Feather degrading bacterium and application thereof

Abstract

The invention provides a feather degrading bacterium and application thereof, and belongs to the technical field of microorganisms. The feather degrading bacterium provided by the invention is identified as bacillus subtilis NCS630-18, the strain is preserved in the Guangdong Microbial Culture Collection Center, the address is the 5th floor of the building 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, the preservation date is October 10, 2024, and the preservation number is GDMCC NO: 65237. According to the strain provided by the invention, the feather degradation process is more efficient and environment-friendly, a green and sustainable solution is provided for treatment of biomass resources such as feathers, and a new thought and a new method are also provided for development and utilization of related microbial resources.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a feather-degrading bacterium and application thereof. Background Art

[0002] With the development of animal husbandry, feather production, a byproduct of poultry farming and slaughtering, has continued to increase. Feathers contain over 90% keratin, making them a naturally high-protein resource. However, feathers' complex structure, characterized by numerous hydrogen bonds, disulfide bonds, and hydrophobic interactions, makes them difficult to degrade. This characteristic also means that waste feathers, if not handled promptly or properly, can easily produce a foul odor, polluting the environment and wasting protein resources.

[0003] At present, the degradation methods of feathers mainly include physical degradation, chemical degradation, enzymatic degradation and microbial degradation.

[0004] Physical degradation: This involves breaking down the feather structure through mechanical forces, shear forces, or heat treatment. The main purpose of physical degradation is to break the feathers into smaller particles for easier processing.

[0005] Chemical degradation: Using chemical reagents such as acids, alkalis, and oxidants to break down the peptide and disulfide bonds in feather keratin, leading to degradation. However, this method has drawbacks such as disrupting the amino acid structure, producing a single product, and poor product stability.

[0006] Enzymatic degradation: This method uses enzymes such as keratinase to degrade feathers. Enzymatic degradation has mild reaction conditions and is environmentally friendly, but the enzymes are expensive and have limited degradation efficiency.

[0007] Microbial degradation method: feather degradation is achieved through the joint action of a certain microorganism or multiple microorganisms. The microbial degradation method has the advantages of mild reaction conditions, no environmental pollution, low damage rate of the obtained product and high product utilization rate. The prior art CN103642735A discloses a keratinase-producing Bacillus pumilus and its application method, which discloses a keratinase-producing strain Bacillus pumilus. Wool is used as a fermentation substrate and fermented at 35°C and 220r / min for 48 hours. The keratinase activity is 214.5U / mL; CN105112344A discloses a keratinase-producing Bacillus parabrevis and its application method, which discloses a keratinase-producing strain Bacillus parabrevis. acillus parabrevis), using wool as the fermentation substrate, fermented under fermentation conditions of 40°C and 220 r / min for 16 hours, and the keratinase activity reached 412 U / mL; CN107699553A "An alkaline keratinase KerT derived from Bacillus amyloliquefaciens and its dehairing use" discloses a keratinase KerT derived from Bacillus amyloliquefaciens CGMCCNO.11218, which achieved a keratinase activity of 445.37 U / mL after shake flask fermentation in a culture medium containing feathers for 48 hours. It can be seen that the keratinase activity of the strain fermentation broth provided by the prior art is around 400 U / mL. Feathers are rich in protein and amino acids and are a potential high-quality protein resource. However, the keratinase activity produced by existing strains is low, and the feather decomposition efficiency is not ideal. Therefore, it is particularly important to provide a more efficient and environmentally friendly feather-degrading bacterium. Summary of the Invention

[0008] The purpose of the present invention is to provide a feather-degrading bacterium to improve feather degradation efficiency and more fully utilize protein and amino acid resources in feathers.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a feather-degrading bacterium, characterized in that the feather-degrading bacterium is Bacillus subtilis NCS630-18, which is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: October 10, 2024, deposit number: GDMCC NO: 65237.

[0011] The present invention also provides an application of the feather-degrading bacteria in degrading feathers.

[0012] The present invention also provides a method for degrading feathers using the feather-degrading bacteria, which is characterized in that the feather-degrading bacteria are inoculated into a feather culture medium and fermented to achieve feather degradation and production of keratinase.

[0013] Preferably, the feather culture medium uses feathers as the sole carbon and nitrogen source, and the concentration of feathers is 18-22 g / L.

[0014] Preferably, the inoculum amount of the feather degrading bacteria is 200-250ul, and the concentration is OD 600 =0.6~0.8.

[0015] Preferably, the fermentation temperature is 28-40° C., the fermentation time is 130-140 hours, and the rotation speed is 180-200 r / min.

[0016] Preferably, the pH of the feather culture medium is 6.5-7.5.

[0017] The present invention also provides a fermentation liquid containing keratinase prepared by the method.

[0018] Preferably, the activity of keratinase in the fermentation broth is 1117.5 U / mL.

[0019] The present invention also provides an application of the fermentation liquid in degrading feathers.

[0020] The present invention successfully isolated a strain of Bacillus subtilis NCS630-18 from deep-sea sediments, which is highly effective in feather degradation. This strain secretes a highly effective keratinase enzyme, effectively breaking down the keratin component of feathers. The present invention comprehensively optimized the enzyme production conditions of the strain. Under conditions of a feather concentration of 20g / L, a fermentation temperature of 37°C, an initial pH of 7.0, and without the addition of additional nitrogen and carbon sources, the strain achieved higher keratinase production and degradation activity, making the feather degradation process more efficient and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the standard curve of tyrosine;

[0022] Figure 2 This is a diagram of the separation and purification of the strain in the primary screening culture medium;

[0023] Figure 3 This is a graph showing the effect of different feather concentrations on enzyme production of the strain in Example 1;

[0024] Figure 4 This is a diagram showing the effect of different temperatures on enzyme production of the strain in Example 2;

[0025] Figure 5This is a diagram showing the effect of different pH values ​​on enzyme production by the strain in Example 3;

[0026] Figure 6 This is a graph showing the effect of different carbon sources on enzyme production of the strain in Example 4;

[0027] Figure 7 This is a diagram showing the effect of different nitrogen sources on enzyme production of the strain in Example 5.

[0028] Preservation Instructions

[0029] Bacillus subtilis NCS630-18, the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the deposit date is: October 10, 2024, and the deposit number is: GDMCC NO: 65237. DETAILED DESCRIPTION

[0030] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Experimental materials, reagents, and instruments

[0033] 1.1 Experimental Materials

[0034] Deep-sea sediment samples were obtained from the deep-sea sediments of the Hippocampus cold seep;

[0035] The feathers were obtained from chickens in Ronghua Vegetable Market, cleaned with distilled water, and dried in an oven at 60°C to constant weight before use.

[0036] 1.2 Preparation of experimental culture medium and reagents

[0037] All culture media were prepared with distilled water and sterilized by moist heat sterilization at 121°C for 20 minutes. (Pre-screened milk culture media and liquid milk culture media were sterilized at 115°C for 15 minutes.)

[0038] Table 1 Culture medium preparation table

[0039]

[0040]

[0041] 2. Screening of feather-degrading bacteria

[0042] 2.1 Preliminary screening of feather-degrading bacteria

[0043] The deep-sea sediment sample was weighed and diluted with artificial seawater at a mass-to-volume (M / V) ratio of 1:10 to prepare a sediment suspension. 1 mL of the sediment suspension was then pipetted into 25 mL of enrichment medium and mixed thoroughly. The culture tube was placed on a shaker and incubated at 30°C and 150 rpm for 48 h. The culture solution was then graded diluted with sterile distilled water (10 -1 ~10 -6 ), vortex and mix well, then spread on milk plate culture medium, culture at 30℃ for 24 hours and observe, and select the strain that can produce transparent bacterial ring on milk plate culture medium (such as Figure 2 The strain was isolated and purified according to the above method until a single colony was obtained, and finally 83 monoclonal strains were obtained. At the same time, the isolated and purified single colony was inoculated into a 40% glycerol tube and stored in a -80°C refrigerator.

[0044] 2.2 Feather-degrading bacteria rescreening

[0045] A single colony isolated and purified from the initial screening of feather-degrading bacteria in 2.1 was expanded using liquid milk culture plates at 30°C and 200 rpm for 48 hours to obtain an expanded culture. Subsequently, 1 mL of the expanded culture was inoculated into a secondary screening medium. Culture was continued at the same temperature and speed for 30 days, and feather degradation was observed. The results showed that 71 secondary screening cultures showed varying degrees of feather degradation: 3 cultures showed complete feather degradation; 3 cultures showed a small amount of incomplete feather degradation; and 6 cultures showed no feather degradation at all.

[0046] 2.3 Determination of keratinase activity

[0047] The enzyme activity of the strains whose feathers were completely degraded in the three rescreened culture media in part 2.2 and the strains whose feather fermentation liquid still had a small amount of feathers that were not completely degraded in the three rescreened culture media were selected. The details are as follows:

[0048] The method for determining enzyme activity was carried out according to the Chinese industry standard for determining protease activity and the method for determining keratinase activity in reference to related studies, with slight modifications. Specifically, the substrate in the original reaction system was replaced with soluble keratin. Operationally, 0.1000 g of tyrosine that had been dried to a constant weight was first accurately weighed, and 100 mL of distilled water was added to prepare a 1000 μg / mL tyrosine solution. 10 mL of the 1000 μg / mL tyrosine solution was then pipetted to a fixed volume of 100 mL, and then diluted to 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL tyrosine solutions of different concentrations, respectively. Each group of experiments was run in triplicate, and the absorbance at a wavelength of 275 nm was then measured using a UV spectrophotometer. The concentration of tyrosine was used as the horizontal axis, and OD 275 The value is used as the vertical axis to draw the standard curve (such as Figure 1 shown).

[0049] 200 μL of fermentation supernatant from 6 rescreened culture media was diluted 50 times with 10 mL of distilled water, and 2 mL was aspirated and mixed with 3 mL of 2% casein substrate at pH 7.2, and reacted at 40°C for 10 minutes. After the reaction was completed, 5 mL of 0.4 mol / L trichloroacetic acid was immediately added, the reaction solution was filtered with filter paper, and the absorbance at 275 nm was detected with a UV spectrophotometer. In the control experiment, the experimental procedure was consistent with the conventional operation, except that trichloroacetic acid was added before the introduction of casein to inactivate the enzyme, and then the casein was added. The keratinase activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μg of tyrosine from casein per minute at 40°C.

[0050] 2.4 Identification of strains by sequencing 16s rDNA sequences

[0051] Crude bacterial DNA was extracted from single colonies using a DNAChelex-100 resin-based cell wall disruption method. After 10 minutes of filtration in a metal bath, the supernatant was centrifuged at low speed for 10 minutes. Universal primers for the 16S rRNA gene (27F and 1522R) were added to the supernatant. PCR reaction conditions were set to amplify the 16S rRNA gene of the strain. The PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3. The PCR amplification products were electrophoresed (110V, 30 minutes) using a UV transilluminator to observe the electrophoretic bands. After ensuring that the samples were qualified, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The resulting gene sequences were then compared against the NCBI database to confirm the strain species.

[0052] Table 2 PCR reaction system

[0053]

[0054]

[0055] Table 3 PCR reaction program

[0056] step time Pre-denaturation 95℃ 7min Denaturation 94℃ 1min Annealing 58℃ 45s Extension 72°C 1 minute 30 seconds Final extension 72°C 10min

[0057] Results: Through enrichment culture, primary screening, purification and re-screening culture, strains with feather degradation effect were screened out, and the activity of keratinase in the fermentation broth was measured. A strain with high efficiency in feather degradation was screened out from deep-sea sediments. The strain was identified as Bacillus subtilis, numbered NCS630-18, with an enzyme activity of 111.75U / mL by 16S rRNA species identification.

[0058] Table 4 Degradation effect of each strain, feather effect and enzyme activity

[0059]

[0060] Example 2

[0061] Optimization of keratinase production conditions

[0062] The NCS630-18 strain with the highest keratinase activity obtained by screening with milk medium and rescreening medium was selected, and the single-factor conditions for enzyme production of the strain were optimized. The initial fermentation medium was milk medium containing feathers, the feather concentration was 10g / L, the culture conditions were 30℃, 200r / min, the inoculation volume of the strain was 200μL, and the concentration was OD 600 =0.7, liquid volume 25mL, fermentation time 132 hours. This study investigated the effects of various factors on the strain's ability to produce keratinase by varying a single variable. Three parallel sets of experiments were conducted for each condition. After fermentation, the keratinase activity of each fermentation broth was measured, and statistical analysis was performed to determine the optimal enzyme production conditions. The details are as follows:

[0063] (1) Effect of feather concentration on enzyme production

[0064] Weigh 0.125g, 0.250g, 0.375g, 0.5g, and 0.625g of feathers after constant weight, respectively, and add them into 25mL of rescreened culture medium for fermentation. The optimal feather concentration is determined according to the enzyme activity.

[0065] The results are as follows Figure 3As shown, differences in each column of data are indicated by lowercase letters, and differences between different letters indicate highly significant statistical differences (p < 0.001). Experimental data show that keratinase activity increases with increasing feather content, indicating that the presence of feathers effectively promotes keratinase production in the strain. At a feather concentration of 20 g / L, the enzyme activity peaked at 92.45 U / mL. Decreased keratinase activity above 20 g / L may be related to the excessive addition of feathers to the culture medium, which increases the viscosity of the fermentation broth and leads to a decrease in the relative proportion of water and dissolved oxygen levels. Therefore, the optimal feather concentration for enzyme production by this strain was determined to be 20 g / L.

[0066] (2) Effect of fermentation temperature on enzyme production

[0067] Fermentation was carried out at different temperatures of 28°C, 30°C, 37°C and 40°C, and the most suitable fermentation temperature was determined by measuring the enzyme activity in the rescreened culture medium.

[0068] The results are as follows Figure 4 The data in each column are shown in lowercase letters, and the differences between different letters represent extremely significant statistical differences (p < 0.001). Experimental data showed that the enzyme activity of the strain reached a maximum of 87.79 U / mL when fermented at 37 degrees Celsius. The experimental results show that the strain has the highest efficiency in producing keratinase at 37 degrees Celsius.

[0069] (3) Effect of initial pH on enzyme production

[0070] The initial pH values ​​were set to 5, 6, 7, 8, 9 and 10, and the optimal initial pH conditions were determined by comparing the activity of the enzymes in the rescreened culture medium at each pH value.

[0071] The results are as follows Figure 5 As shown, differences in each column of data are indicated by lowercase letters, and differences between different letters represent extremely significant statistical differences (p < 0.001). The initial pH range is between 5 and 9. At an initial pH of 7.0, the enzyme activity of the strain is 80.34 U / mL. Experimental data show that the enzyme activity is low at initial pH 5 and pH 6. As the pH value exceeds 7, the enzyme activity decreases significantly with increasing pH. Based on these results, the optimal initial pH for enzyme production by this strain is determined to be 7.0.

[0072] (4) Effect of external carbon source on enzyme production

[0073] The cells were cultured in a basic salt solution (1.4 g / L dipotassium hydrogen phosphate, 0.7 g / L potassium dihydrogen phosphate, 5 g / L sodium chloride, 0.1 g / L magnesium sulfate, pH 7.0) using 10 g / L glucose, sucrose, potato starch, and feathers as carbon sources, respectively. After a certain period of culture, the keratinase activity between the different carbon source culture media was compared, and the optimal carbon source was determined based on the enzyme activity.

[0074] The results are as follows Figure 6 The data in each column are shown in lowercase letters, and differences between different letters indicate highly significant statistical differences (p < 0.001). The addition of potato starch and glucose did not significantly affect keratinase activity. In contrast, the addition of sucrose decreased keratinase activity, likely because the strain preferentially utilizes sucrose as a carbon source, thereby reducing feather degradation. Based on this finding, the strain does not require an additional carbon source for keratinase production.

[0075] (5) Effect of external nitrogen source on enzyme production of strains

[0076] The experimental strains were cultured in a basic salt solution (1.4 g / L dipotassium hydrogen phosphate, 0.7 g / L potassium dihydrogen phosphate, 5 g / L sodium chloride, 0.1 g / L magnesium sulfate, pH 7.0) with 10 g / L casein and skimmed milk powder as nitrogen sources, respectively. After a certain period of culture, the supernatant of the bacterial solution was collected and the enzyme activity in the supernatant was measured to determine the optimal nitrogen source by comparing the enzyme activity of the bacterial solution in the groups with different carbon sources.

[0077] The results are as follows Figure 7 The data in each column are shown in lowercase letters. Data with different letters indicate highly significant statistical differences (p < 0.001). The experimental data show that the addition of skim milk powder and casein significantly reduced keratinase activity. This nitrogen source may be preferentially utilized by the strain, reducing the strain's use of feathers for keratinase production. Therefore, the strain does not require the addition of other nitrogen sources for keratinase production.

[0078] From the above examples, it can be seen that the effects of feather concentration, fermentation temperature, initial pH, carbon source, and nitrogen source on the enzyme production of the strain were studied through a single-factor enzyme production condition optimization experiment. The results showed that when the feather concentration was 20 g / L, the enzyme activity was as high as 91.45 U / mL; when the strain was fermented at 37°C, the enzyme activity was as high as 89.25 U / mL; under the condition of an initial pH of 7.0, the enzyme activity of the strain was 81.6 U / mL. When the strain was not added with a carbon source or a nitrogen source, the enzyme activity was as high as 81.6 U / mL, indicating that no other carbon source needs to be added for enzyme production.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A feather-degrading bacterium, characterized in that: The feather-degrading bacteria is Bacillus subtilis NCS630-18, which is deposited in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the deposit date is: October 10, 2024, and the deposit number is: GDMCC NO: 65237.

2. Use of the feather-degrading bacteria according to claim 1 in degrading feathers.

3. A method for degrading feathers using the feather-degrading bacteria according to claim 1, characterized in that: The feather-degrading bacteria are inoculated into a feather culture medium and fermented to achieve feather degradation and production of keratinase.

4. The method according to claim 3, wherein The feather culture medium uses feathers as the only carbon and nitrogen source, and the concentration of feathers is 18-22 g / L.

5. The method according to claim 4, wherein The inoculum volume of the feather degrading bacteria is 200-250ul, and the concentration is OD 600 =0.6~0.

8.

6. The method according to claim 5, wherein The fermentation temperature is 28-40° C., the fermentation time is 130-140 hours, and the rotation speed is 180-200 r / min.

7. The use according to claim 6, characterized in that The pH of the feather culture medium is 6.5-7.

5.

8. A fermentation broth containing keratinase prepared by the method according to any one of claims 3 to 7.

9. The fermentation broth according to claim 8, wherein The activity of keratinase in the fermentation broth is 1117.5 U / mL.

10. Use of the fermentation liquid according to claim 8 or 9 in degrading feathers.

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