Feather keratin degrading strain and screening method

Abstract

The invention discloses a feather keratin degradation strain and a screening method, and relates to the technical field of bioengineering, the strain obtained through enrichment, primary screening and secondary screening is identified by morphological and molecular biological levels, the strain is Ochrobactrum intermedium 0123, the classification name is Ochrobactrum intermedium 0123, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20231151, and the preservation number is CCTCC NO: M 20231151. The preservation date is June 30, 2023. Through optimization of culture conditions, the optimal pH value of the strain is 8, and the optimal salinity is 0.4 g / L. By measuring the degradation rate of the strain on feather meal keratin, the degradation rate of the strain within 7 days is 48.90%. The strain obtained by screening has regional specificity in northwest regions, can provide suitable strains for degradation of feather keratin under salinization conditions, and further enriches a feather keratin degrading bacterium strain resource library.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a feather keratin-degrading strain and a screening method thereof. Background Art

[0002] In recent years, the aquaculture industry in China has gradually become more large-scale. As a by-product of the poultry breeding and slaughter industry, the output of poultry feathers and down keratin waste is increasing. The keratin in the feather waste has become an important material resource because it is rich in carbon, sulfur, and nitrogen elements and can be converted into other products. However, keratin is a fibrous protein with the properties of being insoluble in water and resistant to decomposition. After the action of intermolecular disulfide bonds, hydrogen bonds, and salt bonds, it has high stability, is insoluble under general conditions, and cannot be hydrolyzed by general proteases. The main industrial methods for degrading and utilizing keratin include physical methods (high-pressure and high-temperature hydrolysis method), chemical methods (acid-breaking method), as well as landfill and incineration. These treatment methods not only have high energy consumption, but also produce a large amount of wastewater and waste gas, which will cause secondary pollution. More importantly, they will also destroy the nutrients in the feathers, resulting in low solubility and animal digestibility of the feather keratin products, which greatly limits the large-scale recycling and processing of feather waste.

[0003] The microbial and enzymatic degradation method is the most economical and effective means for utilizing keratin. At present, more than 30 kinds of microorganisms that can degrade keratin, such as fungi, actinomycetes, and bacteria, have been screened out, with bacteria being the majority. The degradation rate of most keratin-degrading bacteria for feather meal can reach between 50% and 85%, and the degradation time is mostly 2 to 6 days. For example, Streptomyces albidoflavus Fea-10 can completely degrade 1 g / 100 mL of feather keratin in 3 days; the Bacillus subtilis BS8 strain, which can efficiently degrade feathers, can completely degrade a whole feather within 48 h; Xanthomonas DHHJ can completely degrade feathers in about 6 days; Bacillus pumilus WHK4, after fermentation for 48 h, has a feather degradation rate of 85.76%. Bacillus licheniformis is the most reported keratin-degrading bacterium at present. Fungi are mainly Candida and Streptomyces, and actinomycetes are mainly concentrated in the genus Thermomonospora.

[0004] At present, although there are many strains of keratin-degrading bacteria that have been studied, and the degradation rate of the strains for feather keratin is relatively high in a short period of time, most of the bacterial species are single. Most of the important keratin-degrading bacteria are screened and isolated from rotten feathers or the soil where they have been piled up for a long time (most of the degradation rates are obtained by degrading intact feathers), and most of the sources of feather keratin-degrading bacteria are in East China and South China. After comparing the optimization results of the optimal culture conditions, it is found that the optimal initial pH is mostly weakly alkaline, while the soil in the northwest region is relatively alkaline, providing the innate environmental conditions for the growth of the strains. Exploring highly efficient keratin-degrading bacteria with regional specificity is an important way to solve the resource utilization of local feather keratin and is also a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of the above problems, the present invention provides a feather keratin-degrading strain and a screening method, which solve the problems of single keratin-degrading bacterial species, low salt and alkali tolerance of the strain, and lack of regional specificity in the northwest region in the prior art.

[0006] In order to achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] The present invention provides a feather keratin-degrading strain, which is Ochrobactrum intermedium 0123, classified and named as Ochrobactrum intermedium 0123, deposited with the China Center for Type Culture Collection, the deposit address is Wuhan, China - Wuhan University, the deposit number is CCTCC NO: M 20231151, and the deposit date is June 30, 2023. Hereinafter, it is simply denoted as the O strain.

[0008] The present invention also provides a screening method for a feather keratin-degrading strain, which specifically includes the following steps:

[0009] S1: Enrichment culture: Take the supernatant of the soil suspension and place it in an enrichment medium, and culture it in a constant temperature shaking incubator to obtain a culture solution;

[0010] S2: Primary screening culture: Take the culture solution obtained in step S1 and place it in a liquid screening medium, and culture it in a constant temperature shaking incubator to obtain a culture solution;

[0011] S3: Re-screening culture: Take the culture solution obtained in step S2, add it to sterile water for gradient dilution to obtain a dilution solution; spread the dilution solution on a solid screening medium respectively, and culture it in a constant temperature incubator;

[0012] S4: Separation and purification: Observe the growth of bacteria on the solid screening medium. Pick single colonies with different morphologies and perform streak plating on nutrient agar medium, Gause's No. 1 medium, and fungal medium respectively. Incubate them in a constant temperature incubator and observe their growth status. The colonies obtained by streaking the well-grown strains on a slant are the intermediate Ochrobactrum anthropi 0123 that can degrade feather keratin, and store them in the refrigerator.

[0013] Furthermore, in step S1, the volume of the supernatant of the soil suspension is 1 mL, and the volume of the enrichment medium is 25 mL. The components of the medium are: 5.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 1.0 g of dipotassium hydrogen phosphate. Add distilled water to 1000 mL, mix evenly, adjust the pH to 7.4 - 7.6, sterilize at 121 °C under high-pressure wet heat for 20 min. The temperature of the constant temperature shaking incubator is 37 °C, the rotation speed is 160 r / min, and the incubation time is 24 h.

[0014] Furthermore, in step S2, the volume of the culture solution is 1 mL, and the volume of the liquid screening medium is 100 mL. The components are: 0.5 g of sodium chloride, 0.4 g of dipotassium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 10 g of feather meal, 1000 mL of distilled water, adjust the pH to 8.0 - 8.5, sterilize at 121 °C under high-pressure wet heat for 20 min. The temperature of the constant temperature shaking incubator is 37 °C, the rotation speed is 160 r / min, and the incubation time is 24 h.

[0015] Furthermore, in step S3, the volume of the culture solution is 0.1 mL, the volume of the sterile water is 0.9 mL, the dilution factor is 10 -4 -10 -8 times. The components of the solid screening medium are: 0.5 g of sodium chloride, 0.3 g of dipotassium hydrogen phosphate, 0.4 g of potassium dihydrogen phosphate, 10 g of feather meal, 20.0 g of agar, 1000 mL of distilled water, adjust the pH to 8.0 - 8.5, sterilize at 121 °C under high-pressure wet heat for 20 min. The temperature of the constant temperature incubator is 37 °C.

[0016] Further, the components of the beef extract peptone agar medium in step S4 are: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 20.0 g of agar, 1000 mL of distilled water, pH 7.4 - 7.6, sterilized at 121 °C under high-pressure wet heat for 20 min; the components of the Gao's No. 1 medium are: 20.0 g of soluble starch, 1.0 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20.0 g of agar, 1000 mL of distilled water, the pH is adjusted to 7.4 - 7.6, and sterilized at 121 °C under high-pressure wet heat for 20 min; the components of the fungal medium are: 5.0 g of peptone, 2.0 g of yeast extract powder, 20.0 g of glucose, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 20.0 g of agar, 1000 mL of distilled water, sterilized at 121 °C under high-pressure wet heat for 20 min. The culture temperatures of the constant temperature incubator are 37 °C, 30 °C, and 30 °C respectively, and the storage temperature is 5 °C.

[0017] Furthermore, the preparation method of the soil suspension is as follows: Take 2 g of soil sample and place it in a conical flask containing 20 mL of normal saline, and mix it evenly in a constant temperature shaking incubator at 160 r / min for 20 - 30 min to prepare the soil suspension.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The strain obtained by the present invention through enrichment, primary screening, and secondary screening is identified at the morphological and molecular biological levels. This strain is Ochrobactrum intermedium 0123, and its taxonomic name is Ochrobactrum intermedium 0123. By optimizing the culture conditions of the keratin-degrading strain, it is found that the optimal pH of this strain is 8, and the optimal salinity is 0.4 g / L. By measuring the degradation rate of the strain on feather meal keratin under the optimal culture conditions, it is obtained that the degradation rate of this strain within 7 days is 48.90%. The strain screened by the present invention has regional specificity in the northwest region, can provide a suitable strain for the degradation of feather keratin under saline-alkali conditions, and further enriches the strain resource library of feather keratin-degrading bacteria. Description of the Drawings

[0020] Figure 1 It is the appearance morphology diagram of strain O of the present invention;

[0021] Figure 2 It is the Gram staining diagram of strain O of the present invention;

[0022] Figure 3 It is the PCR amplification result diagram of strain O of the present invention;

[0023] Figure 4 Phylogenetic tree of the 16S rDNA sequence of O strain based on the present invention;

[0024] Figure 5 Effect of temperature on the growth of O strain based on the present invention;

[0025] Figure 6 Effect of initial pH on the growth of O strain based on the present invention;

[0026] Figure 7 Effect of salinity on the growth of O strain based on the present invention;

[0027] Figure 8 Degradation rate of O strain for 7 consecutive days based on the present invention;

[0028] In the figure: Different lowercase letters indicate significant differences (P < 0.05). Specific embodiments

[0029] In order to make the objectives and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The culture media and their components in this embodiment:

[0031] Enrichment medium: 5.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 1.0 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, mix evenly, pH 7.4 - 7.6, sterilize at 121 °C under high-pressure wet heat for 20 min;

[0032] Liquid screening medium: 0.5 g of sodium chloride, 0.4 g of dipotassium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 10 g of feather meal, 1000 mL of distilled water, pH 8.0 - 8.5, sterilize at 121 °C under high-pressure wet heat for 20 min;

[0033] Solid screening medium: 0.5 g of sodium chloride, 0.3 g of dipotassium hydrogen phosphate, 0.4 g of potassium dihydrogen phosphate, 10 g of feather meal, 20.0 g of agar, 1000 mL of distilled water, pH 8.0 - 8.5, sterilize at 121 °C under high-pressure wet heat for 20 min;

[0034] Beef extract peptone agar medium: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 20.0 g of agar, 1000 mL of distilled water, pH 7.4 - 7.6, sterilize at 121 °C under high-pressure wet heat for 20 min;

[0035] Gause's No. 1 medium: soluble starch 20.0 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, agar 20.0 g, distilled water 1000 mL, pH adjusted to 7.4 - 7.6, sterilized at 121 °C for 20 min by high-pressure moist heat;

[0036] Fungal medium: peptone 5.0 g, yeast extract powder 2.0 g, glucose 20.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, agar 20.0 g, distilled water 1000 mL, sterilized at 121 °C for 20 min by high-pressure moist heat;

[0037] LB liquid medium: yeast powder 5.0 g, tryptone 10.0 g, sodium chloride 5.0 g, distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 20 min by high-pressure moist heat;

[0038] Fermentation culture medium: dipotassium hydrogen phosphate 1.4 g, potassium dihydrogen phosphate 0.7 g, sodium chloride 0.5 g, magnesium sulfate heptahydrate 0.1 g, feather meal 10 g, sterilized at 121 °C for 20 min by high-pressure moist heat.

[0039] In this example, the soil sample and the feather meal were both provided by Belke Bioproducts Co., Ltd. in Zhangjiachuan Hui Autonomous County, Gansu Province.

[0040] Example 1

[0041] This example provides a feather keratin-degrading strain, which is Ochrobactrum intermedium 0123, classified and named as Ochrobactrum intermedium 0123, deposited with the China Center for Type Culture Collection, the deposit address is Wuhan, China - Wuhan University, the deposit number is CCTCC NO: M 20231151, and the deposit date is June 30, 2023, hereinafter simply denoted as O strain.

[0042] Example 2

[0043] This example provides a method for screening a feather keratin-degrading strain, which specifically includes the following steps:

[0044] S1: Enrichment culture: Prepare a soil suspension. Take 2 g of the soil sample and place it in a conical flask containing 20 mL of normal saline. Mix it evenly in a constant temperature shaking incubator at 160 r / min for 20 min to make a soil suspension; Take 1 mL of the supernatant of the soil suspension and place it in 25 mL of enrichment medium. Culture it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution;

[0045] S2: Primary screening culture: Take 1 mL of the culture solution obtained in step S1 and place it in 100 mL of a liquid screening medium. Incubate it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution;

[0046] S3: Secondary screening culture: Take 0.1 mL of the culture solution obtained in step S2 and add it to 0.9 mL of sterile water for gradient dilution. The dilution factor is 10 -4 -10 -8 times to obtain a dilution solution; Spread the dilution solutions on a solid screening medium and incubate them in a constant temperature incubator at 37 °C;

[0047] S4: Isolation and purification: Observe the growth of bacteria on the solid screening medium. Pick single colonies with different morphologies and perform streak plating on nutrient agar medium, Czapek medium, and fungal medium respectively. Incubate them in a constant temperature incubator at 37 °C, 30 °C, and 30 °C respectively, observe their growth states, and streak the well-grown strains on a slant and store them in a refrigerator at 5 °C.

[0048] Example 3

[0049] This example provides a method for screening feather keratin-degrading strains, which specifically includes the following steps:

[0050] S1: Enrichment culture: Prepare a soil suspension. Take 2 g of soil sample and place it in a conical flask containing 20 mL of normal saline. Mix it evenly in a constant temperature shaking incubator at 160 r / min for 25 min to make a soil suspension; Take 1 mL of the supernatant of the soil suspension and place it in 25 mL of an enrichment medium. Incubate it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution;

[0051] S2: Primary screening culture: Take 1 mL of the culture solution obtained in step S1 and place it in 100 mL of a liquid screening medium. Incubate it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution;

[0052] S3: Secondary screening culture: Take 0.1 mL of the culture solution obtained in step S2 and add it to 0.9 mL of sterile water for gradient dilution. The dilution factor is 10 -4 -10 -8 times to obtain a dilution solution; Spread the dilution solutions on a solid screening medium and incubate them in a constant temperature incubator at 37 °C;

[0053] S4: Isolation and purification: Observe the growth of bacteria on the solid screening medium. Pick single colonies with different morphologies and perform streak plating on nutrient agar medium, Czapek-Dox agar medium, and fungal medium respectively. Incubate them in a constant temperature incubator at 37 °C, 30 °C, and 30 °C respectively, observe their growth status, and streak the strains with good growth on a slant and store them in a 5 °C refrigerator.

[0054] Example 4

[0055] This example provides a method for screening feather keratin-degrading strains, which specifically includes the following steps:

[0056] S1: Enrichment culture: Prepare a soil suspension. Take 2 g of soil sample and place it in a conical flask containing 20 mL of normal saline. Mix it evenly in a constant temperature shaking incubator at 160 r / min for 30 min to make a soil suspension. Take 1 mL of the supernatant of the soil suspension and place it in 25 mL of enrichment medium. Incubate it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution.

[0057] S2: Primary screening culture: Take 1 mL of the culture solution obtained in step S1 and place it in 100 mL of liquid screening medium. Incubate it in a constant temperature shaking incubator at 37 °C and 160 r / min for 24 h to obtain a culture solution.

[0058] S3: Re-screening culture: Take 0.1 mL of the culture solution obtained in step S2 and add it to 0.9 mL of sterile water for gradient dilution. The dilution factor is 10 -4 -10 -8 times to obtain a dilution solution. Spread the dilution solution on the solid screening medium respectively and incubate it in a 37 °C constant temperature incubator.

[0059] S4: Isolation and purification: Observe the growth of bacteria on the solid screening medium. Pick single colonies with different morphologies and perform streak plating on nutrient agar medium, Czapek-Dox agar medium, and fungal medium respectively. Incubate them in a constant temperature incubator at 37 °C, 30 °C, and 30 °C respectively, observe their growth status, and streak the strains with good growth on a slant and store them in a 5 °C refrigerator.

[0060] Through the above screening method, a total of 6 strains that can use feather meal as the sole carbon source and nitrogen source were screened from the soil samples provided by Zhangjiachuan Hui Autonomous County Berke Bioproducts Co., Ltd. The 6 initially screened strains were inoculated on the solid screening medium, and their growth was initially observed. Select 1 strain with better growth and perform streak plating operation to gradually form obvious single colonies. And these strains were respectively inoculated on bacterial, fungal, and actinomycete media to observe the growth status of the strains and initially judge the types of the strains. After preliminary judgment, it was found that the strain is a bacterium.

[0061] (1) Morphological identification: The obtained microorganisms were subjected to Gram staining, and the morphological, size, edge, color, transparency, etc. of the colonies were observed under a microscope. The specific results are shown in Table 1 and the appendix Figure 1-2 .

[0062] Table 1 Morphological identification results of strain O

[0063]

[0064]

[0065] Note: "+" indicates positive and "-" indicates negative

[0066] (2) Molecular biology identification: The genomic DNA of the degrading strain was extracted using the SK8255 (bacteria) kit. Primers were designed and synthesized according to the conserved region sequence in bacterial 16S rDNA, and a PCR amplification experiment was carried out on the extracted genomic DNA. The purification and sequencing of the PCR products were completed by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 2 or Sequence Listing SEQ ID NO:1 - SEQ ID NO:4, and the amplification reaction system and procedure are shown in Tables 3 - 4. The PCR amplification results are shown in the appendix Figure 3 . The test results were analyzed and processed using MEGA11.

[0067] Table 2 Primer sequences for bacterial identification

[0068]

[0069] Table 3 PCR amplification reaction system

[0070] Reagent Volume / (μl) Template (Genomic DNA 20 - 50 ng / μl) 0.5 <![CDATA[10×Buffer(with Mg 2+ )]]> 2.5 dNTP (each 2.5 mM) 1 Enzyme 0.2 F (10 uM) 0.5 R (10 uM) 0.5 <![CDATA[Add double-distilled H2O to]]> 25

[0071] Table 4 PCR amplification reaction procedure

[0072]

[0073] The 16S rDNA sequence of strain O obtained after sequencing by Sangon Biotech (Shanghai) Co., Ltd. was subjected to a homology search in the NCBI system using BLAST. According to the homology comparison results, the 16S rDNA sequences of some similar strains were selected and downloaded. The sequences were aligned using MEGA11 software and a phylogenetic tree based on 16S rDNA was constructed according to the Neighbor - Joining method. The results are shown in the appendix Figure 4 As shown. It can be seen from the figure that strain O is Ochrobactrum (Ochrobactrum genus) and is suspected to be Ochrobactrum intermedium (Intermediate Ochrobactrum).

[0074] (3) Optimization of the growth conditions of the strain

[0075] a. Optimal culture temperature

[0076] Prepare the bacterial solution. Inoculate strain O into LB liquid medium and incubate it with shaking at 37 °C and 150 r / min for 24 h, then take it out. Take 1 mL of the bacterial solution and inoculate it into 50 mL of LB liquid medium. Set three parallels. After inoculation, place it in an incubator at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 50 °C and 150 r / min for 24 h of shaking culture. Use the blank medium as the control group and measure its optical density value at a wavelength of 600 nm.

[0077] Temperature is one of the most important factors affecting the growth, reproduction and survival of microorganisms. Within a certain temperature range, the metabolic activities and growth and reproduction of cells increase with the increase of temperature. When the temperature rises to a certain extent, it begins to have an adverse effect on cells. The proteins, nucleic acids and cell components in microbial cells will be irreversibly damaged. If the temperature continues to rise, the cell function will decline sharply until death. As shown in Figure 5 the appendix, the most suitable culture temperature for strain O is within 30 °C - 40 °C, and the optimal culture temperature is 38 °C. When the temperature reaches 45 °C, the survival rate of the strain drops suddenly, indicating that the strain is not heat-tolerant.

[0078] b. Optimal initial pH

[0079] Take 1 mL of the bacterial solution and inoculate it into 50 mL of LB liquid medium. Set three parallels. Use hydrochloric acid to adjust the initial pH value of the medium so that the initial pH values are 4 - 11 respectively. Incubate it with shaking at 37 °C and 150 r / min for 24 h. Use the blank medium as the control group and measure its optical density value at a wavelength of 600 nm.

[0080] The pH range for microbial growth is extremely wide. For different microorganisms, there is their respective optimal growth pH range. The optimal initial pH range for the growth of strain O is 4 - 9, as shown in Figure 6 the appendix. Among them, the optimal initial pH of strain O is 8, indicating that strain O has the characteristic of being alkali-tolerant.

[0081] c. Optimal salinity

[0082] Prepare LB medium with NaCl as the inorganic salt. After sterilization, dilute it according to the concentration ratio into LB liquid media with salt contents of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2 g / L respectively. Add 1 mL of the bacterial solution to it and incubate it in a constant temperature shaking incubator at 37 °C and 150 r / min for 24 h. Use the LB medium without NaCl as the control group and measure its optical density value at a wavelength of 600 nm.

[0083] NaCl mainly changes the activity of microorganisms by altering the osmotic pressure. As shown in the appendix Figure 7 It can be seen that the optimal salinity for the growth of strain O is 0.4 g / L. Evidently, strain O has a relatively strong salt tolerance ability.

[0084] (4) Determination of the degradation rate of the strain on feather meal keratin

[0085] The isolated and purified strain O was fermented and cultured in the fermentation culture medium. Every day for 7 consecutive days, 1 mL of the bacterial solution and 19 mL of sterile water were respectively taken, and after thoroughly mixing them, the ultraviolet absorption optical density values were measured at wavelengths of 260 nm and 280 nm. By calculating its protein concentration, the degradation rate of the strain on feather keratin can be obtained.

[0086] Protein mass concentration (mg / mL) = (1.45 × A280 - 0.74 × A260) × N

[0087] Where: A280—the absorbance measured for the test solution at 280 nm;

[0088] A260—the absorbance measured for the test solution at 260 nm;

[0089] N—dilution factor.

[0090] Degradation rate = (protein mass concentration of the final bacterial solution - protein mass concentration of the initial bacterial solution) × volume of the bacterial solution / mass of the feather meal.

[0091] In the experiment to explore the degradation rate of the strain on feather meal keratin, the experimental conditions were all the optimal culture conditions of the strain. As shown in the appendix Figure 8 It can be seen that the degradation rate increases with the passage of time. The degradation rate of strain O can reach 48.90% within 7 days.

[0092] The results of the above examples show that the strain obtained through enrichment, primary screening, and secondary screening, identified at the morphological and molecular biological levels, is Ochrobactrum intermedium 0123, classified and named as Ochrobactrum intermedium 0123. The degradation rate of this strain can reach 48.90% within 7 days. In addition, the optimal culture temperature range of this strain is between 30 - 40 °C, the optimal pH is 8, and the optimal salinity is 0.4 g / L. The strain obtained in this study is from the northwest region and has a high alkali tolerance, which is more suitable for the natural soil conditions of this region. Moreover, studies have shown that an alkaline environment is beneficial for keratin-degrading bacteria to produce keratinase.

[0093] In view of this, the strain screened in the present invention has the regional specificity of the northwest region, can provide a suitable strain for the degradation of feather keratin under saline-alkali conditions, and further enriches the strain resource library of feather keratin-degrading bacteria.

[0094] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A feather keratin-degrading strain, characterized in that: The strain is Ochrobactrum intermedium 0123, classified and named as Ochrobactrum intermedium 0123. The preservation unit is the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20231151, and the preservation date is June 30, 2023.

2. A screening method for a feather keratin-degrading strain as described in claim 1, characterized in that: Specifically, it includes the following steps: S1: Enrichment culture: Take the supernatant of the soil suspension and place it in an enrichment medium, and culture it in a constant temperature shaking incubator to obtain a culture solution. S2: Primary screening culture: Take the culture solution obtained in step S1 and place it in a liquid screening medium, and culture it in a constant temperature shaking incubator to obtain a culture solution. S3: Re-screening culture: Take the culture solution obtained in step S2, add it to sterile water for gradient dilution to obtain a dilution solution; spread the dilution solution on a solid screening medium respectively, and culture it in a constant temperature incubator. S4: Isolation and purification: Observe the growth of bacteria on the solid screening medium, pick single colonies with different morphologies and perform streak plating on nutrient agar medium, Gao's No. 1 medium, and fungal medium respectively, and culture them in a constant temperature incubator. Observe their growth status, streak the well-growing strains on a slant, and the cultured colonies are the feather keratin-degrading strain Ochrobactrum intermedium 0123, which is stored in the refrigerator.

3. The screening method of a feather keratin-degrading strain according to claim 2, wherein: In step S1, the volume of the soil suspension supernatant is 1 mL, the volume of the enrichment medium is 25 mL, and the medium components are: 5.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 1.0 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, mix evenly, pH 7.4 - 7.6, sterilize at 121 °C for 20 min. The temperature of the constant temperature shaking incubator is 37 °C, the rotation speed is 160 r / min, and the culture time is 24 h.

4. The screening method of a feather keratin-degrading strain according to claim 2, characterized in that: In step S2, the volume of the culture solution is 1 mL, the volume of the liquid screening medium is 100 mL, and the components are: 0.5 g of sodium chloride, 0.4 g of dipotassium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 10 g of feather meal, 1000 mL of distilled water, pH 8.0 - 8.5, sterilize at 121 °C for 20 min. The temperature of the constant temperature shaking incubator is 37 °C, the rotation speed is 160 r / min, and the culture time is 24 h.

5. The screening method of a feather keratin-degrading strain according to claim 2, wherein: In step S3, the volume of the culture solution is 0.1 mL, the volume of the sterile water is 0.9 mL, the dilution factor is 10 - 4 - 10 - 8 times, and the components of the solid screening medium are: 0.5 g of sodium chloride, 0.3 g of dipotassium hydrogen phosphate, 0.4 g of potassium dihydrogen phosphate, 10 g of feather meal, 20.0 g of agar, 1000 mL of distilled water, pH 8.0 - 8.5, sterilize at 121 °C for 20 min. The temperature of the constant temperature incubator is 37 °C.

6. The screening method of a feather keratin-degrading strain according to claim 2, characterized in that: In step S4, the components of the nutrient agar medium are: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, 20.0 g of agar, 1000 mL of distilled water, pH 7.4 - 7.6, sterilize at 121 °C for 20 min; the components of the Gao's No. 1 medium are: 20.0 g of soluble starch, 1.0 g of KNO3, 0.5 g of K2HPO4 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20.0 g of agar, 1000 mL of distilled water, the pH is adjusted to 7.4 - 7.6, and autoclaved at 121 °C for 20 min; the components of the fungal medium are: 5.0 g of peptone, 2.0 g of yeast extract powder, 20.0 g of glucose, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 20.0 g of agar, 1000 mL of distilled water, autoclaved at 121 °C for 20 min, the culture temperatures of the constant temperature incubator are 37 °C, 30 °C, 30 °C respectively, and the storage temperature is 5 °C.

7. The screening method of a feather keratin-degrading strain according to claim 3, characterized in that: The preparation method of the soil suspension is: take 2 g of soil sample and place it in a conical flask containing 20 mL of normal saline, and mix it evenly in a constant temperature shaking incubator at 160 r / min for 20 - 30 min to prepare the soil suspension.

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