CN115992065A - Method for preparing animal feed by bioconversion of waste feathers - Google Patents
Method for preparing animal feed by bioconversion of waste feathers Download PDFInfo
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Abstract
The invention discloses a method for preparing animal feed by bioconversion of waste feathers. The method utilizes bacillus subtilis (Bacillus subtilis) HC2 (with the preservation number of GDMCC No. 62466) of a strain of high-yield alkaline protease obtained by screening to carry out high-density fermentation on the puffed waste feather, so as to obtain a high-density thallus and a mixture of degraded feather thereof; adding 8-12% of bagasse, 9-15% of wheat bran and 8-12% of starch into the mixture, and granulating the mixture after compounding to prepare the animal feed. The feed has high soluble protein content (20.2 g/L) and contains high density of active probiotics (2.13×10) 8 cfu/g) with modulation ofPotential for reducing intestinal immunity and enhancing disease resistance. The method has simple operation, no need of centrifugal sterilization or disinfection, and low cost. The invention not only converts the waste feathers into animal feed for reuse, but also can promote the green cyclic development of the livestock and poultry raising industry.
Description
Technical Field
The invention belongs to the technical field of feeds, and particularly relates to a method for preparing animal feeds by bioconversion of waste feathers.
Background
Feathers are the main byproduct of the livestock breeding industry, and the crude protein content is about 90 percent. The feather with better quality is used for clothes and sports goods, and most of the feather is discarded, buried or burnt, so that environmental pollution, disease transmission and resource waste are easily caused. With the intensive development of the aquaculture industry, millions of tons of waste feathers are produced annually in China. The waste feather is a cheap high-protein resource, and the waste feather can be changed into animal feed, so that the waste feather can be recycled, and adverse effects on the environment can be avoided.
There are a large number of disulfide bonds in the feather structure, and cross-linking of disulfide bonds promotes folding of the feather structure, resulting in difficulty in being degraded. The current report mainly adopts a high-temperature puffing method, an enzymolysis method and a microorganism method to treat waste feathers. The feather meal obtained by high-temperature puffing can be used for feeding animals, but the digestibility of the animal is low (< 30%); the composite enzyme preparation can improve the degradation efficiency of feathers, but the preparation process of the enzyme is complex and the cost is high. In recent years, an increasing number of feather degrading microorganisms have been discovered. The microbiological method is the most potential method for green manufacture, but the problem of long time for microbial degradation of feathers is common.
Probiotics are active microorganisms which produce antibacterial substances through metabolism and have beneficial functions on hosts (such as enhancing disease resistance of the hosts), have the potential of replacing antibiotics, and have great application value in the fields of feed, food, medicine and the like. At present, probiotics are mainly applied as feed additives in the aspect of feed, but research on degrading waste feathers and preparing animal feed by adopting probiotics is not reported yet. Therefore, the screening of probiotics to degrade waste feathers and the preparation of the probiotic animal feed can assist the high-value utilization of the waste feathers and the green cyclic development of the livestock and poultry raising industry.
Disclosure of Invention
A first object of the present invention is to provide bacillus subtilis (Bacillus subtilis) HC2, deposited at the cantonese collection of microorganisms (GDMCC), address: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070 accession number GDMCC No:62466.
the second object of the invention is to provide the application of the bacillus subtilis HC2 in degrading feather meal.
Preferably, the feather meal is obtained by puffing waste chicken feather, duck feather and goose feather.
A third object of the present invention is to provide a method for preparing animal feed by bioconversion of waste feathers, comprising the steps of:
waste feather meal is added into bacillus subtilis HC2 fermentation broth for fermentation and biodegradation of the feather meal, a mixture of degraded feathers is obtained, bagasse, wheat bran and starch are added into the mixture, and after uniform compounding, granulation is carried out, so that the animal feed is obtained.
Preferably, the specific steps are as follows:
1) Inoculating bacillus subtilis HC2 into a liquid LB culture medium for culturing for 10-18 hours to prepare seed liquid;
2) Inoculating seed liquid into a fermentation culture medium according to the inoculum size of 4-8% by volume ratio, culturing for 2-4 days at 37 ℃ and 160-220 r/min, and during the period, supplementing glucose and feather meal in batches, wherein the glucose is supplemented according to 0.5% (mass-volume ratio, w/v) of the total volume of the fermentation liquid, and the feather meal is supplemented according to 5-8% (mass-volume ratio, w/v) of the total volume of the fermentation liquid, so as to perform high-density fermentation and biodegradation of feathers, thus obtaining a mixture for degrading feathers;
the fermentation medium comprises the following components: comprises NaCl 0.02-0.08% and KH by mass 2 PO 4 0.02～0.06％、K 2 HPO 4 0.01～0.05％、MgSO 4 ·7H 2 0.015 to 0.055 percent of O, 6 to 8 percent of feather meal and pH of 8.5 to 9.5;
3) Uniformly compounding the mixture obtained in the step 2) with bagasse accounting for 8-12% of the mixture mass, wheat bran accounting for 9-15% of the mixture mass and starch accounting for 8-12% of the mixture mass, and granulating under the condition of 230-240 r/min to obtain the animal feed.
Preferably, the fermentation medium is: based on mass fraction, naCl 0.05%, KH 2 PO 4 0.04％、K 2 HPO 4 0.03％、MgSO 4 ·7H 2 O0.025%, feather powder 6% and water the rest, pH 9.0, and the preparation method comprises mixing the above components uniformly, sterilizing at 115 deg.C for 30min.
The invention combines a puffing method and a microbiological method to degrade waste feathers to quickly prepare animal feed, adopts a Kjeldahl nitrogen determination method (GB/T6432-2018) to measure crude protein and soluble protein in the feed, and adopts a filtration method (GB/T6434-2006) to measure the crude fiber content. The prepared feed has 34.6% of crude protein, 6.1% of crude fiber and 22.2g/L of soluble protein. Alkaline protease activity (QB 747-80) and keratinase activity (whole-feeding standard [2021] in the supernatant of the fermentation broth were examined by Folin-phenol method and spectrophotometry]52). The activity of keratinase is about 591.4U/mL, and the activity of alkaline protease is about 697.5U/mL, as shown in FIG. 2. They are higher than the primary and even optimized enzyme activities reported in many studies. And detecting the quantity of active probiotics in the fermentation liquor and the feed by a dilution gradient method. The number of probiotics in the fermentation liquor is 3.75X10 8 cfu/mL or so; the number of probiotics in the granulated feed is 2.13×10 8 About cfu/g, the number of the probiotics living bacteria is kept to be higher, and the number of the probiotics in the Q/HBMSY001-2019 enterprise standard is obviously higher.
Compared with the prior art, the invention has the following advantages:
1. bacillus subtilis HC2 has a relatively high alkaline protease activity.
2. The probiotics and the fermentation liquor thereof are directly added with auxiliary materials and then granulated, so that centrifugal sterilization or disinfection is not needed, and the cost is saved.
3. The prepared feed has high crude protein and soluble protein content and contains high-density active probiotics.
4. The probiotics of the feed and the antibacterial substances produced by fermentation of the probiotics can inhibit the growth of harmful microorganisms in animal intestinal tracts, regulate intestinal immunity and strengthen disease resistance, and have the potential of replacing antibiotics.
Bacillus subtilis HC2, which was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 2022, 5/12, address: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070 accession number GDMCC No:62466.
drawings
FIG. 1 is a transparent circle produced by the strain screened.
FIG. 2 shows the activities of alkaline protease and keratinase in fermentation broth of Bacillus subtilis HC2 fed-batch fermentation with feather meal as substrate.
Fig. 3 is a prepared animal feed.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
EXAMPLE 1 screening of high alkaline protease producing bacteria
Collecting a sample from a soil sample environment, taking 5g of the soil sample, placing the soil sample in 100mL of liquid LB culture medium, and enriching for about 20 hours at 37 ℃ and 180 r/min; take 10 -5 、10 -6 、10 -7 Three gradient bacterial suspensions 50-100 μl are respectively coated on solid LB culture medium, and cultured for 20h at 37 ℃. Single colonies were picked and transferred to skim milk medium and cultured at 37℃for 25-30 hours, and strain HC2 having the largest ratio of diameter of transparent circle to diameter of bacteria (this ratio is 2.12) was selected, and the result is shown in FIG. 1. Identified as bacillus subtilis (Bacillus subtilis) by 16S rDNA sequencing and Blast alignment, designated bacillus subtilis (Bacillus subtilis) HC2, deposited at the cantonese collection of microorganisms and cell cultures (GDMCC), 5/12, 2022, address: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070 accession number GDMCC No:62466.
liquid LB medium: the yeast powder is 0.5 percent, the tryptone is 1 percent, the sodium chloride is 1 percent, the solvent is water, the pH value is 7.0, and the preparation method comprises the steps of uniformly mixing the components and sterilizing for 30 minutes at 115 ℃.
Solid LB medium: according to mass fraction, yeast powder 0.5%, tryptone 1%, sodium chloride 1%, agar 1.5%, water as solvent and pH 7.0, the preparation method comprises mixing the components uniformly, sterilizing at 115 ℃ for 30min.
Skim milk medium: according to mass fraction, 4% of skim milk, 1.5% of agar and pH of 8.3, the preparation method comprises uniformly mixing the components, sterilizing at 115 ℃ for 30min.
The strain produced a relatively large transparent circle under alkaline conditions, indicating that it produced alkaline protease with relatively high enzymatic activity; the research shows that the alkaline protease can promote the degradation of the feathers, which shows that HC2 bacteria have the potential of degrading the feathers/feather meal.
Example 2 high Density fermentation preparation of feed
The waste chicken feathers (water content about 16%) are puffed by a twin-screw extrusion puffing machine under the conditions of 175r/min, 160 ℃ and 0.4MPa to obtain feather meal. Culturing bacillus subtilis HC2 in a liquid LB culture medium for 16h at 37 ℃ and 180r/min as seed liquid, transferring the seed liquid into a shake flask containing 500mL of fermentation culture medium in an inoculum size of 5% by volume, and supplementing glucose and feather meal in batches under the conditions of 37 ℃ and 180r/min, wherein the glucose is supplemented according to 0.5% (mass volume ratio, w/v) of the volume of the fermentation liquid, and the feather meal is supplemented according to 6% (mass volume ratio, w/v) of the volume of the fermentation liquid, and carrying out high-density fermentation and degradation of the feather meal for 3.5 days to obtain the bacillus subtilis HC2 and the mixture of the feather meal degraded by the same. The supernatant of this mixture had a keratinase activity of about 591.4U/mL and an alkaline protease activity of about 697.5U/mL, as shown in FIG. 2. They are higher than the primary and even optimized enzyme activities reported in many studies.
Adding bagasse accounting for 10% of the mixture mass, wheat bran accounting for 12% of the mixture mass and starch accounting for 10% of the mixture mass into the mixture, uniformly mixing, and granulating under the condition of 235r/min to obtain the feed. The feed had a diameter of about 4mm and a length of 5 to 20mm, see FIG. 3. The crude protein content of the feed is 34.6%, the crude fiber content is 6.1%, and the soluble protein content is 22.2g/L. The number of probiotics in the mixture of the feather meal degradation is 3.75X10 8 cfu/mL, and the number of probiotics in the granulated feed is 2.13 multiplied by 10 8 cfu/g still keeps higher concentration of active probiotics, and is beneficial to realizing the probiotic function.
The detection of the relevant indexes of the feed and the preparation process is to measure the crude protein and the soluble protein in the feed by adopting a Kjeldahl nitrogen determination method (GB/T6432-2018) and measure the crude fiber content by adopting a filtration method (GB/T6434-2006). And detecting the quantity of active probiotics in the fermentation liquor and the feed by a dilution gradient method. The fermentation broth was assayed for alkaline protease activity (QB 747-80) and keratinase activity (full-feed label [2021] 52) using Folin-phenol method and spectrophotometry.
LB medium: the yeast powder is 0.5% by mass, the tryptone is 1% by mass, the sodium chloride is 1%, the pH value is 7.0 (1.5% agar is added when solid LB culture medium is needed), and the balance is water.
Fermentation medium: based on mass fraction, naCl 0.05%, KH 2 PO 4 0.04％、K 2 HPO 4 0.03％、MgSO 4 ·7H 2 O0.025%, feather powder 6% and water the rest, pH 9.0, and the preparation method comprises mixing the above components uniformly, sterilizing at 115 deg.C for 30min.>HC2 Strain 16S RNA sequence (SEQ ID NO. 1)
GGGCGCTGTGTAGAGTTTGATCCTGGCTCAGTAAGACGTAACAAGGGTATCCGTGAATGGTTTGATCGTGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGTCCTGGTACACCCCGCCCGTCTCAGCACGACAATGTAGCCGTAGAATTTGATCCTGGCTCAGTAAGTCGTAACAAGGTAGCCGTGGAGTCACG
Claims (8)
1. Bacillus subtilis (Bacillus subtilis) HC2, accession No. GDMCC No:62466.
2. the use of bacillus subtilis HC2 according to claim 1 for degrading feather meal.
3. The use according to claim 2, wherein the feather meal is obtained by puffing discarded chicken, duck and goose hairs.
4. A method for preparing animal feed by bioconversion of waste feathers, which is characterized by comprising the following steps:
adding feather meal into the bacillus subtilis HC2 fermentation broth of claim 1 for biological fermentation and degradation to obtain a mixture of degraded feathers, adding bagasse, wheat bran and starch into the mixture, uniformly compounding, and granulating to obtain the animal feed.
5. The method according to claim 4, characterized by the specific steps of:
1) Inoculating bacillus subtilis HC2 into a liquid LB culture medium for culturing for 10-18 hours to prepare seed liquid;
2) Inoculating seed liquid into a fermentation culture medium in an inoculum size of 4-8% by volume, culturing for 2-4 days at 37 ℃ and 160-220 r/min, and during the period, supplementing glucose and feather meal in batches, wherein the glucose is supplemented according to 0.5% (mass-volume ratio, w/v) of the volume of the fermentation liquid, and the feather meal is supplemented according to 5-8% (mass-volume ratio, w/v) of the volume of the fermentation liquid, so as to perform high-density fermentation and biodegradation of feathers, thereby obtaining a mixture for degrading feathers;
the fermentation medium comprises the following components: comprises NaCl 0.02-0.08% and KH by mass 2 PO 4 0.02～0.06％、K 2 HPO 4 0.01～0.05％、MgSO 4 ·7H 2 0.015-0.055% of O, 6-8% of feather meal, pH of 8.5-9.5, and sterilizing at 115 ℃ for 30min;
3) Uniformly compounding the mixture obtained in the step 2) with bagasse accounting for 8-12% of the mixture mass, wheat bran accounting for 9-15% of the mixture mass and starch accounting for 8-12% of the mixture mass, and granulating under the condition of 230-240 r/min to obtain the animal feed.
6. The method of claim 4 or 5, wherein the feather meal is puffed chicken, duck and goose feather meal.
7. The method of claim 4 or 5, wherein the fermentation medium is: based on mass fraction, naCl 0.05%, KH 2 PO 4 0.04％、K 2 HPO 4 0.03％、MgSO 4 ·7H 2 O0.025%, feather meal 6%, water the rest, pH 9.0, sterilizing at 115 ℃ for 30min.
8. The method according to claim 4 or 5, wherein the feed has a crude protein content of 34.6%, a soluble protein content of more than 20g/L, a crude fiber content of less than 7% and a probiotic viable count of 2.13×10 8 cfu/g。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116831178A (en) * | 2023-06-28 | 2023-10-03 | 广东省科学院生物与医学工程研究所 | Application of feather degradation liquid in orange fresh-keeping |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116831178A (en) * | 2023-06-28 | 2023-10-03 | 广东省科学院生物与医学工程研究所 | Application of feather degradation liquid in orange fresh-keeping |
| CN116831178B (en) * | 2023-06-28 | 2024-03-12 | 广东省科学院生物与医学工程研究所 | Application of feather degradation liquid in orange fresh-keeping |
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