CN104388331A - Brevibacillus brevis H3 and application thereof - Google Patents
Brevibacillus brevis H3 and application thereof Download PDFInfo
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- CN104388331A CN104388331A CN201410519415.XA CN201410519415A CN104388331A CN 104388331 A CN104388331 A CN 104388331A CN 201410519415 A CN201410519415 A CN 201410519415A CN 104388331 A CN104388331 A CN 104388331A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses brevibacillus brevis H3 and application thereof to prepare a composting inoculant. The brevibacillus brevis H3 is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M2013481, the preservation data is 21rt, October, 2013, and the address is Wuhan University, Wuhan City, Hubei province. The brevibacillus brevis H3 is resistant to high temperature, is survival at a temperature scope of 30 DEG C-60 DEG C, and is relatively high in lipase activity and relatively rapid in growth. The composting inoculant prepared by utilizing the bacterial strain has the advantages of high temperature resistance and capability of secreting protease and lipase by efficiently fermenting animal wastes. By utilizing the composting inoculant with the advantages of being small in environmental pollution, economic, practicable and the like, after a product subjected to compost processing is subjected to subsequent processing, the processed product can be used as an organic fertilizer and the purpose of changing waste into valuables is reached.
Description
(1) technical field
The present invention relates to strain Brevibacillus brevis and an application, particularly a strain Brevibacillus brevis H3 and preparing the application in decomposing microbial inoculum.
(2) background technology
Organic waste is often taken as rubbish and sends refuse tip, incinerator to or arbitrarily abandon.The method developing organic waste is very important, not luminous energy protection of the environment, and can turn waste into wealth.Currently developed the method much utilizing organic waste, but major part is all the utilization to Mierocrystalline cellulose, waste that starch content is high, then less to the Application way containing the protein waste higher with fat.
Rich in proteins and the waste (as animal class waste) of fat, if directly abandoned, will produce a large amount of sewage, foul smell, contaminate environment, and likely cause pathogen transmission to spread, there is great potential safety hazard.In current world wide, the method for process animal class waste mainly comprises buried method, incineration method and oil-refining method.But these methods exist that these some such as costs are high, efficiency is low all separately or the weak point such as poor stability.Just based on this starting point, application composting process process animal class waste has entered into the sight line of people gradually.Composting process can be defined as the microorganism (bacterium, actinomycetes, fungi etc.) or commercial strain that utilize nature extensively to exist, controlledly promotes the Biochemical processes that biodegradable organism transforms to stable soil ulmin.Because composting process is comparatively large by the impact of the factor such as microbe species, envrionment temperature, so compost fermentation efficiency is lower when artificially not adding microbial inoculum.So urgently develop the high microbial inoculum of a kind of fermentation efficiency with the economic again process animal class waste of safety and environmental protection.
Microbial inoculum provided by the invention has high temperature resistant, and extracellular proteinase, lipase are with the advantage of high-efficiency fermenting animal class waste.Utilize this microbial inoculum compost to have the advantages such as environmental pollution is little, economy and facility, the product after compost treatment, through subsequent disposal, can reach as organic fertilizer the object turned waste into wealth.
(3) summary of the invention
The object of the invention is to provide a kind of new strains-Brevibacillus brevis (Brevibacillus brevis) H3 and is preparing the application in decomposing microbial inoculum, the decomposing microbial inoculum utilizing H3 bacterial strain to prepare is under suitable Culture and fermentation conditions, energy efficient-decomposition protein, fat, have good application in process animal class waste.
The technical solution used in the present invention is:
The invention provides a strain new strains--Brevibacillus brevis (Brevibacillus brevis) H3, be preserved in China typical culture collection center, preservation date is on October 21st, 2013, and deposit number is CCTCC NO:M 2013481, and address is Wuhan, Hubei Wuhan University.
Brevibacillus brevis H3 physiological and biochemical property is as follows: on LB substratum, bacterium colony is in white, and matt, edge is irregular, and there is gauffer on surface.Aerobic respiration, well-grown under aerobic conditions; Yielding lipase, the high temperature that ability is 60 DEG C.
The invention provides described Brevibacillus brevis H3 and prepare the application in decomposing microbial inoculum, described decomposing microbial inoculum is made up of Brevibacillus brevis H3 lyophilized powder, subtilis H4 lyophilized powder, subtilis H8-3 lyophilized powder and starch, and described Brevibacillus brevis H3 lyophilized powder and subtilis H4 lyophilized powder, subtilis H8-3 lyophilized powder and starch weight are than being 1:1:1:97;
Described Brevibacillus brevis H3 lyophilized powder is that the fermented liquid obtained through fermentation culture by Brevibacillus brevis H3 is centrifugal, gets that pellet frozen drying (-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h, more preferably-50 DEG C of lyophilize 24h) obtains;
Described subtilis H8-3 lyophilized powder is that the fermented liquid obtained through fermentation culture by subtilis H8-3 is centrifugal, get pellet frozen drying (-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h, more preferably-50 DEG C of lyophilize 24h) obtain, described subtilis (Bacillus subtilis) H8-3, be preserved in China typical culture collection center, preservation date is on October 21st, 2013, deposit number is CCTCC NO:M 2013483, and address is Wuhan, Hubei Wuhan University;
Described subtilis H4 lyophilized powder is that the fermented liquid obtained through fermentation culture by subtilis H4 is centrifugal, get pellet frozen drying (-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h, more preferably-50 DEG C of lyophilize 24h) obtain, described subtilis (Bacillus subtilis) H4, be preserved in China typical culture collection center, preservation date is on October 21st, 2013, deposit number is CCTCC NO:M 2013482, and address is: Wuhan, Hubei Wuhan University.
The present invention also provides the application of a kind of described decomposing microbial inoculum in the waste containing protein and/or fat of degrading, concrete described being applied as: joined by decomposing microbial inoculum in the waste containing protein and/or fat, DeR 1 ~ 4 day (mixing rear placement by decomposing microbial inoculum with waste can degrade for 1 ~ 4 day) at 30 DEG C ~ 60 DEG C, as plant organic fertilizer recycling after degraded product process, the preferred normal temperature compost of described process 1 ~ 4 week.
Further, preferred described decomposing microbial inoculum and waste weight ratio are 0.1 ~ 5:100.
Further, the temperature of preferred described degraded is 50 DEG C ~ 60 DEG C.
Described subtilis H4 physiological and biochemical property is as follows: on LB substratum, bacterium colony is creamy white, and matt, edge is irregular, and there is gauffer on surface.Aerobic respiration, well-grown under aerobic conditions; Gram-positive; Acid can be produced in glucose, sucrose, wood sugar, seminose, maltose fermentation test; Energy gelatin hydrolysate, starch and casein; Reduction litmus milk; V-P measures the positive; Methyl red measures negative.Produce proteolytic enzyme, the high temperature that ability is 70 DEG C.
Subtilis H8-3 physiological and biochemical property is as follows: on LB substratum, bacterium colony is creamy white, and matt, edge is irregular, and there is gauffer on surface.Aerobic respiration, well-grown under aerobic conditions; Gram-positive; Acid can be produced in glucose, sucrose, wood sugar, seminose, maltose fermentation test; Energy gelatin hydrolysate, starch and casein; Reduction litmus milk; V-P measures the positive; Methyl red measures negative.Produce proteolytic enzyme, the high temperature that ability is 60 DEG C.
Brevibacillus brevis H3 physiological and biochemical property is as follows: on LB substratum, bacterium colony is in white, and matt, edge is irregular, and there is gauffer on surface.Aerobic respiration, well-grown under aerobic conditions; Yielding lipase, the high temperature that ability is 60 DEG C.
Subtilis H4 of the present invention has high temperature resistant (can survive at 70 DEG C of high temperature), protease activity is higher, and growth advantage more rapidly.
The preparation method of subtilis H4 lyophilized powder of the present invention is:
(1) slant culture
Subtilis H4 is seeded to slant medium, cultivates 1 day for 30 DEG C ~ 50 DEG C, obtain inclined-plane thalline; Described slant medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, agar powder 15g/L, and solvent is deionized water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 1 day at 30 DEG C ~ 70 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, and solvent is deionized water, and pH value is 7.0;
(3) fermentation culture
Seed liquor is seeded to fermention medium with the inoculum size of volumetric concentration 0.1% ~ 1% (preferably 0.1%), 30 DEG C ~ 70 DEG C, 100 ~ 300rpm fermentation culture 1 ~ 2 day, obtain fermented liquid, fermented liquid is centrifugal, collecting precipitation, at-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h (preferably-50 DEG C of lyophilize 24h), obtains described subtilis H4 lyophilized powder; Described fermention medium final concentration consists of: glucose 30g/L, peptone 10g/L, yeast extract paste 10g/L, sodium-chlor 2g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 1g/L, and solvent is deionized water, and pH value is 7.2.
The preparation method of subtilis H8-3 lyophilized powder of the present invention is:
(1) slant culture
Subtilis H8-3 is seeded to slant medium, cultivates 1 day for 30 DEG C ~ 50 DEG C, obtain inclined-plane thalline; Described slant medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, agar powder 15g/L, and solvent is deionized water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 1 day at 30 DEG C ~ 60 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, and solvent is deionized water, and pH value is 7.0;
(3) fermentation culture
Seed liquor is seeded to fermention medium with the inoculum size of volumetric concentration 0.1% ~ 1% (preferably 0.1%), 30 DEG C ~ 60 DEG C, 100 ~ 300rpm fermentation culture 1 ~ 2 day, obtain fermented liquid, fermented liquid is centrifugal, collecting precipitation, at-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h (preferably-50 DEG C of lyophilize 24h), obtains described subtilis H8-3 lyophilized powder; Described fermention medium final concentration consists of: glucose 30g/L, peptone 10g/L, yeast extract paste 10g/L, sodium-chlor 2g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 1g/L, and solvent is deionized water, and pH value is 7.2.
The preparation method of Brevibacillus brevis H3 lyophilized powder of the present invention is:
(1) slant culture
Brevibacillus brevis H3 is seeded to slant medium, cultivates 1 day for 30 ~ 50 DEG C, obtain inclined-plane thalline; Described slant medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl10g/L, agar powder 15g/L, and solvent is deionized water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 1 day at 30 ~ 60 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, and solvent is deionized water, and pH value is 7.0;
(3) fermentation culture
Seed liquor is seeded to fermention medium with the inoculum size of volumetric concentration 0.1% ~ 1% (preferably 0.1%), 30 ~ 60 DEG C, 100 ~ 300rpm fermentation culture 1 ~ 2 day, obtain fermented liquid, fermented liquid is centrifugal, collecting precipitation, at-50 DEG C ~-10 DEG C lyophilize 12 ~ 24h (preferably-50 DEG C of lyophilize 24h), obtains described Brevibacillus brevis H3 lyophilized powder; Described fermention medium final concentration consists of: glucose 30g/L, peptone 10g/L, yeast extract paste 10g/L, sodium-chlor 2g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 1g/L, and solvent is deionized water, and pH value is 7.2.
Waste containing protein and/or fat of the present invention refers to the domestic animals and fowls of natural death, as chicken, duck, pig etc., and they butcher after waste.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides a strain new strains--subtilis H3, and bacterial strain H3 is high temperature resistant, and all can survive at 30 DEG C-60 DEG C, lipase activity is higher, and growth is rapider; The decomposing microbial inoculum utilizing this bacterial strain to prepare has high temperature resistant, extracellular proteinase, lipase are with the advantage of high-efficiency fermenting animal class waste, described decomposing microbial inoculum compost is utilized to have the advantages such as environmental pollution is little, economy and facility, product after compost treatment, through subsequent disposal, can reach as organic fertilizer the object turned waste into wealth.
(4) accompanying drawing explanation
Lab diagram that Fig. 1 subtilis H4 is high temperature resistant.
Fig. 2 subtilis H4 proteolysis circle lab diagram.
Fig. 3 subtilis H4 protease activity lab diagram.
Lab diagram that Fig. 4 subtilis H8-3 is high temperature resistant.
Fig. 5 subtilis H8-3 protease hydrolysis circle lab diagram.
Fig. 6 subtilis H8-3 protease activity lab diagram.
Lab diagram that Fig. 7 Brevibacillus brevis H3 is high temperature resistant.
Fig. 8 Brevibacillus brevis H3 lipase hydrolysis circle lab diagram.
Fig. 9 Brevibacillus brevis H3 lipase activity lab diagram.
Figure 10 pork is comparison diagram before and after decomposing microbial inoculum process.
Figure 11 decomposing microbial inoculum digestion pork experiment Free Proteins content figure.
Crude fat content variation diagram in the experiment of Figure 12 decomposing microbial inoculum digestion pork.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1 bacterial strain screening and qualification
1, the screening of bacterial strain H4 and qualification
(1) substratum and agent prescription:
SOC liquid nutrient medium final concentration consists of: tryptone 20g/L, yeast extract 5g/L, NaCl 0.5g/L, KCl 2.5mmol/L, MgCl
20.01mol/L, glucose 0.02mol/L, solvent is deionized water, pH value 7.0.
SOC solid medium final concentration consists of: SOC liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
SOC liquid nutrient medium compound method: configure often liter of substratum, adds tryptone 20g, yeast extract 5g, NaCl 0.5g shake container and solute is dissolved completely in 965ml deionized water.Add the 10ml 250mmol/L KCl aqueous solution, adjust pH to 7.0, at 15psi (1.05kg/cm with 5mol/L NaOH
2) steam sterilizing 20min under high pressure.Solution after sterilizing adds the 2mol/L MgCl of 5ml filtration sterilization
2, be cooled to less than 60 DEG C or 60 DEG C, add the 1mol/L D/W of 20ml filtration sterilization.
Skim-milk substratum final concentration forms: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, skim-milk 20g/L, 15g/L agar powder, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.
LB liquid nutrient medium final concentration forms: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.
The dull and stereotyped final concentration composition of LB: LB liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
(2) soil sample collection
Be stored in the soil sample at Hangzhou Pedagogic University's biological medicine and health research center: Rucheng, Chenzhou hot spring soil.
(3) bacterial classification primary dcreening operation
Preparation SOC liquid nutrient medium, packing after sterilizing cooling, every root test tube adds 10ml substratum, adds pedotheque 0.5g, mixes rear 60 DEG C of shaking baths and shakes 3 hours.Sterilized water dilution 10
3doubly, coating SOC solid medium is dull and stereotyped, cultivates 20 hours for 37 DEG C.
(4) bacterial classification sieves again
The good colony inoculation of picking primary dcreening operation plated growth, in skim-milk substratum, is cultivated 20 hours for 37 DEG C.Whether observe has transparent circle to produce.Obtain the bacterial strain that a strain can produce obvious transparent circle, be labeled as bacterial strain H4, preserve.
(5) high temperature resistant experiment
Bacterial strain H4 is inoculated in LB liquid nutrient medium, cultivates 6 hours for 37 DEG C.Be sub-packed in 10ml test tube, respectively the shaking bath concussion half an hour of 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, take out cooling, sterilized water dilution 10
5doubly, get 200 μ l and coat on LB flat board, cultivate 18 hours for 37 DEG C, the bacterium colony (see Fig. 1) of counting growth.The high temperature of visible bacterial strain H4 ability 70 DEG C.
(6) protease activity experiment
Bacterial strain H4 is inoculated in skim-milk substratum, cultivates 24 hours for 37 DEG C, with the intestinal bacteria without protease activity in contrast.The results are shown in Figure 2, show that H4 has protease activity.
Bacterial strain H4 is incubated at LB liquid nutrient medium, and cultivate 18 hours for 37 DEG C, medium centrifugal, gets supernatant.Get BSA (1mg/ml) (being purchased from precious biotechnology Dalian company limited) 100 microlitres, add 100 microlitre supernatants, hatch 0,5,10,15,20,25,30 minute for 50 DEG C, add 5ml coomassie brilliant blue staining liquid termination reaction.Spectrophotometric measurement OD
600absorption value.The results are shown in Figure 3, show that bacterial strain H4 can produce proteolytic enzyme.
(7) 16S rDNA checks order and identifies bacterial classification
Use DNA extraction kit (purchased from the Hangzhou biological company limited of treasured match) to extract the DNA of bacterial strain H4, with 16S rDNA universal primer for primer, carry out pcr amplification.Amplification system is 16S rDNA universal primer 1 μ l, DNA profiling 0.1 μ l, dnTP2 μ l, buffer 2 μ l, and rTaq enzyme 0.2 μ l, sterilized water supplies 20 μ l.Amplification condition is 95 DEG C of denaturation 5min, 32 circulation (98 DEG C of sex change 10sec; 58 DEG C of annealing 30sec; 72 DEG C extend 2min), 16 DEG C of maintenances.Extension increasing sequence is measured by Nanjing Genscript Biotechnology Co., Ltd., and the 16S rDNA total order of bacterial strain H4 is classified as shown in SEQ ID.NO1.According to the 16S rRNA the sequencing results of bacterial strain H4, in conjunction with physiological and biochemical test result, after the comparison of NCBI gene pool, determine that bacterial strain H4 is subtilis (Bacillus subtilis), called after subtilis (Bacillus subtilis) H4.
Primer:
27F：AGAGTTTGATCCTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
atgctcccgg ccgccatggc ggccgcggga attcgattgg ttaccttgtt acgacttcaccccaatcatc ggtcccacct tcggcggctg
gctcctaaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc
gcggcatgct gatccgcgat tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga tttgtgggat
tggcttaacc tcgcggtttc gttgcccttt gttctgtcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc
atccccacct tcctcctgtt tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg cgctcgttgc
gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca cctgtcactc tgcccccgaa ggggacgtcc catctctaga
attgtcagag gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat
tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac
ttagcactca tcgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacgctttcgctcc tcagcgtcag ttacagacca
gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagt
tccccagttt ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg agccctttac gcccaataat
tccggacaac gctcgccacc tacgtattac cgcggctgct ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtaccgcc
ctattcgaac ggtacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc gtcagacttt
cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg
gctacgcatc gtcgccttgg tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagccga agctaccttt
tatgtttgaa ccatgcggtt caaacaacca tccggtatta gccccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg
ttactcaccc gtccgccgct aacatcaggg agcaagctcc catctgtccg ctcgacttgc atgtattagg cacgccgcca gcgttcgtcc
tgagccagga tcaaactcta atcactagtg aattcgcggc cgcctgcagg tcgaccatat gggagagctc ccaacgcgtt ggatgcatag
cttgagtatt ctataggtca c。
2, bacterial strain H8-3 screens and qualification
(1) substratum and agent prescription:
SOC liquid nutrient medium final concentration consists of: tryptone 20g/L, yeast extract 5g/L, NaCl 0.5g/L, KCl 2.5mmol/L, MgCl
20.01mol/L, glucose 0.02mol/L, solvent is deionized water, pH value 7.0.
SOC solid medium final concentration consists of: SOC liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
SOC liquid nutrient medium compound method: configure often liter of substratum, adds tryptone 20g, yeast extract 5g, NaCl 0.5g shake container and solute is dissolved completely in 965ml deionized water.Add the 10ml 250mmol/L KCl aqueous solution, adjust pH to 7.0, at 15psi (1.05kg/cm with 5mol/L NaOH
2) steam sterilizing 20min under high pressure.Solution after sterilizing adds the 2mol/L MgCl of 5ml filtration sterilization
2, be cooled to less than 60 DEG C or 60 DEG C, add the 1mol/L D/W of 20ml filtration sterilization.
Skim-milk substratum final concentration forms: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, skim-milk 20g/L, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.
LB liquid nutrient medium final concentration forms: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.
The dull and stereotyped final concentration composition of LB: LB liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
(2) soil sample collection
Be stored in the soil sample at Hangzhou Pedagogic University's biological medicine and health research center: Fujian Qingliu County
High bad hot spring soil.
(3) bacterial classification primary dcreening operation
Preparation SOC liquid nutrient medium, packing after sterilizing cooling, every root test tube adds 10ml substratum, adds pedotheque 0.5g, mixes rear 60 DEG C of shaking baths and shakes 3 hours.Sterilized water dilution 10
3doubly, coating SOC solid medium is dull and stereotyped, cultivates 20 hours for 37 DEG C.
(4) bacterial classification sieves again
The good colony inoculation of picking primary dcreening operation plated growth, in skim-milk substratum, is cultivated 20 hours for 37 DEG C.Whether observe has transparent circle to produce.Obtain the bacterial strain that a strain can produce obvious transparent circle, be labeled as bacterial strain H8-3, preserve.
(5) high temperature resistant experiment
Bacterial strain H8-3 is inoculated in LB liquid nutrient medium, cultivates 6 hours for 37 DEG C.Be sub-packed in 10ml test tube, respectively the shaking bath concussion half an hour of 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, take out cooling, sterilized water dilution 10
5doubly, get 200 μ l and coat on LB flat board, cultivate 18 hours for 37 DEG C, the bacterium colony (see Fig. 4) of counting growth.The high temperature of visible bacterial strain H8-3 ability 60 DEG C.
(6) protease activity experiment
Bacterial strain H8-3 is inoculated in skim-milk substratum, cultivates 24 hours for 37 DEG C, with the intestinal bacteria without protease activity in contrast.The results are shown in Figure 5, show that bacterial strain H8-3 has protease activity.
Bacterial strain H8-3 is incubated at LB liquid nutrient medium, and cultivate 18 hours for 37 DEG C, medium centrifugal, gets supernatant.Get BSA (1mg/ml) (being purchased from precious biotechnology Dalian company limited) 100 microlitres, add 100 microlitre supernatants, hatch 0,5,10,15,20,25,30 minute for 50 DEG C, add 5ml coomassie brilliant blue staining liquid termination reaction.Spectrophotometric measurement OD
600absorption value.The results are shown in Figure 6, show that bacterial strain H8-3 can produce proteolytic enzyme.
(7) 16s rRNA checks order and identifies bacterial classification
Use DNA extraction kit (purchased from the Hangzhou biological company limited of treasured match) to extract the DNA of bacterial strain H8-3, with 16S rDNA universal primer for primer, carry out pcr amplification.Amplification system is 16SrDNA universal primer 1 μ l, DNA profiling 0.1 μ l, dnTP2 μ l, buffer 2 μ l, and rTaq enzyme 0.2 μ l, sterilized water supplies 20 μ l.Amplification condition is 95 DEG C of denaturation 5min, 32 circulation (98 DEG C of sex change 10sec; 58 DEG C of annealing 30sec; 72 DEG C extend 2min), 16 DEG C of maintenances.Extension increasing sequence is measured by Nanjing Genscript Biotechnology Co., Ltd., and the 16S rDNA total order of bacterial strain H8-3 is classified as shown in SEQ ID.NO2.According to the 16S rRNA the sequencing results of bacterial strain H8-3, in conjunction with physiological and biochemical test result, after the comparison of NCBI gene pool, determine that bacterial strain H8-3 is subtilis (Bacillus subtilis), called after subtilis (Bacillus subtilis) H8-3.
Primer:
27F：AGAGTTTGATCCTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
SEQ ID.NO2：
AATAcATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCG
3, bacterial strain H3 screens and qualification
(1) substratum and agent prescription:
SOC liquid nutrient medium final concentration consists of: tryptone 20g/L, yeast extract 5g/L, NaCl 0.5g/L, 2.5mmol/L KCl, 0.01mol/L MgCl
2, glucose 0.02mol/L, solvent is deionized water, pH value 7.0.
SOC solid medium final concentration consists of: SOC liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
SOC liquid nutrient medium compound method: configure often liter of substratum, adds tryptone 20g, yeast extract 5g, NaCl 0.5g shake container and solute is dissolved completely in 965ml deionized water.Add the 10ml 250mmol/L KCl aqueous solution, adjust pH to 7.0, at 15psi (1.05kg/cm with 5mol/L NaOH
2) steam sterilizing 20min under high pressure.Solution after sterilizing adds the 2mol/L MgCl of 5ml filtration sterilization
2, be cooled to less than 60 DEG C or 60 DEG C, add the 1mol/L D/W of 20ml filtration sterilization.
Sweet oil screening culture medium final concentration forms: yeast extract paste 2g/L, peptone 2g/L, NaCl 5g/L, sweet oil emulsion 12g/L, agar powder 15g/L, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.Wherein sweet oil emulsion compound method is: measure sweet oil 25ml and volumetric concentration 2% polyvinyl alcohol water solution 75ml respectively, mixing, whirlpool concussion emulsification 3 times, each 1 minute.
LB liquid nutrient medium final concentration forms: extractum carnis 5g/L, peptone 10g/L, NaCl 10g/L, solvent is deionized water, and pH value is 7.0.At 15psi (1.05kg/cm
2) steam sterilizing 20min under high pressure.
The dull and stereotyped final concentration composition of LB: LB liquid nutrient medium adds the agar powder of quality final concentration 1.5%.
(2) soil sample collection
Be stored in the soil sample at Hang Shi great biological medicine and health research center: Rucheng, Chenzhou hot spring soil.
(3) bacterial classification primary dcreening operation
Preparation SOC liquid nutrient medium, packing after sterilizing cooling, every root test tube adds 10ml substratum, adds pedotheque 0.5g, mixes rear 60 DEG C of shaking baths and shakes 3 hours.Sterilized water dilution 10
3doubly, coating SOC solid medium is dull and stereotyped, cultivates 20 hours for 37 DEG C.
(4) bacterial classification sieves again
The colony inoculation that picking primary dcreening operation plated growth is good is dull and stereotyped in sweet oil screening culture medium, cultivates 20 hours for 37 DEG C.Observe and whether have hydrolysis circle to produce.Obtain the bacterial strain that a strain can produce obviously hydrolysis circle, be labeled as bacterial strain H3, preserve.
(5) high temperature resistant experiment
Bacterial strain H3 is inoculated in LB liquid nutrient medium, cultivates 6 hours for 37 DEG C.Be sub-packed in 10ml test tube, respectively the shaking bath concussion half an hour of 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, take out cooling, sterilized water dilution 10
5doubly, get 200 μ l and coat on LB flat board, cultivate 18 hours for 37 DEG C, the bacterium colony (see Fig. 7) of counting growth.The high temperature of visible bacterial strain H3 ability 60 DEG C.
(6) lipase activity experiment
Bacterial strain H3 is inoculated in sweet oil screening culture medium dull and stereotyped, cultivates 24 hours for 37 DEG C, with the intestinal bacteria of fat-free enzymic activity in contrast.The results are shown in Figure 8, show that H3 has lipase activity.
Bacterial strain H3 is incubated at LB substratum, cultivates 18 hours for 37 DEG C, centrifuging and taking supernatant.Get 4ml p-NP cetylate substrate (0.09mg/ml, PH=7.0) 1ml bacterial strain H3 supernatant (to add 1ml distilled water in contrast) is added after solution preheating 5min, hatch 0,5,10,15,20,25,30 minute for 50 DEG C, add 5ml dehydrated alcohol termination reaction immediately.Spectrophotometer OD
405survey absorption value.The results are shown in Figure 9.Show that H3 can yielding lipase.
(7) 16s rRNA checks order and identifies bacterial classification
Use DNA extraction kit (purchased from the Hangzhou biological company limited of treasured match) to extract the DNA of bacterial strain H3, with 16S rDNA universal primer for primer, carry out pcr amplification.Amplification system is 16S rDNA universal primer 1 μ l, DNA profiling 0.1 μ l, dnTP2 μ l, buffer 2 μ l, and rTaq enzyme 0.2 μ l, sterilized water supplies 20 μ l.Amplification condition is 95 DEG C of denaturation 5min, 32 circulation (98 DEG C of sex change 10sec; 58 DEG C of annealing 30sec; 72 DEG C extend 2min), 16 DEG C of maintenances.Extension increasing sequence is measured by Nanjing Genscript Biotechnology Co., Ltd., and the 16S rDNA total order of bacterial strain H3 is classified as shown in SEQ ID.NO3.According to the 16S rRNA the sequencing results of bacterial strain H3, in conjunction with physiological and biochemical test result, after the comparison of NCBI gene pool, determine that bacterial strain H3 is Brevibacillus brevis (Brevibacillus brevis), called after Brevibacillus brevis (Brevibacillus brevis) H3.
Primer:
27F：AGAGTTTGATCCTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
SEQ ID.NO3：
GGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGTTACCTTGTTACGACTTCACCCCAGTCATCTACCCCACCTTCGGCGGCTGGCTCCTTGCGGTTACCTCACCGACTTCGGGTGTTGCAAACTCCCGTGGTGTGACGGGCGGTGTGTACAGGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGTTTTAAGAGATTGGCGTCCTCTCGCGAGGTAGCATCCCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCGCCTTCCTCCGTCTTGTCGACGGCAGTCTCTCTAGAGTGCCCAACTGAATGCTGGCAACTAAAGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGCTGCCCCGAAGGGAAGCTCTGTCTCCAGAGCGGTCAGCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGGCACTGAGGGTATTGAAACCCCCAACACCTAGCACTCATTGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATACCGCTTTCCTCTTCTGCACTCAAGCTACACAGTTTCCGATGCGAaCCGGGGtTGAGCCCcGgGCTTTAACAcCAGACTTACATAGCcGCcTGCgcGCGCTTtACGCCcaatAAGTCcGgACaACGCtTGCCACcTACGTATTACCGCgGCTGCTGgCACGTAGtTAGcCGTGGCTTTCTCGTCAgGTAcCG。
Embodiment 2 decomposing microbial inoculum
(1) subtilis H4 lyophilized powder
A, slant culture
Subtilis H4 is seeded to inclined-plane LB substratum, cultivates 1 day at 37 DEG C, obtain inclined-plane thalline.
B, seed culture
Picking one transfering loop bacterial classification from inclined-plane thalline, be seeded in 100ml LB liquid nutrient medium, 50 DEG C of shaking tables cultivate 24 hours, obtain seed liquor.
C, fermentation culture
Be seeded in fermention medium with the ratio of volume ratio 1:1000 by seed liquor, 50 DEG C ferment 24 hours, fermentor tank tank pressure: 0.04MPa, air flow: 0.2v/v.min, rotating speed 200rpm.Get fermented liquid centrifugal, collect wet thallus ,-50 DEG C of lyophilize 24h, obtain subtilis H4 lyophilized powder.
Fermention medium final concentration consists of: glucose 30g/L, peptone 10g/L, yeast extract paste 10g/L, sodium-chlor 2g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 1g/L, potassium primary phosphate 1g/L, and solvent is deionized water, and pH is 7.0.
(2) subtilis H8-3 lyophilized powder
The preparation of subtilis H8-3 lyophilized powder is with subtilis H4 lyophilized powder, and substratum and culture condition are all identical.
Subtilis (Bacillus subtilis) H8-3 used, is preserved in China typical culture collection center, and preservation date is on October 21st, 2013, and deposit number is CCTCC M2013483, and address is Wuhan, Hubei Wuhan University.
(3) Brevibacillus brevis H3 lyophilized powder
The preparation of Brevibacillus brevis H3 lyophilized powder is with subtilis H4 lyophilized powder, and substratum and culture condition are all identical.
Brevibacillus brevis (Brevibacillus brevis) H3 used, is preserved in China typical culture collection center, and preservation date is on October 21st, 2013, and deposit number is CCTCC M2013481, and address is Wuhan, Hubei Wuhan University.
(4) decomposing microbial inoculum
The Brevibacillus brevis H3 lyophilized powder prepared step (1) ~ (3) mixes with mass ratio 1:1:1:97 with subtilis H4 lyophilized powder, subtilis H8-3 lyophilized powder and starch, make decomposing microbial inoculum, the packing of 500g sealing bag, 4 DEG C of preservations.
The character of embodiment 3 decomposing microbial inoculum
(1) protease activity
The TYR reference liquid of preparation 10,20,30,40,50,60 μ g/ml, not contain the distilled water of tyrosine for blank, ultraviolet spectrophotometer 275nm wavelength measures absorbancy.Take absorbancy as ordinate zou, the concentration of tyrosine is X-coordinate, and drawing standard curve, obtains equation y=0.0074x, and extinction constant K is 135.1.
Decomposing microbial inoculum 10g prepared by Example 2 method, adds 100ml 0.1mol/L phosphoric acid buffer (pH=7.5), 200rpm shaking table concussion 30min.3000rpm is centrifugal, gets decomposing agent supernatant.
Get decomposing agent supernatant 1.0ml, 40 DEG C are incubated 2 minutes, add the 1.0ml casein aqueous solution (10mg/ml), mix rear 40 DEG C of reaction 20min, add 2ml trichloroacetic acid solution (0.4mol/L) termination reaction, 3000rpm is centrifugal, gets supernatant, and after distilled water dilutes 10 times, ultraviolet spectrophotometer 275nm wavelength measures absorbancy.Parallel laboratory test 3 times, obtaining sample average absorbancy is 0.271.
With following method in contrast: get decomposing agent supernatant 1.0ml, 40 DEG C are incubated 2 minutes, add 2ml trichloroacetic acid solution (0.4mol/L) and make supernatant liquor inactivation, add the 1.0ml casein aqueous solution (10mg/ml), mix rear 40 DEG C of reactions 30min, 3000rpm centrifugal, get supernatant, after distilled water dilutes 10 times, ultraviolet spectrophotometer 275nm wavelength measures absorbancy, parallel laboratory test 3 times, and obtaining contrast mean light absorbency is 0.134.
X=(A – B) × K × 4/1 × 1/20 × n formula (1)
In formula (1): the enzyme activity of X-sample, U/g; A-sample average absorbancy; B-contrast mean light absorbency; K-extinction constant; The cumulative volume of 4-reaction reagent, mL; 1-draw enzyme liquid 1.0mL, in lmL; 1/20-reaction times 20min, in lmin; N-diluted sample multiple,
The protease activity calculating this decomposing microbial inoculum is 369U/g.
Proteinase activity unit definition: 1g decomposing microbial inoculum, under certain temperature and pH value condition, it is an enzyme activity unit that 1min hydrolyzed casein produces 1 μ g tyrosine, represents with U/g.
(2) lipase activity
Preparation sweet oil PVA emulsion: get mass concentration 4% polyvinyl alcohol (PVA) aqueous solution 150ml, add sweet oil 50ml, at a high speed concussion 2 times, each 2min.
Decomposing microbial inoculum 10g prepared by Example 2 method, adds 100ml 0.1mol/L phosphoric acid buffer (pH=7.5), 200rpm shaking table concussion 30min.3000rpm is centrifugal, gets supernatant.
Sweet oil PVA emulsion 4ml and phosphoric acid buffer (0.1mol/L, pH=7.5) 5ml mixes, and 40 DEG C of preheating 5min, add supernatant 1.0ml, and 40 DEG C are reacted 15 minutes, add 15ml volumetric concentration 95% aqueous ethanolic solution termination reaction.Drip phenolphthalein indicator 2, with the titration of 0.05mol/L standard solution of sodium hydroxide, until system is rendered as redness and keeps 30s not take off for its terminal, the volume of the 0.05mol/L standard solution of sodium hydroxide that record consumes, parallel test 3 times, obtaining average sodium hydroxide solution consumption is 1.23ml.
With following method in contrast: sweet oil PVA emulsion 4ml and phosphoric acid buffer (0.1mol/L, pH=7.5) 5ml mixes, 40 DEG C of preheating 5min, add 15ml volumetric concentration 95% aqueous ethanolic solution, add supernatant 1.0ml, 40 DEG C are reacted 15 minutes.Drip phenolphthalein indicator 2, with the titration of 0.05mol/L standard solution of sodium hydroxide, until system is rendered as redness and keeps 30s not take off for its terminal, the volume of the 0.05mol/L standard solution of sodium hydroxide that record consumes.Parallel test 3 times, obtaining average sodium hydroxide solution consumption is 0.21ml.
X=A × c/0.05 × 50 × 1/15 × n formula (2)
In formula (2): the enzyme activity of X-sample, U/g; The volume differences of standard solution of sodium hydroxide is consumed, mL during A-titration sample; C-Concentration of Sodium Hydroxide Solution Standard, mol/L; 0.05 one Concentration of Sodium Hydroxide Solution Standard reduction factor; 50-0.05mol/L sodium hydroxide solution 1.00mL is equivalent to lipid acid 50 μm of ol; 1/15-reaction times 15min, in 1min; N-diluted sample multiple.
Through calculating, obtaining lipase activity is 34U/g.
Lipase activity unit definition: 1g decomposing microbial inoculum, under 40 DEG C of conditions with pH=7.5, hydrolysed fat per minute produces the lipid acid of 1 μm of ol, is an enzyme activity unit, represents with U/g.
The application of embodiment 4 decomposing microbial inoculum
(1) get 1000g fresh pork, mincer stirs into minced meat.120 DEG C of high-temperature heat sterilization 20min.Add decomposing microbial inoculum prepared by 10g embodiment 2 method after cooling, 60 DEG C of constant temperature stir and carry out DeR, at cultivation 0,6,12,18,24 hours each sample thief 50g, and 0 DEG C of preservation, working sample lipid content and free aminoacid content.
Observe after 10 hours, fragment all digests and becomes meat soup.Observe after 24 hours, in meat soup, have a large amount of bacterium ball to occur.After degraded cultivation terminates, in DeR thing, add 500g wheat bran, normal temperature compost treatment one week, become organic fertilizer (sample is as Figure 10).
Free protein assay: get each degradation time section sample 1g, centrifuging and taking supernatant 25 microlitre, adds water and supply 100 microlitres, adds 5ml coomassie brilliant blue staining liquid.Spectrophotometer OD600 surveys absorption value.The results are shown in Figure 11.Show the protein that decomposing microbial inoculum can be degraded in pork.
The determination of fat: get each degradation time section sample 10g respectively, petroleum ether extraction 3 times, revolves after steaming removes solvent, crude fat content of weighing.The results are shown in Figure 12.Show that decomposing microbial inoculum can effective hydrolysed fat.
(2) get 500KG pig slaughterhouse waste, blender is pulverized.120 DEG C of high-temperature sterilizations 2 hours.Add decomposing microbial inoculum prepared by 1KG embodiment 2 method when being cooled to below 60 DEG C, 60 DEG C of constant temperature stir and carry out DeR 24 hours, and add 200Kg wheat bran stirring and evenly mixing, normal temperature compost becomes organic fertilizer in 2 weeks.Sent by this organic fertilizer Bao Sai bio tech ltd, Hangzhou to detect, detected result is as shown in table 1:
Table 1 organic fertilizer Indexs measure
Claims (2)
1. Brevibacillus brevis (Brevibacillus brevis) H3, is preserved in China typical culture collection center, and preservation date is on October 21st, 2013, and deposit number is CCTCC NO:M 2013481, and address is Wuhan, Hubei Wuhan University.
2. described in a claim 1, Brevibacillus brevis H3 is preparing the application in decomposing microbial inoculum.
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