CN114540252B - Microbacterium P6 for converting livestock and poultry breeding waste and application - Google Patents
Microbacterium P6 for converting livestock and poultry breeding waste and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
-
- 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
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention belongs to the field of recycling harmless utilization of agricultural and forestry wastes, and particularly relates to a microbacteriumExiguobacterium sp.P6, and also relates to the application of the bacillus pumilus P6 in degrading livestock and poultry breeding waste and recycling the waste. The microbacterium P6 is preserved in China general microbiological culture Collection center (CGMCC) on 03.01.2022, with the preservation number of CGMCC No.24453, and the preservation address is No. 3 of Beijing Korean Chen Xilu No. 1. The invention mainly protects the micro bacillus P6 strain, the application thereof in degrading livestock and poultry breeding waste, the method for obtaining the keratinase by fermenting and culturing the strain, and the application of the obtained keratinase in degrading the livestock and poultry breeding waste. The bacillus pumilus P6 provided by the invention can efficiently degrade waste such as feathers, has a feather degradation rate as high as 98%, and has a wide application prospect in the aspect of recycling livestock and poultry breeding waste and waste.
Description
Technical Field
The invention belongs to the field of recycling harmless utilization of agricultural and forestry wastes, and particularly relates to a microbacteriumExiguobacterium sp. P6, and also relates to the application of the bacillus pumilus P6 in degrading livestock and poultry breeding waste and recycling the waste.
Background
The protein content in the poultry feather is over 80 percent, the essential amino acid composition is rich, and the poultry feather is rich in macroelements, microelements, vitamins and growth factors, and is considered as high-quality protein feed with unique value. The main component of poultry feather, wool, animal fur and hoof and horn is keratin. Keratin is a hard protein with a compact and complex structure, is difficult to hydrolyze by common proteases such as trypsin, pepsin and papain, and can only be hydrolyzed by a protease called "keratinase". Thus, keratinases play an important role in the degradation and recycling of keratin.
Research shows that the keratinase is used as a feed additive, so that unconventional protein feed derived from waste such as feather and the like can be effectively degraded, and hydrolysate can improve the nutritional value of the feed and the digestion and absorption efficiency of animals; in addition, the keratinase can degrade prion protein, so that the keratinase can be used as a high-grade feed additive, eliminate prion protein pollution in feed and produce safe feed. The development of the high-efficiency keratinase preparation aiming at the treatment of the livestock and poultry breeding waste can not only realize the harmless treatment of the waste and reduce the environmental problems, but also realize the high-value utilization of the waste and relieve the dilemma of the shortage of protein feed resources.
Although a large number of microorganisms capable of degrading feather keratin are screened at present, the enzyme production efficiency of the strains is relatively low, and the yield of the original enzyme is far from meeting the requirements of the fields of industry, agriculture and medicine, so that high-yield strains are required to be further screened, the fermentation conditions are optimized, and the yield and the activity of keratinase are improved; most of enzyme preparation products sold in the market are expensive, the enzyme activity is low, the action site is single, and the requirement of efficient conversion of wastes such as feathers, fur, hoofs and the like cannot be met. In order to improve the conversion efficiency of the livestock and poultry breeding waste, novel keratinase resources with higher activity need to be screened and identified urgently.
Micro-bacterium (A), (B)Exiguobacterium spp.) Is a gram-positive, spore-free and facultative anaerobic bacterium, has wide distributed habitat and has various unique properties. Comparative genomic analysis shows that the microbacterium can utilize a series of complex polysaccharides and proteins to survive in different environments. The applications of the micro-bacillus and the extracellular protease and other enzymes produced by the micro-bacillus in the technical field of microorganisms and the processing field of aquatic products are disclosed as follows: chitinase is produced by psychrophile Antarctic bacterium DW2 (preservation number CCTCC number M2019536), and can be used for preparing chitosan oligosaccharide (CN 113862192A); the deep sea micro bacillus mutant MS10017 (the preservation number is CCTCC number M2019795) and lactobacillus acidophilus are used together, so that the chitin (CN 111304110A) in the shrimp shells or crab shells can be extracted by high-efficiency decalcification and deproteinization; similarly, a strain of the tiny acetobacter can also be used for treating calcium carbonate and protein of waste shrimp and crab shells to extract chitin (CN 110128568A); the utilization rate of feed protein can be improved in mariculture by using the microbacterium PT3 separated from the shrimp intestinal tract (CN 111334454A); deep sea micro bacillus FELA1 (preservation number GDMCC number 61793) separated from shrimp paste can produce lipase, protease and amylase, and can remarkably enhance the content of flavor substances such as ketones, alcohols, esters and the like in aquatic seasoning (CN 114181874A); the microbacterium WHX-1 (with the preservation number of CGMCC No. 16733) disclosed in CN11214367A can quickly and efficiently degrade proteins in domestic sewage, and has good application prospect and research value in domestic sewage treatment (CN 112143671A). However, it is possible to use a single-layer,feather keratin is a special protein, has compact and complex structure and is rich in disulfide bonds, and is difficult to hydrolyze by common protease. The above-mentioned publications disclose whether the microorganism has a certain degradation effect on common protein substrates, and whether the above-mentioned strain has the same effect and can achieve a desired degradation effect on keratin having a dense and complex structure and rich disulfide bonds, and the above-mentioned patent documents do not disclose.
In the field of agricultural microorganism application, the Microbacterium aurantiacum 8220605 (with the preservation number of CGMCC number 14676, CN 108102992A) and Microbacterium acetobacter AMCC 101217 (with the preservation number of CGMCC number 16305, CN 110951656A) have obvious effects of preventing and treating tomato root-knot nematodes, and besides, the disclosure of related documents for realizing resource transformation and utilization of low-value protein wastes generated in the field of livestock and poultry breeding and processing by utilizing the Microbacterium aurantiacum is not disclosed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a microbacterium P6 strain and application thereof in degrading livestock and poultry breeding wastes. The inventor separates a strain of micro-bacillus P6 from the biogas residue of long-term stacking feather waste near slaughterhouses, and the micro-bacillus P6 can grow in a culture medium with the feather as the only energy source and efficiently degrade the feather. The strain is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms (CCM) at 03-01.2022, the preservation number of the strain is CGMCC number 24453, the preservation address is No. 3 of the Xilu No.1 Hospital of Chaozhou, Chaoyang, Beijing, and the classified name is microbacteriumExiguobacterium sp.。
The sequence of the microbacterium P6 is shown in a sequence table SEQ ID NO. 1.
After the microbacterium P6 is cultured on an LB (LB) plate at 25 ℃ for 48 hours, the colonies on the plate are observed to be light orange with the diameter of about 3 mm (about 2.8-3.2 mm), the colonies are flat, gram staining is positive, and the bacteria are observed to be rod-shaped by a scanning electron microscope and have the size of 0.71 mu m multiplied by 1.36-2.21 mu m.
When the microbacterium P6 is cultured in a fermentation way, the formula of a fermentation medium is measured by taking the g/L as a unit and comprises the following components: feather 15, NaCl 0.5, K 2 HPO 4 1 .0，KH 2 PO 4 0 .4，MgCl 2 ·7H 2 O 0.1，CaCl 2 0.06, adjusting the pH value of the fermentation medium to 7.5 by adopting NaOH; inoculating according to the inoculation amount of 1.5-2.5% in volume ratio, wherein the culture time is 46-50 h (preferably 48 h), and the fermentation culture temperature is 20-30 ℃.
The keratinase obtained by fermenting the bacillus pumilus P6, the method for obtaining the keratinase and the application of the keratinase in degrading livestock and poultry wastes are also the contents to be protected in the important aspect of the invention.
The invention also provides the application of the composition containing at least one of the following components in degrading livestock and poultry wastes:
bacillus pumilus P6;
or, a microbial inoculum containing microbacterium P6;
or, a keratinase enzyme preparation produced by fermentation of microbacterium sp 6;
or viable/dead bacterial suspension produced during fermentation of the microbacterium sp 6;
or, an extract/metabolite of microbacterium sp 6.
The preservation number of the Microbacterium P6 strain is CGMCC number 24453.
The method for obtaining the keratinase by the fermentation culture of the microbacterium P6 comprises the following steps:
(1) preparing fermentation medium, wherein the fermentation medium comprises 15 parts of feather, 0.5 part of NaCl and K, and the unit is g/L 2 HPO 4 1.0，KH 2 PO 4 0.4，MgCl 2 ·7H 2 O 0.1，CaCl 2 0.06, adjusting the pH value of the fermentation medium to 7.5 by adopting NaOH; LB liquid medium: peptone 10, NaCl 10, yeast powder 10;
(2) taking soil as a seed inoculant, and culturing in a feather liquid culture medium;
(3) coating the bacterial liquid in the step (2) on an LB flat plate, inverting the flat plate in an incubator for culture, selecting bacterial colonies with good growth vigor on the flat plate, purifying the bacterial strains on the LB flat plate, eliminating the bacterial strains with weak growth capacity in the passage process, inoculating the purified bacterial strains to a feather liquid culture medium, and culturing;
(4) centrifuging, and collecting the supernatant of the fermentation liquor to obtain keratinase;
preferably, the first and second liquid crystal materials are,
(1) preparing a fermentation medium: feather 15, NaCl 0.5, K in g/L 2 HPO 4 1.0，KH 2 PO 4 0.4，MgCl 2 ·7H 2 O 0.1，CaCl 2 0.06, adjusting the pH value of the fermentation medium to 7.5 by adopting NaOH; LB liquid medium: peptone 10, NaCl 10, yeast powder 10;
(2) taking 1 g of soil as a seed inoculant, and culturing in 100 mL of feather liquid culture medium for 7 d; the culture conditions are as follows: 25 ℃, 180 rpm;
(3) coating 100 mu L of bacterial liquid on an LB (lysogeny broth) flat plate, inversely placing the flat plate in an incubator at 25 ℃ for culturing for 24 hours, selecting bacterial colonies with good growth vigor on the flat plate, continuously streaking for 3 times to purify the bacterial strains, eliminating the bacterial strains with weak growth capacity in the passage process, inoculating the purified bacterial strains to a feather liquid culture medium according to the inoculation amount of 1.5-2.5% in volume ratio, and culturing for 46-50 hours at 20-30 ℃;
(4) centrifuging at 10000-12000 rpm for 12-18 min, and collecting supernatant of fermentation liquor to obtain keratinase.
Preferably, centrifugation is carried out for 15 min at 11000 rpm.
More preferably, the supernatant of the fermentation broth is collected as follows: filtering out residues of the fermented culture medium with gauze, centrifuging at 11000 rpm for 15 min, and collecting fermentation liquor to obtain keratinase;
through detection, the keratinase obtained by the method can efficiently degrade livestock and poultry wastes (particularly feather keratin of livestock and poultry). Of course, the keratinase is purified, and the purified enzyme solution or enzyme preparation can also be applied to the degradation of livestock and poultry wastes, including but not limited to feather keratin, and keratin in other parts of livestock and poultry, and the above improvements also fall into the protection scope of the invention.
The application of the keratinase obtained by the method and/or the purified enzyme liquid and enzyme preparation in degrading livestock and poultry wastes, particularly the application of the keratinase obtained by the method in degrading livestock and poultry feathers is the important protection content of the invention.
Specifically, the keratinase is directly used for degrading the feathers of the livestock and poultry; or purifying the keratinase to degrade the feather of livestock and poultry; wherein, when the keratinase degrades feather keratin for hydrolysis, the optimal action temperature of enzyme liquid when the feather keratin is degraded is kept to be 45-55 ℃ (certainly, the enzyme liquid can play the activity in the range of 35-55 ℃), and the pH value is 7.5-8.5; preferably, the temperature at which the enzyme solution acts to degrade feather keratin is maintained at 50 ℃ and pH 8.0 (i.e., the optimum temperature and pH for enzyme action).
The invention has the beneficial effects that:
(1) a brand-new keratin degrading bacterium, namely microbacterium P6 capable of efficiently degrading feather keratin waste is obtained, and no report that the bacterium from the genus degrades feather keratin substrate is found at present;
(2) the micro-bacillus P6 has excellent degradation effect on keratin, and the degradation rate of the micro-bacillus P6 on feathers is about 98%; has huge application potential and prospect in the degradation and high-efficiency transformation of livestock and poultry wastes, especially livestock and poultry feathers;
(3) the keratinase produced by the fermentation of the microbacterium P6 has excellent degradation capability on keratin in feathers, the degradation rate is about 85 percent, and the application of the enzyme produced by the microbacterium P6 is also provided.
Drawings
FIG. 1 is a graph of the degradation of feathers by Microbacterium P6 in a medium using feathers as a sole carbon and nitrogen source;
FIG. 2 shows the morphology of Microbacterium P6 in visual observation (A), optical observation (B) and electron microscope observation (C);
FIG. 3 is a phylogenetic tree analysis of the evolution position of Microbacterium P6;
FIG. 4 is a graph of the degradation of feather keratin by the keratinase of Microbacterium P6.
Detailed Description
The present invention will now be further described with reference to specific embodiments in order to enable those skilled in the art to better understand the present invention.
Example 1
The inventors isolated and purified the microorganism bacillus P6, specifically as follows:
the method for separating and purifying the bacillus pumilus P6 comprises the following steps:
(1) feather liquid medium (g/L): feather 15, NaCl 0.5, K 2 HPO 4 1.0， KH 2 PO 4 0.4，MgCl 2 ·7H 2 O 0.1，CaCl 2 0.06, adjusting the pH value to 7.5 by NaOH;
LB liquid Medium (g/L): peptone 10, NaCl 10, yeast powder 10;
(2) taking 1 g of soil as a seed inoculant, and culturing in 100 mL of feather liquid culture medium for 7 d; the culture conditions are as follows: 25 ℃ and 180 rpm;
(3) coating 100 mu L of bacterial liquid on an LB flat plate, inverting the flat plate in an incubator at 25 ℃ for culturing for 24 h, selecting bacterial colonies with good growth vigor on the flat plate, continuously marking for 3 times on the LB flat plate, and purifying the strains, so that strains with weak growth capacity in the passage process are eliminated;
(4) inoculating the purified strain into a feather liquid culture medium, culturing at 25 ℃ and 180 rpm for 48 h, visually observing the feather degradation degree, selecting the strain with strong feather degradation capability after the culture is finished, measuring the keratinase enzyme activity of fermentation liquor of the strain, and calculating the feather degradation rate.
The enzyme activity determination method of the keratinase is slightly modified according to the Fang method, and the increase of the absorbance value of 660 nm per minute is defined as 1 enzyme activity unit U (Fang et al, International Biodetermination & Biodetermination 2013, 82: 166-); feather degradation rates were determined according to the Jaouadi method (Jaouadi et al, PloS ONE, 2013, 8(10): e 76722).
The bacterial strain P6 capable of efficiently degrading keratin is obtained by screening through the method, the total enzyme activity of 1L fermentation liquor is 20000U, and the feather degradation rate of the bacterial strain is 98% (shown in figure 1).
Example 2
Species identification for the strain P6 in example 1
(1) Morphological characteristics of the strains
After culturing at 25 ℃ for 48 hours on LB plates, colonies on the plates were observed to be light orange with a diameter of about 3 mm, the colonies were flat (FIG. 2A), and gram-positive (FIG. 2B). The bacteria are observed to be rod-shaped by a scanning electron microscope, and the size of the bacteria is 0.71 mu m multiplied by 1.36-2.21 mu m (figure 2C).
(2) Phylogenetic analysis
A single colony of the P6 strain was inoculated into liquid LB medium and cultured overnight at 37 ℃.1 mL of the cultured bacterial liquid is taken, and the total DNA of the bacteria is extracted according to the instruction of the 'Baitach bacteria genome extraction kit'.
16S Universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3') were designed. Using bacterial genome as template, DNA fragments were obtained by PCR according to the PCR system and procedure described in the specification of I-5. sup.2 × High-Fidelity Master Mix (TP 001, Okagaku, Beijing), BioChemica 1995, 2, 34-35; Chester, N. and Marshak, D.R., Analytical biochemistry 1993, 209, 284-290). The DNA fragments were recovered using a DNA gel recovery kit (Omega) and the procedures were carried out according to the kit instructions. The recovered PCR products were sequenced in Botanhei Biotechnology (Shanghai) Ltd.
Sequencing determines the full length of 1436 bp of the 16S rRNA sequence. BLAST alignment analysis of sequences in NCBI database:
all of the Bacillus pumilus with high sequence identityExiguobacterium sp.With Acetobacter asiaticumExiguobacterium acetylicumstrain AMCC 101217 sequence identity 99.86%. The 16S rRNAs of the strains with higher sequence consistency are subjected to multiple alignment by using Clustalx X1.83 software, and a sequence evolutionary tree is drawn by using a Neighbor-Joining Method (FIG. 3) of MEGA 6.0 software.
The separated and purified microbacterium P6 is preserved in China general microbiological culture Collection center (CGMCC) on the day 01 of 03.2022, the preservation number is CGMCC number 24453, and the preservation address is No. 3 of Xilu No.1 Beichen of the rising area of Beijing.
Example 3
The keratinase is obtained by fermentation culture of Microbacterium P6 as follows:
in example 1, in the process of separation, purification, fermentation and culture of microbacterium P6, (4) after the culture is finished, the dregs of the culture medium are filtered out by gauze, and the culture medium is centrifuged for 15 min at 11000 rpm, and the supernatant of the fermentation liquid is collected, namely keratinase.
With respect to the effect of the keratinase on the degradation of feather keratin obtained by the above method, the present inventors carried out the following experiments:
the keratinase obtained by the method is used for degrading feather substrate (1%) under the conditions of 37 ℃ and 150 rpm, the enzyme activity is 10U, and the result shows that the keratinase produced by the strain can efficiently degrade feathers, the degradation rate of the feathers under the experimental enzymolysis condition is 85% (figure 4), and the method indicates that the novel keratinase produced by the microbacterium P6 has huge application potential in the aspect of efficient conversion of livestock and poultry wastes.
In examples 3-1 to 3-4, the conditions of the keratinase action such as temperature and rotational speed were different;
the specific degradation effect is as follows:
as can be seen from the data in the above table, the keratinase produced by the Microbacterium P6 of the present invention in the fermentation process has excellent effect of degrading keratin, and the degradation rate reaches more than 85%.
Sequence listing
<110> Shandong province academy of agricultural sciences
<120> microbacterium P6 for converting livestock and poultry breeding wastes and application thereof
<141> 2022-03-31
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1436
<212> DNA
<213> Microbacterium (Exiguobacterium)
<400> 1
ttcgacggct ggctccttgc ggttacctca ccggcttcgg gtgttgcaaa ctctcgtggt 60
gtgacgggcg gtgtgtacaa gacccgggaa cgtattcacc gcagtatgct gacctgcgat 120
tactagcgat tccgacttca tgcaggcgag ttgcagcctg caatccgaac tgggaacggc 180
tttatgggat tggctccacc tcgcggtctc gctgcccttt gtaccgtcca ttgtagcacg 240
tgtgtagccc aactcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300
tgtcaccggc agtctcccta gagtgcccaa ctgaatgctg gcaactaagg ataggggttg 360
cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420
cctgtcacca ttgtccccga agggaaaact tgatctctca agcggtcaat gggatgtcaa 480
gagttggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gtccccgtca attcctttga gtttcagcct tgcggccgta ctccccaggc ggagtgctta 600
atgcgttagc ttcagcactg aggggcggaa accccccaac acctagcact catcgtttac 660
ggcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc 720
agttacagac caaagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780
ccgctacacg tggaattcca ctcttctctt ctgtactcaa gccttccagt ttccaatggc 840
cctccccggt tgagccgggg gctttcacat cagacttaaa aggccgcctg cgcgcgcttt 900
acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960
gttagccgtg gctttctcgt aaggtaccgt caaggtacga gcattccctc tcgtacgtgt 1020
tcttccctta caacagagtt ttacgatccg aaaaccttca tcactcacgc ggcgttgctc 1080
catcagactt tcgtccattg tggaagattc cctactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctatgcat cgtcgccttg 1200
gtgggccgtt accccaccaa ctagctaatg caccgcaagg ccatctcaag gtgacgccga 1260
agcgcctttc atcagcggac catgcggtcc gttgaactat ccggtattag ctccgatttc 1320
tcggagttat cccaatcctt gaggcaggtt ccttacgtgt tactcacccg tccgccgctc 1380
attccgctgc cttccctccg aagagttccg tcagttcctg cgctcgactt gcatgt 1436
Claims (4)
1. Micro-bacterium (A), (B)Exiguobacterium sp.) P6, wherein said bacterium is Microbacterium (Microbacterium sp.) (Exiguobacterium sp.) P6 was collected in China general microbiological culture Collection center (CGMCC) on the day 01 of 03.2022, the collection number of the strain was CGMCC 24453, and the collection address was No. 3 of Xilu No.1, North Chen, the area facing the Yangyang, Beijing.
2. Micro-bacterium (A), (B)Exiguobacterium sp.) The application of P6 in degrading livestock and poultry waste is characterized in that the micro bacillus (Microbacterium), (Microbacterium sp.)) is)Exiguobacterium sp.) The preservation number of the P6 is CGMCC number 24453, and the livestock and poultry waste is keratin-containing livestock and poultry waste.
3. The use according to claim 2, wherein said use is the administration of a micro-bacterium (Microbacterium sp.) (Exiguobacterium sp.) Keratinase produced by P6 fermentation is applied to degrading livestock and poultry waste.
4. The use according to claim 2, wherein said use is the administration of a micro-bacterium (Microbacterium sp.) (Exiguobacterium sp.) The keratinase produced by P6 fermentation is applied to degrading livestock and poultry waste after being purified.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372678A (en) * | 2008-10-22 | 2009-02-25 | 南京农业大学 | Keratin degrading bacteria NJY1 |
| CN102154144A (en) * | 2010-12-06 | 2011-08-17 | 天津科技大学 | Strain capable of degrading feather keratin efficiently and screening method thereof |
| CN104619838A (en) * | 2012-08-22 | 2015-05-13 | 诺维信公司 | Metalloprotease from exiguobacterium |
| CN105821023A (en) * | 2016-05-04 | 2016-08-03 | 山东省农业科学院生物技术研究中心 | Separating and purifying method and application of keratinase |
| CN107868762A (en) * | 2017-11-17 | 2018-04-03 | 山东省农业科学院生物技术研究中心 | Produce bacillus cereus and its application of keratinase |
| WO2021212674A1 (en) * | 2020-04-20 | 2021-10-28 | Hebei Monband Water Soluble Fertilizer Co., Ltd | Exiguobacterium undae mb338 and use thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015050723A1 (en) * | 2013-10-03 | 2015-04-09 | Danisco Us Inc. | Alpha-amylases from exiguobacterium, and methods of use, thereof |
| WO2015050724A1 (en) * | 2013-10-03 | 2015-04-09 | Danisco Us Inc. | Alpha-amylases from a subset of exiguobacterium, and methods of use, thereof |
| CN108795814B (en) * | 2018-06-25 | 2021-08-27 | 信阳师范学院 | Bacterial strain capable of degrading waste feather, screening method and application thereof |
-
2022
- 2022-03-31 CN CN202210343514.1A patent/CN114540252B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372678A (en) * | 2008-10-22 | 2009-02-25 | 南京农业大学 | Keratin degrading bacteria NJY1 |
| CN102154144A (en) * | 2010-12-06 | 2011-08-17 | 天津科技大学 | Strain capable of degrading feather keratin efficiently and screening method thereof |
| CN104619838A (en) * | 2012-08-22 | 2015-05-13 | 诺维信公司 | Metalloprotease from exiguobacterium |
| CN105821023A (en) * | 2016-05-04 | 2016-08-03 | 山东省农业科学院生物技术研究中心 | Separating and purifying method and application of keratinase |
| CN107868762A (en) * | 2017-11-17 | 2018-04-03 | 山东省农业科学院生物技术研究中心 | Produce bacillus cereus and its application of keratinase |
| WO2021212674A1 (en) * | 2020-04-20 | 2021-10-28 | Hebei Monband Water Soluble Fertilizer Co., Ltd | Exiguobacterium undae mb338 and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| ISOLATION, IDENTIFICATION AND DEHAIRING ACTIVITY OF INDONESIAN NATIVE KERATINOLYTIC BACTERIA EXIGUOBACTERIUM SP.DG1;Jajang Gumilar et al.;《Pak.J.Biotechnol.》;20151231;第12卷(第1期);摘要部分，第42页，第46页右栏第2段，第47页讨论部分 * |
| 一株家禽羽毛角蛋白降解菌的分离与鉴定;周童娜等;《家畜生态学报》;20151130;第36卷(第11期);第37-41页 * |
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