CN112080435A

CN112080435A - Purple lilac spore strain and application thereof in degrading chicken feather

Info

Publication number: CN112080435A
Application number: CN202011006413.2A
Authority: CN
Inventors: 韩燕峰; 董醇波; 牟泉蓉; 鲁小晓; 张玲雪; 陈万浩; 梁宗琦
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2020-12-15

Abstract

The invention discloses a purple lilac spore strain and application thereof in degrading chicken feather, wherein the strain is obtained by separating soil in Kunming city of Yunnan province. The strain is GZAC18-2JMP, is preserved in China general microbiological culture Collection center on 27 th of 08 th of 2020, and has a preservation number: CGMCC No. 20249. Researches show that the purple lilac spore is a mesophilic fungus and can degrade chicken feather, and the optimal culture method for degrading the chicken feather is to inoculate the GZAC18-2JMP strain into basic fermentation liquor added with the chicken feather, to stand for 48 hours at 25 ℃, to perform shaking culture at 110rpm/min for 96 hours, wherein the chicken feather in the fermentation liquor is in a crushed powder shape, the soluble protein is 175.7 mu g/mL, the dry weight of the filter residue of the chicken feather is 0.36g, and the degradation rate of the chicken feather is 64%. The strain and the chicken feather degradation culture method thereof can be used for biodegradation of chicken feather.

Description

Purple lilac spore strain and application thereof in degrading chicken feather

Technical Field

The invention belongs to the field of biodegradation, and particularly relates to a purple lilac spore bacterium and application thereof in degrading chicken feather.

Background

The chicken feather is a fibrous and insoluble structural protein, is rich in keratin, and is the third most abundant polymer which is inferior to cellulose and chitin in nature. When the keratin waste such as chicken feather is naturally accumulated, the degradation is very slow, a large amount of accumulation is easy to cause, and meanwhile, the protein which is rich in the keratin waste and the attached nitrogen and phosphorus-containing substances such as excrement, blood and the like are also easy to become nutrient sources of other pathogenic bacteria, so that the secondary pollution to the ecological environment and the harm to the human health are caused. Keratin waste is usually treated by landfill, incineration and other methods, but the incineration brings potential safety hazards to the environment and human health, so that the application of a green and healthy method to degrade the keratin is particularly important. And the degraded chicken feather is rich in protein or amino acid source, and can be used for producing feed or coating film materials, biomedical materials and the like. The method for degrading the chicken feather by using the microorganisms is a mild, efficient and green method, and has wide development potential and high practical value. The common microbial degradation of chicken feather is mainly bacteria, yeast and filamentous fungi. In Chinese patent application, 6 patents are related to the degradation of keratin by microorganisms, and the degradation strains are all bacteria, namely: the keratin degrading bacteria NJY1(CN200810155958.2) is bacillus thuringiensis, the immobilization method (CN200810039457.8) of the feather keratin degrading bacteria is bacteria stenotrophomonas maltophilia, the strain for efficiently degrading feather keratin and the screening method (CN201010574642.4) thereof are bacillus licheniformis, and the method (CN201110245892.8) for anaerobically degrading feather keratin by utilizing the microbial strain 18D-TA and the strain 18D-TA (CN201110245892.8) for anaerobically degrading feather keratin are anaerobic bacteria and the keratin degrading bacteria NJK4(CN201310577095.9) are bacillus pumilus. The degradation of the chicken feather by the fungus is not disclosed in Chinese invention patent. Compared with bacteria and yeasts, the filamentous fungi have many advantages in the degradation of chicken feather, such as the capability of generating rich degradation enzyme systems, high-activity keratinase, more rich and thorough degradation products and high degradation efficiency at proper temperature. At present, the reported filamentous fungi for degrading chicken feather mainly comprise paecilomyces, chrysosporium, myceliophthora and other fungi. The neurospora fungi are mainly applied to the biological control of nematodes, and have not been reported in the degradation of chicken feather.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a new bacterial strain capable of degrading chicken feather and a degradation method are provided.

The technical scheme of the invention is as follows: a purple lilac spore (purple lilacinum) GZAC18-2JMP, strain collection number: CGMCC No.20249, deposited in China general microbiological culture Collection center on 08-27.2020, with the deposition address: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.

Application of purple lilac spore (Purpureocillium lilacinum) GZAC18-2JMP in degrading chicken feather.

Specifically, the purple lilac spore bacteria is inoculated into the chicken feather degradation fermentation liquid, and is firstly kept stand for 48 hours at 25 ℃, and then is cultured for 96 hours by shaking.

Further, the chicken feather degradation fermentation liquor comprises the following components: 10g/L of chicken feather, 0.5g/L of monopotassium phosphate, 1.2g/L of sodium chloride, 0.05g/L of zinc sulfate, 0.2/L g of calcium chloride and pH 7.0.

Compared with the prior art, the invention has the following beneficial effects:

the purple lilac spore fungus GZAC18-2JMP is obtained from soil through separation, is a Chinese native strain, is not introduced from abroad, and can adapt to local natural environment. Experiments show that the purple lilac spore has excellent chicken feather degradation capability, can be used for biodegradation of chicken feather, and has potential application value in production of microbial fertilizer and amino acid feed and ecological environment protection.

Preservation information:

purple violet (purple spore bacillus) GZAC18-2JMP, strain accession number: CGMCC No.20249, deposited in China general microbiological culture Collection center on 08-27.2020, with the deposition address: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.

Drawings

FIG. 1 shows the colony morphology and spore-forming structure of the purple lilac spore bacterium GZAC18-2JMP on the PDA culture medium;

FIG. 2 is a phylogenetic tree of purple lilac spore and related strains constructed based on ITS rDNA sequence;

FIG. 3 shows a protease transparent circle produced by the purple lilac GZAC18-2JMP on the skim milk powder culture medium;

FIG. 4 is a comparative graph of fermentation broth of purple lilac spore bacterium GZAC18-2JMP before and after fermentation in the culture method of the present study;

FIG. 5 is a plotted protein standard curve;

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1 sample Collection and isolation of strains

1. Experimental materials and methods

1.1, collecting a soil sample: collecting soil from flower bed soil of Kunming city, Yunnan province, with a collection depth of 3-5cm, packaging the collected soil sample in sterile plastic packaging bag, and taking back to laboratory for use.

1.2 culture Medium for experiments

Martin PDA medium: weighing 200 g of peeled and cleaned potato, cutting into pieces, boiling in water for 30min, filtering, collecting filtrate, adding 20g of glucose and 20g of agar powder, fully and uniformly stirring, carrying out autoclaving at 121 ℃ for 30min, cooling to about 40 ℃, and adding 3mL of 1% sterilized Mongolian red water solution, 3mL of 1% penicillin solution and 3mL of 1% streptomycin solution.

1.3 enrichment and isolation of the strains: mixing a soil sample and 10g of chicken feather powder, placing the mixture in a culture dish, preserving moisture at the constant temperature of 25 ℃ for 1 month, picking out the mycelia from another clean culture dish in a super clean workbench for purification after the mycelia grow on the chicken feather is observed, culturing for 5d, transferring the single colonies after the single colonies grow out to a new PDA test tube slant culture medium for preservation, obtaining a purified strain GZAC18-2JMP, and preserving for later use.

1.4 DNA extraction and PCR amplification of the Strain

Scraping the GZAC18-2JMP strain culture cultured to produce spores, putting the culture into a sterilized mortar, adding liquid nitrogen capable of covering the culture to grind the culture into fine powder, putting the fine powder into a 1.5mL centrifuge tube, and extracting DNA according to the operation flow of a fungus DNA extraction kit (Beijing Baitaike biotechnology limited). Storing the extracted fungal DNA in a refrigerator at-20 ℃ for later use. The fungal ITS-5.8S rDNA region was amplified using primers ITS4: 5'-TCCTCCGCTTATTGATATGG-3' and ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' (synthesized by Kunming Shuichi Biotech, Inc.). The PCR amplification reaction system is a 25 mu L system: DNA template 2. mu.L, mix 21. mu.L, primers ITS5 and ITS4 each 1. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, and extension at 72 ℃ for 1min for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃. And sending the qualified PCR product to Kunming Shuichi Biotech limited for sequencing.

2 results of the experiment

The colony morphology was described after the purified GZAC18-2JMP strain was inoculated into PDA medium and cultured at 25 ℃ for 14 days (3 replicates), and the microscopic morphology was measured and described after flaking, as follows:

morphological description of GZAC18-2JMP Strain: as shown in figure 1, the culture is carried out on a PDA culture medium for 14 days at 25 ℃, the diameter of a colony is 60-65mm, the colony is flat, sparse villous, purple gray, has flesh-colored fine particles on the surface, has 3 wheel layers and has radial grains; the back is white. The hypha has width of 0.5-2 μm, and is transparent and smooth. Conidiophores are 17-35 mu m in length, can reach 65 mu m individually, and have larger length difference; the crop rotation body consists of 3-6 bottle stalks. The base part of the bottle stalk is expanded in a columnar shape and is suddenly thinned upwards, and the diameter of the bottle stalk is 7-9 multiplied by 1.5-3 mu m; the neck is short and the diameter is less than 0.5 μm. The conidia are transparent, smooth, with 1.3-3.0 × 1.0-2.2 μm core shape and long chain arrangement. The morphological characteristics of the strain are consistent with those of the reported purple rhodosporidium.

Based on ITS rDNA sequence, downloading highly similar Porphyra strain sequence, using ClustaXI 5.3 software to compare the strain GZAC18-2JMP with the downloaded sequence, using RxAML software to establish ML phylogenetic tree (see figure 2) for GZAC18-2JMP strain and related strain by using Isaria farinosa as ectogroup. The results show that the sequence of the strain is well clustered with the other 4 sequences of the purple lilac spore bacterium with high support rate.

By combining morphological characteristics and molecular data, the GZAC18-2JMP strain is identified as the purple lilac spore bacterium.

EXAMPLE 2 Primary screening of the strains for protease production

1. Experimental materials and methods

1.1 culture Medium for experiments

Solid culture medium of skimmed milk powder (g/L): 5g of skimmed milk powder, 20g of agar powder and 1000mL of water.

1.2 Experimental methods

Inoculating GZAC18-2JMP strain to the center of skimmed milk powder solid culture medium, and placing in a constant temperature incubator at 25 ℃. The diameter of the transparent circle and the colony was measured, and the ratio of the two was calculated.

2. Results of the experiment

As shown in FIG. 3, the diameter of the transparent circle generated by the GZAC18-2JMP strain on the degradation of the skim milk powder increases with the growth of the colony. And (3) by the day 7, the size of the transparent ring is 41mm, the diameter of the bacterial colony is 35mm, the ratio of the size of the transparent ring to the diameter of the bacterial colony is 1.17, and the results are visually displayed through a culture dish, so that the GZAC18-2JMP bacterial strain can better degrade protein in the skim milk powder, and the research on the degradation of the keratinase can be further carried out.

Example 3 evaluation of the Effect of the Violet purple mold Strain on the degradation of Chicken feather

1. Experimental materials and methods

1.1 treatment of chicken feather: collecting chicken feather in a farm poultry slaughter house in Huaxi district of Guiyang city, cleaning the collected chicken feather with tap water, soaking for 12h, washing with running water, autoclaving at 121 deg.C for 30mins, and oven drying at 50 deg.C.

1.2 preparation of fermentation Medium

10g of chicken feather, 0.5g of monopotassium phosphate, 1.2g of sodium chloride, 0.05g of zinc sulfate and 0.2g of calcium chloride, wherein the volume is 1000mL, the pH value is 7.0, and the chicken feather is sterilized at 121 ℃ for 30mins for later use.

1.3 preparation of the purple Violet spore suspension

In a clean bench, gently scraping hypha of a rhodosporidium lilacinum GZAC18-2JMP strain cultured for 7 days at 25 ℃ on a PDA culture medium, transferring the hypha to a sterilized test tube filled with 10mL of 0.05% Tween-80, placing the test tube and the sterilized test tube on a vortex oscillator, oscillating for 1min, standing, filtering the suspension in the clean bench by using 3 layers of mirror wiping paper, counting the drops of the filtered spore suspension by using a hemocytometer, and adjusting the spore concentration to 1 × 10⁷one/mL, 1mL was inoculated into the fermentation medium.

1.4 cultivation method for degrading chicken feather

The flask containing 100mL of fermentation medium was placed statically in an incubator at 25 ℃ for 48 hours, and then placed in a shaker at 110rpm for 96 hours at 25 ℃.

A comparison of the fermentation broths before and after fermentation for the above 3 replicates is shown in FIG. 4.

1.5 drawing of protein standard curve:

1) coomassie brilliant blue G250, 100mg dissolved in 50mL 95% ethanol, 100mL 85% phosphoric acid added, and diluted to 1L with distilled water.

2)0.2mol/L, pH7.0 phosphate buffer and standard protein solution preparation: weighing KH₂PO₄2.12g and K₂HPO₄5.56g of the mixture was dissolved in distilled water, and the volume was adjusted to 200mL after mixing. 100mg of bovine serum albumin was weighed and prepared into a 1mg/mL standard protein solution using the above buffer.

3) Taking 7 test tubes, respectively adding 0, 10 mu L, 20 mu L, 40 mu L, 60 mu L, 80 mu L and 100 mu L of standard protein solution, respectively adding Coomassie brilliant blue solution to a constant volume of 5mL, shaking up, measuring absorbance at 595nm wavelength, and calculating the content of soluble protein in the sample according to the absorbance value.

The protein standard curve was plotted as shown in FIG. 5.

1.6 determination of soluble protein content in fermentation broth

Taking 100 mu L of fermentation liquor fermented for one week, adding Coomassie brilliant blue to a constant volume of 5mL, shaking up, measuring absorbance at 595nm wavelength, and calculating the content of soluble protein in the sample through a protein standard curve.

1.7 weight of Chicken feather residue in fermentation broth

And filtering the fermentation liquor fermented for one week by using quick qualitative filter paper, drying the filtered chicken feather filter residue by using an oven, and measuring the weight of the chicken feather filter residue.

2. Results of the experiment

2.1 comparison of the integrity of the Chicken feather Structure in the fermentation broth

The chicken feather is rich in keratin matrix, has a complex structure and is difficult to degrade. As can be seen from FIG. 4, after degradation by strain GZAC18-2JMP, the original intact chicken feather structure is degraded into chicken feather powder, which is suspended in the fermentation broth.

2.2 measurement of soluble protein content in fermentation broth

The higher the soluble protein content in the fermentation liquor, the better the degradation effect. The OD value of the fermentation broth prepared according to fig. 5 was determined to be 1.2989, and the increase in soluble protein content was 175.7 μ g/mL, as calculated according to the formula of fig. 5, where y is 0.007x +0.069(y is the OD value), indicating that the chicken feather degradation effect was better.

2.3 determination of the content of Chicken feather residue in the fermentation broth

After fermentation culture for 1 week, the weight of the measured chicken feather filter residue is reduced to 0.36g/100mL from 1g/100mL of the reference, 0.64g is degraded, which indicates that the strain has good effect on degrading chicken feather, and the degradation rate reaches 64%.

Claims

1. A purple lilac spore (purple lilacinum) GZAC18-2JMP, strain collection number: CGMCC No.20249, China general microbiological culture Collection center (CGMCC) on 08-27.2020.

2. Use of the purple lilac fungus of claim 1 for the degradation of chicken feather.

3. The use of claim 2, wherein the fermentation broth is prepared by inoculating the Arthrobotrys lilacinus into a chicken feather degradation fermentation broth, standing the fermentation broth at 25 ℃ for 48 hours, and then performing shake culture for 96 hours.

4. The use of claim 3, wherein the composition of the chicken feather degradation broth is: 10g/L of chicken feather, 0.5g/L of monopotassium phosphate, 1.2g/L of sodium chloride, 0.05g/L of zinc sulfate, 0.2/L g of calcium chloride and pH 7.0.