Pichia kudriavzevii capable of degrading feather meal

Abstract

The invention discloses pichia kudriavzevii capable of degrading feather meal, and belongs to the technical field of application of microorganisms in agriculture and animal husbandry. The strain is preserved in China Center for Type Culture Collection of Microorganisms, the address is China Center for Type Culture Collection, Wuhan University, Wuhan, Hong Mountain area, Wuhan, Hubei Province, the preservation date is April 24, 2025, the preservation number is CCTCC M 2025876, and the strain is classified and named as Pichia kudriavzevii Ker01. The bacterial strain can degrade feather meal keratin through a fermentation method and increase the small peptide content of feather meal, so that the feather meal is promoted to be used as a protein feed raw material, and the bacterial strain is a probiotic with high application value and has important significance on development of animal husbandry and feed fermentation industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and livestock microbial application technology, specifically relating to a biodegradable feather meal Pichia kudriavzevii Ker01 strain. Background Technology

[0002] Corn and soybean meal-based diets are a traditional component of my country's dietary formulas. However, due to issues such as the reduction of arable land per capita, the competition between humans and livestock for grain is severe, leading to a long-standing reliance on soybean imports. Statistics show that my country imported 105 million tons of soybeans in 2024, accounting for 66% of total grain imports. In recent years, influenced by the international situation, international soybean meal prices have risen rapidly and remained unstable, posing challenges to the development of my country's livestock industry. my country's poultry farming industry has developed rapidly, with poultry meat and egg production increasing year by year. In 2024, the total number of poultry slaughtered reached 17.34 billion, a 3.1% increase over the previous year. Feathers account for approximately 5% to 7% of a poultry's total weight and are a major waste product in poultry farming, generating a large amount of waste feathers annually. Improper disposal can cause serious environmental pollution. Studies show that feathers contain about 85% crude protein and are rich in various amino acids, but due to their high keratin content, they are difficult for animals to digest and utilize. Studies have found that after microbial fermentation, the keratin in feather meal decomposes into many small peptides, creating a novel protein feed resource. This increases the utilization value of feather meal and helps advance the process of reducing and replacing soybean meal. Therefore, fully developing and utilizing feather meal can not only effectively reduce and replace soybean meal and alleviate grain pressure, but also, to some extent, solve the environmental pollution problem caused by discarded feathers. Summary of the Invention

[0003] Based on the actual problems and needs in the above-mentioned livestock breeding production process, the present invention provides a biodegradable feather meal Pichia kudriavzevii Ker01 strain. This strain can degrade the keratin in feather meal, increase its acid-soluble protein content, and increase the yield of small peptides.

[0004] The objective of this invention is achieved through the following technical solution: a biodegradable feather meal Pichia kudriavzevii Ker01 strain, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province, 430072, China, with a deposit date of April 24, 2025, accession number CCTCC M 2025876, and classified as Pichia kudriavzevii Ker01. The colonies are oval, with a raised, wrinkled surface and irregular, serrated edges. The cells are opaque and milky white, and under a microscope, they appear ellipsoidal or rod-shaped.

[0005] Furthermore, the growth conditions of *Kudriazwibichthys* as described above are as follows: temperature 30-40℃, pH 3-7, and NaCl concentration 1%-4%.

[0006] Furthermore, the preferred temperature is 37°C and the pH is 5.

[0007] Furthermore, the cultivation method of *Pichia pastoris* as described above is as follows: Take OD... 600 =1.0 seed culture was inoculated into the culture medium at a volume ratio of 1%, and cultured on a shaker at a temperature of 37℃, pH=5, and 220r / min.

[0008] Furthermore, the culture medium used is YPD liquid culture medium.

[0009] Another object of the present invention is to provide the application of the above-described strain of biodegradable feather powder, *Kudriazwibichthys*, as a degrading agent in the degradation of keratin in feather powder.

[0010] Furthermore, as described above, the method is as follows: after sterilizing the feather powder at 121°C for 15 minutes, it is placed in a vacuum-sealed fermentation bag containing a one-way breathing valve, with an inoculum content of 6%, a moisture content of 55%, a fermentation temperature of 38°C, and a fermentation time of 7 days.

[0011] Advantages and beneficial effects of the present invention: The biodegradable feather meal yeast provided by the present invention can degrade keratin in feather meal, increase its acid-soluble protein content and increase the yield of small peptides. After solid-state fermentation using this strain, the acid-soluble protein content of feather meal is 13.24% and the yield of small peptides reaches 14.88%, which are 186.46% and 165.52% higher than before fermentation, respectively. This effectively solves the problem of the difficulty in utilizing keratin in feather meal, promotes the use of feather meal as a protein feed raw material, develops new protein feed resources and realizes the reduction and substitution of soybean meal. Attached Figure Description

[0012] Figure 1 This is a colony morphology diagram of strain Ker01;

[0013] Figure 2 This is a microscopic image of the cell morphology of strain Ker01 under an optical microscope.

[0014] Figure 3 Phylogenetic tree diagram of strain Ker01;

[0015] Figure 4 The growth curve of strain Ker01;

[0016] Figure 5The graph shows the growth of strain Ker01 at different temperatures;

[0017] Figure 6 This is a graph showing the growth of strain Ker01 under different pH conditions.

[0018] Figure 7 The figure shows the growth of strain Ker01 under different NaCl concentrations.

[0019] Figure 8 This is a diagram showing the growth of strain Ker01 under different bile salt concentrations. Detailed Implementation

[0020] The following embodiments provide a more detailed description of the present invention. However, it should be understood that the specific embodiments described herein are only for illustrating and explaining the purpose, technical solutions, and advantages of the present invention, and do not constitute any limitation on the present invention.

[0021] Test materials: provided by the Institute of Animal Nutrition, Northeast Agricultural University; Lindian chicken manure was collected from the Acheng Experimental Base of Northeast Agricultural University.

[0022] Example 1:

[0023] The specific steps for strain screening and identification are as follows:

[0024] 1. Enrichment and initial screening of strains: Accurately weigh 1.00g of fecal sample, add 9ml of sterile physiological saline, shake in a shaker at 37℃ for 30min, take 2ml of supernatant and add it to 100ml of enrichment medium, and enrich incubate in a shaker at 37℃ and 200rpm for 3-10 days. Observe the feather degradation in the enrichment medium. When feather degradation is obvious, perform serial dilutions of the enrichment solution and spread it on feather powder plates (10... -7 10 -8 10 -9 The culture was incubated upside down at 37℃ for 3 days. Strains with different morphologies were picked and purified by streaking on YPD plates. Single colonies of the purified culture were then incubated in YPD liquid for 24 hours, transferred to YPD plates, and streaked twice. The morphology of the strains on the two plates was observed to ensure consistency; if identical, purification was successful. The strains were then inoculated onto milk agar and feather meal plates using a disposable inoculation loop, and the presence of hydrolysis zones was observed.

[0025] Select strains with obvious hydrolysis and measure the diameter of the hydrolysis zone.

[0026] 2. Re-screening and preservation of strains: Purified strains with obvious hydrolysis zones obtained from the initial screening were selected, and seed culture was prepared. This seed culture was inoculated into liquid fermentation medium at a 10% inoculum concentration and fermented at 37℃ and 220 rpm for 72 h on a shaker. After 72 h, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min. The supernatant was the crude enzyme solution. The enzyme activity of the crude enzyme solution was measured, and strains with high enzyme activity were selected for identification. The screened strains were inoculated into slant test tubes and stored at 4℃. Additionally, the strain seed culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80℃ for long-term preservation.

[0027] 3. Determination of keratinase activity: Add 50 μL of crude enzyme solution, 150 μL of pH 8.0 buffer, and 100 μL of 2.5% soluble keratin solution sequentially to an EP tube. Vortex to mix, and incubate at 40℃ for 10 min. Stop the reaction by adding 200 μL of 4% trichloroacetic acid (TCA), vortex to mix, and let stand for 5 min. Centrifuge at 8000 rpm for 5 min. Take 200 μL of the supernatant and add 1 ml of 4% Na2CO3 and 200 μL of Folin-Ciocalteu sequentially. Incubate at 40℃ for 10 min for color development. After cooling to room temperature, measure the OD. 660 In the control group, TCA was added to terminate the reaction for 10 minutes before adding the crude enzyme solution; the remaining procedures were the same as in the experimental group. The experiment was repeated three times, and the average value was taken. OD per unit time 660 Each increase of 0.01 is defined as one keratinase activity unit.

[0028] 4. Fourteen strains exhibiting distinct hydrolysis zones on feather meal plates during the initial screening were selected. Crude enzyme solutions were prepared to determine their keratinase activity. The keratinase activities of the secondary screening strains are shown in Table 1. Strain E showed the highest keratinase activity at 35.16 U / ml. Therefore, strain E was selected as the final screening strain and named Ker01.

[0029] Table 1. Keratinase activity of strains

[0030]

[0031] Note: Data are expressed as mean ± standard error, n = 3.

[0032] 5. Strains Identification: 1) Morphological Examination: Physiological examination: The purified strain was inoculated onto YPD solid medium and cultured at 30℃ for 24 hours. Colony morphology, size, color, moisture content, transparency, edge morphology, and surface wrinkles were observed. Microscopic morphological examination: A drop of sterile water was placed on a clean glass slide. A single colony was picked up with an inoculation loop and spread on the sterile water. A drop of diluted Lugol's iodine solution was added to one side of the slide for staining. Cell morphology was observed under a light microscope. 2) Physiological and Biochemical Identification: Referring to the characteristics and identification manuals for yeasts and fungi, the screened strains underwent physiological and biochemical identification, including sugar fermentation tests, carbon source assimilation tests, nitrogen source assimilation tests, amyloid compound formation tests, urease tests, and catalase tests. 3) Molecular Biological Identification: Finally, the ITS gene sequence of the strains was determined. A phylogenetic tree was constructed using MEGA11 to identify the species of the screened strains.

[0033] 6. The biological characteristics of the selected strains are as follows: Figure 1 and 2 As shown, the colonies are oval, with a raised and wrinkled surface and irregular, serrated edges. The bacteria are opaque and milky white, and their microscopic morphology is either ellipsoidal or rod-shaped.

[0034] 7. The physiological and biochemical identification results of the strain are shown in Table 2-5. The results indicate that strain Ker01 has good utilization of glucose, sucrose, and cellobiose, and can utilize soluble starch, lactose, xylose, and glycerol. Strain Ker01 cannot use nitrates and urea as carbon sources.

[0035] Table 2. Physiological and biochemical identification results of strain Ker01

[0036]

[0037] Note: "+" indicates positive, and "-" indicates negative.

[0038] Table 3. Sugar fermentation results of strain Ker01

[0039]

[0040] Note: “acid” indicates acid production, “0” indicates gas production, “+” indicates both acid and gas production, and “-” indicates no change.

[0041] Table 4 Results of carbon source assimilation test of strains

[0042]

[0043] Note: "+" indicates that it can be used, "-" indicates that it cannot be used, and the more "+" signs there are, the better the effect of using it.

[0044] Table 5 Results of nitrogen source assimilation test of strains

[0045]

[0046] Note: "+" indicates that it can be used, "-" indicates that it cannot be used, and the more "+" signs there are, the better the effect of using it.

[0047] 7. The strain identification process and results are as follows: The screened strains were identified using ITS sequencing, and the ITS primer information is shown in Table 6. Then, the strain sequences were compared with the NCBI database using the Blast program to determine the species. Figure 3 As shown, a phylogenetic tree of the strains was drawn.

[0048] Table 6 ITS Primer Sequences

[0049]

[0050] Based on morphological observation, physiological and biochemical reactions, and ITS sequencing molecular identification, the strain with the ability to inhibit the growth of pathogenic bacteria was identified as Pichia kudrica.

[0051] Example 2:

[0052] Growth characteristics analysis of strain Ker01

[0053] 1. Seed culture preparation: A single colony of Ker01 was picked and inoculated into YPD liquid medium and cultured at 37℃ and 220 r / min for 24 h to obtain the seed culture. The seed culture was adjusted to OD using sterilized YPD liquid medium. 600 =1.0 is reserved for later use.

[0054] 2. Growth Curve Determination: The prepared seed culture was inoculated into 5 mL of YPD liquid medium at an inoculum rate of 1% and cultured at 37℃ and 220 r / min for 24 h. The inoculated seed culture was then placed in an incubator for further cultivation, and samples were taken every 2 h to determine the OD of each culture. 600 The values ​​were used to plot the growth curve of the strain from 0 to 24 hours.

[0055] 3. Determination of optimal growth temperature: The prepared seed culture was inoculated into 5 mL of YPD liquid medium at an inoculum volume of 1%. The incubator temperature was adjusted to 20℃, 30℃, 37℃, 45℃, 50℃ and 55℃, and the incubation speed was 220 r / min for 6 h. The OD of each culture was measured. 600 The absorbance values ​​were used to analyze the growth of the selected strains under different temperature conditions.

[0056] 4. Determination of optimal growth pH: The initial pH of the YPD liquid medium was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1 mol / L HCl and 1 mol / L NaOH. The prepared seed culture was inoculated into 5 mL of YPD liquid medium at a 1% inoculum and cultured at 37℃ and 220 rpm for 6 h. The OD of each culture was measured. 600 The absorbance values ​​were used to analyze the growth of the selected strains under different pH conditions.

[0057] 5. Test results are as follows Figure 4-6 As shown.

[0058] 6. The results showed that the growth of the strain followed an S-shaped curve, entering the logarithmic growth phase after 4 hours and the stationary phase after 12 hours. Growth capacity experiments showed that 37℃ was the optimal temperature for growth; before 37℃, the growth capacity gradually increased with rising temperature; above 37℃, the growth capacity gradually decreased with increasing temperature, with 30℃-40℃ being the most suitable temperature range for growth. Furthermore, the strain can...

[0059] It can be grown under conditions of pH 3-7, with the optimal pH being 5.

[0060] Example 3

[0061] Analysis of growth characteristics of strain Ker01 under different concentrations of NaCl

[0062] 1. Activation of the strain: The experimental procedure is the same as in Example 3;

[0063] 2. Growth determination of the strain under corresponding NaCl conditions: The activated bacterial culture was inoculated at a rate of 1% into YPD liquid medium containing 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, and 9% (w / v) NaCl. An uninoculated culture medium of the same type served as a control. The cultures were incubated at 37℃ and 220 rpm for 3 hours. After incubation, the OD of the cultures was measured. 600 The nm value was determined, and 100 μL of the dilution was inoculated onto a solid culture medium and incubated at 37°C for 12 h to determine the growth of the selected strain.

[0064] 3. For example Figure 7 The results of the strain growth ability experiment are shown in the figure.

[0065] 4. The results of strain growth show that the strain has a certain tolerance to NaCl. As the NaCl concentration increases, the strain's growth ability decreases. When the sodium chloride concentration exceeds 5%, the strain almost stops growing.

[0066] Example 4

[0067] Growth characteristics of strain Ker01 under different concentrations of bile salts

[0068] 1. Seed liquid preparation: The experimental procedure is the same as in Example 2;

[0069] 2. Growth assay of the strain under corresponding bile salt conditions: Activated bacterial culture was inoculated at a rate of 1% into YPD liquid medium containing 0%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 2%, and 3% (w / v) bile salts. An uninoculated culture medium of the same type served as a control. The cultures were incubated at 37℃ and 220 rpm for 3 hours. After incubation, the OD of the cultures was measured. 600 The nm value was determined, and 100 μL of the dilution was inoculated onto a solid culture medium and incubated at 37°C for 12 h to determine the growth of the selected strain.

[0070] 3. For example Figure 8 The results of the strain growth ability experiment are shown in the figure.

[0071] 4. The results of the strain growth capacity experiment showed that the strain grew well without the addition of bile salts. When the concentration of bile salts exceeded 0.3%, the strain's growth capacity decreased, and it could hardly grow at all.

[0072] Example 5

[0073] Strain Ker01's ability to degrade feather meal keratin through solid-state fermentation

[0074] 1. Seed liquid preparation: The experimental procedure is the same as in Example 2;

[0075] 2. Solid-state fermentation: After autoclaving at 121℃ for 15 minutes, Ker01 seed liquid with a water content of 55% was inoculated at a rate of 6%. The sample was placed in a vacuum-sealed fermentation bag with a one-way breathing valve and fermented in a constant temperature and humidity incubator at 38℃ for 7 days. After 7 days, the bag was removed, the sample was dried at 65℃ for 72 hours, pulverized and passed through a 40-mesh sieve for the determination of acid-soluble proteins in the sample.

[0076] 3. Determination of acid-soluble protein content: The content of small peptides in the sample was determined using the trichloroacetic acid method. Accurately weigh 3.00 g of sample, add 15 ml of 15% TCA, mix well, and let stand for 5 min. Then add 25% TCA, mix well, and let stand for 20 min. Centrifuge at 4000 rpm for 10 min. Filter the supernatant, accurately pipette 10 ml of the filtrate into a digestion tube, add sulfuric acid and catalyst, and digest in a digestion furnace. Analyze using a Kjeldahl nitrogen analyzer.

[0077] Determine its acid-soluble protein content

[0078] Small peptide yield % = acid-soluble protein content (g) / crude protein content (g) * 100%;

[0079] 4. The experimental results are shown in Table 7. After solid-state fermentation with strain Ker01, the acid-soluble protein content of the feather meal was 13.24%, and the yield of small peptides reached [missing information].

[0080] The percentages were 14.91%, representing increases of 173.70% and 165.39% respectively compared to before fermentation.

[0081] Table 7. Solid-state fermentation keratin degradation ability of strain Ker01

[0082]

[0083]

[0084] Note: Data values ​​are expressed as mean ± standard error, n = 6.

Claims

1. A biodegradable feather meal Pichia kudriavzevii Ker01 strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province, 430072, China. The deposit date is April 24, 2025, the accession number is CCTCC M 2025876, and the classification name is Pichia kudriavzevii Ker01.

2. The *Pichia kudrica* strain of biodegradable feather meal according to claim 1, characterized in that: The growth conditions are as follows: temperature 30-40℃, pH 3-7, NaCl concentration 1%-4%.

3. The *Pichia kudrica* strain of biodegradable feather meal according to claim 1, characterized in that: The optimal temperature for growth is 37°C, and the pH value is 5.

4. The method for culturing a strain of biodegradable feather meal Pichia pastoris according to claim 1, characterized in that: The method is as follows: Take OD 600 =1.0 seed culture was inoculated into the culture medium at a volume ratio of 1%, and cultured on a shaker at a temperature of 37℃, pH=5, and 220r / min.

5. The method for culturing a strain of biodegradable feather meal Pichia pastoris according to claim 1, characterized in that: The culture medium used is YPD liquid medium.

6. The application of the *Kudriazwibichthys* strain of biodegradable feather meal as described in claim 1 as a degrading agent for degrading keratin in feather meal.

7. The application according to claim 6, characterized in that: The method is as follows: After sterilizing the feather powder at 121℃ for 15 minutes, it is placed in a vacuum-sealed fermentation bag with a one-way breathing valve. The inoculum content is 6%, the moisture content is 55%, the fermentation temperature is 38℃, and the fermentation time is 7 days.

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