A kind of degradation bacteria and its separation and screening method and application

Abstract

The present application relates to a degrading bacterium and its separation and screening method and application, belonging to the field of microbial technology. A degrading bacterium, the degrading bacterium is Bacillus velezensis, named ZKHW 1.021, and the deposit number is GDMCC NO.63679. The strain was isolated and screened from soil samples of discarded pig hair that had been piled up for a long time around a pig slaughterhouse. The Bacillus velezensis ZKHW 1.021 of the present application can quickly degrade pig hair under high temperature conditions. It has been determined that in a liquid culture medium of pure pig hair, after the bacteria was cultured for 72 hours, the keratinase activity reached 48.66U / mL, the fermentation broth had a complete range of amino acids, the amino acid content reached 2.7435g / kg, the polypeptide content was 3.1548g / kg, and the degradation rate of pig hair reached 88.34%.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a degradation bacterium and a separation and screening method and application thereof. Background Art

[0002] With the rapid development of my country's pig industry, the number of pigs slaughtered in my country reached 688.61 million in 2019. After the pigs were slaughtered, a considerable amount of by-products such as pig hair and hoofs were left behind. One pig can produce about 1 kg of wet pig hair. Most of the pig hair is discarded as scraps. This not only pollutes the environment but also causes a waste of resources. The mass fraction of crude protein in pig hair is as high as 80%, which contains a variety of essential amino acids and is rich in trace elements, growth factors and vitamins. It is an animal protein resource with potential value. However, the protein in pig hair is mainly composed of keratin, which contains more cysteine. A large number of disulfide bonds are formed between them, and hydrogen bonds and intermolecular forces make it difficult for ordinary proteases (such as trypsin, pepsin, papain, etc.) to hydrolyze. Traditional methods of processing waste pig hair, such as high-temperature and high-pressure cooking, acid-base hydrolysis, and the use of oxidants or reduction, result in serious damage to amino acids, high energy consumption, and severe pollution. Using microorganisms to degrade keratin can increase the nutritional value of the hydrolyzate, reduce energy consumption, costs, and environmental pollution, and is an environmentally friendly way to utilize waste resources.

[0003] At present, there are not many strains isolated and screened, and the efficiency is low, and most of them are mesophilic bacteria. Since the optimal reaction temperature of keratinase is generally 40-60℃, and the optimal growth temperature of mesophilic bacteria is generally 28-37℃, the two do not match. Therefore, screening pig hair degrading bacteria that are stable and efficient under high temperature conditions is also a research hotspot. After proper treatment of pig hair waste, the discarded pig hair keratin can be directly converted into complex amino acids, peptides or polypeptides, thus turning waste into treasure, which can not only protect the environment but also enhance resource utilization, which will have very significant economic benefits and far-reaching social benefits. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the embodiments of the present application includes providing a degrading bacterium to produce keratinase to efficiently degrade pig hair.

[0005] In the first aspect, the present invention provides a degradation bacterium, which is Bacillus velez Bacillus velezensis , named ZKHW 1.021, and the deposit number is GDMCC NO. 63679.

[0006] In some embodiments of the present application, the 16S rDNA nucleotide sequence of the degrading bacteria is shown as SEQ ID NO.1.

[0007] In the second aspect, an embodiment of the present application provides a method for isolating and screening the above-mentioned degrading bacteria, comprising: mixing the collected soil sample with water to obtain a mixed solution; adding the mixed solution to a selective culture medium for cultivation I to obtain a bacterial solution; spreading the bacterial solution on a solid selective culture medium plate for cultivation II, screening well-growing single colonies in the degradation culture medium, and cultivating III, while using a degradation culture medium without inoculation of single colonies as a control, observing the degradation of pig hair, and screening out degrading bacteria that meet the conditions.

[0008] In some embodiments of the present application, the composition of the selection medium includes: 0.5-10 g / L pig hair, 0.1-1.0 g / L sodium chloride, 0.05-0.2 g / L sodium dihydrogen phosphate and 0.5-2 g / L disodium hydrogen phosphate.

[0009] In some embodiments of the present application, the conditions of culture I include: shaking culture at 150-200 r / min, culture temperature of 24-60°C, and culture time of 80-110 h.

[0010] In some embodiments of the present application, the conditions of culture II include: culture temperature of 24-60°C and culture time of 65-75h.

[0011] In some embodiments of the present application, the conditions for culture III include: a culture temperature of 24-60° C. and a culture time of 48-96 h.

[0012] In a third aspect, an embodiment of the present application provides an application of the above-mentioned degrading bacteria in degrading pig hair.

[0013] In some embodiments of the present application, the method of using degrading bacteria to degrade pig hair includes: mixing the pretreated pig hair sample with an inorganic salt solution, and then adding a seed solution containing degrading bacteria for culturing to degrade the pig hair.

[0014] In some embodiments of the present application, the pretreatment includes: washing and removing impurities from the pig hair, steaming at 110-130° C. for 50-70 min, drying and crushing at 40-50° C. to obtain a pretreated pig hair sample.

[0015] In some embodiments of the present application, the mass ratio of pig hair to inorganic salt solution is (0.1-1):100.

[0016] In some embodiments of the present application, the components of the inorganic salt solution include: 0.1-1.0 g / L NaCl, 0.05-0.2 g / L NaH2PO4 and 0.5-2.0 g / L Na2HPO4.

[0017] In some embodiments of the present application, the preparation of seed solution includes: inoculating the degrading bacteria onto a NA solid culture medium plate, culturing at 24-60°C for 15-20 hours to activate them; transferring the activated degrading bacteria to NB liquid seed culture medium, and culturing to the logarithmic growth phase.

[0018] In some embodiments of the present application, the conditions for culturing the degrading bacteria include: culturing at 24-60° C. for 65-80 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the ZKHW 1.021 colony map in this application;

[0021] Figure 2 The Gram staining diagram of ZKHW 1.021 in this application;

[0022] Figure 3 This is the phylogenetic tree diagram of ZKHW 1.021 in this application;

[0023] Figure 4 This is the effect diagram of ZKHW 1.021 degrading pig hair in this application; wherein, Figure 4 A is the experimental group, i.e., ZKHW 1.021 was added to the degradation medium; Figure 4 B is the blank control group, i.e., no ZKHW 1.021 was added to the degradation medium;

[0024] Figure 5 The amino acid spectrum of the enzymatic hydrolyzate obtained by degrading pig hair with ZKHW 1.021 in this application.

[0025] Description of biological material deposit:

[0026] Biomaterial ZKHW 1.021, taxonomic nomenclature: Bacillus velezensis Bacillus velezensi , deposited on July 24, 2023 in the Guangdong Microbiological Culture Collection Center (GDMCC), located at Guangdong Institute of Microbiology, Guangzhou, Guangdong, China, with the deposit number GDMCC NO. 63679. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0028] The present invention provides a degradation bacterium, which is Bacillus velez Bacillus velezensis , named ZKHW 1.021, and its 16S rDNA nucleotide sequence is shown in SEQ ID NO. 1. ZKHW 1.021 was obtained by the inventors through separation and screening from soil samples where discarded pig hair had been accumulated for a long time around a pig slaughterhouse.

[0029] The separation and screening method of the degradation bacteria comprises: weighing 10.0 g of a soil sample from a long-term accumulation of discarded pig hair around a pig slaughterhouse, placing it in a conical flask containing 90 mL of sterile water, and fully shaking and mixing to obtain a mixed solution. Take 5 mL of the mixed solution and place it in a conical flask containing 100 mL of selective culture medium, and shake and culture it at 24-60°C and 150-200r / min. Take the bacterial solution that has been acclimated and cultured for 80-110 hours, dilute it gradiently, spread it on a solid selective culture medium (2% agar is added to the selective culture medium) plate, and culture it for 65-75 hours (24-60°C), screen single colonies with good growth, further purify it by using the four-zone streaking method, and store each strain at low temperature on the slant of a NA culture medium test tube. Select the single colony that has been screened and purified well in the degradation culture medium, place it in a shaking table at 24-60°C, and culture it at 150-200r / min. Use the uninoculated degradation culture medium as a control to observe the degradation of pig hair. After further cultivation in degradation medium, the thermophilic strain with the highest efficiency in decomposing pig hair was selected. After morphological, physiological, biochemical and DNA identification, it was named Bacillus Velezii ( Bacillus velezensis )ZKHW 1.021.

[0030] Among them, the NA medium formula: peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L. The selection medium formula: pig hair 0.5-10 g / L, sodium chloride 0.1-1.0 g / L, sodium dihydrogen phosphate 0.05-0.2 g / L and disodium hydrogen phosphate 0.5-2 g / L, pH natural. The degradation medium formula: pig hair 10 g / L, sodium chloride 0.5 g / L, sodium dihydrogen phosphate 0.1 g / L, disodium hydrogen phosphate 1.3 g / L, pH natural.

[0031] Methods for using ZKHW 1.021 to degrade pig hair include:

[0032] (1) Preparation of ZKHW 1.021 seed solution: The selected Bacillus Velezii ( Bacillus velezensis ) ZKHW 1.021 was inoculated onto NA solid culture medium plates and cultured at 24-60°C for 15-20 hours to activate them; the activated seeds were transferred to NB liquid seed culture medium and cultured to the logarithmic growth phase.

[0033] (2) Pretreatment of pig hair: Wash and remove impurities from the pig hair, steam at 110-130°C for 50-70 min, dry and crush at 40-50°C to obtain the pretreated pig hair sample.

[0034] (3) Degrading pig hair using ZKHW 1.021: Mix the pig hair sample pretreated in step (2) with an inorganic salt solution at a mass ratio of (0.1-1):100, and then add the seed solution prepared in step (1) for culturing to degrade the pig hair. The inorganic salt solution comprises: 0.1-1.0 g / L NaCl, 0.05-0.2 g / L NaH2PO4, and 0.5-2.0 g / L Na2HPO4.

[0035] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0036] Example 1

[0037] (1) Separation and screening ZKHW 1.021

[0038] Weigh 10.0 g of soil sample from the area around the pig slaughterhouse where discarded pig hair has been piled up for a long time, place it in a conical flask containing 90 mL of sterile water, and shake it thoroughly to mix. Take 5 mL of the mixed solution and place it in a conical flask containing 100 mL of selective culture medium, and shake and culture it at 45°C and 180 r / min. Take the bacterial solution that has been acclimated and cultured for 96 hours and dilute it gradiently. Then spread it on a solid selective culture medium (2% agar is added to the selective culture medium) and culture it for 72 hours. Screen single colonies with good growth, and further purify them by the four-zone streak method. Store each strain at low temperature on the NA culture medium test tube slope. Select the single colonies that have been screened and purified in the degradation culture medium, place them in a shaker at 45°C and culture them at 180r / min. Use the uninoculated degradation culture medium as a control to observe the degradation of pig hair. After further cultivation in the degradation culture medium, the high-temperature strain with the highest efficiency in decomposing pig hair was screened. After morphological, physiological, biochemical and DNA identification, it was named Bacillus Velezii ( Bacillus velezensis )ZKHW 1.021.

[0039] NA medium formula: peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L.

[0040] The medium formula was selected as follows: 1 g / L pig hair, 0.5 g / L sodium chloride, 0.1 g / L sodium dihydrogen phosphate, 1.3 g / L sodium hydrogen phosphate, and natural pH.

[0041] Degradation culture medium formula: pig hair 10 g / L, sodium chloride 0.5 g / L, sodium dihydrogen phosphate 0.1 g / L, sodium hydrogen phosphate 1.3 g / L, natural pH.

[0042] (2) Preparation of pig hair fermentation liquid

[0043] The Bacillus Velezii ( Bacillus velezensis ) ZKHW 1.021 was inoculated onto NA solid culture medium plates and cultured at 45°C for 18 h to activate it; the activated seeds were transferred to NB liquid seed culture medium (NA culture medium with 1% agar added), and cultured to the logarithmic growth phase to obtain ZKHW 1.021 seed solution.

[0044] The pig hair was washed and cleaned, steamed at 121°C for 60 minutes, dried and crushed at 45°C to obtain a pretreated pig hair sample; the pretreated pig hair sample was mixed with an inorganic salt solution at a mass ratio of 1:100, and then the prepared ZKHW 1.021 seed solution (4%) was added, and cultured at 45°C and 180r / min for 72 hours to obtain a pig hair fermentation liquid. The components of the inorganic salt solution include: NaCl 0.5g / L, NaH2PO4 0.1035 g / L and Na2HPO4 1.3277 g / L.

[0045] Test Example 1

[0046] This test example is to screen out the Velezella bacillus in Example 1 ( Bacillus velezensis )ZKHW1.021 was used for biological traits, physiological and biochemical characteristics and molecular identification tests.

[0047] (1) Morphological characteristics

[0048] Bacillus Velez Bacillus velezensis ) After culturing ZKHW 1.021 on NA medium for 24 h, the colonies were slightly yellow and opaque, round, moist and not smooth, with regular edges, slightly raised, and about 1.8 mm in diameter. Gram staining was positive, and the bacteria were short rods arranged in short chains.

[0049] The colony map of ZKHW 1.021 is shown in Figure 1 Gram staining is shown in Figure 2 .

[0050] (2) Physiological and biochemical characteristics

[0051] The following experiments were performed on ZKHW 1.021:

[0052] 1. Contact enzyme test:

[0053] The contact enzyme test is usually used to test whether the microbial strain has specific collagenase (also called gelatinase) activity. These enzymes can break down collagen, resulting in the dissolution of the gel. The following are the basic steps of the contact enzyme test:

[0054] Preparation of agar gel plates: Prepare agar gel plates containing collagen. Collagen at a certain concentration can be added to the agar gel medium.

[0055] Inoculation strain: Use the culture of Bacillus Velez ZKHW 1.021. Take a certain amount of bacterial suspension and spread it evenly on the surface of the agar gel plate.

[0056] Incubation: The agar gel plate is incubated under appropriate conditions, usually at an appropriate temperature and humidity, for a period of time to allow the strain to grow and produce enzymes.

[0057] Observation: Observe the growth of colonies on the surface of the agar gel plate. If the strains have catalase activity, they will decompose collagen, resulting in clear areas of the agar gel, which are collagen degradation areas.

[0058] Result analysis: Based on the size and shape of the transparent area and the environment around the colony, it can be preliminarily determined whether the strain has catalase activity. The larger the transparent area, the stronger the collagen decomposition ability.

[0059] 2. Voges-Proskauer experiment (VP experiment)

[0060] The VP test is a microbiological test used to detect the presence of acetate in microbial metabolites. This test is commonly used to identify Gram-negative bacteria, especially Enterobacteriaceae ( Enterobacteriaceae ) members to determine the presence of acetate in their metabolites.

[0061] The process of VP experiment involves two main steps: 1. Addition of Voges-Proskauer reagent: First, Voges-Proskauer reagent needs to be added to the culture medium. The reagent usually consists of two components: α-naphthol and ketone reagent (usually a 5% soap water solution, such as 40% sodium hydroxide solution). The addition of these two reagents helps to detect metabolites that produce acetate. 2. Oxidation reaction of ketone reagent: Ketone reagent reacts with acetate in microbial metabolites and forms ketone bodies through oxidation reaction. Ketone bodies undergo condensation reaction under the catalysis of α-naphthol to produce red products. The appearance of this red product indicates that there is a metabolic pathway that produces acetate in the sample.

[0062] The results of the VP experiment are usually judged by the color change produced. If a red product is produced, it means that there are metabolites that produce acetate in the sample, which can be judged as a positive reaction; if no color change occurs, it is a negative reaction.

[0063] 3. Anaerobic growth experiment: Culture Bacillus Velez ZKHW 1.021 in an anaerobic environment, such as a sealed container, and observe whether there is any growth.

[0064] 4. Nitrate reduction experiment: (Use the nitrate reduction kit produced by Shanghai Yuanye Biotechnology Co., Ltd.).

[0065] 5. Starch hydrolysis experiment: Bacillus Velez ZKHW 1.021 was cultured in NA medium containing starch. After a period of culture, iodine solution was used to detect whether starch hydrolysis products were formed.

[0066] 6. Casein hydrolysis experiment: Culture Bacillus Velez ZKHW 1.021 in a culture medium containing casein. After a period of culture, observe whether casein degradation occurs and whether specific products are produced.

[0067] 7. Gelatin hydrolysis experiment: Culture Bacillus velezensis ZKHW 1.021 in NA medium containing gelatin. After a period of culture, observe whether gelatin degradation occurs and whether specific products are produced.

[0068] 8. Cellulose decomposition experiment: Bacillus velezensis ZKHW 1.021 was cultured in NA medium containing cellulose. After a period of culture, the degradation of cellulose and the production of specific products were observed.

[0069] 9. Indole experiment: Culture Bacillus velezensis ZKHW 1.021 in NA medium containing indole. After a period of culture, observe whether indole ketone is produced. If indole ketone is produced, the color of the culture medium will change from yellow or colorless to blue.

[0070] 10. Lecithinase experiment: Streak or spot-inoculate Bacillus Velez subtilis ZKHW 1.021 on an egg yolk agar plate. After culturing for a period of time, observe whether a milky white turbid circle is produced.

[0071] 11. Tyrosine hydrolysis experiment: Culture Bacillus velezensis ZKHW 1.021 in NA medium containing tyrosine. After a period of culture, observe whether a transparent zone is produced.

[0072] 12. Phenylalanine dehydrogenase experiment: Culture Bacillus velezensis ZKHW 1.021 in NA medium containing phenylalanine and incubate at 37°C for 18-24h. After growth, take out and inject 0.2mL (or 4-5 drops) of 10% FeCl3 aqueous solution on the growth surface to observe whether it turns green.

[0073] The physiological and biochemical characteristics of Bacillus velez ZKHW 1.021 are shown in Table 1.

[0074] Table 1 Physiological and biochemical characteristics of ZKHW 1.021

[0075]

[0076] Note: + indicates positive; - indicates negative.

[0077] Through physiological and biochemical determination, the Velez Bacillus provided by the present application ( Bacillus velezensis )The contact enzyme and VP tests, anaerobic growth, nitrate reduction, starch hydrolysis, casein hydrolysis, gelatin hydrolysis, cellulose decomposition tests of ZKHW1.021 were all positive, and the indole, lecithinase, tyrosine hydrolysis, phenylalanine dehydrogenase and other tests were all negative; the growth temperature range was 20-60°C, and the maximum tolerance to NaCl was 10%. The above test results show that the strain has a variety of metabolic pathways and can use a variety of substrates for growth and metabolism in different environments, which may have potential application value in the fields of environmental remediation and waste degradation. Moreover, since it showed positive results in the metabolic experiments of multiple substrates, it shows that Bacillus Velezii ZKHW 1.021 may have ecological functions such as decomposing organic matter, degrading waste, and participating in environmental remediation.

[0078] (3) Bacillus Velez Bacillus velezensis ) Sequence determination and analysis of 16S rDNA of ZKHW 1.021.

[0079] 1. DNA extraction: Bacillus Velezii ( Bacillus velezensis )ZKHW 1.021 was streaked on a NA medium plate and cultured overnight at 45°C. A single colony was picked and placed in a 1.5 mL EP tube, and bacterial DNA was extracted using the CTAB method.

[0080] The following are the general steps of the CTAB method:

[0081] Materials and reagents preparation:

[0082] Single colony of Bacillus velezensis ZKHW 1.021 after culture.

[0083] CTAB extraction buffer: A buffer containing CTAB (n-hexadecyltrimethylammonium bromide). Common components include CTAB, NaCl, Tris-HCl, etc.

[0084] Proteinase K Solution: An enzyme used to remove proteins, used to digest cell membranes and proteins.

[0085] Chloroform: used to extract DNA.

[0086] Isomorphic phenol / chloroform mixture: used to separate the aqueous and organic phases in chloroform.

[0087] Steps:

[0088] Pick a single colony: Use a sterile toothpick or cotton swab to pick a single colony on the NA medium plate and transfer it to a 1.5 mL centrifuge tube.

[0089] Bacterial lysis: Add an appropriate amount of CTAB extraction buffer to a 1.5 mL centrifuge tube containing bacteria, using an appropriate volume of buffer to completely soak the bacteria. Then add a certain amount of proteinase K solution and gently invert to mix.

[0090] Incubation: Place the centrifuge tube containing bacteria and buffer in a 65°C water bath and incubate for 30-60 minutes to promote cell lysis.

[0091] Extract DNA: Add an equal volume of chloroform and gently invert to mix, allowing the mixture to separate into two phases. Centrifuge the tube to separate the two phases. DNA is enriched in the upper aqueous phase.

[0092] Precipitate DNA: Transfer the upper aqueous phase to a new centrifuge tube, add isopropanol, and gently invert to mix to precipitate the DNA. Centrifuge the tube to precipitate the DNA.

[0093] Wash DNA: Pour off the upper layer of liquid, add 75% ethanol to wash, gently invert to mix, centrifuge the tube, pour off the washing solution, and repeat the washing steps once.

[0094] Dry DNA: Invert the tube onto a paper towel and allow the DNA pellet to dry at room temperature, avoiding over-drying which may affect solubility.

[0095] Dissolve DNA: Dissolve the DNA in an appropriate amount of deionized water to concentrate it and restore it to a suitable concentration. The quality and concentration of DNA can be tested by UV spectrometer or agarose gel electrophoresis.

[0096] 2. 16S rDNA gene PCR amplification

[0097] PCR primers were synthesized by Shanghai Bioengineering Company.

[0098] Primer A: 27F 5'-AGAGTTTTGATCCTGGCTCTAG-3'

[0099] Primer B:1492R 5'-GGTTACCTTGTTACGACTT-3'

[0100] The PCR reaction system is shown in Table 2:

[0101] Table 2

[0102] 10×PCR buffer 5μL 2 mmol / Ld NTP 4μL 10 pmol / L Primer A 1μL 10 pmol / L Primer B 1μL 2 U / L Taq enzyme 0.4μL DNA template 1μL Sterile ddH2O 37.4μL Total volume 50μL

[0103] PCR amplification conditions: 95℃ 5 min; 95℃ 50 s, 56℃ 50 s, 72℃ 90 s, 30 cycles; 72℃ 10 min; 4℃ storage. PCR amplification products were detected by 1% agarose gel electrophoresis, and PCR products were recovered and purified using an agarose gel DNA recovery kit (spin column type).

[0104] 3. Sequencing

[0105] After the PCR product was purified, it was sent to Shanghai Meiji Company for sequencing. Its 16S rDNA nucleotide sequence is shown in SEQ ID NO: 1. Comparison of this sequence with known standard strains in the NCBI database showed that the strain Bacillus velezensis strain LB 67 had the highest homology, reaching 99%. The 16S rDNA phylogenetic tree was drawn using the Neighbor-Joining method using MEGA7.0 software (see Figure 3 ), determine its evolutionary status, and combined with its morphological and physiological and biochemical characteristics, the strain is most likely to be Bacillus licheniformis and is named Bacillus velezensis ZKHW 1.021, the 16S rDNA nucleotide sequence of ZKHW 1.021 is shown in SEQ ID NO.1.

[0106] Test Example 2

[0107] In this test example, the pig hair fermentation liquid prepared in Example 1 was used to determine the degradation rate of pig hair, the activity of keratinase in the pig hair fermentation liquid, the water-soluble free amino acids in the pig hair fermentation liquid, and the polypeptide content in the pig hair fermentation liquid.

[0108] Determination of pig hair degradation rate: Filter the pig hair fermentation liquid with filter paper, collect the filter residue and dry it at 105℃ to constant weight, and calculate the degradation rate. Pig hair degradation rate (%) = (control sample dry weight - experimental sample dry weight) / control sample dry weight × 100%. Among them, the control sample dry weight is the weight of the final product obtained by fermenting and culturing the mixed solution of pig hair and inorganic salts without adding ZKHW 1.021 seed liquid.

[0109] Keratinase activity determination: Filter the fermentation solution with 2 layers of gauze, remove the filtrate, centrifuge at 12000g, 4℃ for 10min, take 1.0mL of the supernatant, add 2.0mL 0.05mol / L Tris / HCl buffer (pH7.5) and 10mg keratin substrate, react in a constant temperature water bath at 37℃, and keep warm at 100r / min for 1h; at the end of the reaction, add 2.0mL 10% trichloroacetic acid (TCA) to terminate the reaction, and centrifuge at 10000g for 15min. The absorbance of the supernatant is measured at 595nm, and the reaction tube with TCA added before insulation is used as a control. Enzyme activity unit definition: Under experimental conditions, the amount of enzyme produced by unit mL of keratin decomposition is Δ OD595 The amount of enzyme required to increase the value by 0.001 units is one activity unit (U).

[0110] Determination of water-soluble free amino acids in pig hair fermentation broth: Take 5mL of pig hair fermentation broth to a 10mL colorimetric tube, add ddH2O to make up to 10mL, and let stand overnight. Take 2mL of supernatant to a 10mL centrifuge tube, add 2mL of sulfosalicylic acid, mix well, and let stand for 1h to precipitate the protein; add 1mL of EDTA and 1mL of hydrochloric acid, and centrifuge at 4℃, 6000g for 15min (or filter with a 0.22μm microporous filter membrane). Take 1mL of supernatant to a culture dish and evaporate to dryness; add 1mL of 20mmol / L HCl solution, filter with a 0.22μm microporous filter membrane, and take at least 500μL to the injection bottle; determine with L-8900 fully automatic amino acid analyzer.

[0111] Determination of polypeptide content in pig hair fermentation broth: Accurately weigh 6.000g of pig hair fermentation broth into a 50mL volumetric flask, add 15% trichloroacetic acid solution to 50mL, mix well, and let stand for 5min. Dry filter with medium-speed qualitative filter paper, discard a small amount of initial filtrate, and transfer the filtrate to a centrifuge tube. Centrifuge at 4000r / min for 10min, accurately transfer 10mL of the supernatant into a digestive tube, and determine the protein content by Kjeldahl method. The polypeptide content in pig hair fermentation broth is the protein content determined above minus the free amino acid content in pig hair fermentation broth.

[0112] Figure 4 The triangular flask in A contains the fermentation liquid to which ZKHW 1.021 has been added. Figure 4 B is the blank control group. Figure 4 A and Figure 4 B It can be seen that ZKHW 1.021 can effectively degrade pig hair, and after testing, it was found that the degradation rate of pig hair reached 88.34% after 3 days of fermentation, and the keratinase activity in the fermentation broth reached 48.66 U / mL, indicating that the Bacillus Velezii ( Bacillus velezensis )ZKHW 1.021 has the ability to quickly degrade pig hair. After determination, the fermentation liquid obtained after 72 hours of fermentation has a complete range of amino acids, and the amino acid content reaches 2.7435 g / kg. The amino acid spectrum is shown in Figure 5 , the polypeptide content is 3.1548 g / kg. Therefore, Bacillus Velezii ( Bacillus velezensis )ZKHW 1.021 is used for the degradation and resource utilization of waste pig hair in the livestock and poultry breeding industry.

[0113] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A degradation bacterium, characterized in that: The degrading bacteria is Bacillus Velez Bacillus velezensis ), named ZKHW 1.021, and the deposit number is GDMCC NO. 63679.

2. The degrading bacteria according to claim 1, characterized in that The 16S rDNA nucleotide sequence of the degrading bacteria is shown in SEQ ID NO.

1.

3. Use of the degrading bacteria as claimed in claim 1 or 2 in degrading pig hair.

4. The use according to claim 3, characterized in that: The method for degrading pig hair using the degrading bacteria comprises: The pretreated pig hair sample is mixed with an inorganic salt solution, and then a seed solution containing the degradation bacteria is added for cultivation to degrade the pig hair.

5. The use according to claim 4, characterized in that: The pretreatment comprises: washing and removing impurities from the pig hair, steaming at 110-130° C. for 50-70 minutes, and drying and crushing at 40-50° C. to obtain the pretreated pig hair sample.

6. The use according to claim 4, characterized in that: The mass ratio of the pig hair to the inorganic salt solution is (0.1-1):

100.

7. The use according to claim 4, characterized in that: The components of the inorganic salt solution include: 0.1-1.0 g / L NaCl, 0.05-0.2 g / L NaH2PO4 and 0.5-2.0 g / L Na2HPO4.

8. The use according to claim 4, characterized in that: The preparation of the seed solution includes: inoculating the degrading bacteria onto a NA solid culture medium plate, culturing at 24-60° C. for 15-20 hours to activate them; transferring the activated degrading bacteria to a NB liquid seed culture medium, and culturing them to a logarithmic growth phase.

9. The use according to claim 8, characterized in that: The culture conditions include: culturing at 24-60° C. for 65-80 h.

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