CN104357346B - The protease producing strains of one plant of hydrolysis rice residue: bacillus subtilis and its screening and application method - Google Patents
The protease producing strains of one plant of hydrolysis rice residue: bacillus subtilis and its screening and application method Download PDFInfo
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Abstract
The invention discloses the protease producing strains that one plant hydrolyzes rice residue: bacillus subtilis and its screening and application method, the bacillus subtilis (Bacillus subtilis) bacterial strain pure culture on May 13rd, 2014 is preserved in Wuhan China typical culture collection center, address: Wuhan, China Wuhan University, postcode 430072, abbreviation bacillus subtilis HP90, deposit number CCTCC NO.M 2014201.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 subtilis HP90 is hydrolyzed for rice residue protein, and degree of hydrolysis is up to 19.83%.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, and therefore, which has important application prospect, apply also for 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 the protease producing strains of one plant of hydrolysis rice residue: bacillus subtilis enables for rice
The bioanalysis of residue protein hydrolyzes.
It is yet another object of the invention to provide the screening technique of above-mentioned bacillus subtilis and application methods, it is made to have spy
Fixed industrial use.
In order to achieve the above objectives, the protease producing strains that inventor provides one plant of hydrolysis rice residue are bacillus subtilises
(Bacillus subtilis), the bacterial strain pure culture are preserved in Wuhan Chinese Typical Representative culture on May 13rd, 2014
Collection, depositary institution address: Wuhan, China Wuhan University, postcode 430072, deposit number are CCTCC NO.M
2014201.Bacterial strain abbreviation bacillus subtilis HP90, is isolated from Guizhou In China tradition sausage.
Above-mentioned bacillus subtilis HP90 morphological feature and physiological and biochemical property are close to bacillus, bacillus subtilis
The 16S rRNA sequence of HP90 and 6051 (AJ276351) sequence homology of Bacillus subtilis ATCC 99% with
On, house-keeping gene recA sequence and Bacillus subtilis subsp.subtilis RO-NN-1 (CP002906.1) sequence
Homology is up to 99%.According to the classification overview of≤primary Jie Shi systematic bacteriology handbook >=middle bacillus, HP90 belongs to gemma bar
Gammaproteobacteria (Bacilli), bacillus head (Bacillales), Bacillaceae (Bacillaceae), bacillus
(Bacillus), bacillus subtilis strain (Bacillus subtilis).
The screening technique for the bacillus subtilis HP90 bacterial strain that inventor provides is: first sampled from sausage, it will be in sample
After protease producing strains distinguish enriched culture, use casein for substrate, dilution spread is 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 of transparent circle
It falls in three rides on casein plate to purify, single colonie transposing slant medium after purification saves, to realize primary dcreening operation；It
Afterwards using rice residue as substrate, shake flask fermentation secondary screening, with microorganism shake flask fermentation rice residue, centrifugation, supernatant infuses casein plate hole, permanent
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, centrifugation
Supernatant protein enzyme activity is surveyed afterwards, using enzyme activity as the secondary secondary screening of index；The higher strain fermentation rice residue of last enzyme activity, centrifugation, on
Clear 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, CaCl20.01~
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 bacterium is bacterium
Complete medium, fungi are PDA culture medium；Ingredient of the seed culture medium with fermentation medium in terms of g/100mL: rice residue 1.2~
1.8, NaCl 0.5~1.0, MgSO4·7H2O0.01~0.02, CaCl20.01~0.02, K2HPO40.5~1.0, pH 6.5~
7.5。
In order to verify bacterial strain of the invention, following experiment has been carried out:
(1) sample acquires:
23 separation samples are collected in October, 2012 respectively, from Guizhou Province tradition bacon, fermented soya bean, sour meat, fermented meat,
The sampling such as bean product, distiller's yeast, fermented grain, is sealed with Polythene Bag；In November, 2012 is from Guizhou southeast of Guizhou Province slaughterhouse and the Guizhou Hua Xi
Topsoil, mud sampling near university, dining room, South, 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；The pH 6.5~7.5 of culture medium.
(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 HP90 is formed is maximum.
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, MgSO4·7H2O0.01~0.02, CaCl20.01~0.02,
NaCl 0.5~1.0, agar powder 2.0；The pH 6.5~7.5 of culture medium.
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 being cultivated for 24 hours under 30 DEG C, 180r/min,
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 maximum enzyme solution institute of transparent loop 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, the transparent loop diameter that bacterial strain HP90 is formed is maximum, be 2.75 ±
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；The pH 6.5 of culture medium~
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 the phosphate buffer of pH7.5, for test.By 1% junket egg
White 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 examinations
Test): it draws the diluted crude enzyme liquid of 1m and test tube is added, 40 DEG C of water-baths preheat 2min, add 1% casein solution 1mL to shake up, 40 DEG C of standards
True timing 10min adds 0.4mol/L trichloroacetic acid 2mL to shake up termination reaction, takes out and stands 10min, and filtering takes 1mL filtrate, adds
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-
The mean light absorbency of sample 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 the protease of bacterial strain HP90
Vigor is higher than other 7 plants, up to 354 ± 1.5u/mL.
(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/L
NaOH 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/L
NaOH standard solution solution is equivalent to the g number of nitrogen.
Through third time secondary screening, bacterial strain HP90 fermentation liquid ammoniacal nitrogen is apparently higher than other 7 plants, and arrangement the 1st is 22.20mg/
100mL, corresponding rice residue protein degree of hydrolysis are (19.83 ± 0.5) %.
The identification of bacterial strain HP90:
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, carrying out morphological observation, physiological and biochemical property, 16S to bacillus subtilis HP90 bacterial strain
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 HP90 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 subtilis HP90 is short straight rod shape under an optical microscope, and Gram's staining is all
It is purple, it is Gram-positive bacillus.Physiological and biochemical property is automatically analyzed using API 20NE, Biolog GP microbial identification
System, Physiology and biochemistry the results are shown in Table 1, table 2.
1 bacterial strain HP90 physiological and biochemical property of table-produces acid using carbon source
Note :+: it is positive ,-: negative, w: weakly positive
2 bacterial strain HP90 physio-biochemical characteristics of table -- enzyme activity produces acid
Note :+: it is positive ,-: negative, w: weakly positive
It is preliminary to reflect according to the above form and physiological and biochemical property as a result, bacterial strain HP90 feature meets bacillus feature
Surely belong to 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 HP90, 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.CCTCC NO.M 2014201
16S rRNA sequence B LAST search the bacterial strain of 43 with the higher genetic fragment of the sequence homology, the bacterial strain and solution altogether
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens subsp.plantarum str.FZB42) (NC
009725.1) 16S rRNA sequence homology chooses the higher 35 plants of bacterial strains of sequence homology, application 99% or more
MEGA5.0 analyzes software building systematic evolution tree, as a result sees Fig. 8.As shown in Figure 8, bacterial strain HP90 and Bacillus subtilis
ATCC 6051 (AJ276351) is belonged in a minimum branch, shows that the affinity between them 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, and wherein homology 99% has 1 plant
For Bacillus subtilis subsp.subtilis RO-NN-1 (CP002906.1), homology 98% have 4 plants such 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 plants such as Bacillus
SubtilisPY79 (CP006881.1), Bacillus subtilis QB928 (CP003783.1) etc., homology 94% has 2
Strain, such as Bacillus subtilis subsp.spizizenii TU-B-10 (CP002905.1), Bacillus subtilis
subsp.spizizenii str.W23(CP002183.1)。
Therefore, it is analyzed and identified according to its morphological feature, physiological and biochemical property, 16S rRNA and house-keeping gene recA, bacterial strain
HP90 is closer to bacillus subtilis (Bacillus subtilis), according to≤primary Jie Shi systematic bacteriology handbook >=middle gemma
The classification overview of Bacillus belongs to bacillus guiding principle (Bacilli), bacillus head (Bacillales), Bacillaceae
(Bacillaceae), bacillus (Bacillus), bacillus subtilis strain (Bacillus subtilis)
The bacillus subtilis HP90 application method that inventor provides is: taking slant preservation strains B. amyloliquefaciens
HP602 ring 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；The pH 6.5 of culture medium~
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 subtilis HP90 screened has the property for producing protease, is directly used in the hydrolysis of rice residue protein.The bacterial strain is used
In hydrolyze rice residue protein the advantages of be it 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, present invention bacillus subtilis HP90 isolated from traditional sausage exists
Hydrolyzing has important application prospect on rice residue protein.Meanwhile the bacterial strain is equally applicable for the hydrolysis of other protein.
Detailed description of the invention
Fig. 1 is that the hydrolysis transparent circle plate that bacterial strain HP90 of the present invention is formed on casein Selective agar medium observes photo；
Fig. 2 is that the plate that bacterial strain HP90 is isolated and purified on casein Selective agar medium observes photo；Fig. 3 is that the test tube of bacterial strain HP90 is oblique
Face preservation photo；Fig. 4 is that the observation of the first time secondary screening plate well test result of bacterial strain HP90 is shone；Fig. 5 is bacterial strain HP90 thin
Colony morphological observation in bacterium complete medium flat board shines；Fig. 6 is bacterial strain HP90 1000 times of gram under an optical microscope
Staining cell morphologic observation is shone；Fig. 7 be bacterial strain HP90 under an optical microscope 1600 times Gram's staining cellular morphology observation
According to；Fig. 8 is the phylogenetic tree constructed according to HP9016S 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 sample 80g, are sealed with Polythene Bag；From Guizhou southeast of Guizhou Province slaughterhouse and the Guizhou Hua Xi
Topsoil, mud near university, dining room, South sample 80g, are sealed with Polythene Bag.After fetching sample, that is, set about separating work
Make.
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.
Enrichment culture based component (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；Culture
The pH 6.5~7.5 of base；
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；Medium 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 maximum bacterial strain of diameter carries out following secondary screening.
The same fermentation medium of seed culture medium, ingredient (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；The pH 6.5~7.5 of culture medium.
(2) it chooses plate well test and hydrolyzes the transparent maximum bacterial strain of loop diameter with the obtained supernatant enzyme of plate well test method
Liquid, upper clear enzyme solution survey 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 highest ammonia nitrogen content is final goal bacterial strain HP90.
Embodiment 2: hydrolyzing the application method of the extracellular protease producing strains of rice residue, takes slant preservation bacterial strain HP902 ring point
Be not connected 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) connects
In kind amount access fermentation medium, shaking table culture for 24 hours, is directly used in the hydrolysis of protein in rice residue, degree of hydrolysis under similarity condition
Up to 19.83%.
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；The pH 6.5~7.5 of culture medium.
Claims (4)
1. the protease producing strains of one plant of hydrolysis rice residue: bacillus subtilis, it is characterised in that the bacillus subtilis
(Bacillus subtilis) bacterial strain pure culture is preserved in Wuhan China typical culture collection on May 13rd, 2014
Center, depositary institution address: Wuhan, China Wuhan University, postcode 430072, abbreviation bacillus subtilis HP90, deposit number
For CCTCC NO. M 2014201.
2. bacillus subtilis as described in claim 1, it is characterised in that the 16S rRNA of the bacillus subtilis HP90
Sequence and 6051 sequence homology of Bacillus subtilis ATCC 99% or more, house-keeping gene recA sequence with
Bacillus subtilis subsp. subtilis RO-NN-1 sequence homology is up to 99%；According to " primary Jie Shi Systems Bacterial
Learn to do volume " in bacillus categorised regulation, HP90 belong to bacillus guiding principle (Bacilli), bacillus head
(Bacillales), Bacillaceae (Bacillaceae), bacillus (Bacillus), bacillus subtilis strain
(Bacillus subtilis).
3. the application method of bacillus subtilis as claimed in claim 1 or 2, it is characterised in that: take slant preservation bacterial strain withered
Careless bacillus HP902 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 be respectively connected in fermentation medium, under similarity condition shaking table culture for 24 hours, i.e., for rice residue protein hydrolyze.
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, MgSO47H2O 0.01~0.02, CaCl2 0.01~0.02,
K2HPO4 0.5~1.0；Its pH 6.5~7.5.
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| Enzymatic hydrolysis of rice dreg protein： Effects of enzyme type on the functional properties and antioxidant activities of recovered proteins;Qiang Zhao等;《Food Chemistry》;20120319;第134卷;1360-1367 * |
| 双酶法水解米渣蛋白工艺研究_李绮丽;李绮丽;《粮食与油脂》;20110110(第1期);19-22 * |
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