A method for bioconverting waste feathers to prepare animal feed

Abstract

This invention discloses a method for preparing animal feed through the bioconversion of waste feathers. The method utilizes a screened strain of Bacillus subtilis HC2 (accession number GDMCC No. 62466) that produces a high amount of alkaline protease to perform high-density fermentation of puffed waste feathers, obtaining a mixture of high-density bacterial cells and the degraded feathers. Then, 8-12% sugarcane bagasse, 9-15% wheat bran, and 8-12% starch are added, and the mixture is granulated to obtain animal feed. This feed has a high soluble protein content (20.2 g / L) and contains a high density of active probiotics (2.13 × 10⁻⁶). 8 This method (using cfu / g) has the potential to regulate intestinal immunity and enhance disease resistance. It is simple to operate, requires no centrifugation or sterilization, and is low-cost. This invention not only converts waste feathers into animal feed for reuse but also promotes the green and circular development of the livestock and poultry farming industry.

Description

Technical Field

[0001] This invention belongs to the field of feed technology, and specifically relates to a method for preparing animal feed through the bioconversion of waste feathers. Background Technology

[0002] Feathers are a major byproduct of livestock farming, containing approximately 90% crude protein. Except for a small amount of higher-quality feathers used for clothing and sporting goods, most feathers are discarded, landfilled, or incinerated, easily causing environmental pollution, disease transmission, and resource waste. With the intensive development of animal husbandry, my country generates millions of tons of waste feathers annually. Waste feathers are an inexpensive, high-protein resource; converting them into animal feed can both turn waste into treasure, achieving resource utilization of waste feathers, and avoiding their adverse environmental impact.

[0003] Feathers contain numerous disulfide bonds, and the cross-linking of these bonds causes the feather structure to fold, making it difficult to degrade. Current reports mainly employ high-temperature puffing, enzymatic hydrolysis, and microbial methods to treat waste feathers. Feather powder obtained through high-temperature puffing can be used to feed animals, but its digestibility is low (<30%). Compound enzyme preparations can improve the degradation efficiency of feathers, but the enzyme preparation process is complex and costly. In recent years, an increasing number of feather-degrading microorganisms have been discovered. Microbial methods are the most promising approach for green manufacturing, but the degradation of feathers by microorganisms generally suffers from a long timeframe.

[0004] Probiotics are a class of live microorganisms that produce antimicrobial substances through metabolism and provide beneficial functions to the host (such as enhancing the host's disease resistance). They have the potential to replace antibiotics and are of great application value in feed, food, and pharmaceutical fields. Currently, probiotics are mainly used as feed additives in feed, and research on using probiotics to degrade waste feathers and prepare animal feed has not been reported. Therefore, screening for probiotics to degrade waste feathers and prepare probiotic animal feed can help promote the high-value utilization of waste feathers and the green and circular development of livestock and poultry farming. Summary of the Invention

[0005] The first objective of this invention is to provide a Bacillus subtilis HC2 with high alkaline protease and keratinase activity, which was deposited on May 12, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China, with accession number GDMCCNo: 62466.

[0006] A second objective of this invention is to provide the application of the aforementioned Bacillus subtilis HC2 in the degradation of feather meal.

[0007] Preferably, the feather powder is obtained by puffing up waste chicken feathers, duck feathers, and goose feathers.

[0008] A third objective of this invention is to provide a method for the bioconversion of waste feathers to prepare animal feed, comprising the following steps:

[0009] Waste feather meal was added to the Bacillus subtilis HC2 fermentation broth for fermentation and biodegradation of the feather meal, resulting in a mixture of degraded feathers. Sugarcane bagasse, wheat bran, and starch were added to the mixture, and after being mixed evenly, the mixture was granulated to obtain animal feed.

[0010] Preferably, the specific steps are as follows:

[0011] 1) Inoculate Bacillus subtilis HC2 into liquid LB medium and culture for 10-18 h to obtain seed culture;

[0012] 2) Inoculate the seed culture into the fermentation medium at a volume ratio of 4-8% and culture at 37℃ and 160-220 r / min for 2-4 days. During this period, glucose and feather meal are added in batches, with glucose added at 0.5% (w / v) of the total volume of the fermentation broth and feather meal added at 5-8% (w / v) of the total volume of the fermentation broth. High-density fermentation and biodegradation of feathers are carried out to obtain a mixture of degraded feathers.

[0013] The fermentation medium, by mass fraction, comprises 0.02–0.08% NaCl, 0.02–0.06% KH₂PO₄, 0.01–0.05% K₂HPO₄, 0.015–0.055% MgSO₄·7H₂O, 6–8% feather meal, and has a pH of 8.5–9.5.

[0014] 3) Mix the mixture from step 2) with 8-12% by weight of sugarcane bagasse, 9-15% by weight of wheat bran, and 8-12% by weight of starch, and granulate at 230-240 r / min to obtain animal feed.

[0015] Preferably, the fermentation medium is composed of the following components by mass fraction: 0.05% NaCl, 0.04% KH2PO4, 0.03% K2HPO4, 0.025% MgSO4·7H2O, 6% feather meal, and the balance being water, with a pH of 9.0. The preparation method is to mix all components evenly and sterilize at 115°C for 30 minutes.

[0016] This invention utilizes a combination of extrusion and microbial degradation methods to rapidly prepare animal feed from waste feathers. The Kjeldahl method (GB / T6432-2018) was used to determine the crude protein and soluble protein content of the feed, and the filtration method (GB / T 6434-2006) was used to determine the crude fiber content. The prepared feed contained 34.6% crude protein, 6.1% crude fiber, and 22.2 g / L soluble protein. The activities of alkaline protease (QB 747-80) and keratinase (Quansibiao

[2021] No. 52) in the supernatant of the fermentation broth were detected using the Folin-phenol method and spectrophotometry. The keratinase activity was approximately 591.4 U / mL, and the alkaline protease activity was approximately 697.5 U / mL. (See...) Figure 2 These values ​​are higher than the enzyme activities reported in many studies, even after initial screening and optimization. The number of viable probiotics in the fermentation broth and feed was determined using a dilution gradient method. The viable probiotic count in the fermentation broth was 3.75 × 10⁻⁶. 8 Approximately CFU / mL; the viable probiotic count in the granulated feed is 2.13 × 10⁻⁶. 8 It maintains a high number of live probiotics at around CFU / g, significantly higher than the number of probiotics specified in the enterprise standard Q / HBMSY001-2019.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. Bacillus subtilis HC2 has relatively high alkaline protease activity.

[0019] 2. Probiotics and their fermentation broth can be directly added to excipients and then granulated, eliminating the need for centrifugation or sterilization, thus saving costs.

[0020] 3. The resulting feed has a high content of crude protein and soluble protein, and contains a high density of active probiotics.

[0021] 4. The probiotics in the feed itself and the antibacterial substances produced by fermentation can inhibit the growth of harmful microorganisms in the animal's intestines, regulate intestinal immunity, and enhance disease resistance, thus having the potential to replace antibiotics.

[0022] Bacillus subtilis HC2 was deposited on May 12, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China. Accession number: GDMCC No.: 62466. Attached Figure Description

[0023] Figure 1 It is the transparent zone produced by the selected strain.

[0024] Figure 2The activity of alkaline protease and keratinase in the fermentation broth of Bacillus subtilis HC2 fed fermentation with feather meal as substrate.

[0025] Figure 3 It is prepared animal feed. Detailed Implementation

[0026] The following embodiments are for further illustration of the present invention, but not for limiting the present invention.

[0027] Example 1: Screening of bacteria producing high levels of alkaline protease

[0028] Soil samples were collected from the environment. 5g of soil sample was placed in 100mL of liquid LB medium and enriched at 37℃ and 180r / min for approximately 20h. 10g of soil sample was then collected. -5 10 -6 10 -7 Three gradient bacterial suspensions (50–100 μl each) were spread onto solid LB agar and incubated at 37°C for 20 h. Single colonies were picked and transferred to skim milk agar and incubated at 37°C for 25–30 h. The strain HC2, which produced the largest ratio of clear zone diameter to bacterial diameter (2.12), was screened. Results are shown in [Figure number missing]. Figure 1 Identified as Bacillus subtilis by 16S rDNA sequencing and BLAST comparison, it was named Bacillus subtilis HC2 and deposited on May 12, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No. 62466.

[0029] Liquid LB medium: by mass fraction, yeast extract 0.5%, tryptone 1%, sodium chloride 1%, solvent is water, pH 7.0. The preparation method is to mix all components evenly and sterilize at 115℃ for 30 min.

[0030] Solid LB medium: by mass fraction, yeast extract 0.5%, tryptone 1%, sodium chloride 1%, agar 1.5%, solvent is water, pH 7.0. The preparation method is to mix all components evenly and sterilize at 115℃ for 30 min.

[0031] Skim milk culture medium: by mass fraction, skim milk 4%, agar 1.5%, pH 8.3. The preparation method is to mix all components evenly and sterilize at 115℃ for 30 min.

[0032] The strain produces a relatively large clear zone under alkaline conditions, indicating that it produces alkaline protease with relatively high enzyme activity. Studies have found that alkaline protease can promote the degradation of feathers, suggesting that HC2 bacteria has the potential to degrade feathers / feather powder.

[0033] Example 2: High-density fermentation for feed preparation

[0034] Feather meal was obtained by puffing waste chicken feathers (approximately 16% moisture content) using a twin-screw extruder at 175 r / min, 160 °C, and 0.4 MPa. Bacillus subtilis HC2 was cultured in liquid LB medium at 37 °C and 180 r / min for 16 h as a seed culture. This seed culture was then transferred at a 5% (v / v) inoculation rate to a shake flask containing 500 mL of fermentation medium. The mixture was then fermented at 37 °C and 180 r / min with glucose and feather meal added in batches. Glucose was added at 0.5% (w / v) of the fermentation broth volume, and feather meal at 6% (w / v) of the fermentation broth volume. High-density fermentation and feather meal degradation were carried out for 3.5 days to obtain a mixture of Bacillus subtilis HC2 and its degraded feather meal. The supernatant of this mixture showed approximately 591.4 U / mL keratinase activity and approximately 697.5 U / mL alkaline protease activity. (See [link to relevant information]). Figure 2 These values ​​are higher than the enzyme activities reported in many studies, both in the initial screening and even after optimization.

[0035] Add 10% by weight of sugarcane bagasse, 12% by weight of wheat bran, and 10% by weight of starch to the mixture, mix well, and granulate at 235 r / min to obtain feed. The feed has a diameter of approximately 4 mm and a length of 5–20 mm. See [link to relevant documentation]. Figure 3 The feed contained 34.6% crude protein, 6.1% crude fiber, and 22.2 g / L soluble protein. The live probiotic count in the feather meal degradation mixture was 3.75 × 10⁻⁶. 8 The cfu / mL concentration of probiotics in the granulated feed was 2.13 × 10⁻⁶. 8 Even at cfu / g, it still maintains a high concentration of active probiotics, which is beneficial for achieving probiotic functions.

[0036] The detection of feed and related indicators in the preparation process was carried out using the Kjeldahl method (GB / T 6432-2018) to determine crude protein and soluble protein in the feed, and the filtration method (GB / T 6434-2006) to determine crude fiber content. The number of active probiotics in the fermentation broth and feed was detected by the dilution gradient method. The activity of alkaline protease (QB747-80) and keratinase (Quansibiao

[2021] No. 52) in the fermentation broth was detected by the Folin-phenol method and spectrophotometry.

[0037] LB medium: by mass fraction, yeast extract 0.5%, tryptone 1%, sodium chloride 1% (1.5% agar needs to be added for solid LB medium), pH 7.0, the remainder is water. The preparation method is to mix all the components evenly and sterilize at 115℃ for 30 min.

[0038] Fermentation medium: By mass fraction, NaCl 0.05%, KH₂PO₄ 0.04%, K₂HPO₄ 0.03%, MgSO₄·7H₂O 0.025%, 6% feather meal, balance water, pH 9.0. The preparation method is to mix all components thoroughly and sterilize at 115℃ for 30 min. >HC2 strain 16S RNA sequence (SEQ ID NO.1)

[0039]

Claims

1. Bacillus subtilis HC2, with the preservation number of GDMCC No: 62466.

2. The application of Bacillus subtilis HC2 in claim 1 in degrading feather meal.

3. Use according to claim 2, characterized in that, The feather meal is obtained by expanding waste chicken feathers, duck feathers and goose feathers.

4. A method for preparing animal feed through the bioconversion of waste feathers, characterized in that, The method comprises the following steps: The Bacillus subtilis HC2 fermentation broth in claim 1 is added with feather meal for biological fermentation and degradation, and a feather-degraded mixture is obtained, and then sugarcane residue, wheat bran and starch are added to the mixture, uniformly compounded, and granulated to prepare animal feed.

5. The method of claim 4, wherein, The specific steps are as follows: 1) The Bacillus subtilis HC2 is inoculated in a liquid LB culture medium and cultured for 10-18 h to prepare a seed liquid; 2) The seed liquid is inoculated into a fermentation medium at a volume ratio of 4-8%, and cultured at 37°C and 160-220 r / min for 2-4 days, during which high-density fermentation and biological degradation of feathers are carried out by adding glucose and feather meal in batches, wherein the glucose is added at a volume ratio of 0.5% (mass / volume ratio, w / v) based on the volume of the fermentation broth, and the feather meal is added at a volume ratio of 5-8% (mass / volume ratio, w / v) based on the volume of the fermentation broth, to obtain a feather-degraded mixture; The fermentation medium comprises, by mass fraction, NaCl 0.02-0.08%, KH2PO4 0.02-0.06%, K2HPO4 0.01-0.05%, MgSO4·7H2O 0.015-0.055%, 6-8% feather meal, pH 8.5-9.5, and is sterilized at 115°C for 30 min; 3) The mixture of step 2) is uniformly compounded with 8-12% sugarcane residue based on the mass of the mixture, 9-15% wheat bran based on the mass of the mixture, and 8-12% starch based on the mass of the mixture, and granulated at 230-240 r / min to prepare animal feed.

6. The method according to claim 4 or 5, characterized in that, The feather meal is expanded waste chicken feather powder, duck feather powder and goose feather powder.

7. The method of claim 5, wherein, The fermentation medium comprises, by mass fraction, NaCl 0.05%, KH2PO4 0.04%, K2HPO4 0.03%, MgSO4·7H2O 0.025%, 6% feather meal, and the balance is water, pH 9.0, and is sterilized at 115°C for 30 min.

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