CN104450554B - Protease producing strains --- bacillus amyloliquefaciens and its screening and the application method of one plant of hydrolysis rice residue - Google Patents
Protease producing strains --- bacillus amyloliquefaciens and its screening and the application method of one plant of hydrolysis rice residue Download PDFInfo
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Abstract
The invention discloses protease producing strains --- bacillus amyloliquefaciens and its screening and application method that one plant hydrolyzes rice residue, the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain pure culture is preserved in Wuhan China typical culture collection center on May 13rd, 2014, abbreviation bacillus amyloliquefaciens HP60, deposit number is CCTCC NO.M 2014200.It screens to obtain aimed strain by primary dcreening operation and secondary screening.After taking slant preservation strain culturing, that is, it is directly used in the hydrolysis of protein in rice residue.Bacillus amyloliquefaciens HP60 is hydrolyzed for rice residue protein, and degree of hydrolysis is up to 15.63%.The bacterial strain is at low cost, can recycle, stability is good, it is green safe, biocatalysis can be directly used in, raw material sources are extensive, method simple practical.Therefore, which has important application prospect, which can be applied to the hydrolysis of other protein.
Description
Technical field
The present invention relates to microorganism or enzymes, in particular to protease producing strains.
Background technique
Paddy belongs to the common rice subspecies of grass family Oryza common cultivated rice subgenus kind in botany, is that the whole world first is big
Grain variety.The average annual yield of paddy has about accounted for the 2/5 of national total output of grain, has reached 1.90 hundred million t.Rice residue is with morning
Long-grained nonglutinous rice, to crack rice, be aged rice etc. be that raw material is fermented and produced and is filtered Rice & peanut milk during syrup, monosodium glutamate, biochemical drug etc.
Remaining residue afterwards, cheap, yield is huge, and every consumption 7t rice will generate 1t in the production process of rice starch sugar
Rice residue.Protein content is about 40%~70% in rice residue, is equivalent to 5~8 times of protein content in paddy, and content is close
Soybean, and amino acid ratio meets the idealized model of WHO/FAO recommendation, and quality is acknowledged as in foodstuff seed albumen most
Good person.But these good protein resources are most of that all the cheap feed factory that has been sold to makees feed in the form of dry powder, and
Deep development and utilization are not obtained, if by these rice residue protein resource conversions at amino acid and polypeptide with high added value,
Its biological value, economic value can be not only improved, to the technology content for improving the deep processing of China's paddy, and promotes entire grain
The development of industry suffers from far-reaching influence.
Protein can get amino acid and polypeptide through acid, alkali or Protease Treatment.Acid, alkaline hydrolysis can destroy strongly
Some sensitivity amino acid, and generate toxic chloropropyl alcohol substance.Enzyme hydrolysis condition is mild, to sensitive amino acid without destruction,
It maximum can retain raw material physicochemical property, flavor, functional characteristic, green safe, energy consumption is small.But it is most at present to utilize commercially available protein
Enzymatic protein, this makes industrial production cost higher, and therefore, screening protease producing strains are particularly important.
At present in Chinese patent database, the patent application for being related to protease producing strains is few, only
No. ZL2010101038046 " gene of a kind of organic solvent tolerant protease producing strains and its produced organic solvent tolerant protease and
Using ", No. 2007101919691 " a kind of Organic solvent-tolerant alkali protease producing strains and the organic solvent-resistant basic proteins
The gene of enzyme and application " and No. 2013101991045 " a kind of collagenase producing bacteria ".Up to now, it there is no and be related to hydrolyzing
The application part of the protease producing strains of rice residue.
Summary of the invention
The present invention is intended to provide protease producing strains --- the bacillus amyloliquefaciens of one plant of hydrolysis rice residue, enable to use
It is hydrolyzed in the bioanalysis of rice residue protein.
It is yet another object of the invention to provide the screening technique of above-mentioned bacillus amyloliquefaciens and application methods, have it
Specific industrial use.
The protease producing strains for the hydrolysis rice residue that inventor provides are bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens), which has been preserved in Wuhan Chinese Typical Representative culture guarantor on May 13rd, 2014
Hiding center, abbreviation bacillus amyloliquefaciens HP60, depositary institution address: Wuhan, China Wuhan University 430072, deposit number
For CCTCC NO.M 2014200.The bacterial strain system is isolated from Guizhou In China tradition fermented soya bean.
The morphological feature and physiological and biochemical property of above-mentioned bacillus amyloliquefaciens HP60 solves starch bud close to bacillus
It is known in the 16S rRNA sequence and Genbank of spore bacillus HP60Bacillus amyloliquefaciens subsp.plantarum str.FZB42009725.1 sequence homology of NC 99% or more, house-keeping gene recA withBacillus amyloliquefaciens IT-45(CP004065.1),Bacillus amyloliquefaciens LFB112(CP006952.1) etc. 9 plants of homologys are up to 99%.According to≤primary Jie Shi systematic bacteriology handbook >=middle bacillus
Classification overview, HP60 belong to bacillus guiding principle (Bacilli), bacillus head (Bacillales), Bacillaceae
(Bacillaceae), bacillus (Bacillus), bacillus amyloliquefaciens kind (Bacillus amyloliquefaciens).
The screening technique for the bacillus amyloliquefaciens HP60 that inventor provides is: first sampling from fermented soya bean, by the egg in sample
After white enzyme producing strains distinguish enriched culture, use casein for substrate, dilution spread is flat in casein after enrichment culture
On plate Selective agar medium, using the ability of microorganism decomposition casein as primary dcreening operation index, picking has the biggish bacterium colony of transparent circle
Three rides purify on casein plate, and single colonie transposing slant medium after purification saves, to realize primary dcreening operation；Later
Using rice residue as substrate, shake flask fermentation secondary screening, with microorganism shake flask fermentation rice residue, centrifugation, supernatant infuses casein plate hole, constant temperature
Culture, using plate bore dia as index first time secondary screening；Bacterial strain of the plate through being relatively large in diameter is again through shake flask fermentation rice residue, after centrifugation
Supernatant protein enzyme activity is surveyed, using enzyme activity as the secondary secondary screening of index；The higher strain fermentation rice residue of last enzyme activity, centrifugation, supernatant
Liquid boiling water enzyme deactivation surveys ammoniacal nitrogen in supernatant, using ammoniacal nitrogen as index third time secondary screening.
The condition of the enrichment culture are as follows: ingredient of the enriched medium in terms of g/100mL: beef extract 0.5~1.0, albumen
Peptone 0.5~1.0, (NH4)2SO40.1~0.2, K2HPO40.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl2 0.01
~0.02, NaCl 0.5~1.0, pH 6.5~7.5；Ingredient of the casein Selective agar medium in terms of g/100mL: beef extract
0.5~1.0, casein 0.5~1.0, (NH4)2SO40.1~0.2, K2HPO40.5~1.0, MgSO4·7H2O 0.01~
0.02, CaCl20.01~0.02, NaCl 0.5~1.0, agar powder 2.0, pH 6.5~7.5；Slant preservation culture medium is thin
Bacterium is bacterium complete medium, and fungi is PDA culture medium；Ingredient of the seed culture medium with fermentation medium in terms of g/100mL: rice
Slag 1.2~1.8, NaCl 0.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, K2HPO40.5~
1.0, pH 6.5~7.5.
In order to verify bacterial strain of the invention, inventor has carried out following experiment:
(1) sample acquires:
23 separation samples are collected in October, 2012 respectively, from Guizhou Province tradition bacon, fermented soya bean, sour meat, fermented meat,
Bean product, distiller's yeast, fermented grain sampling, are sealed with Polythene Bag；In November, 2012 is big from Guizhou southeast of Guizhou Province slaughterhouse and the Guizhou Hua Xi
Topsoil, mud sampling near South dining room, are sealed with Polythene Bag；After fetching sample, that is, set about mask work.
(2) enrichment culture:
5.0g sample is weighed, is added in 50mL sterile saline, 4 small beades are placed into, in 30 DEG C, 180r/
Shaken cultivation 30min under min takes 5mL to be added in enriched medium, and 30 DEG C, shaking table enrichment culture is for 24 hours under 180r/min；
The ingredient of above-mentioned enriched medium be (in terms of g/100mL): beef extract 0.5~1.0, peptone 0.5~1.0,
(NH4)2SO40.1~0.2, K2HPO40.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, NaCl
0.5~1.0, pH 6.5~7.5.
(3) primary dcreening operation:
The enrichment bacterium solution for taking above-mentioned 0.2mL to dilute appropriate gradient is coated on casein Selective agar medium plate, stands 5min,
It is inverted plate, is cultivated in 30 DEG C.Picking forms the biggish single bacterium colony of transparent circle, and scribing line is drawn on test tube slant Z-shaped after purification
Line is cultivated to after there is plentiful bacterium colony under the conditions of 30 DEG C, 4 DEG C of Storage in refrigerator.
Primary dcreening operation obtains altogether forms 115 plants of bacterial strain of obvious transparent circle, and the transparent circle that wherein bacterial strain HP60 is formed is larger.
The ingredient of above-mentioned primary dcreening operation casein Selective agar medium is (in terms of g/100mL): beef extract 0.5~1.0, casein
0.5~1.0, (NH4)2SO40.1~0.2, K2HPO40.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~
0.02, NaCl 0.5~1.0, agar powder 2.0, pH 6.5~7.5.
Slant preservation culture medium: bacterium bacterium complete medium, fungi PDA culture medium.
(4) first time secondary screening:
It is punched on primary dcreening operation casein Selective agar medium plate with punch, every plate uniformly makes a call to 3 holes, and (three holes are one group flat
Row), aperture 0.4cm.2 ring of picking primary dcreening operation test tube bacterium accesses in seed culture medium, after 30 DEG C, 180r/min culture for 24 hours, with
6%~8%(1.235 × 106Cfu/mL) in inoculum concentration access fermentation medium, after being cultivated for 24 hours under similarity condition, in 4
DEG C, 10000r/min be centrifuged 10min, supernatant, that is, crude enzyme liquid.30 μ L crude enzyme liquids are taken to inject plate well, plate is trained in 30 DEG C of constant temperature
After supporting 12h, measurement hydrolyzes transparent loop diameter d as first time secondary screening index；Selection forms the enzyme solution institute that transparent circle is relatively large in diameter
Corresponding bacterial strain is in case second of secondary screening.
Through first time secondary screening, 115 plants of bacterial strain crude enzyme liquids all form transparent circle, transparent loop diameter from 1.0cm to 2.8cm not
Deng, totally 26 plants of the bacterial strain of transparent loop diameter d >=2.4cm, bacillus amyloliquefaciens HP60 formed transparent circle be relatively large in diameter, be
2.70±0.05cm。
Above-mentioned seed culture medium is (in terms of g/100mL): rice residue 1.2~1.8, NaCl 0.5 with the ingredient of fermentation medium
~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, K2HPO40.5~1.0, pH 6.5~7.5.
(5) second of secondary screening:
The bacterial strain for hydrolyzing transparent loop diameter d >=2.4cm is chosen, using its crude enzyme liquid of Folin- phenol reagent determination of color
Prolease activity in (crude enzyme liquid preparation is prepared with crude enzyme liquid in first time secondary screening).
The drafting of standard curve: 9 10mL volumetric flask number consecutivelies 0 to 8 are taken, the tyrosine mark of 100 μ g/mL is separately added into
Quasi- solution 0,1,2,3,4,5,6,7,8mL, with distilled water constant volume, obtained concentration is followed successively by 0,10,20,30,40,50,60,70,
The tyrosine solution of 80 μ g/mL.Above-mentioned gradient tyrosine solution 1mL is taken respectively, respectively plus 0.4mol/L Na2CO3Solution 5mL,
Folin- phenol reagent 1mL is placed in 40 DEG C of water-baths the 20min that develops the color, and takes out in 680nm wavelength, 10mm cuvette, to be free of junket ammonia
No. 0 pipe of acid is blank, measures its absorbance respectively；Again using absorbance as ordinate, the concentration of tyrosine is abscissa, is drawn
Absorbance value-tyrosine concentration standard curve.
Enzyme activity determination: crude enzyme liquid dilutes multiple appropriate with pH7.5 phosphate buffer, for test.By 1% casein
Solution is put into 40 DEG C of waters bath with thermostatic control and preheats 5min, then operates by following processes: 1. test group (need to make 3 parallel tests):
It draws the diluted crude enzyme liquid of 1m and test tube is added, 40 DEG C of water-baths preheat 2min, 1% casein solution 1mL is added to shake up, 40 DEG C of accurate meters
When 10min, add 0.4mol/L trichloroacetic acid 2mL shake up termination reaction, take out stand 10min, filtering, take 1mL filtrate, add
0.4mol/LNa2CO35mL adds Folin- phenol reagent 1mL, 40 DEG C of colour developing 20min to survey it with 10mm cuvette in 680nm wavelength
Absorbance.2. blank group: same test group only exchanges the addition sequence of casein solution and solution of trichloroacetic acid.
Enzyme activity calculates: enzyme-activity unit is defined as 1mL crude enzyme liquid under the conditions of 40 DEG C, and 1min caseinhydrolysate generates 1 μ g
The enzyme amount of tyrosine is an enzyme activity unit (u).Fermentation liquid prolease activity (u/mL)=A × K × 4n/10, in formula, A-sample
The mean light absorbency of product parallel test；K-extinction constant；The total volume (mL) of 4-reaction solutions；10-the reaction time (min)；
N-crude enzyme liquid extension rate.
Fermentation liquid enzyme activity is obtained at 8 plants of bacterial strain of 190u/mL or more through second of secondary screening, wherein bacillus amyloliquefaciens
The prolease activity arrangement the 2nd of HP60, up to 263 ± 7.0u/m.
(6) third time secondary screening:
It chooses above-mentioned prolease activity to be centrifuged after 8 plants of strain fermentations of 190u/mL or more, be surveyed after supernatant boiling water enzyme deactivation
Fixed wherein amino acid nitrogen content；Preparation of the supernatant preparation with first time secondary screening supernatant.
The measurement of amino-acid nitrogen: drawing the fermented supernatant fluid of the above-mentioned sterilized enzyme of 5mL, be placed in 100mL volumetric flask, distills
Water constant volume.Then it draws 20mL to be placed in 200mL beaker, adds 60mL distilled water, start magnetic stirring apparatus, use 0.05mol/
LNaOH standard solution is titrated to acidometer instruction pH=8.2.The mixing of 10mL formalin is added, then uses 0.05mol/LNaOH
Standard solution is titrated to pH=9.2, writes down ml of consumption 0.05mol/LNaOH standard solution.Reagent blank examination is done simultaneously
It tests.
Amino-acid nitrogen calculates: X=[(V1-V2)·C×0.014] ×100/[5×(V3/100)]
In formula: the content of ammoniacal nitrogen, g/100mL in X-sample；V1After formalin is added in-test sample dilution
Consume the volume of NaOH standard solution, mL；V2The body of formalin post consumption NaOH standard solution is added in-practical blank test
Product, mL；V3- sample diluting liquid the amount of taking, mL；The concentration of C-NaOH standard solution, mol/L；0.014—1mL1mol/
LNaOH standard solution solution is equivalent to the g number of nitrogen.
Other 6 plants are apparently higher than through third time secondary screening bacterial strain HP60 fermentation liquid ammoniacal nitrogen, arranges the 2nd, for (17.50 ±
0.35) mg/100mL, corresponding rice residue protein degree of hydrolysis are respectively (15.63 ± 0.30) %.
The identification of bacterial strain HP60:
The category kind for identifying strain, understands its basic physiological and biochemical property, to effectively utilizing existing document, instruct into one
Step experiment important role.For this purpose, to bacillus amyloliquefaciens HP60 bacterial strain progress morphological observation, physiological and biochemical property,
16S rRNA and house-keeping gene recA Sequence Identification.
Morphological feature: by strain streak inoculation in bacterium complete medium solid plate in 30 DEG C, 18h is cultivated.In bacterium
The growth of bacillus amyloliquefaciens HP60 bacterium colony is very fast on complete medium, and bacterium colony is in faint yellow, translucent, round or subcircular,
Edge is neat, smooth semi-moist, slightly swells, 1.5~3.5mm of colony diameter, not chromogenesis, sticky, easy picking.Inclined-plane is taken to protect
It deposits young age bacterium and carries out Gram's staining, bacillus amyloliquefaciens HP60 cellular morphology is long, direct rod shape, leather under an optical microscope
Blue Albert'stain Albert is purple, is Gram-positive bacillus.Physiological and biochemical property uses API 20NE, Biolog GP microbial identification
Automatic analysis system, Physiology and biochemistry the results are shown in Table 1, table 2.
1 bacterial strain HP60 physiological and biochemical property of table-utilization of carbon source
| Serial number | Detection project | As a result | Serial number | Detection project | As a result |
| A1 | Water | - | E1 | D-Tag | - |
| A2 | Alpha-cyclodextrin | - | E2 | D- trehalose | + |
| A3 | A- cyclodextrin | - | E3 | Turanose | - |
| A4 | Dextrin | + | E4 | Xylitol | - |
| A5 | Glycogen | - | E5 | D- xylose | + |
| A6 | Inulin | - | E6 | Acetic acid | w |
| A7 | Mannosan | - | E7 | Alpha-hydroxybutyric acid | - |
| A8 | Polysorbate40 | - | E8 | Beta-hydroxy-butanoic acid | w |
| A9 | Tween 80 | - | E9 | γ-hydroxybutyric acid | w |
| A10 | N- acetyl group-D galactosamine | w | E10 | P- hydroxyl phenylacetic acid | - |
| A11 | N- acetyl-D-glucose amine | w | E11 | α-ketoglutaric acid | w |
| A12 | Amarogentin | w | E12 | α -one valeric acid | - |
| B1 | L-arabinose | w | F1 | Lactamide | - |
| B2 | D-arabinose | - | F2 | D-ALPHA-Hydroxypropionic acid methyl esters | - |
| B3 | Arbutin | + | F3 | Pfansteihl | - |
| B4 | D- cellobiose | + | F4 | D-malic acid | - |
| B5 | D-Fructose | + | F5 | L MALIC ACID | - |
| B6 | L- fucose | - | F6 | Methyl pyruvate | + |
| B7 | D- galactolipin | - | F7 | Monomethyl succinate | w |
| B8 | D- galacturonic acid | - | F8 | Propionic acid | - |
| B9 | Gentiobiose (16 Portugal's disaccharides of β) | + | F9 | Pyruvic acid | + |
| B10 | Maltonic acid | w | F10 | Succinamic acid | w |
| B11 | Alpha-D-glucose | + | F11 | Succinic acid | w |
| B12 | M- inositol | - | F12 | N- acetyl group-Pidolidone | w |
| C1 | α-D- lactose | - | G1 | L-Alanine amine | - |
| C2 | Lactulose | - | G2 | D-alanine | - |
| C3 | Maltose | w | G3 | L-Alanine | w |
| C4 | Maltotriose | w | G4 | L- alanyl-glycine | w |
| C5 | PEARLITOL 25C | w | G5 | Altheine acid | w |
| C6 | D-MANNOSE | w | G6 | Pidolidone | w |
| C7 | D- melezitose | - | G7 | Glycyl-L-glutamic acid | - |
| C8 | D- melibiose | - | G8 | L-Glutimic acid | w |
| C9 | Alpha-Methyl-D- galactoside | - | G9 | Serine | w |
| C10 | Beta-methyl-D- galactoside | - | G10 | Butanediamine | w |
| C11 | 3- methyl-D-glucose | + | G11 | 2,3- butanediol | + |
| C12 | Alpha-Methyl-D-Glucose glycosides | w | G12 | Glycerine | + |
| D1 | Beta-methyl-D-Glucose glycosides | w | H1 | Adenosine | w |
| D2 | Alpha-Methyl-D-MANNOSE glycosides | - | H2 | 2'-deoxyadenosine | - |
| D3 | 6-O-D- glucopyranose acyl-D- fructofuranose | w | H3 | Inosine | w |
| D4 | D-Psicose | w | H4 | Thymidine | w |
| D5 | D- melitriose (gossypose) | w | H5 | Uridine | w |
| D6 | L- rhamnose | - | H6 | 5'-monophosphate adenosine | - |
| D7 | D-ribose | + | H7 | 5 '-monophosphate thymidines | w |
| D8 | Salicin | w | H8 | 5 '-uridine monophosphate | - |
| D9 | Sedoheptulosan | - | H9 | 6- phosphoric acid-D-Fructose | - |
| D10 | D-glucitol | w | H10 | 1- phosphoric acid-alpha-D-glucose | - |
| D11 | Stachyose | - | H11 | 6- phosphoric acid-D-Glucose | w |
| D12 | Sucrose | + | H12 | D-L- alpha-phosphate glycerol | + |
Note :+: it is positive ,-: negative, w: weakly positive
2 bacterial strain HP60 physio-biochemical characteristics of table-enzyme activity produce acid
| Detection project | As a result | Detection project | As a result |
| NO3 nitrate reduction is to nitrite | + | MNE assimilates mannose | + |
| TRP indoles | - | MAN assimilates mannitol | + |
| GLU is acidified glucose | - | NAG assimilates N- acetyl-gucosamine | + |
| The double water Jie's enzymes of ADH arginine | - | MAL assimilates maltose | + |
| URE urase | - | GNT assimilates gluconate | + |
| ESC hydrolyzes aesculin (β-glucosaccharase) | + | CAP assimilates capric acid | - |
| GEL gelatin hydrolysate (protease) | + | ADI assimilates hexanedioic acid | - |
| PNPG beta galactoside enzyme | - | MLT assimilates malic acid | + |
| GLU assimilates glucose | + | CIT assimilates citric acid | + |
| ARA assimilates arabinose | + | The same phenylacetic acid of PAC | + |
Note :+: it is positive ,-: negative, w: weakly positive
According to the above form and physiological and biochemical property as a result, feature meets bacillus feature, Preliminary Identification category gemma
Bacillus.
16S rRNA sequence and house-keeping gene recA sequence are by Wuhan China typical culture collection center (China
Center for Type Culture Collection, CCTCC) identification；16S rRNA and the recA gene obtained after sequencing
Sequence logs in the website http://blast.ncbi.nlm.nih.gov/Blast.cgi, in Blast program and Genbank
Know that sequence is compared.
Phylogenetic tree building: 16S rRNA of bacterial strain HP60, house-keeping gene recA sequence are logged in into NCBI(National
Center for Biotechnology Information) database, pass through online BLAST(Basic Local
Alignment Search Tool) program is compared with known array in Genbank carries out analysis.The 16S rRNA of bacterial strain HP60
Sequence B LAST searches the bacterial strain of 43 with the higher genetic fragment of the sequence homology, the bacterial strain and solution starch gemma bar altogether
Bacterium (Bacillus amyloliquefaciens subsp.plantarum str.FZB42) (NC 009725.1) 16S
RRNA sequence homology chooses the higher 35 plants of bacterial strains of sequence homology 99% or more, analyzes software structure using MEGA5.0
Systematic evolution tree is built, as a result sees Fig. 8.As shown in Figure 8, bacterial strain HP60 withBacillus amyloliquefaciens subsp.plantarum str. FZB42(NC 009725.1) is belonged in a minimum branch, shows the parent between them
Edge is nearest.House-keeping gene recA sequence B LAST searches the bacterial strain of 17 with the higher genetic fragment of the sequence homology altogether,
Wherein homology up to 99% bacillus amyloliquefaciens (Bacillus amyloliquefaciens) 9 plants, such asBacillus amyloliquefaciens IT-45(CP004065.1),Bacillus amyloliquefaciens LFB112
(CP006952.1) etc., homology is up to 4 plants of bacillus amyloliquefaciens of 98%, e.g.,Bacillus amyloliquefaciens subsp.plantarum NAU-B3(HG514499.1),Bacillus amyloliquefaciens Y2(CP003332.1)
Deng, homology up to 4 plants of bacillus amyloliquefaciens of 94%, such asBacillus amyloliquefaciens XH7
(CP002927.1),Bacillus amyloliquefaciens DSM7(FN597644.1) etc..
Therefore, it is analyzed and is reflected according to bacterial strain HP60 morphological feature, physiological and biochemical property, 16S rRNA and house-keeping gene recA
It is fixed, bacterial strain HP60 closer to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), according to " primary Jie Shi system is thin
Mycology handbook " in bacillus classification overview, bacterial strain HP60 belong to bacillus guiding principle (Bacilli), bacillus head
(Bacillales), Bacillaceae (Bacillaceae), bacillus (Bacillus), bacillus amyloliquefaciens kind
(Bacillus amyloliquefaciens).
The bacillus amyloliquefaciens HP60 application method that inventor provides is: taking slant preservation strains B. amyloliquefaciens
2 ring of HP60 is connected in seed culture medium respectively, 30 DEG C, 180r/min shaking table culture for 24 hours after, with 6%~8%(0.1~5 ×
106Cfu/mL) inoculum concentration is respectively connected in fermentation medium, and shaking table culture for 24 hours, that is, is used for rice residue protein water under similarity condition
Solution.
Above-mentioned seed culture medium is (in terms of g/100mL): rice residue 1.2~1.8, NaCl 0.5 with the ingredient of fermentation medium
~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, K2HPO40.5~1.0, pH 6.5~7.5.
Rice residue protein is hydrolyzed with bioprotein enzyme process, will be the development trend using proteolytic enzyme protolysate matter.The present invention
The bacillus amyloliquefaciens HP60 screened has the property for producing protease, and for the hydrolysis of rice residue protein, degree of hydrolysis is reachable
15.63%.The bacterial strain for hydrolyze rice residue protein the advantages of be it is at low cost, can recycle, stability is good, green safe, energy
It is directly used in biocatalysis, raw material sources are extensive, method simple practical.Therefore, the present invention is isolated from traditional fermented soya bean
Bacillus amyloliquefaciens HP60 has important application prospect on hydrolysis rice residue protein.Meanwhile the bacterial strain is equally applicable for other
The hydrolysis of protein.
Detailed description of the invention
Fig. 1 is that the hydrolysis transparent circle plate that bacterial strain HP60 of the present invention is formed on casein Selective agar medium observes photo；
Fig. 2 is that the plate that bacterial strain HP60 is isolated and purified on casein Selective agar medium observes photo；Fig. 3 is that the test tube of bacterial strain HP60 is oblique
Face preservation photo；Fig. 4 is that the observation of the first time secondary screening plate well test result of bacterial strain HP60 is shone；Fig. 5 is bacterial strain HP60 thin
Colony morphological observation in bacterium complete medium flat board shines；Fig. 6 is bacterial strain HP60 1000 times of gram under an optical microscope
Staining cell morphologic observation is shone；Fig. 7 be bacterial strain HP60 under an optical microscope 1600 times Gram's staining cellular morphology observation
According to；Fig. 8 is the phylogenetic tree constructed according to HP60 16S rRNA sequence homology.
Specific embodiment
Embodiment 1: hydrolyzing the screening of the protease producing strains of rice residue, from Chinese Huaxi, Guiyang tradition bacon, fermented soya bean, acid
Meat, fermented meat, bean product, distiller's yeast, fermented grain etc. sample each 80g, are sealed with Polythene Bag；From Guizhou southeast of Guizhou Province slaughterhouse and Hua Xi
Topsoil, mud near Guizhou University, dining room, South sample each 80g, are sealed with Polythene Bag.After fetching sample, that is, set about
Mask work.
5.0g sample is weighed, is added in 50mL sterile saline, 4 small beades, 30 DEG C, 180r/min are placed into
Shaken cultivation 30min, take 5mL be added enriched medium in, 30 DEG C, 180r/min shaking table enrichment culture for 24 hours.
Enriched medium (g/100mL): beef extract 0.5~1.0, peptone 0.5~1.0, (NH4)2SO40.1~0.2,
K2HPO40.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, NaCl 0.5~1.0, pH 6.5
~7.5；
Primary dcreening operation: the enrichment bacterium solution for taking above-mentioned 0.2mL to dilute appropriate gradient is coated on casein Selective agar medium plate, stands
5min is inverted plate, cultivates in 30 DEG C.Picking forms the biggish single bacterium colony of transparent circle, and scribing line is after purification on test tube slant
Z-shaped line is drawn, is cultivated under the conditions of 30 DEG C to after there is plentiful bacterium colony, 4 DEG C of Storage in refrigerator.
Casein Selective agar medium ingredient (in terms of g/100mL): beef extract 0.5~1.0, casein 0.5~1.0, (NH4)2SO40.1~0.2, K2HPO40.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, NaCl 0.5
~1.0, agar powder 2.0, pH 6.5~7.5.
Slant preservation culture medium: bacterium bacterium complete medium, fungi PDA culture medium.
Secondary screening: (1) plate well is tested: the strain inoculated for taking 2 rings to be just sieved to is in seed culture medium, and 30 DEG C, 180r/min is trained
It supports for 24 hours, with 6%~8%(0.1~5 × 106Cfu/mL) inoculum concentration is respectively connected in fermentation medium, is cultivated under similarity condition
After for 24 hours, 4 DEG C, 10000r/min centrifugation 10min, supernatant infuse casein plate hole, and 30 DEG C of culture 12h choose hydrolysis
The big bacterial strain of diameter the 2nd carries out following secondary screening.
The same fermentation medium components of seed culture medium (in terms of g/100mL): rice residue 1.2~1.8, NaCl 0.5~1.0,
MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, K2HPO40.5~1.0, pH 6.5~7.5.
(2) it chooses the plate well test hydrolysis big bacterial strain of transparent loop diameter the 2nd and upper clear enzyme solution is made with plate well test method,
Upper clear enzyme solution surveys prolease activity according to forint phenol development process, and the high bacterial strain of enzyme activity is carrying out next step secondary screening.
(3) upper clear enzyme solution is made according still further to plate well test method in the high bacterial strain of enzyme activity, and upper clear enzyme solution goes out in 100 DEG C of waters
Enzyme 5min surveys its ammoniacal nitrogen amount, and higher ammonia nitrogen content is final goal bacterial strain.
Embodiment 2: hydrolyzing the application method of the protease producing strains of rice residue, and 2 ring of slant preservation bacterial strain HP60 is taken to connect respectively
In seed culture medium, 30 DEG C, 180r/min shaking table culture for 24 hours after, with 6%~8%(0.1~5 × 106Cfu/mL it) is inoculated with
In amount access fermentation medium, shaking table culture for 24 hours, is directly used in the hydrolysis of protein in rice residue under similarity condition, and degree of hydrolysis reaches
15.63%.
The same fermentation medium components of seed culture medium (in terms of g/100mL) used: rice residue 1.2~1.8, NaCl 0.5~
1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02, K2HPO40.5~1.0, pH 6.5~7.5.
Claims (4)
1. protease producing strains --- the bacillus amyloliquefaciens of one plant of hydrolysis rice residue, it is characterised in that the bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) bacterial strain pure culture has been preserved in the training of Wuhan Chinese Typical Representative on May 13rd, 2014
Object collection, abbreviation bacillus amyloliquefaciens HP60 are supported, deposit number is CCTCC NO.M 2014200.
2. bacillus amyloliquefaciens as described in claim 1, it is characterised in that the form of the bacillus amyloliquefaciens HP60
Feature and physiological and biochemical property are close to bacillus, known Bacillus in 16S rRNA sequence and Genbank
009725.1 sequence homology of amyloliquefaciens subsp.plantarum str.FZB42 NC 99% or more,
House-keeping gene recA and Bacillus amyloliquefaciens IT-45, Bacillus amyloliquefaciens
LFB112 homology is up to 99%；According to the classification overview of≤primary Jie Shi systematic bacteriology handbook >=middle bacillus, HP60 belongs to
Bacillus guiding principle (Bacilli), bacillus head (Bacillales), Bacillaceae (Bacillaceae), bacillus
(Bacillus), bacillus amyloliquefaciens kind (Bacillus amyloliquefaciens).
3. the application method of bacillus amyloliquefaciens described in one of claims 1 or 2, it is characterised in that: take slant preservation bacterium
Bacillus amyloliquefaciens HP602 ring is connected in seed culture medium respectively, in 30 DEG C, 180r/min shaking table culture for 24 hours after, with
6%~8% inoculum concentration is respectively connected in fermentation medium, and shaking table culture for 24 hours, that is, is used for rice residue protein water under similarity condition
Solution.
4. application method as claimed in claim 3, it is characterised in that: the ingredient of the seed culture medium is in terms of g/100mL
Ingredient are as follows: rice residue 1.2~1.8, NaCl 0.5~1.0, MgSO4·7H2O 0.01~0.02, CaCl20.01~0.02,
K2HPO40.5~1.0, pH 6.5~7.5.
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| 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 |
| CN106676023A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens highly yielding neutral protease and application thereof |
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