CN108004169A - Bacillus licheniformis ZL-1 and its application - Google Patents
Bacillus licheniformis ZL-1 and its application Download PDFInfo
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Abstract
The invention discloses bacillus licheniformis Bacillus licheniformis ZL 1 and its application.Bacillus licheniformis Bacillus licheniformis ZL 1 of the present invention separation screenings from the high temperature nuclear reactor body of feather joint edible fungi residues obtain, it is preserved in Guangdong Province's Culture Collection, deposit number is GDMCC No.60284, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is on November 24th, 2017.Through measuring, bacillus licheniformis Bacillus licheniformis ZL 1 of the present invention can be cultivated 3 days in the fluid nutrient medium of pure feather, its keratinase activity reaches 52.14U/mL, reach 90.15% to the degradation rate of feather.In addition, bacillus licheniformis Bacillus licheniformisZL 1 of the present invention have good tolerance to high temperature and salinity, can well it be grown at 55 DEG C, and it is resistant to 14% salinity, therefore During High-Temperature Composting fermentation, production compound amino acid fertilizer and production keratinase are carried out for raw material available for degradation of feather, production compositing acid solution, using feather, had a good application prospect and market value.
Description
Technical field
The invention belongs to microorganism and its applied technical field, it is more particularly related to bacillus licheniformis
Bacillus licheniformis ZL-1 and its application.
Background technology
Feather is remaining discarded object after poultry processing, in global scope, has millions of tons feather to discard every year
Thing produces, and the waste feathers that China produces every year nearly 700,000 t, and the problem of management of solid waste gets married fowl meat producing plant
Urgent problem to be solved.But feather waste is a kind of abundant native protein resource, its crude protein content is up to 85%-
90%, also contain vitamin B12With some unidentified growth factors.In feather protein except lysine, methionine content substantially compared with
Low outer, the composition of other animal essential amino acids is slightly above fish meal, it means that produces compound ammonia using the hydrolysis of feather protein matter
Base acid has potential economic benefit.But the protein in poultry feather is mainly made of keratoprotein, in keratin contain compared with
More cystines, forms substantial amounts of disulfide bond between them, along with hydrogen bond and intermolecular force are difficult by common proteases
(such as trypsase, pepsin, papain) hydrolyzes.Utilize high temperature and pressure boiling, enzymatic isolation method, puffing, acid and alkali hydrolysis
With using conventional method working process waste feathers such as oxidant or reduction, amino acid destruction is serious, enzyme preparation is of high cost, energy
The problems such as consumption is high, pollution is big, and the nutritive value of hydrolysate can be improved by carrying out degraded using microorganism keratin, be reduced
Energy consumption, cost and the pollution problem to environment, are a kind of environmentally friendly utilization of waste as resource modes.
There is multiple-microorganism to report fungi horse onyx group from Ward in 1899 with degradation of feather keratin in nature
Since capsule bacterium can decompose keratin, kind more than 30 has been had found so far, the main bacillus including bacterium class, Mycophyta
Aspergillus and the streptomyces of actinomyces.
China lags behind foreign countries in the screening of feather keratin decomposer and the research of application aspect, the height separated
Effect bacterial strain is simultaneously few, and is fungi and actinomyces mostly, and keratin degrading bacterias of these reports are grown under normal temperature environment mostly
(25~35 DEG C), the keratin degrading bacteria report on (more than 45 DEG C) under hot conditions are few.It is most suitable due to keratinase
Reaction temperature generally at 40 DEG C~60 DEG C, using feather as raw material compost fermentation in heap temperature generally more than 50 DEG C, so as to give
Conventional biologic treating technique brings larger difficulty.Therefore, screening has the angle egg of stability and high efficiency under the high temperature conditions
White degradation bacteria is also a research hotspot.
The content of the invention
It is an object of the invention to：The deficiencies of overcoming the keratin degrading bacteria lacked in the prior art under hot conditions, carries
For a kind of keratin degrading bacteria bacillus licheniformis Bacillus licheniformis stable and efficient under the high temperature conditions
ZL-1 and its application.
In order to realize foregoing invention purpose, the present invention provides a kind of bacillus licheniformis Bacillus
Licheniformis ZL-1, it is that separation screening obtains from the high temperature nuclear reactor body of feather joint edible fungi residues, is preserved in Guangdong
Culture Collection is saved, deposit number is GDMCC No.60284, and preservation address is No. 100 big for Xianlie Middle Road, Guangzhou City
5 building, the building of institute the 59th, preservation date are on November 24th, 2017.
Identify, tie through colonial morphology after bacillus licheniformis (Bacillus licheniformis) ZL-1 separation of the present invention
Fruit is as follows：After cultivating 24h on NA culture mediums, bacterium colony is coarse, opaque, and it is in tree root shape that edge is irregular, easy picking, and bacterium colony is straight
Footpath is 15mm, and bacillus, thalline is single, paired or catenation；Gram-positive；Gemma ellipse, middle life are held life, are had partially
Pod membrane.
Bacillus licheniformis (Bacillus licheniformis) ZL-1 of the present invention is through physiological and biochemical test, as a result such as
Under：The catalase of bacillus licheniformis (Bacillus licheniformis) ZL-1, V-P experiments, anaerobic growth, nitrate are also
The experiments such as original, Starch Hydrolysis, casein hydrolysis, gelatin hydrolysis, cellulose decomposition are the positive, indoles, lecithinase, tyrosine
The experiments such as hydrolysis, Phenylalanine dehydrogenase are feminine gender；25~60 DEG C of growth temperature range, the maximum tolerance to NaCl are
14%.
Bacillus licheniformis (Bacillus licheniformis) ZL-1 of the present invention, the nucleotide sequence of its 16S rDNA
Such as SEQ ID NO:Shown in 1, fragment length 1455bp, compares with ncbi database and shows, the bacterial strain and bacillus licheniformis
The homology highest of (Bacillus licheniformis) ATCC 14580, up to 99%.Used using 7.0 softwares of MEGA
Neighbor-Joining methods draw 16S rDNA phylogenetic trees, its evolutionary degree are determined, with reference to its morphological feature and physiology
Biochemical character, the bacterial strain most likely bacillus licheniformis category, and be named as Bacillus licheniformis ZL-1.
The cultivation temperature of the seed liquor of bacillus licheniformis (Bacillus licheniformis) ZL-1 of the present invention is 45
~55 DEG C.
It is verified by experiments, ZL-1 is under the high temperature conditions for bacillus licheniformis (Bacillus licheniformis) of the present invention
It can stablize and keratin of efficiently degrading, therefore, bacillus licheniformis (Bacillus licheniformis) ZL-1 of the present invention
It is compound available for fermentative degradation feather, production compositing acid solution, the fermented abandoned feather of During High-Temperature Composting and edible fungi residues, production
Amino acid fertilizer and production keratinase.
In order to realize foregoing invention purpose, present invention also offers a kind of degradation of feather, compositing acid solution is produced
Method, it includes the following steps：
By feather and inorganic salt solution according to 1:100 mass ratio mixing, adds 5wt% bacillus licheniformis
(Bacillus licheniformis) ZL-1 seed liquors, are cultivated 3 days under 45~55 DEG C, 200rpm, complete feather degraded, and
Obtain compositing acid solution；The inorganic salt solution component includes：NaCl 0.5g/L、NaH2PO4 0.1035g/L、
Na2HPO41.3277g/L；The preparation method of bacillus licheniformis (Bacillus licheniformis) the ZL-1 seed liquors
It is：Bacillus licheniformis (Bacillus licheniformis) ZL-1 is inoculated on NA solid plate culture mediums, 45 DEG C of trainings
Foster 24h makes its activation, is transferred in NB liquid seed culture mediums and is cultivated to exponential phase after activation；The bacillus licheniformis
(Bacillus licheniformis) ZL-1, is preserved in Guangdong Province's Culture Collection, deposit number GDMCC
No.60284, preservation address are 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is on November 24th, 2017.
After measured, keratinase activity reaches 52.14U/ml to gained compositing acid solution in zymotic fluid, compound in zymotic fluid
Amino acid classes are complete, and amino acid content reaches 3.4278g/kg, reach 90.15% to the degradation rate of feather.
Preferably, the feather also includes by pretreatment, preprocess method：Feather is broken into it is fluffy cotton-shaped, then
With water according to 1:40 mass ratio mixing, adjusts pH to 10, and 180s is heated under conditions of microwave energy is 700W.
In order to realize foregoing invention purpose, present invention also offers a kind of fermented abandoned feather of During High-Temperature Composting and edible mushroom
Slag, the method for producing compound amino acid fertilizer, it includes the following steps：
According to carbon-nitrogen ratio it is 20 by feather and edible fungi residues:1~25:1 ratio is mixed to get mixture, adds mixture
Bacillus licheniformis (Bacillus licheniformis) the ZL-1 microbial inoculums and mixture quality 0.1 of quality 1~2%~
0.2% aspergillus niger microbial inoculum, obtains total mixture, adjusts the water content of total mixture to being put into solid-state fermentation tank after 70~75%
In, it is 50~55 DEG C to adjust temperature, and ventilate 30~60min daily, and continuous compost cools the temperature to natural temperature, make it after 7 days
Natural compost ferments 14 days, obtains compound amino acid fertilizer；
The preparation method of bacillus licheniformis (Bacillus licheniformis) the ZL-1 microbial inoculums is：By lichens bud
Spore bacillus (Bacillus licheniformis) ZL-1 is inoculated into fermentation culture and ferments, at 45~50 DEG C, stirring
Speed is 180~200rpm, and throughput cultivates 36~48h under conditions of being 0.5~2vvm, obtains bacillus licheniformis
(Bacillus licheniformis) ZL-1 microbial inoculums；The fermented and cultured formula of liquid is：Glucose 1.5g/L, corn flour
1.5g/L, dregs of beans 2.0g/L, sodium chloride 0.5g/L；
The preparation method of the aspergillus niger microbial inoculum is：By aspergillus niger strain (be purchased from Guangdong Province's Culture Collection,
Deposit number is GDMCC No.3.576) it is inoculated into bran mass and ferments, the tray static gas wave refrigerator 3~5 at 37 DEG C
My god, obtain aspergillus niger microbial inoculum；The formula of the bran mass is：Wheat bran：Potassium dihydrogen phosphate：Mass ratio=1000 of water:5:
1000。
Relative to the prior art, the invention has the advantages that and beneficial effect：
Bacillus licheniformis (Bacillus licheniformis) ZL-1 of the present invention is compared to the domestic keratin reported
Degradation bacteria, it is strong to temperature tolerance degree, it can be grown in 20~60 DEG C of temperature conditionss, growth temperature can reach keratinase
40~60 DEG C of optimal reactive temperature, there is high keratinase activity (keratinase activity 52.14U/ under the conditions of 45~55 DEG C
Ml), it is high (degradation rate 90.15%) to the degradation rate of feather, it can be grown in the fermentation of the high temperature high-rate composting of feather and mushroom slag
Well.
Bacillus licheniformis (Bacillus licheniformis) ZL-1 resistance of the present invention is strong, is resistant to 14%
High salinity, and a variety of enzymes such as energy eccrine fiber element enzyme, amylase, protease, lipase.
The present invention is molten using the compound amino acid of bacillus licheniformis (Bacillus licheniformis) ZL-1 productions
Liquid, can be using feather as unique carbon nitrogen source, and production cost is low, and technique is simple, and amino acid classes are neat in compositing acid solution
Entirely.
The present invention utilizes bacillus licheniformis (Bacillus licheniformis) ZL-1 production compound amino acid fertilizer
The fermentation time of feather and edible fungi residues can be greatly shortened, it is cost-effective.
Culture presevation information：
Bacillus licheniformis ZL-1, are preserved in Guangdong Province's Culture Collection, and deposit number is
GDMCC No.60284, preservation address are 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is 2017 11
The moon 24.
Brief description of the drawings
Fig. 1 is bacillus licheniformis (Bacillus licheniformis) ZL-1 systematic growth tree graphs.
Fig. 2 is the design sketch of bacillus licheniformis (Bacillus licheniformis) ZL-1 degradation of feather, wherein boring
Shape bottle ZL-1 is the experimental group for adding bacillus licheniformis (Bacillus licheniformis) ZL-1, and conical flask CK is blank
Control group.
To obtain compound amino acid molten for bacillus licheniformis (Bacillus licheniformis) ZL-1 degradation of feather by Fig. 3
Amino acid spectrogram in liquid.
Embodiment
In order to become apparent from the purpose of the present invention, technical solution and advantageous effects, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
It is bright, be not intended to limit the present invention, the parameter of embodiment, ratio etc. can adaptation to local conditions make a choice and substance had no to result
Influence.In embodiment unless otherwise specified, it is this area conventional reagent and method and step.
The separation of 1 bacillus licheniformis of embodiment (Bacillus licheniformis) ZL-1
Chicken feather is derived from chicken farm under Guangdong Wen'S Foodstuffs Group Co., Ltd., and edible fungi residues are derived from Guangzhou kingly way life
Thing Science and Technology Ltd., mixing are put into unlimited solid-state fermentation tank, and it is 45 DEG C to keep temperature in tank, is collected after feather decomposition
Fermentation residue, dries in the shade naturally, and 4 DEG C save backup.
Take the above-mentioned samples of 10g to be put into the triangular flask equipped with 90mL sterile waters (band bead), shaken under 180rpm rotating speeds
30min is swung, after standing 10min, after taking upper liquid to dilute 10 times, is once made 10-4、10-5、10-6Dilution, takes 0.1mL uniform
It is coated on NA culture medium flat plates, back-off tablet, which is placed in 45 DEG C of incubators, to be cultivated 2 days.The bacterial strain different to each colonial morphology
Make 3 parallel purification tablets.Selecting primary dcreening operation single bacterium to fall within basic salt feather culture medium, be placed in 45 DEG C of shaking tables, 180rpm is cultivated,
Compared with nonvaccinated culture medium, observation feather degraded situation.
NA culture medium prescriptions：Peptone 10.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, agar 15.0g/L.
Basic salt feather culture medium prescription：Natural feather 1g/L, sodium chloride 0.5g/L, sodium dihydrogen phosphate 0.1035g/L, phosphorus
Sour disodium hydrogen 1.3277g/L.
By the further culture of basic salt feather culture medium, filter out and decompose the highest high temperature bacterial strain of feather efficiency, warp
After crossing morphology and Physiology and biochemistry and DNA identifications, bacillus licheniformis (Bacillus licheniformis) ZL-1 is named as.
The biological character and Physiology and biochemistry of 2 bacillus licheniformis of embodiment (Bacillus licheniformis) ZL-1
And Molecular Identification
S1. morphological feature
After bacillus licheniformis (Bacillus licheniformis) ZL-1 cultivates 24h on NA culture mediums, bacterium colony is thick
Rough, opaque, irregular edge is in tree root shape, and easy picking, colony diameter 15mm, bacillus, thalline is single, and in pairs or chain is arranged
Row；Gram-positive；Gemma ellipse, middle life hold life, there is pod membrane partially.
S2. physiological and biochemical property：Through physiological and biochemical test, bacillus licheniformis (Bacillus of the invention
Licheniformis) catalase of ZL-1, V-P experiments, it is anaerobic growth, nitrate reduction, Starch Hydrolysis, casein hydrolysis, bright
The experiments such as glue solution, cellulose decomposition are the experiment such as the positive, indoles, lecithinase, tyrosine hydrolysis, Phenylalanine dehydrogenase
It is feminine gender；25~60 DEG C of growth temperature range, the maximum tolerance to NaCl are 14%.Concrete outcome as shown in table 1
Table 1
Note：+, it is positive；-, it is negative.
S3. the sequencing of the 16S rDNA of bacillus licheniformis (Bacillus licheniformis) ZL-1 is with dividing
Analysis：
(1) DNA is extracted：Bacillus licheniformis (Bacillus licheniformis) ZL-1 streak inoculations are cultivated in NA
On the tablet of base, 45 DEG C of overnight incubations.Picking single bacterium colony is with 1.5mL EP pipes, DNA of bacteria is extracted using CTAB methods.
(2) 16S rDNA gene PCRs extend：
PCR primer is synthesized by Shanghai Sangon Biotech Company.
The nucleotide sequence of Primer A (27F) such as SEQ ID NO:Shown in 2；The nucleotides sequence of Primer B (1492R)
Row such as SEQ ID NO:Shown in 3.
PCR reaction systems such as table 2.
Table 2
PCR amplification condition：95℃5min；95 DEG C of 50s, 56 DEG C of 50s, 72 DEG C of 90s, 30 circulations；72℃10min；4 DEG C of guarantors
Hide.Pcr amplification product is detected using 1% agarose gel electrophoresis, using Ago-Gel DNA QIAquick Gel Extraction Kits
(centrifugation column type) recovery purifying PCR product.
(3) sequencing：PCR product after purification, is served Hai Meiji companies and is sequenced, its sequence such as SEQ ID NO:1
It is shown.The sequence is compared with reference culture known to ncbi database to be shown, the bacterial strain and bacillus licheniformis (Bacillus
Licheniformis) the homology highest of ATCC 14580, up to 99%.Neighbor- is used using MEGA7.0 softwares
Joining methods draw 16S rDNA phylogenetic trees (seeing Fig. 1), its evolutionary degree are determined, with reference to its morphological feature and physiology
Biochemical character, the bacterial strain most likely bacillus licheniformis category, and be named as Bacillus licheniformis ZL-1.
The pure feather production of 3 bacillus licheniformis of embodiment (Bacillus licheniformis) ZL-1 fermentative degradations is compound
Freamine Ⅲ and its index of correlation measure
(1) pretreatment of feather raw material：By feather with crushing crusher machine, into fluffy cotton-shaped.Then by cotton-shaped feather with
Water is according to 1:40 mass ratio mixing, adjusts pH to 10, and 180s is heated under conditions of microwave energy is 700W.
(2) prepared by bacillus licheniformis (Bacillus licheniformis) ZL-1 seed liquors：Embodiment 1 is filtered out
Bacillus licheniformis (Bacillus licheniformis) ZL-1 be inoculated on NA solid medium tablets, 45 DEG C culture
24h, makes its activation；The seed of above-mentioned activation is transferred in NB liquid seed culture mediums, culture 24h to exponential phase.
(3) by pretreated feather and inorganic salt solution according to 1:100 mass ratio mixing, adds 5wt% lichens buds
Spore bacillus (Bacillus licheniformis) ZL-1 seed liquors, at 45 DEG C, cultivate 3 days under 200rpm, obtain compound ammonia
Base acid solution, observes the degraded situation (see Fig. 2) of feather, measures feather degradation rate, keratinase activity and compound amino acid are molten
Amino acid classes and content in liquid.The inorganic salt solution component is as follows：NaCl 0.5g/L, NaH2PO40.1035g/L,
Na2HPO4 1.3277g/L。
(4) feather degradation rate measures：Compositing acid solution is filtered with filter paper, filter residue is collected and is dried to perseverance in 105 DEG C
Weight, calculates degradation rate.
Feather degradation rate (%)=(control sample dry weight-laboratory sample dry weight)/control sample dry weight × 100.
(5) measure of keratinase activity is according to document " Gradisar H, Kern S, Friedrich
J.Keratinase of Doratomyces microsporus.Appl Microbiol Biotechnol[J].2000,53
(2):Based on the described methods of 196-200. ", and changed.Specific method is：By compositing acid solution with 2 layers of yarn
Cloth filters, and goes filtrate, 12,000 × g, 4 DEG C of centrifugation 10min, take supernatant 1.0mL to add 2.0mL 0.05mol/L Tris/
HCl buffer solutions (pH7.5) and 10mg feather meal substrates, 37 DEG C of thermostat water bath reactions, 100rpm isothermal holdings 1h.Reaction terminates
10% trichloroacetic acids of Shi Tianjia 2.0mL terminate reaction, 10,000 × g centrifugations 15min.Supernatant measures its extinction under 595nm
Value, control is used as using the reaction tube that TCA is added before insulation.Enzyme-activity unit defines：Under experiment condition, Unit/mL decomposes keratin
The enzyme amount of generation is ΔOD595Enzyme amount needed for value 0.001 unit of rise is a unit of activity (U).
(6) water-soluble free amine group acidity test in compositing acid solution：5mL compositing acid solutions are taken to 10mL colorimetrics
Guan Zhong, adds ddH2O is settled to 10mL, stands overnight.2mL supernatants are taken into 10mL centrifuge tubes, add 2mL sulfosalicylic acids,
Mix, standing 1h makes albumen precipitation.4 DEG C are added after 1mL EDTA and 1mL hydrochloric acid, 6,000 × g centrifugations 15min is (or with 0.22 μm
Filtering with microporous membrane).Supernatant 1mL is taken to be evaporated into culture dish.1mL 20mmol/L HCl solutions are added, it is micro- with 0.22 μm
Hole membrane filtration.500 μ L are at least taken to sample injection bottle.Measured with the full-automatic amino-acid analyzers of L-8900.
Test result finds that culture medium mesoptile is degraded (see Fig. 2), and the degradation rate of feather is reached after fermenting 3 days
90.15%, while keratinase activity reaches 52.14U/mL in zymotic fluid, shows the bacillus licheniformis of the present invention
(Bacillus licheniformis) ZL-1 has the ability of fast degradation feather, and after measured, ferment the 3 days amino acid obtained
Compound amino acid A wide selection of colours and designs in solution, amino acid content reach 3.4278g/kg, and amino acid spectrogram is shown in Fig. 3.Therefore can be by ground
Clothing bacillus (Bacillus licheniformis) ZL-1 is used for degraded and the recycling of feather, and keratinase
Production.
4 bacillus licheniformis of the embodiment fermented abandoned feather of (Bacillus licheniformis) ZL-1 During High-Temperature Compostings and
Mushroom slag, produces compound amino acid fertilizer
(1) prepared by bacillus licheniformis (Bacillus licheniformis) ZL-1 microbial inoculums：Embodiment 1 is filtered out
Bacillus licheniformis (Bacillus licheniformis) ZL-1 is inoculated on NA solid medium tablets, 45 DEG C of culture 24h,
Make its activation, inoculation will have been activated and fermented into fermentation culture, at 45 DEG C, mixing speed 200rpm, ventilation
Measure to cultivate 48h under conditions of 1vvm, obtain bacillus licheniformis ZL-1 microbial inoculums, the total viable bacteria for the microbial inoculum that liquid fermentation is obtained
Number can reach 1 × 1010cfu.The fermented and cultured formula of liquid is：Glucose 1.5g/L, corn flour 1.5g/L, dregs of beans 2.0g/L,
Sodium chloride 0.5g/L.
(2) prepared by aspergillus niger microbial inoculum：Aspergillus niger strain (is purchased from Guangdong Province's Culture Collection, deposit number
For GDMCC No.3.576) it is inoculated into bran mass and ferments, the tray static gas wave refrigerator at 37 DEG C, cultivates 3-5 days, obtains
To aspergillus niger microbial inoculum.The formula of the bran mass is：Wheat bran：Potassium dihydrogen phosphate：Mass ratio=1000 of water:5:1000.
According to C/N ratios it is 25 by discarded poultry feather and edible fungi residues:1 ratio uniform mixing, into raw mixture
Add bacillus licheniformis (Bacillus licheniformis) the ZL-1 microbial inoculums and 0.2% of raw mixture quality 2%
Aspergillus niger microbial inoculum, then the water content of adjusting raw mixture to 70%, is put into solid-state fermentation tank, adjusts the temperature to 50 DEG C,
Ventilation 30min daily, and suitable quantity of water is spread, continuous compost is after 7 days, closing temperature controller, allows its Natural compost to ferment 14 days, complete
Into the decomposed of material, compound amino acid fertilizer is obtained.
The announcement and guidance of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, but these terms are merely for convenience of description, do not limit the present invention in any way.
Sequence table
<110>Guangzhou kingly way bio tech ltd
<120>Bacillus licheniformis ZL-1 and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1446
<212> DNA
<213>Bacillus licheniformis (Bacillus licheniformisZL-1 16S rDNABacillus
licheniformis)
<400> 1
acgtgcggca tgctatacat gcagtcgagc ggaccgacgg gagcttgctc ccttaggtca 60
gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120
aaccggggct aataccggat gcttgattga accgcatggt tcaatcataa aaggtggctt 180
ttagctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360
ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt gttagggaag 420
aacaagtacc gttcgaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600
gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagcaaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ctctgacaac cctagagata gggcttcccc ttcgggggca gagtgacagg 1020
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080
caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140
aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tgggcagaac aaagggcagc gaagccgcga ggctaagcca atcccacaaa 1260
tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttggagcca gccgccgaag 1440
tgggac 1446
<210> 2
<211> 21
<212> DNA
<213> Primer A(Artificial Sequence)
<400> 2
agagtttgat cctggctcta g 21
<210> 3
<211> 19
<212> DNA
<213> Primer B(Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (9)
1.Bacillus licheniformis ZL-1, are preserved in Guangdong Province's Culture Collection, and deposit number is
GDMCC No.60284, preservation address are 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is 2017 11
The moon 24.
2. the Bacillus licheniformis ZL-1 described in claim 1, it is characterised in that the nucleosides of its 16S rDNA
Acid sequence such as SEQ ID NO:Shown in 1.
3. the Bacillus licheniformis ZL-1 described in claim 1, it is characterised in that the culture temperature of its seed liquor
Spend for 45~55 DEG C.
4. Bacillus licheniformis ZL-1 described in claim 1 are molten in fermentative degradation feather, production compound amino acid
Application in liquid.
5. a kind of degradation of feather, the method for producing compositing acid solution, it is characterised in that include the following steps：
By feather and inorganic salt solution according to 1:100 mass ratio mixing, adds 5wt% bacillus licheniformis (Bacillus
Licheniformis) ZL-1 seed liquors, are cultivated 3 days under 45~55 DEG C, 200rpm, complete feather degraded, and obtain compound ammonia
Base acid solution；
The inorganic salt solution component includes：NaCl 0.5g/L、NaH2PO4 0.1035g/L、Na2HPO41.3277g/L；
The preparation method of the Bacillus licheniformis ZL-1 seed liquors is：By bacillus licheniformis (Bacillus
Licheniformis) ZL-1 is inoculated on NA solid plate culture mediums, and 45 DEG C of culture 24h make its activation, and NB is transferred to after activation
Cultivated in liquid seed culture medium to exponential phase；
The Bacillus licheniformis ZL-1, are preserved in Guangdong Province's Culture Collection, and deposit number is
GDMCC No.60284, preservation address are 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is 2017 11
The moon 24.
6. degradation of feather, the method for producing compositing acid solution according to claim 5, it is characterised in that the feather
Also include by pretreatment, preprocess method：Feather is broken into it is fluffy cotton-shaped, then with water according to 1:40 mass ratio mixes
Close, adjust pH to 10,180s is heated under conditions of microwave energy is 700W.
7. Bacillus licheniformis ZL-1 described in claim 1 are in the fermented abandoned feather of During High-Temperature Composting and edible mushroom
Application in slag, production compound amino acid fertilizer.
8. a kind of fermented abandoned feather of During High-Temperature Composting and edible fungi residues, the method for producing compound amino acid fertilizer, it is characterised in that
Include the following steps：
According to carbon-nitrogen ratio it is 20 by feather and edible fungi residues:1~25:1 ratio is mixed to get mixture, adds mixture quality
1~2% Bacillus licheniformis ZL-1 microbial inoculums and the aspergillus niger microbial inoculum of mixture quality 0.1~0.2%, obtain
To total mixture, the water content of total mixture is adjusted to being put into solid-state fermentation tank after 70~75%, it is 50~55 to adjust temperature
DEG C, ventilate 30~60min daily, and continuous compost cools the temperature to natural temperature after 7 days, its Natural compost is fermented 14 days, obtains
To compound amino acid fertilizer；
The preparation method of the Bacillus licheniformis ZL-1 microbial inoculums is：By Bacillus licheniformis
ZL-1, which is inoculated into fermentation culture, to ferment, and at 45~50 DEG C, mixing speed is 180~200rpm, throughput for 0.5~
36~48h is cultivated under conditions of 2vvm, obtains Bacillus licheniformis ZL-1 microbial inoculums；The fermentation culture is matched somebody with somebody
Fang Wei：Glucose 1.5g/L, corn flour 1.5g/L, dregs of beans 2.0g/L, sodium chloride 0.5g/L；
The preparation method of the aspergillus niger microbial inoculum is：Aspergillus niger strain is inoculated into bran mass and is fermented, at 37 DEG C
Lower tray static gas wave refrigerator 3~5 days, obtains aspergillus niger microbial inoculum；The formula of the bran mass is：Wheat bran：Potassium dihydrogen phosphate：Water
Mass ratio=1000:5:1000.
9. applications of the Bacillus licheniformis ZL-1 described in claim 1 in keratinase is produced.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109370944A (en) * | 2018-11-20 | 2019-02-22 | 江南大学 | One plant of bacillus for producing protease and its application in compost |
| CN110183252A (en) * | 2019-06-05 | 2019-08-30 | 江苏丘陵地区南京农业科学研究所 | The method and application of compound amino acid liquid fertilizer are prepared using biodegradable feather |
| CN110747128A (en) * | 2019-11-18 | 2020-02-04 | 山东隆科特酶制剂有限公司 | Bacillus licheniformis strain capable of producing keratinase in high yield and application thereof |
| CN110862950A (en) * | 2019-12-24 | 2020-03-06 | 烟台富康生物科技有限公司 | Bacillus licheniformis and application thereof |
| CN110923175A (en) * | 2019-12-26 | 2020-03-27 | 江苏大道生物环境科技有限公司 | Bacillus and application thereof in reduction and resource production of organic fertilizer by kitchen waste |
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| CN114395516A (en) * | 2022-03-21 | 2022-04-26 | 河北科技大学 | Bacillus licheniformis TD-4 and method for producing protease and application thereof |
| CN114958668A (en) * | 2022-05-24 | 2022-08-30 | 武汉轻工大学 | Liquid culture medium and method for preparing nano-selenium by microbial fermentation |
| CN116606755A (en) * | 2023-03-15 | 2023-08-18 | 中国国际海运集装箱(集团)股份有限公司 | Bacillus licheniformis and application thereof in degradation of polyolefin |
| CN116656565A (en) * | 2023-06-16 | 2023-08-29 | 天典(广东)生物科技有限公司 | Bacillus licheniformis and application thereof |
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