CN105567598B

CN105567598B - A kind of Tibetan pig source bacillus amyloliquefaciens and its application

Info

Publication number: CN105567598B
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: 2019-03-01
Anticipated expiration: 2036-01-15
Also published as: CN105567598A

Abstract

The invention discloses a kind of Tibetan pig source bacillus amyloliquefaciens and its applications, disclosed Tibetan pig source bacillus is identified through Morphological Identification, biochemical identification and molecules, bacillus amyloliquefaciens BY-5 (Bacillus amyloliquefaciens), deposit number are as follows: CCTCC NO:M 2013739 are named as according to taxology.The bacillus amyloliquefaciens are separated from hiding pig cecal content, can efficiently generate cellulolytic enzyme, the ability with degradation of sodium carboxymethylcellulo, e；Furthermore, external probiotic properties evaluation discovery is carried out to the bacterial strain, the bacterial strain adverse-resistant characteristic is strong, it can not only heat-resisting, acidproof and bile tolerance, and there is certain rejection ability to Escherichia coli, salmonella and staphylococcus aureus, in addition, Tibetan pig source bacillus amyloliquefaciens also have certain rejection ability to sticking for pig intestinal mucosa to 3 kinds of pathogenic bacteria.

Description

A kind of Tibetan pig source bacillus amyloliquefaciens and its application

Technical field

The invention belongs to microorganism fields, and in particular to a kind of Tibetan pig source bacillus amyloliquefaciens and its application.

Background technique

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

In animal husbandry and feed industry, in order to alleviate the situation of current energy feed shortage, application of cellulase and The separation screening of the cellulose-decomposing bacterium of cellulase-producing is taken seriously always.From the point of view of the source of cellulase, microorganism It is the most important source of cellulase.Currently, can be to micro- life that complicated cellulose is effectively degraded in nature Object includes fungi, bacterium, actinomyces, and wherein the degradation capability of fungi is most strong, mainly include trichoderma, aspergillus, Penicillium and The bacterial strain of the mould category of acremonium, paint spot etc..In these fungies, fungus Trichoderma is formed and eccrine fiber element enzyme system because producing Ingredient is most comprehensively, enzyme amount is big, activity is high and is widely used in enzyme preparation production, wherein the active higher strain that represents has: health Family name's trichoderma, aspergillus niger, trichoderma reesei etc.^[1-3].And opposite fungi, bacterium is simple with culture, the speed of growth is fast, fermentation period is short The advantages that, therefore, is filtered out from nature and be capable of the cellulose decomposing bacteria of efficient degradation cellulose and also result in people's Extensive concern^[4].Currently, it is flat to generally use sodium carboxymethylcellulose (CMC-Na) in terms of the separation screening of cellulose-decomposing bacterium Plate just sieve method and measurement cellulase activity secondary screening method combine, wherein bacillus, Ruminococcus, Bacteroides, The cellulose-decomposing bacterium of fusobacterium, Cellulomonas etc. is from the cud of ox^[5], hiding pig^[6]And the excrement of giant panda^[7], grass Fish enteron aisle^[8], soil^[9], goose gastrointestinal tract^[10]And marine bacteria^[11]It has separated and has come out in.

2, the application of cellulase preparation:

Cellulose is the polysaccharose substance for being widely present in nature, and cellulase can effectively hydrolyze cellulosic material At monosaccharide and then fermentative production of ethanol, hydrogen and microbial oil etc., therefore to it, rationally utilization can alleviate global energy Pressure^[12].In animal productiong, cellulase is as a kind of microorganism formulation, mainly to be directly appended to the mode in daily ration It uses.If Lv Donghai (2002) research is pointed out, cellulase is added in the daily ration of meat duck, meat duck early stage can not only be improved Average daily gain, to crude fat utilization rate, to dry matter and crude fiber digestibility, and meat duck can be reduced to some extent and disappeared Change the relative size of organ, increase the duodenal relative length of meat duck Later growth, and makes meat duck intestinal mucosa lower layer, flesh Layer and the thinning trend of overall thickness^[13].Gaoyang etc. (2014) research is pointed out, adds non-starch polysaccharide enzyme in the daily ration of grower pigs (zytase, 1,4 beta-glucanase, cellulase) can effectively improve growth of growing-finishing pigs performance, and improve to a certain extent Meat^[14].In addition, addition cellulase can effectively reduce the damage of ammoniacal nitrogen in excrement and urine in nonruminant It loses^[15], therefore, cellulase has been also used in the composting treatment process such as animal wastes, rubbish by Many researchers, smelly to reduce The source of gas substantially improves farm's feeding environment, improves animal welfare.In addition, in paper-making industry and food industry and weaving In the production such as industry, cellulase also has very extensive purposes and application prospect^[12]。

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

Antibiotic is 20th century most important medical discovery, has played great work in treatment human and animal's disease With^[16].With greatly developing for intensive aquaculture industry, in domestic animal and poultry production, almost 90% antibiotic is with asian treatment Level adds the prevention for being used for Animal diseases in feed and control, promotes growth of animals or poultry, improves food conversion ratio^[17 _, ^18]。 Inappropriate due to antibiotic is abused using with long-term, is inevitably inhibited and has been damaged part profitable strain, lead to animal Gastral dysfunction causes disease；The increase of animal body endurance strain, these endurance strains are also resulted in simultaneously By direct between humans and animals^[19 _, ^20]With it is indirect^[21 _, ^22]Form propagated.In addition, antibiotic is in edible animal body Residual serious threat is also resulted in safely to the health and meat products of the mankind^[18 _, ^23].Therefore, many sections of recent domestic Worker strongly seek and develop to have no toxic side effect, noresidue, the antibiosis that growth of animal but also disease preventing and treating can be promoted Plain substitute.

Probiotics are because having the advantages that other drugs are irreplaceable, i.e., " illness is cured the disease, not sick diseases prevention, disease-free health care " Effect, and increasingly received an acclaim as application of the green feed additive in animal-breeding^[24].Wherein probiotics is One kind generates the additive for microbe feedstuff of the work of beneficial effect by promotion intestinal microbial balance to host animal, is One potential substitute of antibiotic.Studies have shown that probiotics is improving breeding performonce fo animals, improving microbial population of animal intestinal tract And reduction disease generation etc. played an important role^[25-27].Japan begins to use probiotics in nineteen sixty, and China exists Begin to use within 1980 probiotics, Food and Drug Administration (US-FDA)) disclosed 42 kinds in 1989 can Direct-fed Safely and effectively microorganism fungus kind^[26].End on June 30th, 2013, FDA " generally recognized as safe substance " (generally Recognized as safe, GRAS) in inventory, be related to microorganism fungus kind and microorganism fungus kind source has 108 records, contains 1, lid, 40, mesh belongs to 66 kinds of 5 subspecies^[28].And 2008 editions (notification numbers 1126) institute that our countries issue in the Ministry of Agriculture is public On the basis of 16 kinds of feed microbe additives of cloth, the micro- life of feed of the Ministry of Agriculture's (notification number 2045) newly promulgated for 2013 It has increased 19 kinds of microbe additives in object catalogue newly, and subjects has been provided^[29].It can be seen that probiotics is as one The novel green feed additive of kind has become a hot topic of research, and is gradually widely used in animal productiong.It is wide in European Union The general probiotics as feed addictive mainly includes bacillus (Bacillus cercus, bacillus licheniformis and withered grass bud Spore bacillus), enterococcus spp (enterococcus faecium), Lactobacillus (cheese lactic acid bacteria, lactobacterium acidophilus, Lactobacillus farciminis, plant cream Bacillus, Lactobacillus rhamnosus), Mycosphaerella (acidophilus micrococcus), streptococcus and some fungies such as saccharomyces cerevisiae and Crewe dimension Yeast etc.^[30], and the prevention of the growth and disease to animal mostly play the role of it is positive.Wherein bacillus is because of its resistance By force, high temperature high voltage resistant, the easily good characteristics such as storage, and have and adjust intestinal flora balance, enhancing animal immunizing power, improve production The various functions such as performance are considered as optimal microbe additive.Such as Lee (2014) research is pointed out, is increased in pig diet Different amounts of probiotics bacillus subtilis LS1-2 fermentation thalli is mended, can increase different phase being averaged for pig of wean and increase day by day Weight, average daily gain and the digestibility to nutriment；Meanwhile the concentration variation of the IgG and IgA in blood, small intestine suede The variation of hair height and Crypt depth and the cell concentration in diet is in a linear relationship, clostridium and large intestine in caecum Bacillus group number is then reduced with biomass concentration increase in diet^[25].In addition, the evil that probiotics generate farm It is smelly also have certain removal effect and effect it is obvious.Such as Ye Fenxia will be by bacillus, the false silk of streptomyces griseus and the torrid zone The complex microorganism preparations of saccharomycete composition are used for the deodorizing test of hoggery pigsties and dung yard, the results showed that, in pig house NH₃、H₂S and odour concentration reduce 78.4%, 66.7% and 83.3% respectively；NH in pig manure field₃、H₂S and odour concentration 84.4%, 62.1% and 88.5% is reduced respectively^[31].Li Wanjun (2011) research is pointed out, micro- life is added in layer diets The production performance that state preparation (main component is bacillus, lactic acid bacteria and saccharomycete) can significantly improve laying hen, reduces in excrement Ammonia yield improves chicken coop air environment^[32].It can be seen that probiotics probiotics is to animal growth and supports It grows an enhancement of environment and all has certain meaning.However, a kind of research of new probiotic bacterium, first having to the problem of facing is bacterial strain Screening and its functional activity measurement.In screening process, do not require nothing more than that these strain growth speed are fast, are resistant to animal The gastrointestinal tract environment factor of itself has specific Function, and strain source wants consistent with using object, this is to improve strain The effective way of specificity^[33-35]。

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Summary of the invention

The purpose of the present invention is provide a kind of Tibetan pig source bacillus amyloliquefaciens, the bacterial strain for animal productiong and industrial production It is the bacillus for capableing of decomposition of cellulose isolated from hiding pig intestinal contents.Identified, which is a kind of Its classification naming is bacillus amyloliquefaciens BY-5 (Bacillus by new Tibetan pig source bacillus amyloliquefaciens Amyloliquefaciens), sent to China typical culture collection center and carried out preservation, address: the Chinese Wuhan, the Wuhan is big It learns；Preservation date: on December 31st, 2013；Deposit number is CCTCC NO:M 2013739, which can be used for cellulase Extraction, in favor of industrial development；Or the nonruminants such as pig are promoted to digest coarse-fibred micro- life for making in animal productiong State preparation, such as probiotics.

The 16SrRNA gene order of the Tibetan pig source bacillus amyloliquefaciens is as shown in SEQ ID NO:1.The hiding The sequence for the endo cellulase gene that pig source bacillus amyloliquefaciens are cloned is as shown in SEQ ID NO:2.Clone's solution starch Bacillus BY-5 (Bacillus amyloliquefaciens) cellulose enzyme gene, and carried out in prokaryotic expression system Expression.With DNAssist analysis software 499 encoding amino acid sequences analysis hair encoded to cellulose enzyme gene bglc-BY It is existing, the amino acid sequence by a cellulase and N-terminal in glycosyl hydrolase family 5 cellulose binding region (CBM- 3) albumen for forming, and encoding belongs to the family of BglC.Moreover, it has been found that the cellulase protein is made of 20 kinds of amino acid, The molecular weight theoretical value of coded albumen is 55KD, isoelectric point 8.6.

The Tibetan pig source bacillus amyloliquefaciens are used to prepare the application of probiotics.The Tibetan pig source solution starch Bacillus is used for the application that cellulase extracts.The Tibetan pig source bacillus amyloliquefaciens are used for the application of fiber degradation. Tibetan pig source bacillus amyloliquefaciens of the invention have higher cellulase activity, can be used in enteron aisle or cellulose in excrement Degradation improves farm's animal living enviroment；Meanwhile in vitro in the experiment of simulated gastrointestinal tract, there is strong degeneration-resistant spy Property, it can also be used to microorganism formulation is researched and developed, to prevent and treat the generation of animal intestinal tract disease.

Tibetan pig source bacillus amyloliquefaciens have strong tolerance to heat, simulated gastric fluid, simulated intestinal fluid (different gallbladder salinities), And there is certain inhibiting effect to Escherichia coli, salmonella and staphylococcus aureus, to chitterlings epithelial cell ZYM- The small toxicity of SIEC02；But the bacterium to Escherichia coli, salmonella and staphylococcus aureus to cell stick have it is certain Inhibiting effect.

Tibetan pig source bacillus amyloliquefaciens of the invention are to be separated from hiding chitling content, screen and obtain, and existing Technology is compared, and is had the advantage that

1) Tibetan pig source bacillus amyloliquefaciens BY-5 (Bacillus amyloliquefaciens) is from hiding chitling content The pure natural strain filtered out in object, fermented supernatant fluid has very high cellulase activity, and has strong degeneration-resistant spy Property, cytotoxicity are small.Therefore, exploitation, the decomposed processing of muck, cellulase extraction, crude fibre of probiotics are used directly for Degradation etc..

2) from Tibetan pig source bacillus amyloliquefaciens BY-5 (Bacillus amyloliquefaciens) genomic DNA gram Grand cellulose enzyme gene bglc-BY gene can carry out heterogenous expression in pET prokaryotic expression system.

Detailed description of the invention

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

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

The Gram's staining of Fig. 3 cellulose-decomposing bacterium；

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

The agarose gel electrophoresis figure of the 16S rRNA gene of Fig. 5 cellulose-decomposing bacterium, in the figure: M is Marker III, Swimming lane 1,2 is the 16S rRNA gene band of cellulose-decomposing bacterium；

The 16S rRNA genic system of Fig. 6 cellulose-decomposing bacterium Bacillus amyloliquefaciens BY-5 is developed Tree；

Fig. 7 cellulose-decomposing bacterium Bacillus amyloliquefaciens BY-5 growth curve；

Fig. 8 glucose standard curve；

The enzymatic productivity of Fig. 9 cellulose-decomposing bacterium Bacillus amyloliquefaciens BY-5；

The Ago-Gel electricity of Figure 10 Bacillus amyloliquefaciens BY-5 cellulose enzyme gene bglc-BY Swimming is schemed, in figure: M is Marker III, and swimming lane 1,2 is bglc-BY gene band, size 1500bp；

Figure 11 cellulose incision enzyme gene bglc-BY sequence evolution tree；

The sequence of Figure 12 fiber endonuclease protein amino acid is analyzed；

The structure prediction of Figure 13 cellulose endonuclease protein；

The digestion of Figure 14 cellulose enzyme gene pET28 (a+)-bglc-BY expression vector is identified；In the figure: M is MarkerIII, swimming lane 1 are the double digestion segment of pET-bglc-BY expression vector, and swimming lane 2 is pET-bglc-BY expression vector Single endonuclease digestion segment；

Expression of Figure 15 recombinant bacterial strain pET-bglc-BL21 in prokaryotic cell, having transparent circle is recombinant bacterial strain pET- Bglc-BL21, transparent circle is not empty carrier pET-BL21 control；

Figure 16 is the heat resistance 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 bacteriostatic test of the BY-5 to pathogenic bacteria.Figure A is Escherichia coli, and B is salmonella, and C is golden yellow grape Coccus；

Below in conjunction with attached drawing and specific test and the present invention is described in further detail.

Specific embodiment

Probiotics are using normalMicroorganismOr live made of the substance for promoting microorganism to growMicroorganism system Agent.That is, all can promoteNormal microfloraGrowth and breeding and inhibition pathogenic bacteria growth and breeding preparation is referred to as Probiotics.The effect of adjusting enteron aisle because of it is quick, and constructs intestinal microecology balance, no matter in baby, old man, or it is new Livestock fowl all can prevent and treat diarrhea, constipation.Now common people with probiotics have ecological viable bacteria element,Medilac-Vita、Strong spirit Stachyose、Zheng Chang Sheng、Meter YaDeng.

Bacillus of the invention-bacillus amyloliquefaciens BY-5 (Bacillus Amyloliquefaciens), in the deposit number of China typical culture collection center are as follows: CCTCC NO:M 2013739.The solution Bacillus amyloliquefaciens can be separating obtained from the content of hiding pig caecum, small intestine or stomach, can generate cellulase, has drop Solve the ability of sodium carboxymethylcellulose.At 37 DEG C, producing enzyme fermented and cultured is carried out under the conditions of 220rpm, the results show that bacterial strain is being sent out Cellulase-producing amount is high when ferment culture is to 28~44h and stablizes.

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens of the applicant to be issued on NCBI Subsp.plantarum str.FZB42) cellulose enzyme gene bglc be foundation, using primer primer5.0 design draw Object carries out PCR amplification, and Successful amplification has gone out the interior of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5 Cellulase β-Isosorbide-5-Nitrae glucanase gene bglc-BY is cut, and successfully constructs cellulose incision enzyme gene bglc-BY protokaryon table Up to system, when 37 DEG C, 220rpm, bacterium solution OD600 reach 0.8 or so, IPTG induction is carried out, the results show that constructed original Nuclear expression system expresses success in vitro.

Bacillus-bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5 of the invention Show that the bacterial strain has strong heat-resisting, acidproof and bile tolerance ability by external prebiotic evaluation, in addition, having one to pathogenic bacteria Fixed inhibition and inhibition effect on adhesion.

Found out by result above, cellulose-decomposing bacterium-bacillus amyloliquefaciens of separation screening from hiding chitling road (Bacillus amyloliquefaciens) BY-5 can be used to the exploitation of fermenting and producing cellulase and pig source probiotics, To greatly developing, health, section grain pig breeding industry is of great significance and wide application prospect.

It is described in detail below by way of the embodiment that applicant provides.

The separation screening of embodiment 1:BY-5

1.1 test material sources and acquisition

Hiding chitling road contents samples are collected in Yang Ling Hua Yi Industrial Co., Ltd., pig slaughterhouse, hiding, and test pig is August Age health pig, feeding and management condition is consistent, in addition daily ration composition adds 10% or so bean dregs containing 90% green and rough feeds With wheat bran etc..Pig splits abdominal cavity after slaughter for hiding, is rapidly separated Stomach duodenum, jejunum, ileum, caecum, and every section takes 20cm or so, and the exhaust sealing in intestinal tube both ends Thread ligation, cutting, dress sealing polybag, are put into ice chest and take back rapidly Laboratory, and 4 DEG C of refrigerators are put into, carry out the separation of cellulose-decomposing bacterium as early as possible in a short time.

The separation screening of 1.2 bacterial strains

1.2.1 the configuration of culture medium

Sodium carboxymethylcellulose (CMC-Na) screening and culturing medium (g/L): CMC-Na 10g, peptone 5g, yeast powder 0.5g, K₂HPO₄1.5g, MgSO_4·7H₂O 0.2g, sodium chloride 5g, agar powder 15g, distilled water constant volume to 1L.

Basic Medium of shaking flask fermentation: 1% CMC is added in common liq LB culture medium.

LB culture medium: tryptose extract 10g；Yeast extract 5g；Sodium chloride 5g；Distilled water constant volume is to 1L.

The above culture medium is required to high pressure sterilization.

1.2.2 the primary dcreening operation of cellulose-decomposing bacterium

Hiding pig Stomach duodenum, jejunum, ileum, caecum cecal content 1g is weighed respectively to be transferred to and fill accordingly In the triangular flask of 100mL sterile water, vibrate 30min in 80 DEG C of electric heating constant temperature shaking bath pots, 10^-2Intestinal contents are dilute Release liquid, then again by 10 times of dilution methods successively gradient dilution at 10^-3-10^-6Intestinal contents dilution.Screening and culturing medium is made Then solid plate takes 100uL to be applied on the plate accordingly numbered respectively and carries out label with pipette, in 37 DEG C of constant temperature incubations In case be inverted culture 48h, find in the solid medium from stomach and the different gradients of caecum, have a large amount of canescence, The translucent bacterium colony with wrinkle mould, and in the culture medium from small intestinal segment content, the bacterium colony of this type is few.Then Appropriate 0.1% Congo red solution dyeing 1h is added into the culture dish of each gradient for we, discards dye liquor, appropriate 1mol/L is added NaCl solution washing, pours out NaCL solution after l h, finds to have around these whites, translucent wrinkle mould bacterium colony transparent Circle.With the diameter (D) and colony diameter (d) of periphery of bacterial colonies hydrolysis circle on vernier caliper master plate, the straight of the type bacterium colony is found Diameter is 0.7cm or so, and the hydrolytic circle to carboxymethyl cellulose is 2.2cm or so (Fig. 1).Then it is chosen with the oese of sterilizing It fetches and is cultivated in the LB culture medium of liquid respectively from the type bacterium colony of different intestinal segments, and the scribing line point repeatedly on CMC plate From purifying culture 4-5 for the bacterium colony (Fig. 2) up to being purified, and the strain glycerol adding of purifying culture is standby in -80 DEG C of preservations With.

In experiment, inventor simultaneously divides the cellulose of every section of hiding pig Stomach duodenum, jejunum, ileum, caecum content Solution is separated, and discovery is most in stomach and caecum, in small intestinal segment (duodenum, jejunum, ileum) it has also been found that there is cellulose The presence of decomposer.

1.2.3 the preparation and secondary screening of cellulose-decomposing bacterium fermentation crude enzyme liquid

By each bacterial strain filter out, freezen protective, scribing line carries out activation culture in LB solid medium tablets respectively, Then it chooses monoclonal to be inoculated into the fresh LB liquid medium of 50mL, shake culture is to logarithm under the conditions of 37 DEG C, 220rpm Phase (OD=1.0), liquid spawn is made.By the liquid spawn prepared (OD=1.0) respectively with 1% inoculum concentration be inoculated with into In 50mL Medium of shaking flask fermentation, fermented and cultured for 24 hours, takes appropriate fermentation liquid in 5000rpm, 4 DEG C under the conditions of 37 DEG C, 220rpm Under the conditions of be centrifuged 15min, supernatant is crude enzyme liquid, and the cellulase activity by measuring crude enzyme liquid carries out isolated bacterium Secondary screening.The result shows that the bacterial strain that preliminary screening arrives, and according to the measurement result of its cellulase activity, have chosen cellulase (0.23U/mL) highest one plant of bacterium living is used for subsequent research, which is BY-5 cellulose-decomposing bacterium.

Embodiment 2: the identification of cellulose-decomposing bacterium

2.1 morphologic features

BY-5 cellulose-decomposing bacterium, in 37 DEG C, 220rpm shake culture, takes culture to the bacterium of 12h in LB liquid medium Liquid carries out Gram's staining, and coloration result discovery, the thallus is in the shape of a rod, is dyed to purple, belongs to gram-positive bacteria (Fig. 3). Culture is taken to carry out spore staining with malachite green method to bacterium solution for 24 hours, the results show that the gemma of the bacterium is dyed to green, nutrition Body dyes red (Fig. 4).

The Physiology and biochemistry of 2.2 cellulose-decomposing bacteriums is identified

According to the method in " common bacteria system identification handbook " and " Berger bacterial identification manual " to BY-5 cellulose point It solves bacterium and carries out physio-biochemical characteristics index determining.According to the measurement result in table 1 show test strain physio-biochemical characteristics and " common bacteria system identification handbook table " and the characteristic of " Berger bacterial identification manual " bacillus amyloliquefaciens are almost the same.Cause The test strain screened in the test, is initially identified as the bacillus amyloliquefaciens (Bacillus of Bacillus by this ) or its mutation amyloliquefaciens.

The Physiology and biochemistry qualification result of 1 bacterial strain of table

Note: "+" indicates this feature or using the substance, and " one " indicates not having this feature or do not utilize the substance.

The molecular biology identification of 2.3 cellulose-decomposing bacteriums

2.3.1 the 16S rRNA gene fragment order measurement of cellulose-decomposing bacterium

The BY-5 cellulose-decomposing bacterium strain of freezen protective is subjected to activation culture, (the OD when culture to logarithmic phase₆₀₀= 1.0-1.5), thalline were collected by centrifugation, carries out extracting genome DNA.Bacterial genomes DNA extraction method is biochemical referring to Beijing Tiangeng The method that Science and Technology Ltd.'s bacterial genomes kit provides carries out.Then with bacterial universal primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCA-3', downstream primer 1492R:5'-GGTTACCTTGTTACGACTT-3 ') carry out bacterial 16 S The amplification of rRNA gene.PCR 25uL reaction system includes: DNA profiling 0.5uL (guaranteeing 50-100ng/uL), upstream and downstream primer Each 1uL, mixTag enzyme 12.5uL, deionized water supply 25uL.PCR amplification condition are as follows: reaction condition are as follows: 95 DEG C of initial denaturations 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 50s, 35 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of forever.1% agar Sugared detected through gel electrophoresis amplification (Fig. 5), and pcr amplification product glue is recycled, and recovery product is connected in pGEM-T carrier, It is transferred to DH5 α competent escherichia coli cell, the solid LB media plate of Amp resistance, 37 DEG C of mistakes are coated on after renewal cultivation 1h Night culture.Bacterium colony PCR is accredited as positive bacteria and falls behind, and Beijing Hua Da gene biological company is sent to be sequenced.Sequencing result is existed Homology analysis is carried out in GenBank with BLAST software on NCBI, obtains the gene order of close typical strain, is utilized Mega5.0 constructs systematic evolution tree.

Through being sequenced, cellulose-decomposing bacterium 16S rRNA gene order length is 1455bp, and size is consistent with expected results (Fig. 5), gene order (Accession number:KT800418) is:

GATGGCGGGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGT AACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAATCG CATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTA ACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAG GTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAA CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGC GTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGG GGAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGT GGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGG TAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCA CTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGG GGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC CTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTA AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGC GGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCC GAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGACAAGAATAAG

2.3.2 the building of the sequence analysis and systematic evolution tree of separated cellulose-decomposing bacterium

By the 16S rRNA gene order of the BY-5 cellulose-decomposing bacterium of measurement, in GenBank database it is all The prokaryotes 16S rRNA gene of measurement is compared, the results showed that, the cellulose-decomposing bacterium and Bacillus in NCBI The genetic distance of amyloliquefaciens strain FZB42 is nearest (Fig. 6), the phase of the 16S rRNA gene order of the two Reached 99% like property, although and farther out with the genetic distance of other reported bacillus, sequence is similar Property is also 90% or more.Illustrate that the Tibetan pig source cellulose-decomposing bacterium being separated to belongs to the bacillus amyloliquefaciens of bacillus One kind or mutation.In conjunction with the morphological feature of the bacterium, physio-biochemical characteristics and Molecular Identification, this experiment sieving is arrived Cellulose bacterium be named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5.

The analysis of embodiment 3Bacillus amyloliquefaciens BY-5 growth characteristics

By bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5 in LB lining out, in 37 DEG C of items Activation culture under part is chosen monoclonal and is inoculated into 5mL LB liquid medium, and shake culture arrives under the conditions of 37 DEG C, 220rpm OD₆₀₀It between 1.0~1.5, is inoculated with 1% inoculum concentration into 60mL fresh LB, 2h sampling is spaced after 4 hours, until 60h measures its absorbance using spectrophotometer method under 600nm wavelength, draws growth curve of bacteria.Measurement result shows 8~28h is logarithmic phase to bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5 after inoculation, after 28h Initially enter stationary phase, and continue to 36h or so, after 36h, the breeding of thallus progresses into decline phase (Fig. 7).

The measurement of 4 cellulose-decomposing bacterium BY-5 cellulase activity of embodiment

4.1Bacillus amyloliquefaciens BY-5 cellulase activity measuring method

The production of glucose standard curve and the measuring method of cellulase activity refer to The Ministry of Agriculture of the People's Republic of China, MOA The Activity Determination of Cellulase (NY/T 912-2004) of publication carries out, and is changed according to the actual situation.

4.1.1 the configuration of required reagent and buffer

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

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

3, the 5- dinitrosalicylic acid for weighing 6.3g is dissolved with water, and 21.0g NaOH is added, and 182g sodium potassium tartrate tetrahydrate adds 500mL water adds 5.0g re-distilled phenol and 5.0g sodium sulfite after heating for dissolving, and stirring and dissolving is cooling, is settled to 1000mL.

(3) 1.000g glucose (AR) (105 DEG C of dryings to constant weight) glucose standards solution (10mg/mL): are weighed with steaming 100mL is settled to after distilled water dissolution.

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

4.1.2 the production of grape essence standard curve

Standard blank sample: drawing the NaAc_HAc buffer solution 4.0mL of pH=5.5, and DNS reagent 5.0mL, boiling is added Heating water bath 5min.It is cooled to room temperature with tap water, is settled to 25.0mL with water, standard blank sample is made.

1.0mg/mL glucose solution 1.0,2.0,3.0,4.0,5.0,6.0,7.0mL are drawn respectively, use buffer respectively It is settled to 10mL, is configured to the glucose standards solution that concentration is 0.1-0.7mg/mL.Then it is carried out according to table 2:

2 standard solution configuration proportion of table

It by above-mentioned each test tube mixed liquor, gently shakes and mixes, boiling water bath heats 5min, fixed with distilled water after flowing water is cooling Hold to 25mL, shake up, absorbance is surveyed at 540nm absorption peak, using glucose content as ordinate, with OD₅₄₀For abscissa work Figure, obtains concentration of glucose and OD₅₄₀Relation curve (Fig. 8).

4.1.3 the measurement of cellulase activity

A_B: 5mL DNS is added through appropriate diluted enzyme solution in 2mL, mixes, adds 2mL substrate, mixes, and boiling water boils 5min is settled to 25mL, and with standard blank sample (see 4.1.2) for blank, light absorption value A is measured under 540nm wavelength condition_B。

A_E: 2mL 1%CMC is added through appropriate diluted enzyme solution in 2mL, mixes, and 40 DEG C of accurate response 30min add 5mL DNS is mixed, and boiling water boils 5min, it is settled to 25mL, with standard blank sample (see 4.1.2) for blank, in 540nm wavelength condition Lower measurement light absorption value A_E。

Enzyme-activity unit definition: in pH=5.5,40 DEG C, under the conditions of reacting 30min, per minute by substrate carboxymethyl cellulose Enzyme amount needed for sodium degradation generates 1 μm of ol reduced sugar is defined as an enzyme activity unit, is indicated 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 dilution, u/mL；

A_EFor the absorbance of enzyme reaction solution；

A_BFor the absorbance of enzyme blank sample；

K is the slope of standard curve；

C_OFor the intercept of standard curve；

M is the molecular weight (180.2) of glucose；

T is enzyme digestion reaction time, min；

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

X_DValue should between 0.04-0.08U/mL, if not within this range, the dilution of enzyme solution should be reselected, Analysis measurement is carried out again.

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.

Enzymatic productivity of the 4.2Bacillus amyloliquefaciens BY-5 in the different fermentations time

By seed liquor (OD=1.0 of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5) with 1.0% inoculum concentration is inoculated in 100mL Medium of shaking flask fermentation (LB+1%CMC-Na), is issued in 37 DEG C, 220rpm condition Ferment culture.Since the 8h that ferments, 2mL is sampled every 4h, fermentation liquid 15min is centrifuged under the conditions of 5000rpm, 4 DEG C, supernatant is For crude enzyme liquid, the measurement of cellulase activity is then carried out according to 4.1 method to crude enzyme liquid.The result shows that when with fermentation Between extension, the rate of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5 enzymatic production is also gradually Increase.When fermentation is to 36h, the activity of cellulase reaches highest (0.35U/mL), and bacterial strain producing enzyme between 28~44h Kept stable (Fig. 9).

The clone of embodiment 5Bacillus amyloliquefaciens BY-5 cellulose enzyme gene and analysis

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

5.1.1BY-5 the PCR amplification of fiber enzyme gene

It requires to extract solution starch gemma according to Beijing Tiangeng biochemical technology Co., Ltd bacterial genomes DNA extraction kit Bacillus (Bacillus amyloliquefaciens) BY-5 genomic DNA, with the bacillus amyloliquefaciens issued on NCBI The cellulose enzyme gene bglc of (Bacillus amyloliquefaciens subsp.plantarum str.FZB42) be according to According to using primer primer5.0 design primer, adding restriction enzyme site appropriate, expand target fragment bglc-BY, primer sequence Column see the table below 3, and the PCR reaction system of cellulose enzyme gene bglc-BY is shown in Table 4.

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

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

PCR amplification program is as follows: 95 DEG C of reaction 5min, 94 DEG C of reaction 30s, 58 DEG C of reaction 30s, 72 DEG C of reaction 90s, and 72 DEG C 10min is finally placed in 4 DEG C wherein recycling 35 times under the conditions of 94 DEG C, 58 DEG C and 72 DEG C.It is dense using 1.2% after PCR amplification The agarose electrophoresis of degree is detected (Figure 10).

5.1.2PCR product recycles

PCR product is recycled using Bio-Flux plastic recovery kit.Detailed step is as follows:

A: with clean, sharp scalpel, the Ago-Gel containing target DNA fragment being cut, and it is dry to be put into 1.5mL In net centrifuge tube；

B: being added Extraction Buffer in the ratio (gel quality milligram number: sol solutions volume microlitre number) of 1:3, It is incubated in 50 DEG C of water-baths, until gel melts.

C: mixed liquor is transferred completely into Spin column, and 6000g is centrifuged one minute, and discards the liquid in adapter Body.

D: 500 μ L Extraction Buffer being added into Spin column, are centrifuged 30~60s in 12000g, and abandon Remove liquid in adapter.

E: the Wash Buffer of 750 μ L being added into Spin column, is placed at room temperature for 2~5min, is centrifuged in 12000g 30~60s discards liquid in adapter.

F: it is centrifuged 1min in 12000rpm again, then Spin column is transferred in 1.5mL centrifuge tube.To Spin The ddH of 50 μ L is added in column₂O, and it is placed at room temperature for 1min.

G:12000g is centrifuged 1min, contains DNA target fragment in microcentrifugal tube, saves in -20 DEG C.

5.1.3 connection conversion

With the connection kit of Fermentas company, by PCR recovery product and pGEM-T vector under the conditions of 22 DEG C 10min is connected, linked system is as shown in table 5 below:

5 linked system table of table

Connection product is transferred in DH5 α Competent cells, specific step of converting is as follows:

A: taking competent cell in -80 DEG C of refrigerators and melts in ice bath.

B: 10.0 μ L connection products are added, and place 30min on ice.

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

D: connection product is taken out from ice bath, places 2~3min in ice.

E: the LB culture medium of 900 μ L liquid non-resistants is added, and in 37 DEG C, 150rpm renewal cultivation 45min.

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

5.1.4 the PCR detection of Insert Fragment

Monoclonal on picking culture plate is expanded respectively with the upstream and downstream primer of cellulose enzyme gene bglc-BY, PCR amplification system, response procedures, agar sugar detection are identical as target gene amplification condition (table 5), will test as positive colony Bacterium solution saves, and sample presentation is sequenced.

Sequencing shows the cellulose enzyme gene of the Bacillus amyloliquefaciens BY-5 of clone (Accession number:KT800419)) sequence is as follows:

ATGAAACGGTCAATCTCTATTTTTATTACGTGTTTATTGATTGCGGTATTGACAATGGGCGGCTTGCTGCCTTCGCC GGCATCAGCAGCAGGGACAAAAACGCCAGTAGCCAAGAATGGTCAGCTTAGCATAAAAGGTACACAACTTGTAAACC AAGACGGCAAAGCGGTACAACTGAAAGGGATCAGTTCACATGGATTGCAATGGTATGGCGATTTCGTCAATAAAGAC AGCTTAAAATGGCTGAGAGACGATTGGGGCATCACCGTTTTCCGCGCGGCAATGTATACGGCAGACGGCGGTTATAT TGACAACCCGTCCGTGAAAAATAAAGTAAAAGAAGCGGTTGAAGCGGCAAAAGAACTTGGGATATATGTCATCATTG ACTGGCATATCTTAAATGACGGCAACCCAAACCAAAATAAAGAGAAGGCGAAAGAATTTTTCAAGGAAATGTCAAGT CTTTACGGAAACACGCCAAACGTCATTTATGAAATTGCAAATGAACC AAACGGTGACGTGAACTGGAAGCGTGATATTAAACCGTATGCGGAAGAAGTGATTTCCGTTATCCGCAAAAATGACC CCGACAATCCCATCATTGTCGGAACCGGTACATGGAGCCAGGATGTGAATGATGCTGCCGATGACCAGCTAAAAGAT GCAAACGTCATGTACGCGCTTCATTTTTATGCCGGCACACACGGCCAATCTTTACGGGATAAAGCAAACTATGCACT CAGTAAAGGAGCGCCTATTTTCGTGACGGAATGGGGAACAAGCGACGCGTCTGGAAATGGCGGTGTATTCCTTGACC AGTCGCGGGAATGGCTGAATTATCTCGACAGCAAGAAAATCAGCTGGGTGAACTGGAATCTTTCTGATAAGCAGGAA TCATCCTCAGCGTTAAAGCCTGGAGCATCTAAAACAGGCGGCTGGCCGCTGTCAGATTTAACTGCTTCAGGAACCTT CGTAAGAGAAAACATTCGCAGCAACAAAGATTCAACGAAGGACGCCCCTGAAACGCCAGCACAAGATAATCCCACAC AGGAAAAAGGCGTTTCTGTACAATACAAAGCAGGGGATGGGAGTGTGAACAGCAACCAAATCCGCCCGCAGCTTCAC ATAAAAAATAACGGGAATACGACGGTTGATTTAAAAGATGTCACTGCCCGTTACTGGTATAACGCGAAAAACAAAGG CCAAAACTTTGACTGTGACTACGCGCAGATTGGATGCAGCAATCTGACCCACAAGTTTGTGACGCTGCATAAACCTA AGCAAGGCGCAGATACCTATCTGGAACTGGGGTTTAAAAAAGGAACACTGTCACCGGGAGCAAGCACAGGGAATATT CAGCTTCGTCTTCACAACGATGACTGGAGCAATTATGCACAAAGCGGCGATTATTCCTTTTTCCAATCAAATACGTT TAAAACAACGAAAAAAATCACATTATATCATCAAGGAAAACTGATTTGGGGAACAGAACCCAATTAG

5.2 cellulose enzyme gene bglc-BY sequence evolution trees

The cellulose enzyme gene bglc-BY sequence of Bacillus amyloliquefaciens BY-5 is committed to Genbank (Accession number:KT800419), is had found, the cellulose for the BY-5 that we are screened by sequence analysis The fiber of enzyme gene bglc-BY and the nearest Bacillus amyloliquefac-iens strain FZB42 of its affiliation The genetic distance of plain enzyme gene is far (Figure 11), and the similar sequence of the two is 96%；And with other bacillus The existing high homology of cellulase gene sequence, and have a certain difference.Illustrate us from separated cellulose point Successful clone has arrived cellulose enzyme gene in solution bacterium Bacillus amyloliquefaciens BY-5, and the gene is the same as other The genetic distance of the cellulose enzyme gene of bacillus is remote, is a new gene.

The bioinformatic analysis of 5.3bglc-BY gene

5.3.1 the amino acid sequence analysis of cellulose endonuclease protein

The further analysis of the amino acid sequence of cellulose enzyme gene bglc-BY is found with DNAssist analysis software, it should Albumen (499aa) is by 20 kinds of Amino acid profiles (Figure 12), the wherein highest three kinds of amino acid of amino acid residue content are as follows: glycine (Glycine), lysine (Lysine), asparagine (Asparagine)；And the least three kinds of amino acid of content are as follows: half Guang Propylhomoserin (cysteine), histidine (histidine), methionine (methionine).According to the amino acid of cellulase protein The molecular weight theoretical value that sequence is calculated is 55KD.In addition, the isoelectric point of cellulose restriction endonuclease is 8.6 by forecast analysis, Therefore, the cellulase that bacillus amyloliquefaciens BY-5 secretes in the test belongs to alkali protease.

5.3.2 cellulase protein structure prediction

Amino acid sequence is by glycosyl hydrolase to be found to 499 further analyses of amino acid sequence of bglc-BY gene coding The cellulose binding region (CBM-3) of a cellulase and N-terminal in enzyme family 5 forms (Figure 13).With bacillus subtilis Fungin enzyme is such as: CAA97610.1, ACK38261.1, AAK94871.1, AAK39540.1, AAO63626.1 etc. have 99% Above similitude belongs to the family of cellulolytic enzyme BglC.

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

The building of 6.1 prokaryotic expression carriers

By the bacterium solution of the pGEM-bglc-BY containing recombinant plasmid of preservation and the bacterium solution of pET28 containing expression vector (a+) respectively at The flat lining out of the LB of ammonia benzyl resistance and kalamycin resistance shakes bacterium culture with the toothpick picking monoclonal of sterilizing, and extracts Plasmid.The plasmid of extraction is all made of BamHI and Hand III in 37 DEG C of water-baths, carries out double digestion.Double digestion system is as follows Shown in table 6:

6 plasmid double digestion system of table

Digestion products are detected with 1% agarose gel electrophoresis.It will identify the target gene fragment of correct (Figure 14) Bglc-BY and the pET28a of linearisation (a+) segment carry out glue recycling, connection, and linked system is shown in Table 7.

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

Connection product is connected into 10min under the conditions of 22 DEG C, is converted to competent cell DH5 α, coated plate (kanamycins Resistance), 37 DEG C of insulating boxs are incubated overnight, and after the screening of blue hickie and bacterium colony PCR detection, are selected positive colony and are extracted plasmid. Then expression vector plasmid pET-bglc being transferred in BL21 (DE3) competent cell, converted product 4000rpm is centrifuged 2min, And appropriate converted product is taken to be coated on the LB plate of kalamycin resistance, 37 DEG C of overnight incubations.Picking monoclonal carries out shaking bacterium training It supports, primer when being cloned using target gene is carried out PCR identification positive colony as primer and will identify that correct recon progress is cold Freeze and saves.Recon positive colony is named as pET-bglc-BL21.PET-28 (a+) empty carrier conversion BL21 is simultaneously, with It compares.

The inducing expression of 6.2 recombinant bacterial strain pET-bglc-BL21

Recombinant bacterium pET-bglc-BL21 is incubated overnight rear picking single bacterium in the flat lining out of LB of kalamycin resistance It falls, is seeded in the LB culture medium containing kanamycins, 37 DEG C, after 220rpm cultivates 12h, in the ratio of 1:50, be inoculated in new In fresh LB (blocking that resistance) fluid nutrient medium, when shake culture to logarithmic growth phase (OD600 is in 0.5-0.8), add dense eventually IPTG (be added IPTG before the sample) induction that degree is 1mmol/L samples when induction is to 8h, and sample spot is connect containing CMC- It is cultivated for 24 hours on the LB plate (containing that resistance 100ug/mL is blocked) of Na, IPTG, whether observe after congo red staining has around thallus Transparent circle, while being control with the empty carrier pET-28 (a+) without containing endo glucanase gene.Result of study shows recombination Bacterium pET-bglc-BL21 bacterium on the LB plate of CMC-Na, IPTG has the generation (Figure 15) of transparent circle, illustrates recombinant bacterium pET- Cellulose enzyme gene in bglc-BL21 can successfully carry out heterogenous expression under the induction of IPTG.

The external prebiotic evaluation of embodiment 7Bacillus amyloliquefaciens BY-5

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

(1) activation culture of bacterial strain: before test, Bacillus amyloliquefaciens BY-5, the large intestine frozen Bacillus and salmonella, staphylococcus aureus are crossed activation culture on sterile LB solid medium, choose monoclonal switching Activation culture 2 times.By the Escherichia coli after activation, salmonella, staphylococcus aureus, Bacillus Amyloliquefaciens BY-5 is seeded in LB liquid medium with 0.1% inoculum concentration respectively, at 37 DEG C, 220rmp item Culture under part (wherein 3 kinds of pathogenic indicator bacterias cultivate 12h, and Bacillus amyloliquefaciens BY-5 is cultivated for 24 hours).It takes Each bacterium solution is centrifuged 15min in right amount under the conditions of 4000rpm and collects thallus, and thallus washs 2 with sterile PBS liquid (pH 7.4) It is secondary, thallus finally is resuspended with isometric sterile PBS liquid.Then Bacillus amyloliquefaciens BY-5 is hanged into bacterium The concentration of liquid is adjusted to OD₆₀₀=0.1 or so, 3 kinds of pathogenic indicator bacterias pass through respectively colony counting method adjust its concentration to 1 × 10⁸Cfu/mL or so, respectively as the seed liquor of follow-up test.

(2) count of bacteria: bacterium solution to be measured is carried out 10 times and is incremented by dilution, the bacterium solution of 3 suitable gradients is chosen, takes 0.1mL is applied to respectively on LB solid plate, 3 repetitions of each gradient.The plate wiped is applied after slightly drying, is inverted, 37 DEG C It cultivates 12h to take out, the specific method of counting detailed rules and regulations of bacterial clump are according to People's Republic of China's food microbiological examination bacterium colony Total (GB/T4789.2-2008) method is measured to carry out.Viable count in every milliliter of bacterium solution to be measured calculates as follows:

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

7.2Bacillus amyloliquefaciens BY-5 degeneration-resistant test in vitro

(1) heat resistance

By BY-5 seed liquor (OD₆₀₀=0.1), be respectively placed in 65 DEG C, 75 DEG C, 85 DEG C, handle 20min in 100 DEG C of water-baths Afterwards, cooling with flowing water rapidly, as control, to calculate BY-5 in not equality of temperature by colony counting method without Overheating Treatment bacterium solution The survival rate of degree condition learns that BY-5 is resistant to certain high temperature, but as the temperature rises, and the survival rate of BY-5 is Decline.Wherein 85% or more, survival rate under the conditions of 85 DEG C and 100 DEG C is respectively the survival rate at 65 DEG C, 75 DEG C 78.67% and 35.45% (Figure 16).

(2) resistance to simulated gastric fluid ability

By BY-5 seed liquor (OD₆₀₀=0.1) it is seeded in the simulated gastric fluid of pH1-4, is mixed, 37 DEG C with 5% inoculum concentration Stationary culture.1h, 2h, 3h, 4h take and cultivate bacterium solution in above-mentioned each processing after being inoculated with initial and inoculation respectively, carry out plate count And the survival rate of each bacterial strain is calculated, evaluate its acid resistance.The result shows that under the conditions of same pH, with the extension of incubation time, The survival rate of BY-5 is on a declining curve.And in same incubation time, under condition of different pH, with the raising of pH, BY-5's is deposited Motility rate is in the trend risen.1-2h is wherein handled under condition of different pH, the survival rate of BY-5 has reached 85% or more；And 1-4h is handled under conditions of pH2-4, the survival rate of BY-5 has reached 80% or more, illustrates that BY-5 is resistant to certain acidity Simulated gastric fluid (Figure 17).

(3) ability of the simulated intestinal fluid of resistance to different gallbladder salinities

The culture for 24 hours of 2mL BY-5 is taken to be centrifuged, PBS liquid washing thalline 2 times, then by bacterial sediment in the artificial stomach of pH2 After handling 2h in liquid, centrifuging and taking bacterial sediment suspends again after then being washed twice again with PBS and suitably dilutes, and adjusts bacterium solution Concentration is OD₆₀₀=0.1, it is suspended in the simulated intestinal fluid of different gallbladder salinities, is mixed, 37 DEG C of stationary cultures with 5% inoculum concentration. After being inoculated with initial and inoculation after 1h, 2h, 3h, 4h, after taking appropriate above-mentioned each bacterium solution gradient dilution respectively, plate count is carried out, Evaluate its resistance to simulated intestinal fluid and bile tolerance.The result shows that BY-5 is in 0.1-0.4% gallbladder salinity in the same processing time In simulated intestinal fluid, with the raising of gallbladder salinity, overall survival rate is slightly in downward trend；In addition, in same gallbladder salinity Under the conditions of, with the extension of processing time, the survival rate of BY-5 is also in downward trend.However, in which kind for the treatment of conditions Under, the survival rate of BY-5 is still up to 90% or more, illustrates there is strong bile tolerance ability (Figure 18).

(4) antagonistic property

A, the culture of Bacillus amyloliquefaciens BY-5 and filtrate preparation

The seed liquor of BY-5 is inoculated in 100mL LB liquid medium with 4% inoculum concentration, bacterium fermentation training is shaken in 37 DEG C 28h is supported, is centrifuged 15min, separating thallus and supernatant under the conditions of 4000rpm, supernatant filters that (filter membrane diameter is through filter 0.22 μm), it is spare to collect filtrate.

B, the thick of Bacillus amyloliquefaciens BY-5 extracellular antiseptic substance mentions: the BY-5 in above-mentioned A is filtered Liquid takes 50mL, and the 50%PEG6000 stirring 1h of 75mL is added, until PEG6000 is completely dissolved, 12,000rpm centrifugation 15min are gone Supernatant mixes the PBS buffer solution of 10 times of volumes of precipitating, using the antibacterial of Odontothrips loti measurement BY-5 antibacterial substance crude extract Ability.

C, the production of antibacterial plate and bacteriostatic test: the solid LB solid medium of sterilizing is cooled to 60 DEG C or so, is used The graduated cylinder of sterilizing accurately measures 10mL, and the culture dish for pouring into sterilizing is interior after its solidification, and Oxford cup is placed on thereon.It is by concentration 1×10⁸The indicator bacteria of cfu/mL is cooled in 50 DEG C or so of LB solid medium with the addition of 1% inoculum concentration, each after shaking up Culture dish falls 25mL or so, removes Oxford cup after its thoroughly solidification (Oxford cup and culture bottle will sterilize in time).Then it is training It supports in each cup aperture in ware, is separately added into each 150 μ L of crude extract of BY-5 extracellular antiseptic substance, is placed in 37 DEG C of constant temperature incubation 12h is cultivated in case, measures the diameter of inhibition zone, evaluates its bacteriostasis.3 repetitions are done in each processing, then take its average value.

It is seen by the antibacterial result of Figure 19, BY-5 shows certain inhibiting effect to 3 kinds of pathogenic bacteria.Wherein BY-5 is to large intestine The fungistatic effect of bacillus and salmonella is close, and the diameter of inhibition zone is respectively 1.3cm and 1.35cm, and to Staphylococcus aureus The bacteriostatic activity of bacterium is higher than the bacteriostatic activity to Escherichia coli and salmonella.

7.3 test cell line

7.3.1 preparation and recovery and culture, the passage of cell before cell culture

Prepare before cell culture: iuntercellular and the superclean bench 1h before on-test answer ultraviolet-sterilization, and divulge information 30min, Guarantee that the environment of cell culture is sterile.In addition, cell culture is at 37 DEG C, CO₂The culture that concentration is 5%, humidity is 95% It is carried out in case, the culture solution and PBS liquid used in the process of cell manipulation should be in cell incubators at preheating before cell manipulation Reason.

Recovery and culture, the passage of cell: chitterlings epithelial cell ZYM-SIEC02 is (by Xibei Univ. of Agricultural & Forest Science & Technology animal Medical college's man of virtue and ability's penetrating judgment, which is awarded, to be given) be removed from liquid nitrogen after, aseptically recovered and cultivated, passed on.Passage 2 times Afterwards, cell concentration is adjusted to 2 × 10⁵For testing, extra cell carries out freezing processing cells/mL.

7.3.2 the MTT test of cell

It is 2 × 10 by concentration⁵The ZYM-SIEC02 cell inoculation of cells/mL is in 96 well culture plates, every 100 μ L of hole, to Cell is long wash cell 2 times to after single layer with the PBS of sterilizing, be then separately added into plate hole 100 μ L concentration for 1 × 10⁸The BY-5 of cfu/mL, Escherichia coli, salmonella, staphylococcus aureus bacterium solution, every kind of bacterium sets 4 repetitions, to add DMEM The plate hole of culture solution is blank control, be placed in 37 DEG C, 2h is incubated in 5%CO2 incubator after inhale and abandon supernatant, wash cell 5 with PBS Secondary, MTT (5mg/mL) 20 μ L is added in every hole, is placed in 37 DEG C, 5%CO₂Cell incubator in continue be incubated for 4h.Careful inhale abandons hole After interior liquid, 150 μ L of DMSO is added into every hole and dissolves insoluble matter, vibrates 10min, its OD is then surveyed in microplate reader₄₉₀.It is logical Crossing calculating test group cell, to judge size of each bacterial strain to cytotoxicity, (cell is opposite relative to the survival rate of cellular control unit Survival rate 100% is 0 grade, is 1 grade between 80-99%；It is 2 grades between 50-79%；30-49% is 3 grades；0-29% is 4 Grade.).Cell survival rate (%)=test group absorbance value/control group absorbance value × 100%.

It being shown by the test result of Fig. 8, BY-5 and staphylococcus aureus are 1 grade to the toxicity of ZYM-SIEC02 cell, And Escherichia coli, salmonella are 2 grades to the toxicity level of cell.

Toxicity test of 8 bacterium of table to cell

7.3.3 adhesion assay

24 orifice plates are taken, it is 2 × 10 that a certain amount of concentration, which is added, in every hole⁵The ZYM-SIEC02 cell of cells/mL, be placed in 37 DEG C, 5%CO₂Constant-temperature incubation in incubator grows to single layer to cell, is rinsed 2 times with the PBS (pH7.4) of sterilizing, cell blank pair According to 1mL DMEM solution is added, it is respectively 1 × 10 that test group, which is separately added into 1mL concentration,⁸Cfu/mL pathogenic bacteria and BY-5 are (with unparalleled Anti- DMEM culture solution suspended bacterial), in 5%CO₂2h is incubated at 37 DEG C of incubator.It is washed cell 5 times with sterile PBS, after Coated plate engineering test is carried out with sterile distilled water lytic cell, and after cell pyrolysis liquid is made gradient dilution, wherein large intestine bar Bacterium, salmonella test group are coated on Mai Kangkai culture medium, and staphylococcus is coated in mannitol culture medium with high salt, and BY-5 is coated in LB On agar plate, is cultivated at 37 DEG C in incubator for 24 hours, count, calculate each bacterium to intestinal epithelial cell ZYM-SIEC02 Adhesion, the results are shown in Table 9.

Stick bacterial population/cell number × 100 for index (cfu/100cells)=stick, wherein cell quantity is by compareing The cell count of group obtains.

Adhesion of 9 bacterium of table to cell

7.3.4 Adherence inhibition is tested

In 24 orifice plates, after cell grows to single layer, rinsed 2 times with the PBS (pH7.4) of sterilizing, to add 1mL concentration point It Wei 1 × 10⁸Cfu/mL causes a disease indicator bacteria for control group, to add 500 μ L concentration for 1 × 10⁸Cfu/mL BY-5 and 500 μ L concentration It is 1 × 10⁸Cfu/mL pathogenic bacteria indicator bacteria mixed culture is test group, (is suspended with DMEM culture solution without double antibody in test Bacterium), in 5%CO₂2h is incubated at 37 DEG C of incubator.Washed cell 5 times with sterile PBS, after cracked with sterile distilled water it is thin Born of the same parents, and cell pyrolysis liquid is serially diluted, wherein Escherichia coli, salmonella test group are coated on Mai Kangkai culture medium, Staphylococcus is coated in mannitol culture medium with high salt, and BY-5 is coated on LB agar plate, cultivates for 24 hours at 37 DEG C in incubator, It counts.BY-5 is calculated to the Adherence inhibition rate of each pathogenic bacteria.Adherence inhibition rate=[(control group bacterium sticks number-test group Bacterium sticks number)/control group bacterium sticks number] × 100%.

The result shows that being seen (table 9) by adhesion of the bacterium to cell, various pathogenic indicator bacterias are equal to the Adhering capacity of cell Higher than BY-5, the BY-5 sticked on 100 ZYM-SIEC02 cells averagely only has 8 or so, and wherein pathogenic species are to cell Adhering capacity size is followed successively by staphylococcus aureus > Escherichia coli > salmonella.Adherence inhibition test result is come from table 10 It sees, BY-5 has inhibiting effect to 3 kinds of sticking for pathogenic indicator bacteria, and Adherence inhibition ability is descending to be followed successively by Escherichia coli > salmonella > staphylococcus aureus.

Inhibition effect on adhesion of the table 10BY-5 to pathogenic bacteria

Claims

1. a kind of Tibetan pig source bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is characterized in that it is classified Be named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BY-5, and deposit number is CCTCC NO:M 2013739。

2. the application that Tibetan pig source bacillus amyloliquefaciens described in claim 1 are used to prepare probiotics.

3. Tibetan pig source bacillus amyloliquefaciens described in claim 1 are used for the application that cellulase extracts.

4. the application that Tibetan pig source bacillus amyloliquefaciens described in claim 1 are used for fiber degradation.