CN104357346A - Rice dreg's protease hydrolysis producing strain bacillus subtilis and screening and application methods thereof - Google Patents
Rice dreg's protease hydrolysis producing strain bacillus subtilis and screening and application methods thereof Download PDFInfo
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Abstract
The invention discloses a rice dreg's protease hydrolysis producing strain bacillus subtilis and its screening and application methods. The bacillus subtilis strain pure culture was preserved in the China Center for Type Culture Collection on May, 13th, 2014. The address is Wuhan University, Wuhan, China. The postal code is 430072. The strain is called bacillus subtilis HP90 for short. Preservation number of the strain is CCTCCNO.M2014201. Through primary screening and secondary screening, the target strain is obtained. After cultivation of the slant preservation strain, the strain is directly used for hydrolysis of protein in rice dreg. The bacillus subtilis HP90 is used for hydrolysis of protein in rice dreg, and degree of hydrolysis reaches 19.83%. The strain is low-cost, can be recycled, has good stability, is green and safe, can directly be used for biological catalysis and has wide sources of raw materials. The methods provided by the invention are simple and practical. Therefore, the strain has an important application prospect and also can be applied in hydrolysis of other proteins.
Description
Technical field
The present invention relates to microorganism or enzyme, particularly protease-producing bacterium.
Background technology
Paddy, phytology belongs to the common rice subspecies of Gramineae Oryza common cultivated rice subgenus kind, is global first grain variety.The average annual output of paddy has accounted for greatly 2/5 of national total output of grain, reaches 1.90 hundred million t.The residue that rice residue is with early rice, crack rice, ageing rice etc. is remaining after being filtered Rice & peanut milk for raw material carries out fermenting and produce in the processes such as syrup, monosodium glutamate, biochemical drug, cheap, output is huge, often consumes 7t rice and will produce 1t rice residue in the production process of Starch rice sugar.In rice residue, protein content is about 40% ~ 70%, is equivalent to 5 ~ 8 times of protein content in paddy, and its content is close to soybean, and amino acid ligand is than the idealized model meeting WHO/FAO recommendation, and its quality is acknowledged as the best in foodstuff seed albumen.But the protein resource major part of these high-qualitys all in the form of dry powder cheap selling gives feed factory and makees feed, do not obtain deep development and utilization, if these rice residue protein resource conversion to be become to have amino acid and the polypeptide of high added value, not only can improve its biological value, economic worth, to the technology content improving China's paddy deep processing, and promote that the development of whole Grain Development has far-reaching influence.
Protein can obtain amino acid and polypeptide through acid, alkali or protease treatment.Acid, alkaline hydrolysis strongly can destroy some responsive amino acid, and generate poisonous propylene chlorohydrin class material.Enzyme hydrolysis condition is gentle, and to responsive amino acid without destruction, energy maximum reservation raw material physico-chemical property, local flavor, functional performance, green safety, energy consumption is little.Majority utilizes commercially available protein enzymatic protein at present, and this makes industrial production cost higher, and therefore, screening protease-producing bacterium is particularly important.
In current Chinese patent database, the patent application relating to protease-producing bacterium is few, only has No. ZL2010101038046 " a kind of organic solvent tolerant protease producing strains and produce gene and the application of organic solvent tolerant protease ", No. 2007101919691 " gene of a kind of Organic solvent-tolerant alkali protease producing strains and this Organic solvent-tolerant alkali protease and application " and No. 2013101991045 " a kind of Collagenase producing strains ".Up to now, there is no the application part of the protease-producing bacterium relating to hydrolysis rice residue.
Summary of the invention
The present invention aims to provide protease-producing bacterium---the subtilis of a strain hydrolysis rice residue, and the biological process enabled for rice residue protein is hydrolyzed.
Another object of the present invention is to provide screening method and the using method of above-mentioned subtilis, makes it possess specific industrial use.
For achieving the above object, contriver provides the protease-producing bacterium of a strain hydrolysis rice residue to be subtilis (Bacillus subtilis), this bacterial strain pure growth is preserved in Wuhan China typical culture collection center on May 13rd, 2014, depositary institution address: Wuhan, China Wuhan University, postcode 430072, deposit number is CCTCC NO.M 2014201.This bacterial strain is called for short subtilis HP90, is to be separated to obtain from Guizhou In China tradition sausage.
Above-mentioned subtilis HP90 morphological specificity and physiological and biochemical property close to bacillus, the 16S rRNA sequence of subtilis HP90 with
bacillus subtilis ATCC 6051(AJ276351) sequence homology is more than 99%, house-keeping gene recA sequence with
bacillus subtilis subsp. subtilis RO-NN-1(CP002906.1) sequence homology reaches 99%.According to the classification overview of≤the primary Jie Shi systematic bacteriology handbook>=middle bacillus, HP90 genus genus bacillus guiding principle (
bacilli), genus bacillus order (
bacillales), Bacillaceae (
bacillaceae), bacillus (
bacillus), bacillus subtilis bacterial classification (
bacillus subtilis).
The screening method of the subtilis HP90 bacterial strain that contriver provides is: first sample from sausage, by the protease-producing bacterium in sample respectively after enrichment culture, employing casein is substrate, after enrichment culture, dilution spread is on casein plate Selective agar medium, using the caseic ability of microbial decomposition as primary dcreening operation index, picking has the larger bacterium colony of transparent circle three ride purifying on casein plate, and the single bacterium colony after purifying moves and connects slant medium preservation, thus realizes primary dcreening operation; Be substrate with rice residue afterwards, shake flask fermentation sieves again, with microorganism shake flask fermentation rice residue, centrifugal, supernatant liquor note casein plate hole, constant temperature culture, with plate well diameter for index first time multiple sieve; The dull and stereotyped bacterial strain larger through diameter is again through shake flask fermentation rice residue, and centrifugal rear survey Supernatant protein enzyme activity, with enzyme work for index secondary sieves again; Last enzyme is lived higher strain fermentation rice residue, centrifugal, and supernatant liquor boiling water goes out enzyme, and surveying ammonia-state nitrogen in supernatant liquor, take ammonia-state nitrogen as index third time multiple sieve.
The condition of described enrichment culture is: enrichment medium is in the composition of g/100mL: extractum carnis 0.5 ~ 1.0, peptone 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, pH 6.5 ~ 7.5; Casein Selective agar medium is in the composition of g/100mL: extractum carnis 0.5 ~ 1.0, casein 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, agar powder 2.0, pH 6.5 ~ 7.5; Slant preservation substratum bacterium is bacterium perfect medium, and fungi is PDA substratum; Seed culture medium with fermention medium in the composition of g/100mL: rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0, pH 6.5 ~ 7.5.
In order to verify bacterial strain of the present invention, carry out following experiment:
(1) sample collecting:
23 sample separation are collected in October, 2012 respectively, from samplings such as Guizhou Province tradition bacon, fermented soya bean, sour meat, fermented meat, bean product, distiller's yeast, wine unstrained spirits, seal with polyethylene bag; In November, 2012, from the topsoil near slaughterhouse, the Guizhou Province southeast and dining room, Hua Xi Guizhou University South, mud sampling, seals with polyethylene bag; After fetching sample, namely set about mask work.
(2) enrichment culture:
Take 5.0g sample, join in 50mL stroke-physiological saline solution, then put into 4 little granulated glass spherees, in 30 DEG C, shaking culture 30min under 180r/min, get 5mL and add in enrichment medium, 30 DEG C, shaking table enrichment culture 24h under 180r/min;
The composition of above-mentioned enrichment medium is (in g/100mL): extractum carnis 0.5 ~ 1.0, peptone 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum.
(3) primary dcreening operation:
Getting enrichment bacterium liquid that above-mentioned 0.2mL dilutes suitable gradient, to coat casein Selective agar medium dull and stereotyped, leaves standstill 5min, be inverted dull and stereotyped, in 30 DEG C of cultivations.Picking forms the larger single bacterium colony of transparent circle, draws Z-shaped line after line purifying on test tube slant, is cultured to after there is plentiful bacterium colony, 4 DEG C of Storage in refrigerator under 30 DEG C of conditions.
Primary dcreening operation obtains bacterial strain 115 strain forming obvious transparent circle altogether, and wherein the transparent circle of bacterial strain HP90 formation is maximum.
The composition of above-mentioned primary dcreening operation casein Selective agar medium is (in g/100mL): extractum carnis 0.5 ~ 1.0, casein 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, agar powder 2.0; The pH 6.5 ~ 7.5 of substratum.
Slant preservation substratum: bacterium bacterium perfect medium, fungi PDA substratum.
(4) first time sieves again:
Punch on primary dcreening operation casein Selective agar medium flat board with punch tool, every dull and stereotyped evenly make a call to 3 holes (three holes be a group parallel), aperture is 0.4cm.Picking primary dcreening operation test tube bacterium 2 articulating enters in seed culture medium, in 30 DEG C, cultivate 24h under 180r/min after, with 6% ~ 8%(1.235 × 10
6cfu/mL) in inoculum size access fermention medium, cultivate 24h under similarity condition after, in 4 DEG C, the centrifugal 10min of 10000r/min, supernatant liquor and crude enzyme liquid.Get 30 μ L crude enzyme liquids and inject plate well, dull and stereotyped after 30 DEG C of constant temperature culture 12h, measure hydrolysis transparent circle diameter d and sieve index again as first time; Choose the bacterial strain corresponding to enzyme liquid forming transparent circle largest diameter to sieve in order to second time is multiple.
Through first time multiple sieve, 115 strain bacterial strain crude enzyme liquids all form transparent circle, and from 1.0cm to 2.8cm not etc., bacterial strain totally 26 strains of transparent circle diameter d >=2.4cm, the transparent circle largest diameter that bacterial strain HP90 is formed is 2.75 ± 0.05cm to transparent circle diameter.
Above-mentioned seed culture medium is (in g/100mL) with the composition of fermention medium: rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum.
(5) the multiple sieve of second time:
Choose the bacterial strain of hydrolysis transparent circle diameter d >=2.4cm, adopt its crude enzyme liquid of Folin-phenol reagent determination of color (crude enzyme liquid preparation in crude enzyme liquid preparation multiple sieve of same first time) middle proteinase activity.
The drafting of typical curve: get 9 10mL volumetric flask number consecutivelies 0 to 8, adds the tyrosine standardized solution 0,1,2,3,4,5 of 100 μ g/mL respectively, 6,7,8mL, uses distilled water constant volume, and obtained concentration is followed successively by 0, and 10,20,30,40,50,60, the tyrosine solution of 70,80 μ g/mL.Get above-mentioned gradient tyrosine solution 1mL respectively, respectively add 0.4mol/L Na
2cO
3solution 5mL, Folin-phenol reagent 1mL, is placed in 40 DEG C of water-baths and develops the color 20min, take out in 680nm wavelength, 10mm cuvette, with not containing No. 0 pipe of tyrosine for blank, measure its absorbancy respectively; Be ordinate zou again with absorbancy, the concentration of tyrosine is X-coordinate, draws absorbance-tyrosine concentration typical curve.
Enzyme activity determination: the phosphoric acid buffer of crude enzyme liquid pH7.5 dilutes suitable multiple, for test.1% casein solution is put into 40 DEG C of water bath with thermostatic control preheating 5min, then by following process operation: 1. test group (3 parallel tests need be done): the crude enzyme liquid drawing 1m dilution adds test tube, 40 DEG C of water-bath preheating 2min, add 1% casein solution 1mL to shake up, 40 DEG C of accurate timing 10min, add 0.4mol/L trichoroacetic acid(TCA) 2mL and shake up termination reaction, take out and leave standstill 10min, filter, get 1mL filtrate, add 0.4mol/LNa
2cO
35mL, adds Folin-phenol reagent 1mL, and 40 DEG C of colour developing 20min, in 680nm wavelength, survey its absorbancy with 10mm cuvette.2. blank group: same to test group, just the addition sequence of casein solution and solution of trichloroacetic acid is exchanged.
Enzyme activity calculate: enzyme live unit definition be 1mL crude enzyme liquid under 40 DEG C of conditions, the enzyme amount that 1min caseinhydrolysate produces 1 μ g tyrosine is an enzyme activity unit (u).Fermented liquid proteinase activity (u/mL)=A × K × 4n/10, in formula, the mean light absorbency of A-sample parallel test; K-extinction constant; The cumulative volume (mL) of 4-reaction solution; 10-reaction times (min); N-crude enzyme liquid extension rate.
Obtain bacterial strain 8 strain of fermentation broth enzyme work at more than 190u/mL through the multiple sieve of second time, wherein the proteinase activity of bacterial strain HP90 is higher than other 7 strains, reaches 354 ± 1.5u/mL.
(6) third time sieves again:
Choose above-mentioned proteinase activity centrifugal after the 8 strain strain fermentations of more than 190u/mL, supernatant liquor boiling water goes out after enzyme and measures wherein amino acid nitrogen content; The preparation of supernatant liquor is sieved in described supernatant liquor preparation again with first time.
The mensuration of amino-acid nitrogen: the fermented supernatant fluid drawing the above-mentioned sterilized enzyme of 5mL, is placed in 100mL volumetric flask, distilled water constant volume.Then draw 20mL and be placed in 200mL beaker, add 60mL distilled water, start magnetic stirring apparatus, be titrated to acidometer instruction pH=8.2 with 0.05mol/L NaOH standardized solution.Add the mixing of 10mL formaldehyde solution again, then be titrated to pH=9.2 with 0.05mol/LNaOH standardized solution, write down the milliliter number consuming 0.05mol/LNaOH standardized solution.Do reagent blank test simultaneously.
Amino-acid nitrogen calculates: X=[(V
1-V
2) C × 0.014] × 100/ [5 × (V
3/ 100)]
In formula: the content of ammonia-state nitrogen in X-sample, g/100mL; V
1-test sample diluent adds the volume of formaldehyde solution post consumption NaOH standardized solution, mL; V
2-actual blank test adds the volume of formaldehyde solution post consumption NaOH standardized solution, mL; V
3-sample diluting liquid the amount of taking, mL; The concentration of C-NaOH standardized solution, mol/L; 0.014-1mL1mol/L NaOH standardized solution solution is equivalent to the g number of nitrogen.
Through third time multiple sieve, bacterial strain HP90 fermented liquid ammonia-state nitrogen is apparently higher than other 7 strains, and arrangement the 1st, be 22.20mg/100mL, corresponding rice residue protein degree of hydrolysis is (19.83 ± 0.5) %.
The qualification of bacterial strain HP90:
The genus kind of qualification bacterial classification, understands the physiological and biochemical property that it is basic, to effectively utilizing existing document, instructing further experiment important role.For this reason, morphological observation is carried out, physiological and biochemical property to subtilis HP90 bacterial strain, 16S rRNA and house-keeping gene recA Sequence Identification.
Morphological specificity: by bacterial classification streak inoculation in bacterium perfect medium solid plate in 30 DEG C, cultivates 18h.On bacterium perfect medium, bacillus amyloliquefaciens HP90 colony growth is very fast, and bacterium colony be faint yellow, translucent, circular or subcircular, and edge is neat, smooth half moistening, slightly swells, colony diameter 1.5 ~ 3.5mm, not chromogenesis, thickness, easy picking.Get inclined-plane preservation children bacterium in age and carry out gramstaining, subtilis HP90 is short straight rod shape under an optical microscope, and gramstaining is all purple, is Gram-positive bacillus.Physiological and biochemical property adopts API 20NE, Biolog GP microorganism identification automatic analysis system, and Physiology and biochemistry the results are shown in Table 1, table 2.
Table 1 bacterial strain HP90 physiological and biochemical property-utilize carbon source to produce acid
| Numbering | Reagent strip respective tube/substrate | Result | Numbering | Reagent strip respective tube/substrate | Result |
| 0 | Contrast | - | 25 | Polychrom | + |
| 1 | Glycerine | + | 26 | Salicin | + |
| 2 | Red tinea alcohol | - | 27 | Cellobiose | + |
| 3 | D-R | - | 28 | Maltose | + |
| 4 | L-arabinose | + | 29 | Lactose | - |
| 5 | Ribose | + | 30 | Melibiose | + |
| 6 | D-wood sugar | + | 31 | Sucrose | + |
| 7 | L-wood sugar | - | 32 | Trehalose | + |
| 8 | Ribitol | - | 33 | Synanthrin | + |
| 9 | Beta-methyl-D-xyloside | - | 34 | Pine three sugar | - |
| 10 | Semi-lactosi | - | 35 | Raffinose | + |
| 11 | Glucose | + | 36 | Starch | + |
| 12 | Fructose | + | 37 | Glycogen | + |
| 13 | Seminose | + | 38 | Xylitol | - |
| 14 | Sorbose | - | 39 | Hold together ox sugar | w |
| 15 | Rhamnosyl | - | 40 | D-turanose | + |
| 16 | Melampyrin | - | 41 | D-lyxose | - |
| 17 | Inositol | + | 42 | D-Tag | - |
| 18 | N.F,USP MANNITOL | + | 43 | D-rock sugar | - |
| 19 | Sorbyl alcohol | + | 44 | L-rock sugar | - |
| 20 | Alpha-Methyl-D-MANNOSE glucoside | - | 45 | D-R alcohol | - |
| 21 | Alpha-Methyl-D-Glucose glucoside | + | 46 | L-arabinose alcohol | - |
| 22 | N-acetyl-glycosamine | + | 47 | Gluconate | + |
| 23 | Vitamin B17 | + | 48 | 2-keto-D-gluconate salt | - |
| 24 | Ursin | + | 49 | 5-keto-D-gluconate salt | - |
Note :+: positive ,-: negative, w: the weak positive
Table 2 bacterial strain HP90 physio-biochemical characteristics--enzyme is lived, produce acid
| Test item | Result | Test item | Result |
| NO3 nitrate reduction is to nitrite | + | MNE assimilates seminose | + |
| TRP indoles | - | MAN assimilates N.F,USP MANNITOL | + |
| GLU acidifying glucose | - | NAG assimilates N-acetyl-glucosamine | + |
| The two water Jie enzyme of ADH arginine | - | MAL assimilates maltose | + |
| URE urase | - | GNT assimilates gluconate | + |
| ESC is hydrolyzed polychrom (β-glucosaccharase) | + | CAP assimilates capric acid | - |
| GEL gelatin hydrolysate (proteolytic enzyme) | + | ADI assimilates adipic acid | w |
| PNPG beta galactoside enzyme | + | MLT assimilates oxysuccinic acid | + |
| GLU assimilates glucose | + | CIT assimilates citric acid | + |
| ARA assimilates pectinose | + | The same phenylacetic acid of PAC | w |
Note :+: positive ,-: negative, w: the weak positive
According to above physiology and morphology biochemical character result, bacterial strain HP90 feature meets bacillus feature, and preliminary evaluation belongs to bacillus.
16S rRNA sequence and house-keeping gene recA sequence are identified by Wuhan China typical culture collection center (China Center for Type Culture Collection, CCTCC); The 16S rRNA obtained after order-checking and recA gene order log in
http:// blast.ncbi.nlm.nih.gov/Blast.cgiwebsite, compares with known array in Blast program and Genbank.
Phylogenetic tree builds: the 16S rRNA of bacterial strain HP90, house-keeping gene recA sequence are logged in NCBI(National Center for Biotechnology Information) database, by online BLAST(Basic Local Alignment Search Tool) known array carries out com-parison and analysis in program and Genbank.The 16S rRNA sequence B LAST of CCTCC NO. M 2014201 searches the bacterial strain of 43 gene fragments higher with this sequence homology altogether, this bacterial strain and bacillus amyloliquefaciens (
bacillus amyloliquefaciens subsp.plantarum str.FZB42) the 16S rRNA sequence homology of (NC 009725.1) is more than 99%, choose the 35 strain bacterial strains that sequence homology is higher, application MEGA5.0 analysis software constructing system evolutionary tree, the results are shown in Figure 8.As shown in Figure 8, bacterial strain HP90 with
bacillus subtilis ATCC 6051(AJ276351) belong in a minimum branch, show that affinity between them is nearest, house-keeping gene recA sequence B LAST searches the bacterial strain of 17 gene fragments higher with this sequence homology altogether, and wherein 1 strain that has of homology 99% is
bacillus subtilis subsp. subtilis RO-NN-1(CP002906.1), homology 98% have 4 strains as
bacillus subtilis subsp. subtilis str. BAB-1(CP004405.1),
bacillus subtilis XF-1(CP004019.1),
bacillus subtilis BSn5(CP002468.1) etc., homology 97% have 10 strains as
bacillus subtilis PY79(CP006881.1),
bacillus subtilis QB928(CP003783.1) etc., homology 94% has 2 strains, as
bacillus subtilis subsp. spizizenii TU-B-10(CP002905.1),
bacillus subtilis subsp. spizizenii str. W23(CP002183.1).
Therefore, according to its morphological specificity, physiological and biochemical property, 16S rRNA and house-keeping gene recA Analysis and Identification, bacterial strain HP90 closer to subtilis (
bacillus subtilis), according to the classification overview of≤the primary Jie Shi systematic bacteriology handbook>=middle bacillus, genus genus bacillus guiding principle (
bacilli), genus bacillus order (
bacillales), Bacillaceae (
bacillaceae), bacillus (
bacillus), bacillus subtilis bacterial classification (
bacillus subtilis).
The subtilis HP90 using method that contriver provides is: get slant preservation strains B. amyloliquefaciens HP60 2 ring and be connected to respectively in seed culture medium, 30 DEG C, after 24h cultivated by 180r/min shaking table, with 6% ~ 8%(0.1 ~ 5 × 10
6cfu/mL) inoculum size accesses in fermention medium respectively, and under similarity condition, 24h cultivated by shaking table, is namely hydrolyzed for rice residue protein.
Above-mentioned seed culture medium is (in g/100mL) with the composition of fermention medium: rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum.
With bioprotein enzyme process hydrolysis rice residue protein, it will be the development trend utilizing proteolytic enzyme protolysate matter.The subtilis HP90 that the present invention screens, has the character of producing proteolytic enzyme, is directly used in the hydrolysis of rice residue protein.This bacterial strain for be hydrolyzed the advantage of rice residue protein be cost low, can recycle, good stability, green safety, can biocatalysis be directly used in, raw material sources are extensive, method simple practical.Therefore, the present invention is separated the subtilis HP90 that obtains and has important application prospect at hydrolysis rice residue protein from traditional sausage.Meanwhile, this bacterial strain also can be applicable to the hydrolysis of other protein.
Accompanying drawing explanation
Fig. 1 is that photo observed by the hydrolysis transparent circle flat board that bacterial strain HP90 of the present invention is formed on casein Selective agar medium; Fig. 2 is that photo observed by the flat board of bacterial strain HP90 separation and purification on casein Selective agar medium; Fig. 3 is the test tube slant preservation photo of bacterial strain HP90; Fig. 4 is that the observation of sieving of bacterial strain HP90 plate well test-results again is for the first time shone; Fig. 5 is that the colony morphological observation of bacterial strain HP90 on bacterium perfect medium flat board shines; Fig. 6 be bacterial strain HP90 under an optical microscope 1000 times gramstaining cellular form observe shine; Fig. 7 be bacterial strain HP90 under an optical microscope 1600 times gramstaining cellular form observe shine; Fig. 8 is the phylogenetic tree built according to HP90 16S rRNA sequence homology.
Embodiment
Embodiment 1: the screening of the protease-producing bacterium of hydrolysis rice residue, from Chinese Huaxi, Guiyang tradition bacon, fermented soya bean, sour meat, fermented meat, bean product, distiller's yeast, wine unstrained spirits sampling 80g, seals with polyethylene bag; From the topsoil near slaughterhouse, the Guizhou Province southeast and dining room, Hua Xi Guizhou University South, mud sampling 80g, seal with polyethylene bag.After fetching sample, namely set about mask work.
Take 5.0g sample, join in 50mL stroke-physiological saline solution, then put into 4 little granulated glass spherees, 30 DEG C, 180r/min shaking culture 30min, get 5mL and add in enrichment medium, 30 DEG C, 180r/min shaking table enrichment culture 24h.
Enrichment culture based component (in g/100mL): extractum carnis 0.5 ~ 1.0, peptone 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum;
Primary dcreening operation: getting enrichment bacterium liquid that above-mentioned 0.2mL dilutes suitable gradient, to coat casein Selective agar medium dull and stereotyped, leaves standstill 5min, be inverted dull and stereotyped, in 30 DEG C of cultivations.Picking forms the larger single bacterium colony of transparent circle, draws Z-shaped line after line purifying on test tube slant, is cultured to after there is plentiful bacterium colony, 4 DEG C of Storage in refrigerator under 30 DEG C of conditions.
Casein Selective agar medium composition (in g/100mL): extractum carnis 0.5 ~ 1.0, casein 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, agar powder 2.0; Medium pH 6.5 ~ 7.5.
Slant preservation substratum: bacterium bacterium perfect medium, fungi PDA substratum.
Multiple sieve: (1) plate well is tested: get the inoculation that is sieved at the beginning of 2 rings in seed culture medium, 30 DEG C, 180r/min cultivates 24h, with 6% ~ 8%(0.1 ~ 5 × 10
6cfu/mL) inoculum size accesses in fermention medium respectively, and after cultivating 24h under similarity condition, 4 DEG C, the centrifugal 10min of 10000r/min, supernatant liquor note casein plate hole, cultivates 12h for 30 DEG C, multiple sieve below the bacterial strain choosing hydrolysis largest diameter carries out.
The same fermention medium of seed culture medium, composition (in g/100mL): rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum.
(2) choose plate well and test the bacterial strain of hydrolysis transparent circle largest diameter with the obtained upper clear enzyme solution of plate well test method, upper clear enzyme solution surveys proteinase activity according to forint phenol development process, and enzyme high bacterial strain alive is carrying out next step multiple sieve.
(3) enzyme lives high bacterial strain again according to the obtained upper clear enzyme solution of plate well test method, and upper clear enzyme solution goes out in 100 DEG C of waters enzyme 5min, and survey its ammonia-state nitrogen amount, what ammonia nitrogen content was the highest is ultimate aim bacterial strain HP90.
Embodiment 2: the using method of the extracellular protease producing strains of hydrolysis rice residue, gets slant preservation bacterial strain HP90 2 ring and is connected to respectively in seed culture medium, 30 DEG C, after 24h cultivated by 180r/min shaking table, with 6% ~ 8%(0.1 ~ 5 × 10
6cfu/mL), in inoculum size access fermention medium, under similarity condition, 24h cultivated by shaking table, and be directly used in the hydrolysis of protein in rice residue, degree of hydrolysis reaches 19.83%.
The same fermentation medium components of seed culture medium used (in g/100mL): rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0; The pH 6.5 ~ 7.5 of substratum.
Claims (6)
1. one strain hydrolysis rice residue protease-producing bacterium: subtilis, it is characterized in that this subtilis (
bacillus subtilis) bacterial strain pure growth is preserved in Wuhan China typical culture collection center, depositary institution address on May 13rd, 2014: Wuhan, China Wuhan University, postcode 430072, be called for short subtilis HP90, deposit number is CCTCC NO. M 2014201.
2. subtilis as claimed in claim 1, it is characterized in that the 16S rRNA sequence of described subtilis HP90 with
bacillus subtilis ATCC 6051sequence homology more than 99%, house-keeping gene recA sequence with
bacillus subtilis subsp. subtilis RO-NN-1sequence homology reaches 99%; According to the categorised regulation of bacillus in " uncle Jie Shi systematic bacteriology handbook ", HP90 genus genus bacillus guiding principle (
bacilli), genus bacillus order (
bacillales), Bacillaceae (
bacillaceae), bacillus (
bacillus), bacillus subtilis bacterial classification (
bacillus subtilis).
3. the screening method of the bacillus amyloliquefaciens as described in claim 1 and 2, it is characterized in that: first sample from sausage, by the protease-producing bacterium in sample respectively after enrichment culture, employing casein is substrate, after enrichment culture, dilution spread is on casein plate Selective agar medium, using the caseic ability of microbial decomposition as primary dcreening operation index, picking has the maximum bacterium colony of transparent circle three ride purifying on casein plate, single bacterium colony after purifying moves and connects slant medium preservation, thus realizes primary dcreening operation; Be substrate with rice residue afterwards, shake flask fermentation sieves again, with microorganism shake flask fermentation rice residue, centrifugal, supernatant liquor note casein plate hole, constant temperature culture, with plate well diameter for index first time multiple sieve; The dull and stereotyped bacterial strain through largest diameter is again through shake flask fermentation rice residue, and centrifugal rear survey Supernatant protein enzyme activity, with enzyme work for index secondary sieves again; Last enzyme is lived higher strain fermentation rice residue, centrifugal, and supernatant liquor boiling water goes out enzyme, and surveying ammonia-state nitrogen in supernatant liquor, take ammonia-state nitrogen as index third time multiple sieve.
4. screening method as claimed in claim 3, is characterized in that: enrichment medium is in the composition of g/100mL: extractum carnis 0.5 ~ 1.0, peptone 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, the pH 6.5 ~ 7.5 of substratum; Casein Selective agar medium is in the composition of g/100mL: extractum carnis 0.5 ~ 1.0, casein 0.5 ~ 1.0, (NH
4)
2sO
40.1 ~ 0.2, K
2hPO
40.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, NaCl 0.5 ~ 1.0, agar powder 2.0, the pH 6.5 ~ 7.5 of substratum; Slant preservation substratum bacterium is bacterium perfect medium, and fungi is PDA substratum; Seed culture medium with fermention medium in the composition of g/100mL: rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0, the pH 6.5 ~ 7.5 of substratum.
5. the using method of the subtilis as described in claim 1 and 2, it is characterized in that: get slant preservation Strains B. subtilis HP90 2 ring and be connected to respectively in seed culture medium, in 30 DEG C, 180r/min shaking table cultivates after 24h, inoculum size with 6% ~ 8% accesses in fermention medium respectively, under similarity condition, 24h cultivated by shaking table, is namely hydrolyzed for rice residue protein.
6. using method as claimed in claim 5, is characterized in that: the composition of described seed culture medium in the composition of g/100mL is: rice residue 1.2 ~ 1.8, NaCl 0.5 ~ 1.0, MgSO
47H
2o 0.01 ~ 0.02, CaCl
20.01 ~ 0.02, K
2hPO
40.5 ~ 1.0; Its pH 6.5 ~ 7.5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962493A (en) * | 2015-06-24 | 2015-10-07 | 贵州大学 | Hydrolysis rice residue protein bacillus subtilis HP 90 fermentation medium and cultivating method |
| CN109536420A (en) * | 2018-12-29 | 2019-03-29 | 贵州大学 | One bacillus subtilis and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488137A (en) * | 2011-11-16 | 2012-06-13 | 江南大学 | Application of bacillus subtilis amino-peptidase in preparing flavor functional nutrient rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102488137A (en) * | 2011-11-16 | 2012-06-13 | 江南大学 | Application of bacillus subtilis amino-peptidase in preparing flavor functional nutrient rice |
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| QIANG ZHAO等: "Enzymatic hydrolysis of rice dreg protein： Effects of enzyme type on the functional properties and antioxidant activities of recovered proteins", 《FOOD CHEMISTRY》 * |
| 李绮丽: "双酶法水解米渣蛋白工艺研究_李绮丽", 《粮食与油脂》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962493A (en) * | 2015-06-24 | 2015-10-07 | 贵州大学 | Hydrolysis rice residue protein bacillus subtilis HP 90 fermentation medium and cultivating method |
| CN109536420A (en) * | 2018-12-29 | 2019-03-29 | 贵州大学 | One bacillus subtilis and its application |
| CN109536420B (en) * | 2018-12-29 | 2022-09-09 | 贵州大学 | Bacillus subtilis and application thereof |
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