Preparation and application of bacillus licheniformis for decomposing pigeon feathers

Abstract

The invention discloses preparation and application of bacillus licheniformis for decomposing pigeon feathers, and relates to the field of environmental resource application in the breeding industry, and the preparation method comprises the following steps: step 1, strain activation: firstly resuscitating a freeze-dried strain, and then culturing and activating by adopting a gradient activation mode; 2, preparing a seed solution, namely culturing the strain activated in the step 1 in a seed culture medium to obtain the seed solution; according to the method, the components of the pre-activation culture medium comprise 1% of corn flour, 0.5% of soybean meal and 0.1% of dipotassium phosphate, the production cost is low, and the preparation feasibility and economic benefits of the bacillus licheniformis are improved; the feather decomposition efficiency is improved by 2-3 times through substrate pretreatment, the residual feather amount is reduced, meanwhile, the fermentation period is shortened to 40-50 hours, and the feather decomposition efficiency of the pigeons is improved; in the fermentation tank, the activity of the keratinase can reach 600U / mL, so that the decomposition efficiency can be improved, subsequent reuse is facilitated, and the utilization rate is increased.

Description

Technical Field

[0001] This invention relates to the field of environmental resource applications in aquaculture, specifically to the preparation and application of a Bacillus licheniformis that decomposes pigeon feathers. Background Technology

[0002] With the intensive development of global poultry farming, the scale of pigeon farming continues to expand (the global pigeon population is projected to exceed 1.2 billion by 2025, generating 3.5 million tons of feather waste annually). As a byproduct of poultry farming, the efficient treatment of feathers has become a significant challenge for the industry's sustainable development. However, existing technologies face significant bottlenecks in the resource utilization and harmless treatment of feathers, specifically manifested in the following ways: 1. Inefficient treatment methods lead to severe environmental pollution: Currently, approximately 65% ​​of feathers are disposed of through landfill or incineration (according to the 2025 Global Poultry By-products White Paper). Landfilling results in the enrichment of heavy metals in the soil (copper and zinc additives remain in the feathers), while incineration releases dioxins and sulfur oxides, directly violating the EU's Waste Framework Directive (2008 / 98 / EC) and China's Solid Waste Pollution Prevention and Control Law. 2. Low biodegradation efficiency and insufficient resource conversion rate: Traditional microbial fermentation methods (such as using Bacillus licheniformis to secrete keratinase) require 72-120 hours to complete degradation, and the keratin extraction rate is less than 40% (because the disulfide bond structure of feathers is dense, making it difficult to overcome the enzymatic hydrolysis barrier).

[0003] According to CN103834603B, "Preparation and Application of Bacillus licheniformis for Decomposing Pigeon Feathers," the use of Bacillus licheniformis for decomposing pigeon feathers facilitates the recycling of waste from pigeons and promotes ecological and circular pigeon farming. However, this technology has the following problems: 1. The manufacturing cost of the inoculant is too high, affecting its practicality; 2. The fermentation cycle is long, requiring 48-72 hours, which easily leads to a longer production cycle and affects production efficiency.

[0004] In summary, this paper describes the preparation and application of Bacillus licheniformis that decomposes pigeon feathers. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings, the present invention provides a method for preparing and applying Bacillus licheniformis that decomposes pigeon feathers.

[0006] The present invention achieves the above objectives through the following technical solutions: The preparation of a Bacillus licheniformis that decomposes pigeon feathers includes the following steps: Step 1: Strain activation. First, revive the freeze-dried strain, and then use a gradient activation method to cultivate and activate it. Step 2: Seed culture preparation. The activated strain from Step 1 is cultured in a seed culture medium to obtain the seed culture. Step 3: Fermentation broth preparation. Inoculate the seed culture into a shake flask containing 45% to 50% of the bottle volume of fermentation medium at an inoculation rate of 2-3% by volume. Stir and culture at 37-39°C for 12-16 hours while simultaneously introducing nitrogen gas to obtain the fermentation broth. Introducing nitrogen gas can optimize the nitrogen source and reduce raw material costs by 50%, while quantitative control can improve the uniformity of the seed culture.

[0007] Preferably, step one specifically includes the following steps: S11. Resuscitate the lyophilized bacterial culture tubes and inoculate them into a pre-activated culture medium. The pre-activated culture medium consists of 1% corn flour, 0.5% soybean meal and 0.1% dipotassium hydrogen phosphate, with water as the solvent. The pH of the pre-activated culture medium is 7.2. S12. Gradient activation culture: First, a primary culture is carried out at 37℃ for 48 hours. After the spore formation rate is >80%, the second culture is carried out. The strain is transferred to agar medium and cultured at 37℃ for 24 hours. The agar medium consists of 5g yeast powder and 10g sodium chloride, with water as the solvent. The agar concentration is 18g / L. The primary culture can reduce the dependence on nutrient agar. Pre-activation can improve the metabolic activity of the strain. The control of the spore formation rate ensures the stability of the strain.

[0008] Preferably, in step two, the seed culture medium is formulated with 10 g / L yeast extract, 5 g / L ammonium nitrate, 2 g / L dipotassium hydrogen phosphate, and 1 g / L magnesium sulfate, with water as the solvent, and the pH of the seed culture medium is 7.0.

[0009] Preferably, in step three, the nitrogen aeration rate is 1:1 vvm, when the spore concentration is ≥ The culture should be terminated at that time.

[0010] The application of a Bacillus licheniformis strain that decomposes pigeon feathers, as described above, includes the following specific steps: Step 4: Substrate pretreatment. Pigeon feather powder is steamed at 110~130℃ for 15~25 minutes. 0.1% sodium sulfate is added to the steam. Sodium sulfate is used to break disulfide bonds to obtain pretreated feather powder. Substrate pretreatment increases the feather decomposition efficiency by 2-3 times. Step 5: Preparation of fermentation medium. The fermentation medium includes the following components in the following proportions: 5% pretreated feather meal, 2% soybean meal, 1-2% corn steep liquor, 0.02% ferric sulfate and 0.01% manganese sulfate, with water as the solvent. Step Six: Fermentation Control Treatment. The fermentation medium from Step Five is placed in a 5-ton tank for fermentation. The temperature is maintained between 36 and 40°C, the stirring speed is 180 rpm, and the stirring time is 36 hours. The composite carbon source system shortens the fermentation cycle to 40-50 hours.

[0011] Preferably, in step four, the pH value of the fermentation medium is maintained between 7 and 8.

[0012] Preferably, in step six, the dissolved oxygen content is >40% between 0-12 hours and maintained between 20-30% between 12-26 hours.

[0013] Preferably, in step six, the container is placed in the container when the keratinase activity reaches 600 U / mL or the residual feather content is <5%.

[0014] The beneficial effects of this invention are: the preparation and application of the Bacillus licheniformis that decomposes pigeon feathers. 1. The pre-activated culture medium consists of 1% corn flour, 0.5% soybean meal and 0.1% dipotassium hydrogen phosphate. Its production cost is low, which improves the feasibility and economic benefits of preparing Bacillus licheniformis. 2. Substrate pretreatment increases feather decomposition efficiency by 2-3 times, reduces residual feathers, and shortens the fermentation cycle to 40-50 hours, thus improving the efficiency of pigeon feather decomposition. 3. In the fermenter, the activity of keratinase can reach 600 U / mL, which not only improves the decomposition efficiency, but also facilitates subsequent reuse and improves the utilization rate. Attached Figure Description

[0015] The present invention will be described by way of example and with reference to the accompanying drawings, wherein: Figure 1 This is a flowchart illustrating the preparation and application of Bacillus licheniformis for decomposing pigeon feathers according to the present invention. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, illustrating only the basic structure of the invention, and therefore only show the components relevant to the invention.

[0017] like Figure 1 As shown, the preparation of a Bacillus licheniformis that decomposes pigeon feathers includes the following steps: Step 1: Strain activation. First, revive the freeze-dried strain, and then use a gradient activation method to cultivate and activate it. Step 2: Seed culture preparation. The activated strain from Step 1 is cultured in a seed culture medium to obtain the seed culture. Step 3: Fermentation broth preparation. Inoculate the seed culture into a shake flask containing 45% to 50% of the bottle volume of fermentation medium at an inoculation rate of 2-3% by volume. Stir and culture at 37-39°C for 12-16 hours while simultaneously introducing nitrogen gas to obtain the fermentation broth. Introducing nitrogen gas can optimize the nitrogen source and reduce raw material costs by 50%, while quantitative control can improve the uniformity of the seed culture.

[0018] Specifically, step one includes the following steps: S11. Resuscitate the lyophilized bacterial culture tubes and inoculate them into a pre-activated culture medium. The pre-activated culture medium consists of 1% corn flour, 0.5% soybean meal and 0.1% dipotassium hydrogen phosphate, with water as the solvent. The pH of the pre-activated culture medium is 7.2. S12. Gradient activation culture: First, a primary culture is carried out at 37℃ for 48 hours. After the spore formation rate is >80%, the second culture is carried out. The strain is transferred to agar medium and cultured at 37℃ for 24 hours. The agar medium consists of 5g yeast powder and 10g sodium chloride, with water as the solvent. The agar concentration is 18g / L. The primary culture can reduce the dependence on nutrient agar. Pre-activation can improve the metabolic activity of the strain. The control of the spore formation rate ensures the stability of the strain.

[0019] Specifically, in step two, the seed culture medium consists of 10 g / L yeast extract, 5 g / L ammonium nitrate, 2 g / L dipotassium hydrogen phosphate, and 1 g / L magnesium sulfate, with water as the solvent, and a pH of 7.0.

[0020] Specifically, in step three, the nitrogen aeration rate is 1:1 vvm, when the spore concentration is ≥ The culture should be terminated at that time.

[0021] The application of a Bacillus licheniformis strain that decomposes pigeon feathers, as described above, includes the following specific steps: Step 4: Substrate pretreatment. Pigeon feather powder is steamed at 110~130℃ for 15~25 minutes. 0.1% sodium sulfate is added to the steam. Sodium sulfate is used to break disulfide bonds to obtain pretreated feather powder. Substrate pretreatment increases feather decomposition efficiency by 2.3 times. Step 5: Preparation of fermentation medium. The fermentation medium includes the following components in the following proportions: 5% pretreated feather meal, 2% soybean meal, 1-2% corn steep liquor, 0.02% ferric sulfate and 0.01% manganese sulfate, with water as the solvent. Step Six: Fermentation Control Treatment. The fermentation medium from Step Five is placed in a 5-ton tank for fermentation. The temperature is maintained between 36 and 40°C, the stirring speed is 180 rpm, and the stirring time is 36 hours. The composite carbon source system shortens the fermentation cycle to 40-50 hours.

[0022] Specifically, in step four, the pH value of the fermentation medium is maintained between 7 and 8.

[0023] Specifically, in step six, the dissolved oxygen level is >40% between 0 and 12 hours, and is maintained between 20% and 30% between 12 and 26 hours.

[0024] Specifically, in step six, the container is placed in a container when the keratinase activity reaches 600 U / mL or the residual feather content is <5%.

[0025] Implementation Case 1 Preparation process: 1. Strain activation (Step 1): S11: Resuscitate the freeze-dried strain (Bacillus licheniformis freeze-dried tube) and inoculate it on a pre-activated medium (components: 1% corn flour, 0.5% soybean meal, 0.1% dipotassium hydrogen phosphate, pH=7.2).

[0026] S12: Gradient activation culture - primary culture (37°C, 48 hours, spore formation rate >80%), secondary culture (transfer to agar medium: 5g yeast powder, 10g sodium chloride, 18g / L agar, 37°C, 24 hours).

[0027] 2. Seed culture preparation (step two): After activation, the inoculum is inoculated into seed culture medium (10g / L yeast extract, 5g / L ammonium nitrate, 2g / L dipotassium hydrogen phosphate, 1g / L magnesium sulfate, pH=7.0) and cultured until the stable phase.

[0028] 3. Fermentation broth preparation (step three): The seed culture was inoculated into a shake flask at a volume ratio of 2.5% (50% of the flask volume of the fermentation medium), and cultured at 37°C with stirring for 14 hours. Nitrogen gas was introduced (aeration rate of 1:1 vvm). Termination condition: spore concentration ≥10^9 CFU / mL.

[0029] Application process: 1. Substrate pretreatment (step four): Pigeon feather powder is steamed at 120°C for 20 minutes, with 0.1% sodium sulfate added to the steam to break disulfide bonds.

[0030] 2. Preparation of fermentation medium (step five): Ingredients: 5% pretreated feather meal, 2% soybean meal, 1% corn steep liquor, 0.02% ferric sulfate, 0.01% manganese sulfate, water as solvent, pH=7.5.

[0031] 3. Fermentation control treatment (step six): 5-ton tank fermentation, temperature 38°C, stirring speed 180 rpm, time 36 hours; dissolved oxygen control: >40% for 0-12 hours, maintain 25% for 12-26 hours.

[0032] Termination condition: Remove the container when the keratinase activity reaches 600 U / mL.

[0033] The following is a comparison between Example 1 and the reference patent: The experimental data for Example 1 are as follows: enzyme activity reached 600 U / mL, residual feather amount <5%, and the cycle was shortened to 42 hours, verifying the low cost (pre-activated culture medium cost only $0.5 / L) and high efficiency (decomposition efficiency increased by 2.8 times) described in the text. Implementation Case 2 Preparation process: Similar to Implementation Case 1 (standard parameters were used for strain activation, seed culture, and fermentation broth preparation) to ensure consistency at the preparation stage.

[0034] Application process: 1. Substrate pretreatment (step four): Steam temperature 110℃, time 15 minutes, add 0.1% sodium sulfate.

[0035] 2. Preparation of fermentation medium (step five): Same as Case 1 (pretreated feather meal 5%).

[0036] 3. Fermentation control treatment (step six): Same as Case 1 (temperature 38°C, stirring 180 rpm, time 36 hours; dissolved oxygen control is the same).

[0037] The following is a comparison between Example 2 and the reference patent: The experimental data for Example 2 are as follows: the decrease in pretreatment temperature and time resulted in a decrease in enzyme activity to 550 U / mL, an increase in residual feathers to 7.0%, and an efficiency increase of only 2.0 times.

[0038] Implementation Case 3: Preparation process: Same as Case 1 (Standard strain activation and seed liquid preparation).

[0039] Application process: 1. Substrate pretreatment (step four): Same as Case 1 (steam treatment at 120°C for 20 minutes).

[0040] 2. Preparation of fermentation medium (step five): Increase corn steep liquor to 2%, keep other components unchanged (pretreated feather meal 5%, soybean meal 2%, etc.), and test carbon source optimization.

[0041] 3. Fermentation control treatment (step six): Same as Case 1 (temperature 38°C, stirring 180 rpm, time 36 hours; dissolved oxygen control is the same).

[0042] The following is a comparison between Example 3 and the reference patent: The experimental data for Example 3 are as follows: when the corn steep liquor ratio was increased to 2%, the enzyme activity increased slightly to 620 U / mL, the cycle was shortened to 38 hours, the residual feather amount decreased to 3.8%, and the efficiency was increased by 3.0 times.

[0043] To summarize the three examples: Efficiency Analysis: The best case is Case 3, which has the shortest cycle (38 hours) and the highest efficiency (3.0 times improvement), but Case 1 is more balanced (cost savings of 50% and residual feathers of 4.5%).

[0044] Key factor: Pretreatment (Case 2) has the greatest impact on efficiency—a decrease in temperature / time leads to a 75% increase in residual feathers, highlighting the necessity of adding sodium sulfate to the steam in the text.

[0045] Robustness: All cases met the requirements of cycle time < 50 hours and residual feather amount < 8%, proving the stability of the invention.

[0046] Economic benefit analysis: Cost calculation: Cost of pre-activated culture medium: The corn flour / soybean meal formulation in Case 1 costs only $0.5 / L, which is 60% lower than that of conventional agar medium ($2.0 / L).

[0047] Total cost: Case 1 saves 50%, while Case 3 saves 40% due to the increase in corn syrup.

[0048] Quantifying benefits: Based on a 5-ton tank scale: Case 1 reduced feather residue to 4.5%, resulting in a waste reduction of 7.5 tons / year compared to the traditional method (12% feather residue) and a reduction in processing costs of $3,000 / ton.

[0049] Shorter cycle time: Case 3's 38-hour cycle increased capacity by 89% compared to the traditional 72-hour cycle, resulting in an annual increase in output value of approximately $50,000.

[0050] Summary from multiple perspectives: From a preparation perspective: gradient activation (first generation + second generation culture) ensures a spore formation rate of >80% and reduces costs; nitrogen gas is used to optimize the nitrogen source and improve the uniformity of the seed culture.

[0051] From an application perspective: Substrate pretreatment (steam + sodium sulfate) is the core of efficiency; staged control of dissolved oxygen (0-12 hours >40%, 12-26 hours 20-30%) maintains enzyme activity.

[0052] Overall advantages: Compared with reference patents and existing technologies, the advantages of this patent are as follows: the decomposition efficiency is increased by 2-3 times, the cycle is shortened to 40-50 hours, and the enzyme activity is above 600U / mL, which meets the environmental protection requirements of the aquaculture industry (reducing landfill / incineration).

[0053] Based on the above description, those skilled in the art can make various changes and modifications without departing from the technical concept of this invention. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing Bacillus licheniformis that decomposes pigeon feathers, characterized in that: Includes the following steps: Step 1: Strain activation. First, revive the freeze-dried strain, and then use a gradient activation method to cultivate and activate it. Step 2: Seed culture preparation. The activated strain from Step 1 is cultured in a seed culture medium to obtain the seed culture. Step 3: Fermentation broth preparation. Inoculate the seed culture into a shake flask containing 45% to 50% of the bottle volume of fermentation medium at an inoculation rate of 2-3% by volume. Stir and culture at 37-39°C for 12-16 hours while purging with nitrogen to obtain the fermentation broth.

2. The preparation of Bacillus licheniformis for decomposing pigeon feathers according to claim 1, characterized in that: Step one specifically includes the following steps: S11. Resuscitate the lyophilized bacterial culture tubes and inoculate them into a pre-activated culture medium. The pre-activated culture medium consists of 1% corn flour, 0.5% soybean meal and 0.1% dipotassium hydrogen phosphate, with water as the solvent. The pH of the pre-activated culture medium is 7.

2. S12. Gradient activation culture: First, perform primary culture at 37℃ for 48 hours. After the spore formation rate is >80%, proceed to secondary culture. Transfer the strain to agar medium and culture at 37℃ for 24 hours. The agar medium consists of 5g yeast powder and 10g sodium chloride, with water as the solvent, and the agar concentration is 18g / L.

3. The preparation of Bacillus licheniformis for decomposing pigeon feathers according to claim 1, characterized in that: In step two, the seed culture medium is formulated with 10 g / L yeast extract, 5 g / L ammonium nitrate, 2 g / L dipotassium hydrogen phosphate and 1 g / L magnesium sulfate, with water as the solvent, and the pH of the seed culture medium is 7.

0.

4. The preparation of Bacillus licheniformis for decomposing pigeon feathers according to claim 1, characterized in that: In step three, the nitrogen gas flow rate is 1:1 vvm, when the spore concentration is ≥ The culture should be terminated at that time.

5. The application of Bacillus licheniformis according to any one of claims 1-4, characterized in that: The specific steps include the following: Step 4: Substrate pretreatment. The pigeon feather powder is steamed at 110~130℃ for 15~25 minutes. 0.1% sodium sulfate is added to the steam to obtain pretreated feather powder. Step 5: Preparation of fermentation medium. The fermentation medium includes the following components in the following proportions: 5% pretreated feather meal, 2% soybean meal, 1-2% corn steep liquor, 0.02% ferric sulfate and 0.01% manganese sulfate, with water as the solvent. Step Six: Fermentation Control Treatment. The fermentation medium from Step Five is placed in a 5-ton tank for fermentation. The temperature is maintained between 36 and 40°C, the stirring speed is 180 rpm, and the stirring time is 36 hours.

6. The application of Bacillus licheniformis for decomposing pigeon feathers according to claim 5, characterized in that: In step four, the pH value of the fermentation medium is maintained between 7 and 8.

7. The application of Bacillus licheniformis for decomposing pigeon feathers according to claim 5, characterized in that: In step six, the dissolved oxygen level is >40% between 0 and 12 hours, and is maintained between 20% and 30% between 12 and 26 hours.

8. The application of Bacillus licheniformis for decomposing pigeon feathers according to claim 5, characterized in that: In step six, the container is removed when the keratinase activity reaches 600 U / mL or the residual feather content is <5%.

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