element enzyme set member the most comprehensively, enzyme amount is large, active high and be widely used in zymin production, wherein the higher representative bacterial classification of activity has: koning trichoderma, aspergillus niger, Trichodermareesei etc. ^[1-3].And relative fungi, bacterium has cultivates the advantages such as simple, fast growth, fermentation period are short, therefore, filters out can also result in the extensive concern of people by the cellulosic cellulose decomposing bacteria of efficient degradation from occurring in nature ^[4].At present, in the separation screening of cellulose-decomposing bacterium, the multiple sieve method of the dull and stereotyped just sieve method of usual employing Xylo-Mucine (CMC-Na) and mensuration cellulase activity combines, and wherein the cellulose-decomposing bacterium of bacillus, Ruminococcus, Bacteroides, fusobacterium, Cellulomonas etc. is all from the cud of ox ^[5], hide pig ^[6]and the ight soil of giant panda ^[7], grass carp intestinal ^[8], soil ^[9], goose gi tract ^[10]and marine bacteria ^[11]deng in be separated out.

2, the application of cellulase preparation:

Mierocrystalline cellulose is extensively present in natural polysaccharose substance, and cellulosic material can be hydrolyzed into monose and then fermentative production of ethanol, hydrogen and microbial oil etc. by cellulase effectively, therefore can alleviate global energy pressure to its Appropriate application ^[12].In animal productiong, cellulase, as a kind of microbial preparation, mainly uses in the mode of directly adding in daily ration.As Lv Donghai (2002) research is pointed out, cellulase is added in the daily ration of meat duck, the early stage average daily gain of meat duck can not only be improved, to crude fat utilization ratio, to dry-matter and crude fiber digestibility, and the Alimentary relative size of meat duck, the duodenal relative length of increase meat duck Later growth can be reduced to some extent, and the trend that Shi Rouya intestinal mucosa lower floor, muscle layer and total thickness are thinning ^[13].Gaoyang etc. (2014) research is pointed out, adds non-starch polysaccharide enzyme (zytase, beta-glucanase, cellulase) and can effectively improve growth of growing-finishing pigs performance, and improve meat to a certain extent in the daily ration of growing swine ^[14].In addition, in monogastric animal, the loss that cellulase effectively can reduce ammonia-state nitrogen in ight soil and urine is added ^[15], therefore, cellulase has also been used in the composting treatment process such as animal excrement, rubbish by Many researchers, to reduce the source of foul smell, greatly improves plant's feeding environment, improves animal welfare.In addition, in paper-making industry and foodstuffs industry and textile industry etc. are produced, cellulase also has purposes and application prospect very widely ^[12].

3, the research and development of probiotics-probiotic bacterium and application:

Microbiotic is 20th century most important medical discovery, in treatment human and animal disease, played great effect ^[16].Along with greatly developing of intensive culture industry, in domestic animal and poultry production, almost 90% microbiotic with sub-treatment level be added in feed for the prevention and corntrol of Animal diseases, promote growth of animals or poultry, improve food conversion ratio ^[17 _, ^18].Due to antibiotic incorrect use and long-term abuse, inevitably suppress and damaged part profitable strain, causing the dysfunction of animal digestive tract, diseases induced; Result also in the increase of animal body endurance strain, these endurance strains pass through directly between humans and animals simultaneously ^[19 _, ^20]indirectly ^[21 _, ^22]form propagate.In addition, the residual health to the mankind of microbiotic in edible animal body and meat product also result in safely serious threat ^[18 _, ^23].Therefore, the many scientific workers of recent domestic do one's utmost to seek and develop have no side effect, noresidue, can promote growth of animal, again can the Substitutes For Antibiotic of disease preventing and treating.

Probiotics because having the irreplaceable advantage of other drug, i.e. the effect of " ill cure the disease, not sick diseases prevention, anosis health care ", and more and more being received an acclaim by as the application of green feed additive in animal cultivation ^[24].Wherein probiotic bacterium is a class produces the work of beneficial effect to host animal microorganism feed addictive by promoting intestinal microbial balance, is an antibiotic potential substitute.Show after deliberation, probiotic bacterium at raising breeding performonce fo animals, improve microbial population of animal intestinal tract and reduce in disease generation etc. and played important effect ^[25-27].Japan brings into use probiotic bacterium in nineteen sixty, and China brought into use probiotic bacterium in 1980, united States food and drug administration (US-FDA)) disclosing 42 kinds in 1989 can the microbial strains safely and effectively of Direct-fed ^[26].End on June 30th, 2013, FDA " generally recognized as safe material " (generallyrecognizedassafe, GRAS), in inventory, what relate to microbial strains and microbial strains source has 108 records, contains 40,1 order and belongs to 66 kinds 5 subspecies ^[28].And on our country basis of 16 kinds of feed microbe additives of announcing at 2008 editions (notification numbers 1126) that the Ministry of Agriculture issues, increase 19 kinds of microbe additives newly in the feed microbe catalogue of the Ministry of Agriculture's (No. 2045, notification number) newly promulgated for 2013, and subjects has been specified ^[29].As can be seen here, probiotic bacterium becomes the focus of research as a kind of novel green feed additive, and is widely used gradually in animal productiong.In European Union, the probiotic bacterium being widely used as fodder additives mainly comprises bacillus (bacillus cereus, Bacillus licheniformis and subtilis), enterococcus spp (faecium), lactobacillus genus (cheese milk-acid bacteria, lactobacterium acidophilus, Lactobacillus farciminis, plant lactobacillus, lactobacillus rhamnosus), Mycosphaerella (addicted to sour micrococcus), streptococcus and some fungies as yeast saccharomyces cerevisiae and kluyveromyces etc. ^[30], and mostly have positive effect to the growth of animal and the prevention of disease.Wherein genus bacillus is because of good characteristics such as its strong stress resistance, high temperature high voltage resistants, easily storage, and has the various functions such as regulating intestinal canal colony balance, enhancing animal immunizing power, raising production performance, is considered to optimal microbe additive.As (2014) researchs such as Lee are pointed out, the probiotic bacterium subtilis LS1-2 fermentation thalli of the different amount of supplement in pig diet, all can increase the average daily gain of different steps wean pig, average daily ingestion amount and the digestibility to nutritive substance; Meanwhile, the change of the cell concentration in the change in concentration of IgG and IgA in blood, small intestinal villous height and Crypt depth and diet is linear, and the clostridium in caecum and coliform number then increase along with biomass concentration in diet and reduce ^[25].In addition, probiotics also has certain removal effect and successful to the stench that plant produces.As the complex microorganism preparations be made up of genus bacillus, streptomyces griseus and Candida tropicalis is used for the deodorizing test of hoggery pigsties and dung yard by Ye Fenxia etc., result shows, NH in pig house ₃, H ₂s and odour concentration reduce 78.4%, 66.7% and 83.3% respectively; NH in pig manure field ₃, H ₂s and odour concentration reduce 84.4%, 62.1% and 88.5% respectively ^[31].Li Wanjun (2011) research is pointed out, the production performance that probiotics (main component is genus bacillus, milk-acid bacteria and yeast) can significantly improve laying hen is added in layer diets, reduce ammonia generation in excrement, improve chicken coop air environment ^[32].As can be seen here, probiotics probiotic bacterium all has certain meaning to animal growth and plant's environmental improvement.But, the research of a kind of new probiotic bacterium, first will faced by problem be the screening of bacterial strain and the mensuration of functionally active thereof.In screening process, not only require that these strain growth speed are fast, can tolerate the gastrointestinal tract environment factor of animal self, have specific Function, and bacterial classification source wants consistent with use object, this is the specific effective way of raising bacterial classification ^[33-35].

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Summary of the invention

The object of the invention is to hide pig source bacillus amyloliquefaciens for animal productiong and industrial production provide a kind of, this bacterial strain be isolated from the pig intestinal contents of Tibetan can cellulolytic genus bacillus.Through qualification, this genus bacillus is a kind of pig source, Tibetan bacillus amyloliquefaciens newly, be bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens) by its Classification And Nomenclature, deliver to China typical culture collection center and carried out preservation, address: China. Wuhan. Wuhan University; Preservation date: on December 31st, 2013; Deposit number is CCTCCNO:M2013739, and this bacterial strain can be used for the extraction of cellulase, is beneficial to commercial exploitation; Or coarse-fibred probiotics is digested, as probiotic bacterium etc. for making monogastric animals such as promoting pig in animal productiong.

The 16SrRNA gene order of described pig source, Tibetan bacillus amyloliquefaciens is as shown in SEQIDNO:1.The sequence of the endo cellulase gene that described pig source, Tibetan bacillus amyloliquefaciens is cloned is as shown in SEQIDNO:2.Clone's bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens) cellulose enzyme gene, and express in prokaryotic expression system.With DNAssist analysis software, 499 encoding amino acid sequence analyses coded by cellulose enzyme gene bglc-BY are found, this aminoacid sequence is made up of Mierocrystalline cellulose binding region (CBM-3) of the cellulase of in glycosyl hydrolase family 5 and N-terminal, and the albumen of coding belongs to the family of BglC.In addition, also find that this cellulase protein is made up of 20 seed amino acids, the molecular weight theoretical value of coded albumen is 55KD, and iso-electric point is 8.6.

Described pig source, Tibetan bacillus amyloliquefaciens is for the preparation of the application of probiotics.Described pig source, Tibetan bacillus amyloliquefaciens is used for the application that cellulase extracts.Described pig source, Tibetan bacillus amyloliquefaciens is used for the application of fiber degradation.Pig source, Tibetan of the present invention bacillus amyloliquefaciens has higher cellulase activity, can be used in enteron aisle or cellulosic degraded in ight soil, improves plant's animal living enviroment; Meanwhile, in the experiment of Gl tract in vitro, it has strong adverse-resistant characteristic, also can be used for researching and developing microbial preparation, to prevent and treat the generation of animal intestinal disease.

Hide pig source bacillus amyloliquefaciens and have strong tolerance to heat, simulated gastric fluid, simulated intestinal fluid (various biliary salt concn), and to intestinal bacteria, Salmonellas and streptococcus aureus, there is certain restraining effect, little to the toxicity of chitterlings epithelial cell ZYM-SIEC02; But this bacterium has certain restraining effect to intestinal bacteria, Salmonellas and streptococcus aureus to sticking of cell.

Pig source, Tibetan of the present invention bacillus amyloliquefaciens obtains through being separated, screening from the chitling content of Tibetan, compared with prior art, has the following advantages:

1) hiding pig source bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens) is from hiding the pure natural bacterial classification filtered out chitling content, its fermented supernatant fluid has very high cellulase activity, and there is strong adverse-resistant characteristic, cytotoxicity is little.Therefore, the exploitation of probiotic bacterium can be directly used in, muck becomes thoroughly decomposed process, cellulase extraction, lignocellulose degradation etc.

2) heterogenous expression can be carried out in pET prokaryotic expression system from hiding the cellulose enzyme gene bglc-BY gene of cloning bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens) genomic dna of pig source.

Accompanying drawing explanation

The hydrolysis circle of Fig. 1 cellulose-decomposing bacterium on CMC-Na plate;

The Morphological Features of Fig. 2 cellulose-decomposing bacterium;

The gramstaining of Fig. 3 cellulose-decomposing bacterium;

The spore staining of Fig. 4 cellulose-decomposing bacterium;

The agarose gel electrophoresis figure of the 16SrRNA gene of Fig. 5 cellulose-decomposing bacterium, in this figure: M is MarkerIII, swimming lane 1,2 is the 16SrRNA gene band of cellulose-decomposing bacterium;

The 16SrRNA Phylogenetic Tree of Fig. 6 cellulose-decomposing bacterium BacillusamyloliquefaciensBY-5;

Fig. 7 cellulose-decomposing bacterium BacillusamyloliquefaciensBY-5 growth curve;

Fig. 8 glucose standard curve;

The enzymatic productivity of Fig. 9 cellulose-decomposing bacterium BacillusamyloliquefaciensBY-5;

The agarose gel electrophoresis figure of Figure 10 BacillusamyloliquefaciensBY-5 cellulose enzyme gene bglc-BY, in figure: M is MarkerIII, swimming lane 1,2 is bglc-BY gene band, and size is 1500bp;

Figure 11 Mierocrystalline cellulose incision enzyme gene bglc-BY sequence evolution is set;

The amino acid whose sequential analysis of Figure 12 fiber endonuclease protein;

The structure prediction of Figure 13 Mierocrystalline cellulose endonuclease protein;

The enzyme of Figure 14 cellulose enzyme gene pET28 (a+)-bglc-BY expression vector cuts qualification; In this figure: M is markerIII, swimming lane 1 is the double digestion fragment of pET-bglc-BY expression vector, and swimming lane 2 is the single endonuclease digestion fragment of pET-bglc-BY expression vector;

The expression of Figure 15 recombinant bacterial strain pET-bglc-BL21 in prokaryotic cell prokaryocyte, have transparent circle for recombinant bacterial strain pET-bglc-BL21, there is no contrasting for empty carrier pET-BL21 of transparent circle;

Figure 16 is the thermotolerance result curve figure of BY-5;

Figure 17 is the power curve of the resistance to simulated gastric fluid figure of BY-5;

Figure 18 is the bile tolerance power curve figure of BY-5;

Figure 19 is the bacteriostatic test of BY-5 to pathogenic bacterium.Figure A is intestinal bacteria, and B is Salmonellas, and C is streptococcus aureus;

Below in conjunction with accompanying drawing and concrete test and the present invention is described in further detail.

Embodiment

Probiotics utilizes normally microorganismor the work of making of the material of accelerate growth of microorganism microorganism system agent.That is, all can promote normal microfloragrowth and breeding and suppress the preparation of pathogenic bacterium growth and breedings to be all called probiotics.Because effect of its regulating intestinal canal is quick, and build intestinal microecology balance, no matter baby, old man, or newborn livestock and poultry all can prevent and treat diarrhoea, constipation.Now conventional people with probiotics have ecological viable bacteria element, medilac-Vita, strong spirit stachyose, zheng Chang Sheng, meter Yadeng.

Bacillus of the present invention-bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens), at the preserving number of China typical culture collection center is: CCTCCNO:M2013739.This bacillus amyloliquefaciens can be separating obtained from the content hiding pig caecum, small intestine or stomach, can produce cellulase, have the ability of degradation of sodium carboxymethylcellulo, e.At 37 DEG C, carry out product enzymic fermentation under 220rpm condition and cultivate, result shows, and bacterial strain cellulase-producing amount when fermentation culture to 28 ~ 44h is high and stable.

Applicant with the cellulose enzyme gene bglc of the bacillus amyloliquefaciens that NCBI issues (Bacillusamyloliquefacienssubsp.plantarumstr.FZB42) for foundation, utilize primerprimer5.0 to design primer and carry out pcr amplification, Successful amplification has gone out the endo cellulase β-1 of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5, 4 glucanase gene bglc-BY, and successfully construct Mierocrystalline cellulose incision enzyme gene bglc-BY prokaryotic expression system, in 37 DEG C, 220rpm, when bacterium liquid OD600 reaches about 0.8, carry out IPTG induction, result shows, constructed prokaryotic expression system is expressed successfully in vitro.

Bacillus-bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 of the present invention is through external prebiotic evaluation display, this bacterial strain has strong heat-resisting, acidproof and bile tolerance ability, in addition, to pathogenic bacterium, there is certain suppression and inhibition effect on adhesion.

Found out by above result, the exploitation of fermentative production cellulase and pig source probiotic bacterium can be used for, to greatly developing the significant and wide application prospect of health, joint grain pig industry from hiding cellulose-decomposing bacterium-bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 of separation screening chitling road.

The embodiment provided below by way of applicant is described in detail.

The separation screening of embodiment 1:BY-5

1.1 test materials source and collections

Chitling road, Tibetan contents samples is collected in Yang Ling Hua Yi Industrial Co., Ltd. and hides pig slaughterhouse, and test pig is 8 monthly age health pig, and its feeding and management condition is consistent, and daily ration composition contains the green and rough feeds of 90%, the bean dregs of interpolation about 10% in addition and wheat bran etc.Hide pig and cut abdominal cavity open after slaughter, rapid separation Stomach duodenum, jejunum, ileum, caecum, and every section is got about 20cm, and at intestinal tube two ends Thread ligation, cut off, exhaust sealing in dress sealing plastics bag, put into ice chest and take back laboratory rapidly, and put into 4 DEG C of refrigerators, carry out the separation of cellulose-decomposing bacterium at short notice as early as possible.

The separation screening of 1.2 bacterial strains

1.2.1 the configuration of substratum

Xylo-Mucine (CMC-Na) screening culture medium (g/L): CMC-Na10g, peptone 5g, yeast powder 0.5g, K ₂hPO ₄1.5g, MgSO ₄7H ₂o0.2g, sodium-chlor 5g, agar powder 15g, distilled water constant volume is to 1L.

Basis Medium of shaking flask fermentation: add the CMC of 1% in common liq LB substratum.

LB substratum: Trypsin extract 10g; Yeast extract 5g; Sodium-chlor 5g; Distilled water constant volume is to 1L.

Above substratum all needs autoclaving.

1.2.2 the primary dcreening operation of cellulose-decomposing bacterium

Take respectively and hide pig Stomach duodenum, jejunum, ileum, caecum cecal content 1g be transferred in the triangular flask filling 100mL sterilized water accordingly, vibrate 30min in 80 DEG C of electric heating constant temperature shaking bath pots, 10 ^-2intestinal contents diluent, and then become 10 by 10 times of dilution methods successively gradient dilution ^-3-10 ^-6intestinal contents diluent.Screening culture medium makes solid plate, then to get respectively on flat board that 100uL is applied to corresponding numbering with transfer pipet and carry out label, be inverted in 37 DEG C of constant incubators and cultivate 48h, find in the solid medium of the different gradients from stomach and caecum, all there are a large amount of canescence, the translucent bacterium colony with wrinkle mould, and in the substratum from little intestinal segment content, the bacterium colony of this type is few.Then we add appropriate 0.1% Congo red solution-dyed 1h in the culture dish of each gradient, discard dye liquor, add appropriate 1mol/LNaCl solution washing, after lh, pour out NaCL solution, find these whites, all there is transparent circle in the surrounding of translucent wrinkle mould bacterium colony.With diameter (D) and the colony diameter (d) of periphery of bacterial colonies hydrolysis circle on vernier callipers master plate, finding that the diameter of the type bacterium colony is about 0.7cm, is about 2.2cm (Fig. 1) to the hydrolytic circle of carboxymethyl cellulose.Then cultivate in the LB substratum of liquid respectively from the type bacterium colony of different intestinal segment with the transfering loop picking of sterilizing, and isolation and purification culture 4-5 generation of repeatedly ruling on CMC flat board is until obtain the bacterium colony (Fig. 2) of purifying, and the bacterial classification glycerol adding that purifying is cultivated is saved backup in-80 DEG C.

In experiment, contriver is separated the cellulose decomposition of Tibetan pig Stomach duodenum, jejunum, ileum, every section, caecum content simultaneously, find in stomach and caecum maximum, also find that there is the existence of cellulose-decomposing bacterium at little intestinal segment (duodenum, jejunum, ileum).

1.2.3 the cellulose-decomposing bacterium fermentation preparation of crude enzyme liquid and multiple sieve

By filter out, each bacterial strain of freezen protective carries out activation culture at the flat lining out of LB solid medium respectively, then choosing mono-clonal is inoculated in the fresh LB liquid medium of 50mL, 37 DEG C, under 220rpm condition concussion be cultured to logarithmic phase (OD=1.0), make liquid spawn.The liquid spawn prepared (OD=1.0) is inoculated in 50mL Medium of shaking flask fermentation with the inoculum size of 1% respectively, at 37 DEG C, 220rpm condition bottom fermentation cultivation 24h, get appropriate fermented liquid centrifugal 15min under 5000rpm, 4 DEG C of conditions, its supernatant liquor is crude enzyme liquid, carries out multiple sieve by the cellulase activity measuring crude enzyme liquid to the bacterium be separated.Result shows, to the bacterial strain that preliminary screening arrives, again according to the measurement result of its cellulase activity, have chosen the highest strain bacterium of cellulase activity (0.23U/mL) and be used for follow-up research, this Strain Designation is BY-5 cellulose-decomposing bacterium.

Embodiment 2: the qualification of cellulose-decomposing bacterium

2.1 morphologic features

BY-5 cellulose-decomposing bacterium in LB liquid medium in 37 DEG C, 220rpm shakes cultivation, gets the bacterium liquid being cultured to 12h and carries out gramstaining, coloration result finds, this thalline is shaft-like, is dyed to purple, belongs to gram-positive microorganism (Fig. 3).Get the bacterium liquid malachite green method being cultured to 24h and carry out spore staining, result shows, and the gemma of this bacterium is dyed to green, and nourishing body dyes redness (Fig. 4).

The Physiology and biochemistry qualification of 2.2 cellulose-decomposing bacteriums

According to the method in " common bacteria system identification handbook " and " the outstanding Bacteria Identification handbook of uncle ", physio-biochemical characteristics index determining is carried out to BY-5 cellulose-decomposing bacterium.Show according to the measurement result in table 1, the characteristic of the physio-biochemical characteristics of test strain and " common bacteria system identification handbook table " and " the outstanding Bacteria Identification handbook of uncle " bacillus amyloliquefaciens is basically identical.Therefore, the test strain screened in this test is initially identified as bacillus amyloliquefaciens (Bacillusamyloliquefaciens) or its mutation of Bacillus.

The Physiology and biochemistry qualification result of table 1 bacterial strain

Note: "+" indicates that this feature maybe can utilize this material, " one " represents do not have this feature or do not utilize this material.

2.3 cellulose-decomposing bacteriums molecular biology identification

2.3.1 the 16SrRNA gene fragment order of cellulose-decomposing bacterium measures

The BY-5 cellulose-decomposing bacterium bacterial classification of freezen protective is carried out activation culture, (the OD when being cultured to logarithmic phase ₆₀₀=1.0-1.5), collected by centrifugation thalline, carries out extracting genome DNA.The method that bacterial genomes DNA extraction method provides with reference to Beijing Tian Gen biochemical technology company limited bacterial genomes test kit is carried out.Then bacterial universal primers (upstream primer 27F:5'-AGAGTTTGATCCTGGCTCA-3', downstream primer 1492R:5'-GGTTACCTTGTTACGACTT-3 ') is used to carry out the amplification of bacteria 16 S rRNA genes.PCR25uL reaction system comprises: DNA profiling 0.5uL (ensureing 50-100ng/uL), upstream and downstream primer each 1uL, mixTag enzyme 12.5uL, deionized water supplies 25uL.Pcr amplification condition is: reaction conditions is: 95 DEG C of denaturation 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 50s, 35 circulations; Last 72 DEG C extend 10min, 4 DEG C of forever.The agarose gel electrophoresis of 1% detects amplification (Fig. 5), and pcr amplification product glue is reclaimed, and recovery product is connected in pGEM-T carrier, proceed to DH5 α competent escherichia coli cell, the solid LB media coating Amp resistance after renewal cultivation 1h is dull and stereotyped, 37 DEG C of incubated overnight.Bacterium colony PCR is accredited as positive bacteria and falls behind, and send Beijing Hua Da gene biological company to check order.Sequencing result is carried out homology analysis with BLAST software on NCBI in GenBank, obtains the gene order of close typical strain, utilize Mega5.0 constructing system evolutionary tree.

Through order-checking, cellulose-decomposing bacterium 16SrRNA gene order length is 1455bp, and its size conforms to expected results (Fig. 5), and its gene order (Accessionnumber:KT800418) is:

GATGGCGGGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAATCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGACAAGAATAAG

2.3.2 the element sequential analysis of decomposer of institute's separated fiber and the structure of systematic evolution tree

By the 16SrRNA gene order of the BY-5 cellulose-decomposing bacterium of mensuration, compare with all prokaryotic organism 16SrRNA genes measured in GenBank database, result shows, in this cellulose-decomposing bacterium and NCBI, the genetic distance of BacillusamyloliquefaciensstrainFZB42 recently (Fig. 6), the similarity of both 16SrRNA gene orders reaches 99%, although and comparatively far away with the genetic distance of other bacillus reported, sequence similarity is also all more than 90%.Illustrate that pig source, the Tibetan cellulose-decomposing bacterium be separated to belongs to a kind or the mutation of the bacillus amyloliquefaciens of bacillus.In conjunction with the morphological feature of this bacterium, physio-biochemical characteristics and Molecular Identification, Cellulose bacterium called after bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 that this experiment sieving is arrived.

Embodiment 3BacillusamyloliquefaciensBY-5 growth characteristics are analyzed

By bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 at LB lining out, activation culture under 37 DEG C of conditions, chooses mono-clonal and is inoculated in 5mL LB liquid medium, 37 DEG C, under 220rpm condition concussion cultivate OD ₆₀₀between 1.0 ~ 1.5, the inoculum size with 1% is inoculated into 60mL fresh LB, and after 4 hours, interval 2h samples, until 60h, adopts spectrophotometer method to measure its absorbancy under 600nm wavelength, draws growth curve of bacteria.Measurement result shows, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 after inoculation 8 ~ 28h is logarithmic phase, starts to enter stationary phase, and continue to about 36h after 28h, after 36h, the breeding of thalline progresses into the paracme (Fig. 7).

Embodiment 4 cellulose-decomposing bacterium BY-5 cellulase activity measures

4.1BacillusamyloliquefaciensBY-5 cellulase activity measuring method

The Activity Determination of Cellulase (NY/T912-2004) that the making of glucose standard curve and the measuring method of cellulase activity are issued with reference to The Ministry of Agriculture of the People's Republic of China, MOA is carried out, and changes to some extent according to practical situation.

4.1.1 the configuration of required reagent and damping fluid

(1) 0.1mol/LpH=5.5 NaAc_HAc buffer solution: add 100mL distilled water by after 65.0mL0.2mol/L sodium acetate soln and the mixing of 35.0mL0.2mol/L acetum.

(2) 3,5 dinitrosalicylic acids (DNS) reagent:

Take 3, the 5-dinitrosalicylic acid water dissolution of 6.3g, add 21.0gNaOH, 182g Seignette salt, add 500mL water, add 5.0g re-distilled phenol and 5.0g S-WAT again, stirring and dissolving after heating for dissolving, cooling, is settled to 1000mL.

(3) Glucose standards solution (10mg/mL): take after 1.000g glucose (AR) (105 DEG C are dried to constant weight) distilled water dissolves and be settled to 100mL.

(4) carboxymethylcellulose sodium solution: claim 2.0gCMC-Na to be dissolved in 200mL distilled water, add hac buffer 100mL.

4.1.2 the making of grape essence typical curve

The blank sample of standard: the NaAc_HAc buffer solution 4.0mL drawing pH=5.5, adds DNS reagent 5.0mL, boiling water bath heating 5min.Be cooled to room temperature with tap water, be settled to 25.0mL with water, the blank sample of standard of making.

Draw respectively 1.0mg/mL glucose solution 1.0,2.0,3.0,4.0,5.0,6.0,7.0mL, be settled to 10mL with damping fluid respectively, be mixed with the Glucose standards solution that concentration is 0.1-0.7mg/mL.Then carry out according to table 2:

Table 2 standardized solution configuration proportion

By above-mentioned each test tube mixed solution, gently shake mixing, boiling water bath heating 5min, be settled to 25mL with distilled water after flowing water cooling, shake up, surveying absorbancy at 540nm absorption peak place, is ordinate zou with glucose content, with OD ₅₄₀for X-coordinate mapping, obtain glucose concn and OD ₅₄₀relation curve (Fig. 8).

4.1.3 the mensuration of cellulase activity

A _b: 2mL adds 5mLDNS through the enzyme liquid of suitably dilution, mixing, then adds 2mL substrate, mixing, and boiling water boils 5min, is settled to 25mL, is blank, measures light absorption value A under 540nm wavelength condition with the blank sample (see 4.1.2) of standard _b.

A _e: 2mL adds 2mL1%CMC through the enzyme liquid of suitably dilution, mixing, 40 DEG C of accurate response 30min, then adds 5mLDNS, mixing, boiling water boils 5min, be settled to 25mL, be blank with the blank sample (see 4.1.2) of standard, under 540nm wavelength condition, measure light absorption value A _e.

Enzyme unit definition alive: at pH=5.5,40 DEG C, under reaction 30min condition, the enzyme amount that per minute is degraded needed for generation 1 μm of ol reducing sugar by substrate carboxymethyl sodium cellulosate is defined as an enzyme activity unit, represents with U/mL.

4.1.4 the calculating of enzyme activity:

Calculation formula: X _d=[(AE-AB) × K+C _o]/(M × t) × 1000 formulas (1)

In formula (1):

X _dfor the cellulase activity in sample diluent, u/mL;

A _efor the absorbancy of enzyme reaction solution;

A _bfor the absorbancy of the blank sample of enzyme;

K is the slope of typical curve;

C _ofor the intercept of typical curve;

M is the molecular weight (180.2) of glucose;

T is the enzyme digestion reaction time, min;

1000 is transforming factor, 1mmol=1000 μm of ol.

X _dvalue between 0.04-0.08U/mL, if not within the scope of this, should should reselect the extent of dilution of enzyme liquid, then carries out analysis mensuration.

X=XDDf formula (2)

In formula (2): X is the vigor of sample fiber element enzyme, U/g; Df is total extension rate of sample.

4.2BacillusamyloliquefaciensBY-5 is at the enzymatic productivity of different fermentations time

Seed liquor (OD=1.0 of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5) inoculum size with 1.0% is inoculated in 100mL Medium of shaking flask fermentation (LB+1%CMC-Na), 37 DEG C, the cultivation of 220rpm condition bottom fermentation.From fermentation 8h, sample 2mL, centrifugal fermented liquid 15min under 5000rpm, 4 DEG C of conditions every 4h, supernatant liquor is crude enzyme liquid, then to crude enzyme liquid according to 4.1 method carry out the mensuration of cellulase activity.Result shows, along with the prolongation of fermentation time, and the speed increase gradually also of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 enzymatic production.When fermenting to 36h, the activity of cellulase reaches the highest (0.35U/mL), and this bacterial strain produces enzyme kept stable (Fig. 9) between 28 ~ 44h.

The cloning and analysis of embodiment 5BacillusamyloliquefaciensBY-5 cellulose enzyme gene

The clone of 5.1 bacillus amyloliquefaciens BY-5 cellulose enzyme genes

5.1.1BY-5 the pcr amplification of cellulase gene

Require to extract bacillus amyloliquefaciens (Bacillusamyloliquefaciens) BY-5 genomic dna according to Beijing Tian Gen biochemical technology company limited bacterial genomes DNA extraction kit, with the cellulose enzyme gene bglc of the bacillus amyloliquefaciens that NCBI issues (Bacillusamyloliquefacienssubsp.plantarumstr.FZB42) for foundation, primerprimer5.0 is utilized to design primer, add suitable restriction enzyme site, amplification object fragment bglc-BY, primer sequence sees the following form 3, the PCR reaction system of cellulose enzyme gene bglc-BY is in table 4.

Table 3 cellulose enzyme gene bglc-BY primer sequence and restriction enzyme site

The PCR reaction system of table 4 cellulose enzyme gene bglc-BY

Pcr amplification program is as follows: 95 DEG C of reaction 5min, 94 DEG C of reaction 30s, 58 DEG C of reaction 30s, and 72 DEG C of reactions 90s, 72 DEG C of 10min, under wherein 94 DEG C, 58 DEG C and 72 DEG C of conditions, circulation 35 times, is finally placed in 4 DEG C.After pcr amplification terminates, the agarose electrophoresis of 1.2% concentration is adopted to carry out detecting (Figure 10).

5.1.2PCR product reclaims

Utilize Bio-Flux glue to reclaim test kit to reclaim PCR primer.Detailed step is as follows:

A: with clean, sharp scalpel, the sepharose containing target DNA fragment is cut, and put into the clean centrifuge tube of 1.5mL;

B: add ExtractionBuffer in the ratio (gel quality milligram number: sol solutions volume microlitre number) of 1:3, hatch in 50 DEG C of water-baths, until gel melts.

Centrifugal one minute of C: mixed solution is all transferred in Spincolumn, 6000g, and discard the liquid in adapter.

D: add 500 μ LExtractionBuffer in Spincolumn, in the centrifugal 30 ~ 60s of 12000g, and discards liquid in adapter.

E: the WashBuffer adding 750 μ L in Spincolumn, room temperature places 2 ~ 5min, in the centrifugal 30 ~ 60s of 12000g, discards liquid in adapter.

F: again in the centrifugal 1min of 12000rpm, then Spincolumn is transferred in 1.5mL centrifuge tube.The ddH of 50 μ L is added in Spincolumn ₂o, and room temperature places 1min.

The centrifugal 1min of G:12000g, containing DNA object fragment in Eppendorf tube, in-20 DEG C of preservations.

5.1.3 conversion is connected

Connect test kit with Fermentas company, PCR is reclaimed product and under 22 DEG C of conditions, be connected 10min with pGEM-Tvector, linked system is as shown in table 5 below:

Table 5 linked system table

Proceeded to by connection product in DH5 α Competentcells, concrete step of converting is as follows:

A: get competent cell and melt in ice bath in-80 DEG C of refrigerators.

B: add 10.0 μ L and connect product, and place 30min on ice.

C: heat shock 60 ~ 90s in 42 DEG C of water-baths.

D: fech connection product from ice bath, places 2 ~ 3min in ice.

E: add the nonresistant LB substratum of 900 μ L liquid, and in 37 DEG C, 150rpm renewal cultivation 45min.

F: by centrifugal for converted product 4000rpm 2min, discard appropriate supernatant, is applied in the LB culture plate of ammonia benzyl resistance, 37 DEG C of incubated overnight in right amount.

5.1.4 the PCR of Insert Fragment detects

Mono-clonal on picking culture plate, increase with the upstream and downstream primer of cellulose enzyme gene bglc-BY respectively, PCR amplification system, response procedures, agarose detect identical with goal gene amplification condition (table 5), the bacterium liquid of test positive being cloned is preserved, and sample presentation checks order.

Order-checking shows, the cellulose enzyme gene (Accessionnumber:KT800419) of the BacillusamyloliquefaciensBY-5 of clone) sequence is as follows:

ATGAAACGGTCAATCTCTATTTTTATTACGTGTTTATTGATTGCGGTATTGACAATGGGCGGCTTGCTGCCTTCGCCGGCATCAGCAGCAGGGACAAAAACGCCAGTAGCCAAGAATGGTCAGCTTAGCATAAAAGGTACACAACTTGTAAACCAAGACGGCAAAGCGGTACAACTGAAAGGGATCAGTTCACATGGATTGCAATGGTATGGCGATTTCGTCAATAAAGACAGCTTAAAATGGCTGAGAGACGATTGGGGCATCACCGTTTTCCGCGCGGCAATGTATACGGCAGACGGCGGTTATATTGACAACCCGTCCGTGAAAAATAAAGTAAAAGAAGCGGTTGAAGCGGCAAAAGAACTTGGGATATATGTCATCATTGACTGGCATATCTTAAATGACGGCAACCCAAACCAAAATAAAGAGAAGGCGAAAGAATTTTTCAAGGAAATGTCAAGTCTTTACGGAAACACGCCAAACGTCATTTATGAAATTGCAAATGAACCAAACGGTGACGTGAACTGGAAGCGTGATATTAAACCGTATGCGGAAGAAGTGATTTCCGTTATCCGCAAAAATGACCCCGACAATCCCATCATTGTCGGAACCGGTACATGGAGCCAGGATGTGAATGATGCTGCCGATGACCAGCTAAAAGATGCAAACGTCATGTACGCGCTTCATTTTTATGCCGGCACACACGGCCAATCTTTACGGGATAAAGCAAACTATGCACTCAGTAAAGGAGCGCCTATTTTCGTGACGGAATGGGGAACAAGCGACGCGTCTGGAAATGGCGGTGTATTCCTTGACCAGTCGCGGGAATGGCTGAATTATCTCGACAGCAAGAAAATCAGCTGGGTGAACTGGAATCTTTCTGATAAGCAGGAATCATCCTCAGCGTTAAAGCCTGGAGCATCTAAAACAGGCGGCTGGCCGCTGTCAGATTTAACTGCTTCAGGAACCTTCGTAAGAGAAAACATTCGCAGCAACAAAGATTCAACGAAGGACGCCCCTGAAACGCCAGCACAAGATAATCCCACACAGGAAAAAGGCGTTTCTGTACAATACAAAGCAGGGGATGGGAGTGTGAACAGCAACCAAATCCGCCCGCAGCTTCACATAAAAAATAACGGGAATACGACGGTTGATTTAAAAGATGTCACTGCCCGTTACTGGTATAACGCGAAAAACAAAGGCCAAAACTTTGACTGTGACTACGCGCAGATTGGATGCAGCAATCTGACCCACAAGTTTGTGACGCTGCATAAACCTAAGCAAGGCGCAGATACCTATCTGGAACTGGGGTTTAAAAAAGGAACACTGTCACCGGGAGCAAGCACAGGGAATATTCAGCTTCGTCTTCACAACGATGACTGGAGCAATTATGCACAAAGCGGCGATTATTCCTTTTTCCAATCAAATACGTTTAAAACAACGAAAAAAATCACATTATATCATCAAGGAAAACTGATTTGGGGAACAGAACCCAATTAG

5.2 cellulose enzyme gene bglc-BY sequence evolution trees

The cellulose enzyme gene bglc-BY sequence of BacillusamyloliquefaciensBY-5 is committed to Genbank (Accessionnumber:KT800419), found by sequential analysis, far (Figure 11), both sequence similarities are 96% to the genetic distance of the cellulose enzyme gene of the cellulose enzyme gene bglc-BY of the BY-5 that we screen and the nearest Bacillusamyloliquefac-iensstrainFZB42 of its sibship; And the existing high homology of cellulase gene sequence with other bacillus, there is again certain difference.We are described, and from be separated cellulose-decomposing bacterium BacillusamyloliquefaciensBY-5, successful clone is to cellulose enzyme gene, and this gene is far away with the genetic distance of the cellulose enzyme gene of other bacillus, is a new gene.

The bioinformatic analysis of 5.3bglc-BY gene

5.3.1 the amino acid sequence analysis of Mierocrystalline cellulose endonuclease protein

Discovery is analyzed further with the aminoacid sequence of DNAssist analysis software to cellulose enzyme gene bglc-BY, this albumen (499aa) is made up of (Figure 12) 20 seed amino acids, three seed amino acids that wherein amino acid residue content is the highest are: glycine (Glycine), Methionin (Lysine,), l-asparagine (Asparagine); And minimum three seed amino acids of content are: halfcystine (cysteine), Histidine (histidine), methionine(Met) (methionine).Be 55KD according to the molecular weight theoretical value that the aminoacid sequence of cellulase protein calculates.In addition, by forecast analysis, the iso-electric point of Mierocrystalline cellulose restriction endonuclease is 8.6, and therefore, the cellulase that in this test, bacillus amyloliquefaciens BY-5 secretes belongs to Sumizyme MP.

5.3.2 cellulase protein structure prediction

Analyze discovery further to 499 aminoacid sequences of bglc-BY genes encoding, aminoacid sequence is made up of (Figure 13) Mierocrystalline cellulose binding region (CBM-3) of the cellulase of in glycosyl hydrolase family 5 and N-terminal.With subtilis cellulase as: CAA97610.1, ACK38261.1, AAK94871.1, AAK39540.1, AAO63626.1 etc. have the similarity of more than 99%, belong to the family of cellulolytic enzyme BglC.

The prokaryotic expression of embodiment 6BacillusamyloliquefaciensBY-5 cellulose enzyme gene bglc-BY

The structure of 6.1 prokaryotic expression carriers

By the bacterium liquid containing recombinant plasmid pGEM-bglc-BY preserved and the bacterium liquid containing expression vector pET28 (a+) respectively at ammonia benzyl resistance and kalamycin resistance the flat lining out of LB, shake bacterium with the toothpick picking mono-clonal of sterilizing to cultivate, and extract plasmid.All adopt BamHI and HandIII in 37 DEG C of water-baths the plasmid of extraction, carry out double digestion.Double digestion system is as shown in table 6 below:

Table 6 plasmid double digestion system

Digestion products 1% agarose gel electrophoresis is detected.The goal gene fragment bglc-BY and linearizing pET28a (a+) fragment that identify correct (Figure 14) are carried out glue recovery, connection, and linked system is in table 7.

The linked system of table 7bglc-BY and pET28a (a+) fragment

Connection product is connected 10min under 22 DEG C of conditions, is converted in competent cell DH5 α, coated plate (kalamycin resistance), 37 DEG C of thermostat container incubated overnight, after blue hickie screening and bacterium colony PCR detect, select positive colony and extract plasmid.Then expression vector plasmid pET-bglc is proceeded in BL21 (DE3) competent cell, the centrifugal 2min of converted product 4000rpm, and get appropriate converted product and be coated onto on the LB flat board of kalamycin resistance, 37 DEG C of overnight incubation.Picking mono-clonal, carries out shaking bacterium and cultivates, and primer when cloning with goal gene is identified positive colony for primer carries out PCR and the correct recon of qualification is carried out freezen protective.By recon positive colony called after pET-bglc-BL21.Do pET-28 (a+) empty carrier simultaneously and transform BL21, to contrast.

The abduction delivering of 6.2 recombinant bacterial strain pET-bglc-BL21

By recombinant bacterium pET-bglc-BL21 picking list bacterium colony after the flat lining out incubated overnight of LB of kalamycin resistance, be seeded in the LB substratum containing kantlex, 37 DEG C, after 220rpm cultivates 12h, in the ratio of 1:50, be inoculated in fresh LB (blocking that resistance) liquid nutrient medium, when concussion is cultured to logarithmic phase (OD600 is at 0.5-0.8), add the IPTG (sampling before adding IPTG) that final concentration is 1mmol/L to induce, sample when being induced to 8h, and sample spot is connected on containing CMC-Na, 24h cultivated by the LB flat board (containing that resistance 100ug/mL of card) of IPTG, observe after congo red staining around thalline and whether have transparent circle, simultaneously not contain the empty carrier pET-28 (a+) of endo glucanase gene for contrast.Result of study shows, recombinant bacterium pET-bglc-BL21 bacterium on the LB flat board of CMC-Na, IPTG has the generation (Figure 15) of transparent circle, illustrates that the cellulose enzyme gene in recombinant bacterium pET-bglc-BL21 successfully can carry out heterogenous expression under the induction of IPTG.

The external prebiotic evaluation of embodiment 7BacillusamyloliquefaciensBY-5

The activation culture of each bacterial strain of 7.1 test and counting

(1) activation culture of bacterial strain: before test, frozen BacillusamyloliquefaciensBY-5, intestinal bacteria and Salmonellas, streptococcus aureus are rule activation culture on aseptic LB solid medium, chooses mono-clonal switching activation culture 2 times.Intestinal bacteria after activation, Salmonellas, streptococcus aureus, BacillusamyloliquefaciensBY-5 are seeded in LB liquid medium with the inoculum size of 0.1% respectively, at 37 DEG C, cultivate under 220rmp condition (wherein 3 kinds of pathogenic indicators are cultivated 12h, BacillusamyloliquefaciensBY-5 and cultivate 24h).Get each bacterium liquid and collect thalline respectively at 15min centrifugal under 4000rpm condition in right amount, thalline washs 2 times with aseptic PBS liquid (pH7.4), finally with the resuspended thalline of isopyknic aseptic PBS liquid.The concentration of then BacillusamyloliquefaciensBY-5 being hanged bacterium liquid is adjusted to OD ₆₀₀about=0.1,3 kinds of pathogenic indicators adjust its concentration to 1 × 10 respectively by colony counting method ⁸about cfu/mL, carrys out the seed liquor respectively as follow-up test.

(2) bacterial count: bacterium liquid to be measured is carried out 10 times and increases progressively dilution, chooses the bacterium liquid of 3 suitable gradient, gets 0.1mL, is applied on LB solid plate respectively, each gradient 3 repetition.The flat board that painting wipes, after omiting drying, is inverted, and cultivate 12h for 37 DEG C and take out, the concrete method of counting detailed rules and regulations of bacterial colony are carried out according to People's Republic of China's food microbiological analysis Detection of colony sum (GB/T4789.2-2008) method.Viable count in every milliliter of bacterium liquid to be measured is calculated as follows:

Bacterial count (cfu/mL)=flat-plate bacterial colony number × extension rate × 10

The inverse test of 7.2BacillusamyloliquefaciensBY-5 In Vitro Anti

(1) thermotolerance

By BY-5 seed liquor (OD ₆₀₀=0.1), be placed in after 65 DEG C, 75 DEG C, 85 DEG C, 100 DEG C water-baths process 20min respectively, rapid flowing water cools, with without thermal treatment bacterium liquid in contrast, calculate BY-5 by colony counting method to learn in the survival rate of condition of different temperatures, BY-5 can tolerate certain high temperature, but along with the rising of temperature, the survival rate of BY-5 declines all to some extent.Survival rate wherein 65 DEG C, 75 DEG C time is all more than 85%, and the survival rate under 85 DEG C and 100 DEG C of conditions is respectively 78.67% and 35.45% (Figure 16).

(2) resistance to simulated gastric fluid ability

By BY-5 seed liquor (OD ₆₀₀=0.1) be seeded in the simulated gastric fluid of pH1-4 with the inoculum size of 5%, mixing, 37 DEG C of quiescent culture.After and inoculation initial in inoculation, 1h, 2h, 3h, 4h get in above-mentioned each process and cultivate bacterium liquid respectively, carry out plate count and calculate the survival rate of each bacterial strain, evaluating its acid resistance.Result shows, under same pH condition, along with the prolongation of incubation time, the survival rate of BY-5 is on a declining curve.And in same incubation time, under condition of different pH, along with the rising of pH, the survival rate of BY-5 is in the trend risen.Wherein under condition of different pH, process 1-2h, the survival rate of BY-5 all reaches more than 85%; And under the condition of pH2-4, processing 1-4h, the survival rate of BY-5 all reaches more than 80%, illustrates that BY-5 can tolerate the simulated gastric fluid (Figure 17) of certain acidity.

(3) ability of the simulated intestinal fluid of resistance to various biliary salt concn

The 24h culture getting 2mLBY-5 is centrifugal, PBS liquid washing thalline 2 times, after then bacterial sediment being processed 2h in pH2 simulated gastric fluid, and centrifuging and taking bacterial sediment, and then wash twice rear Eddy diffusion with PBS and suitably dilution, adjustment bacterial concentration is OD ₆₀₀=0.1, be suspended in the simulated intestinal fluid of various biliary salt concn with 5% inoculum size, mixing, 37 DEG C of quiescent culture.After inoculating initial and inoculation after 1h, 2h, 3h, 4h, after getting appropriate above-mentioned each bacterium liquid gradient dilution respectively, carry out plate count, evaluate its resistance to simulated intestinal fluid and bile tolerance.Result shows, in the same treatment time, BY-5 is in the simulated intestinal fluid of 0.1-0.4% gallbladder salinity, and along with the rising of gallbladder salinity, its survival rate is totally slightly in downward trend; In addition, under same gallbladder salinity condition, along with the prolongation in treatment time, the survival rate of BY-5 is also all in downward trend.No matter but under which kind for the treatment of condition, the survival rate of BY-5, still up to more than 90%, illustrates to have strong bile tolerance ability (Figure 18).

(4) antagonistic property

The cultivation of A, BacillusamyloliquefaciensBY-5 and filtrate preparation

The seed liquor of BY-5 is inoculated in 100mLLB liquid nutrient medium with 4% inoculum size, bacterium fermentation culture 28h is shaken, centrifugal 15min under 4000rpm condition, separating thallus and supernatant liquor in 37 DEG C, supernatant liquor is through frit (filter membrane diameter is 0.22 μm), and collection filtrate is for subsequent use.

Slightly carrying of B, BacillusamyloliquefaciensBY-5 extracellular antiseptic material: the BY-5 filtrate in above-mentioned A is got 50mL, the 50%PEG6000 adding 75mL stirs 1h, until PEG6000 dissolves completely, 12,000rpm is centrifugal, and 15min removes supernatant, precipitation is mixed with the PBS damping fluid of 10 times of volumes, adopts Odontothrips loti to measure the bacteriostasis of BY-5 antibacterial substance crude extract.

The making of C, antibacterial flat board and bacteriostatic test: the solid LB solid medium of sterilizing is cooled to about 60 DEG C, accurately measures 10mL with the graduated cylinder of sterilizing, in the culture dish pouring sterilizing into after it solidifies, put Oxford cup thereon.Be 1 × 10 by concentration ⁸the indicator of cfu/mL adds with 1% inoculum size and is cooled in the LB solid medium of about 50 DEG C, shakes up rear each culture dish and falls about 25mL, remove Oxford cup (timely sterilizing wanted by Oxford cup and culturing bottle) after it thoroughly solidifies.Then in each cup aperture in culture dish, add each 150 μ L of crude extract of BY-5 extracellular antiseptic material respectively, the constant incubator being placed in 37 DEG C cultivates 12h, measures the diameter of inhibition zone, evaluates its bacteriostasis.3 repetitions are done in each process, then get its mean value.

Seen by the antibacterial result of Figure 19, BY-5 all shows certain restraining effect to 3 kinds of pathogenic bacterium.Wherein the fungistatic effect of BY-5 to intestinal bacteria and Salmonellas is close, and the diameter of inhibition zone is respectively 1.3cm and 1.35cm, and to the bacteriostatic activity of streptococcus aureus higher than the bacteriostatic activity to intestinal bacteria and Salmonellas.

7.3 test cell line

7.3.1 prepare before cell cultures and cell recovery and cultivation, go down to posterity

Prepare before cell cultures: iuntercellular and Bechtop 1h before on-test answers ultraviolet-sterilization, ventilation 30min, ensure that the environment of cell cultures is aseptic.In addition, cell cultures at 37 DEG C, CO ₂concentration is 5%, humidity is carry out in the incubator of 95%, nutrient solution used in cell manipulation process and PBS liquid before cell manipulation should in cell culture incubator thermal pretreatment.

Recovery and the cultivation of cell, to go down to posterity: chitterlings epithelial cell ZYM-SIEC02 (by animal medicine institute of Xibei Univ. of Agricultural & Forest Science & Technology open man of virtue and ability's penetrating judgment award be so kind as to give) take out from liquid nitrogen after, aseptically carry out recovering and cultivating, go down to posterity.Go down to posterity after 2 times, cell concn is all adjusted to 2 × 10 ⁵cells/mL is used for test, and unnecessary cell carries out frozen process.

7.3.2 the MTT test of cell

Be 2 × 10 by concentration ⁵the ZYM-SIEC02 cell of cells/mL is inoculated in 96 well culture plates, and every hole 100 μ L, grows to after individual layer until cell, and with the PBS washed cell 2 times of sterilizing, in plate hole, then add 100 μ L concentration is respectively 1 × 10 ⁸the BY-5 of cfu/mL, intestinal bacteria, Salmonellas, streptococcus aureus bacterium liquid, often kind of bacterium establishes 4 repetitions, to add the plate hole of DMEM nutrient solution for blank, be placed in 37 DEG C, 5%CO2 incubator hatch 2h after inhale abandon supernatant, with PBS washed cell 5 times, every hole adds MTT (5mg/mL) 20 μ L, is placed in 37 DEG C, 5%CO ₂cell culture incubator in continue to hatch 4h.Careful suction is abandoned in hole after liquid, adds DMSO150 μ L and dissolve insolubles in every hole, and vibration 10min, then surveys its OD in microplate reader ₄₉₀.Judge that (comparative survival rate of cells 100% is 0 grade to each bacterial strain, is 1 grade between 80-99% to Cytotoxic size by calculating test group cell relative to the survival rate of cellular control unit; Between 50-79% be 2 grades; 30-49% is 3 grades; 0-29% is 4 grades.)。Absorbance × 100% of the absorbance/control group of cell survival rate (%)=test group.

Shown by the test-results of Fig. 8, BY-5 and streptococcus aureus are 1 grade to the toxicity of ZYM-SIEC02 cell, and intestinal bacteria, the toxicity rank of Salmonellas to cell are 2 grades.

Table 8 bacterium is to the toxicity test of cell

7.3.3 adhesion assay

Get 24 orifice plates, it is 2 × 10 that every hole adds a certain amount of concentration ⁵the ZYM-SIEC02 cell of cells/mL, is placed in 37 DEG C, 5%CO ₂constant-temperature incubation in incubator, treats that Growth of Cells is to individual layer, and with PBS (pH7.4) rinsing 2 times of sterilizing, cell blank contrast adds 1mLDMEM solution, and test group adds 1mL concentration respectively and is respectively 1 × 10 ⁸cfu/mL pathogenic bacterium and BY-5 (with without dual anti-DMEM nutrient solution suspended bacterial), at 5%CO ₂2h is hatched at incubator 37 DEG C.With aseptic PBS washed cell 5 times, after use sterile distilled water lysing cell, and carry out coated plate engineering test after cell pyrolysis liquid being made gradient dilution, wherein intestinal bacteria, Salmonellas test group are coated on Mai Kangkai substratum, and staphylococcus is coated in high salt N.F,USP MANNITOL substratum, and BY-5 is coated on LB agar plate, at 37 DEG C, 24h is cultivated in incubator, counting, calculates the adhesion of each bacterium to intestinal epithelial cell ZYM-SIEC02, the results are shown in Table 9.

Stick bacterial count/cell count × 100 of index (cfu/100cells)=stick, wherein, cell quantity is obtained by the cell counting of control group.

Table 9 bacterium is to the adhesion of cell

7.3.4 Adherence inhibition test

In 24 orifice plates, after Growth of Cells to individual layer, with PBS (pH7.4) rinsing 2 times of sterilizing, be respectively 1 × 10 to add 1mL concentration ⁸cfu/mL indicator of causing a disease is control group, to add 500 μ L concentration for 1 × 10 ⁸cfu/mLBY-5 and 500 μ L concentration are 1 × 10 ⁸cfu/mL pathogenic bacterium indicator mixed culture is test group, and (all using in test without dual anti-DMEM nutrient solution suspended bacterial), at 5%CO ₂2h is hatched at incubator 37 DEG C.With aseptic PBS washed cell 5 times, after use sterile distilled water lysing cell, and cell pyrolysis liquid is made serial dilution, wherein intestinal bacteria, Salmonellas test group are coated on Mai Kangkai substratum, staphylococcus is coated in high salt N.F,USP MANNITOL substratum, BY-5 is coated on LB agar plate, in incubator, cultivate 24h at 37 DEG C, counting.Calculate BY-5 to the Adherence inhibition rate of each pathogenic bacterium.Adherence inhibition rate=[the sticking number of (control group bacterium stick number-test group bacterium stick number)/control group bacterium] × 100%.

Result shows, seen (table 9) by the adhesion of bacterium to cell, various pathogenic indicator to the Adhering capacity of cell all higher than BY-5, the BY-5 that 100 ZYM-SIEC02 cells stick on average only has about 8, and wherein the Adhering capacity size of pathogenic species to cell is followed successively by streptococcus aureus > intestinal bacteria > Salmonellas.Adherence inhibition test-results from table 10, BY-5 all has restraining effect to sticking of 3 kinds of pathogenic indicators, and its Adherence inhibition ability is descending is followed successively by intestinal bacteria > Salmonellas > streptococcus aureus.

Table 10BY-5 is to the inhibition effect on adhesion of pathogenic bacterium

Claims

1. hide a pig source bacillus amyloliquefaciens, it is characterized in that, its taxonomy called after bacillus amyloliquefaciens BY-5 (Bacillusamyloliquefaciens), deposit number is CCTCCNO:M2013739.

2. pig source, Tibetan as claimed in claim 1 bacillus amyloliquefaciens, it is characterized in that, the 16SrRNA gene order of described pig source, Tibetan bacillus amyloliquefaciens is as shown in SEQIDNO:1.

3. pig source, Tibetan as claimed in claim 1 bacillus amyloliquefaciens, it is characterized in that, the sequence of the endo cellulase gene that described pig source, Tibetan bacillus amyloliquefaciens is cloned is as shown in SEQIDNO:2.

4. pig source, the Tibetan bacillus amyloliquefaciens described in claim 1,2 or 3 is for the preparation of the application of probiotics.

5. pig source, the Tibetan bacillus amyloliquefaciens described in claim 1,2 or 3 is used for the application that cellulase extracts.

6. pig source, the Tibetan bacillus amyloliquefaciens described in claim 1,2 or 3 is used for the application of fiber degradation.