CN109628534B - Method for degrading keratin waste resources through combined treatment of bacteria and enzymes - Google Patents
Method for degrading keratin waste resources through combined treatment of bacteria and enzymes Download PDFInfo
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- CN109628534B CN109628534B CN201811332921.2A CN201811332921A CN109628534B CN 109628534 B CN109628534 B CN 109628534B CN 201811332921 A CN201811332921 A CN 201811332921A CN 109628534 B CN109628534 B CN 109628534B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
Abstract
The invention discloses a method for treating keratin waste resources by combining bacteria and enzymes, which mainly comprises the following steps: a. preparing a fermentation medium; b. inoculating the strain into a fermentation culture medium to prepare a primary seed solution; c. adding keratin substances into the first-stage seed liquid for fermentation culture; d. adjusting the reaction temperature after the fermentation culture is finished, and maintaining for 2-6 h; according to the method disclosed by the invention, the keratin waste resources can be fully utilized, the final product with higher protein nitrogen content can be obtained, the protein nitrogen recovery rate is improved, the method is an effective way for realizing harmless and high-value utilization of the keratin waste resources, and the method has good application prospects in the fields of livestock and poultry breeding waste resource utilization, amino acid bio-organic fertilizer, protein feed, polypeptide protein amino acid preparation and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for degrading keratin waste resources by combined treatment of bacterial enzymes.
Background
The livestock breeding industry in China is developed, a large amount of waste keratin such as animal hair, bird feather, horn, claw, hoof and the like is generated every year, and according to statistics, only the feather waste reaches millions of tons. These waste keratins are often not utilized effectively and the problem of environmental pollution is increasingly highlighted. Keratin is rich in a large amount of nutrients, such as crude protein content in feathers is usually more than 80%, and the feather is rich in various amino acids such as cystine, lysine, proline, serine, and the like, and if properly treated, the waste is an important protein resource bank. However, at present, only a small part of keratin waste is effectively recycled, and the vast majority of keratin waste is directly discarded, so that not only is the environment pollution caused, but also the resource waste is realized. At present, the waste keratin is applied to the fields of feed, fertilizer, medicine, cosmetics and the like, and the development space is huge.
At present, the methods for processing keratin resources on a large scale mainly comprise a physical and chemical hydrolysis process (a high-temperature high-pressure hydrolysis method and an acid-base hydrolysis method) and a biological fermentation process. The physicochemical hydrolysis method can obtain higher protein recovery rate, but high temperature and high pressure or strong acid and strong base can destroy partial amino acid, so that the content of free amino acid is extremely low. Meanwhile, the acid-base hydrolysis method has high requirements on equipment, and generates a large amount of salt in the acid-base neutralization process in the later period, thereby affecting the product quality.
The biological fermentation process mainly comprises two types of microbial direct fermentation and enzymatic hydrolysis. The microbial fermentation method utilizes bacterial strains with the secretion capacity of keratinase and the like to degrade feathers, thereby obtaining the compound hydrolyzed amino acid. The enzymolysis method is to degrade keratin materials by using specific keratinase, thereby obtaining amino acid products. Compared with a physicochemical hydrolysis process, the energy consumption for hydrolyzing the feather waste is lower and the pollution is less in the biological fermentation process, the enzyme system generated by microbial fermentation promotes keratin to be decomposed into polypeptide and amino acid, the condition is mild, the loss of thermosensitive amino acid is reduced, and the content of free amino acid can be greatly improved.
However, biological fermentation methods also have some disadvantages. In the process of converting keratin by microorganisms, on one hand, keratin is difficult to biodegrade, and the degradation efficiency is low. Therefore, the excellent strain with high keratin degradation rate and high keratinase activity is the key for realizing the transformation of the keratin by the microorganism. Patent CN101372678B discloses a keratin degrading bacterium NJY1, and patent CN102321553B discloses a feather keratin anaerobic degrading strain 18D-TA, and both strains can degrade keratin well. On the other hand, amino acid generated by keratin degradation can be continuously utilized by microorganisms and converted into inorganic nitrogen such as ammonia nitrogen and the like, so that the content of amino acid nitrogen (namely amino nitrogen) in a fermentation product is low, the specific gravity of non-protein nitrogen is high, and the quality of the product can be influenced. Patent CN102326668B discloses a method for anaerobic degradation of feather keratin by 18D-TA, which can rapidly degrade keratin, but the product has low content of amino acid nitrogen and low availability. Therefore, the proper fermentation process is adopted to strictly limit the conversion of protein nitrogen into inorganic nitrogen, and the method is an effective way for improving the product quality. The invention provides a method for effectively converting keratin waste and obtaining protein nitrogen resources by combining the advantages and disadvantages of the prior art and adopting a bacterial enzyme combined treatment process.
Disclosure of Invention
The invention aims to provide a method for degrading keratin by combined treatment of bacteria and enzymes aiming at the problems of low conversion efficiency and low organic nitrogen/inorganic nitrogen ratio in the process of converting keratin substances into protein nitrogen such as polypeptide protein, amino acid and the like, and aims to realize harmless and high-valued utilization of keratin resources.
In order to realize the purpose, the technical scheme is as follows:
a method for degrading keratin waste resources by combined treatment of bacterial enzymes comprises the following steps:
a. preparing keratin substances to be degraded and preparing a fermentation medium;
b. preparing 18D-TA bacteria liquid, and inoculating the 18D-TA bacteria liquid into the fermentation culture medium obtained in the step a to prepare primary fermentation seed liquid;
c. b, adding the primary fermentation seed liquid obtained in the step b into the keratin substances obtained in the step a for secondary fermentation culture, wherein the secondary fermentation culture time is 0-20 h;
d. and after the second-stage fermentation culture is finished, adjusting the temperature to 70-90 ℃ to perform third-stage fermentation culture, wherein the third-stage fermentation culture time is 2-6 h.
As a further optimization scheme of the invention, the fermentation medium in the step a is an anaerobic medium.
As a further optimization scheme of the invention, the 18D-TA strain in the step a has a strain preservation number of CGMCC NO. 4800.
As a further optimization scheme of the invention, the inoculation volume of the bacterial liquid in the step b is 1-10% of the volume of the culture medium; the fermentation temperature is 45-60 ℃, and the fermentation time is 24 h.
As a further optimization of the present invention, the keratin material in step c comprises at least one of poultry feather, animal hair and hoof nail; the keratin substance and the fermentation seed liquid are mixed according to the mass-volume ratio of 1: 10-1:1.
As a further optimization scheme of the invention, the culture temperature of the secondary fermentation in the step c is 45-60 ℃.
As a further optimization scheme of the invention, the temperature rise rate of the temperature adjustment in the step d is higher than 1 ℃/min.
As a further optimization scheme of the invention, the heating mode comprises at least one of gas bath heating, steam heating, water bath heating and oil bath heating.
As a further optimization scheme of the invention, the primary fermentation in the step a and the secondary fermentation in the step b are both anaerobic fermentation, and the tertiary fermentation in the step c is anaerobic fermentation or aerobic fermentation.
According to the method of the invention, the strain 18D-TA depolymerizes the higher order structure of feather equiangular proteins and produces large amounts of keratinase within 0-20 h. After the reaction temperature is adjusted, the growth of thalli is inhibited due to the rise of the temperature, and the consumption of amino acid nitrogen is effectively reduced; meanwhile, the activity of the keratin enzyme is maximized, the keratin molecules are promoted to be rapidly degraded into polypeptide protein and amino acid, and the conversion of organic nitrogen such as the polypeptide protein and the amino acid to inorganic nitrogen can be effectively inhibited, so that the content of the organic nitrogen in a final product can be obviously improved.
The invention has the beneficial effects that:
the method comprehensively utilizes the physiological metabolism characteristics of the strain 18D-TA, utilizes the difference between the growth temperature and the enzyme production temperature of the strain and the activity temperature of the keratinase and the protease, effectively controls the processes of keratin hydrolysis, amino acid deamination and the like through proper temperature regulation and control, realizes the conversion of keratin to protein nitrogen to the maximum extent, has simple operation method, is an effective way for realizing harmless and high-value utilization of keratin waste resources, and has good application prospect in the fields of livestock and poultry breeding waste resource utilization, amino acid bio-organic fertilizer, protein feed, polypeptide protein amino acid preparation and the like.
Drawings
FIG. 1 is a graph showing the contents of components in a fermentation product obtained in example 1;
FIG. 2 is a graph showing the contents of the components of the fermentation product obtained in comparative example 1;
FIG. 3 is a graph showing the content of each component of the fermentation product obtained in example 2;
FIG. 4 is a graph showing the content of each component of the fermentation product obtained in example 3;
FIG. 5 is a graph showing the content of each component of the fermentation product obtained in example 4;
FIG. 6 is a graph showing the contents of the components of the fermentation product obtained in example 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the accompanying drawings.
The strain in the following embodiment is keratin degrading bacteria 18D-TA with the preservation number of CGMCC NO. 4800.
Example 1
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 2% of 18D-TA liquid seed liquid after high-temperature and high-pressure sterilization, and culturing at 55 ℃ for 24h to obtain primary fermentation seed liquid.
(2) Adding 50% (w/v) feather into the primary fermentation seed liquid, and fermenting and culturing at 55 ℃ for 20 h;
(3) and (3) adjusting the temperature of the fermentation liquid after the fermentation culture to 85 ℃ in a preheating mode, and maintaining for 4 hours.
(4) Collecting the secondary fermentation product, drying and crushing into powder.
(5) The content of each component of the secondary fermentation product is determined, and is shown in figure 1.
Comparative example 1
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 2% of 18D-TA liquid seed liquid after autoclaving, and culturing for 24h at 55 ℃ to obtain primary fermentation seed liquid.
(2) Adding 50% (w/v) feather into the primary fermentation seed liquid, and fermenting and culturing at 55 ℃ for 20 h;
(3) keeping the fermentation liquid at 55 deg.C for 4 h.
(3) Collecting the secondary fermentation product, drying and crushing into powder.
(4) The content of each component of the secondary fermentation product is determined, and is shown in figure 2.
Comparing fig. 1 and fig. 2, it can be found that the dry matter in fig. 1 is higher, and all indexes are improved, wherein the recovery rate of keratin nitrogen is improved by 13%, the content of hydrolyzed amino acid is improved by 2%, the amplitude of acid-soluble protein is improved by 144%, the polypeptide protein is improved by 139%, and the growth rate of free amino acid is 48%, indicating that the temperature is increased after the fermentation culture is finished, which is helpful for keeping protein and amino acid in keratin as much as possible, thereby improving the utilization value of the product.
Example 2
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 10% of 18D-TA liquid seed liquid after autoclaving, and culturing for 24h at 55 ℃ to obtain primary fermentation seed liquid.
(2) Adding 10% (w/v) feather into the primary fermentation seed liquid, adjusting the temperature to 70 ℃ in a water bath heating mode, and maintaining for 6 h.
(3) Collecting the secondary fermentation product, drying and crushing into powder.
(4) The content of each component of the secondary fermentation product is determined, and is shown in figure 3.
Example 3
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 5% of 18D-TA liquid seed liquid after autoclaving, and culturing for 24h at 55 ℃ to obtain primary fermentation seed liquid.
(2) Adding 50% (w/v) feather into the primary fermentation seed liquid, and fermenting and culturing at 55 ℃ for 12 h;
(3) adjusting the temperature of the fermentation liquid after the fermentation culture to 85 ℃ in a steam heating mode, and maintaining for 2 h.
(4) Collecting the secondary fermentation product, drying and crushing into powder.
(5) The content of each component of the secondary fermentation product is determined, and is shown in figure 4.
Example 4
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 8% of 18D-TA liquid seed liquid after autoclaving, and culturing for 24h at 55 ℃ to obtain primary fermentation seed liquid.
(2) Adding 25% (w/v) feather into the primary fermentation seed liquid, introducing nitrogen into the fermentation tank, and fermenting and culturing at 45 deg.C for 12 h;
(3) adjusting the temperature of the fermentation liquid after the fermentation culture to 90 ℃ in a preheating mode, and maintaining for 2 hours.
(4) Collecting the secondary fermentation product, drying and crushing into powder.
(5) The content of each component of the secondary fermentation product is determined, and is shown in figure 5.
Example 5
(1) Preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 8% of 18D-TA liquid seed liquid after autoclaving, and culturing for 24h at 55 ℃ to obtain primary fermentation seed liquid.
(2) Adding 25% (w/v) feather into the primary fermentation seed liquid, introducing air into the fermentation tank, and fermenting and culturing at 60 deg.C for 12 hr;
(3) adjusting the temperature of the fermentation liquid after the fermentation culture to 90 ℃ in a preheating mode, and maintaining for 2 hours.
(4) Collecting the secondary fermentation product, drying and crushing into powder.
(5) The content of each component of the secondary fermentation product is determined, and is shown in figure 6.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A method for degrading feathers through bacterium and enzyme combined treatment is characterized by comprising the following steps:
(1) preparing a fermentation culture medium by adopting a Hencatel anaerobic operation technology, inoculating 2% of strain 18D-TA liquid seed solution after high-temperature and high-pressure sterilization, and culturing at 55 ℃ for 24h to obtain primary fermentation seed solution; the preservation number of the strain 18D-TA is CGMCC NO. 4800;
(2) adding 50% (w/v) feather to be degraded into the primary fermentation seed liquid for secondary fermentation, and performing fermentation culture at 55 ℃ for 20 h;
(3) and (3) adjusting the temperature of the fermentation liquid after the fermentation culture to 85 ℃ in a preheating mode, and performing three-stage fermentation for 4 hours.
2. The method for degrading feather through combined bacterial and enzyme treatment according to claim 1, wherein the concentration of seed liquid obtained in the primary fermentation in step (1) is 108-109And each m L.
3. The method for degrading feathers through combined bacterial and enzyme treatment as claimed in claim 1, wherein the temperature-adjusting temperature rise rate in the step (3) is higher than 1 ℃/min.
4. The method for degrading feathers through combined bacterial and enzyme treatment according to claim 3, wherein the heating manner comprises at least one of gas bath heating, steam heating, water bath heating and oil bath heating.
5. The method for degrading feathers through combined treatment of bacterial and enzyme as claimed in claim 1, wherein the primary fermentation in step (1) and the secondary fermentation in step (2) are both anaerobic fermentation, and the tertiary fermentation in step (3) is anaerobic fermentation or aerobic fermentation.
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| CN101285086A (en) * | 2007-12-29 | 2008-10-15 | 青岛康地恩生物科技有限公司 | Feather products, processing technology and use thereof |
| CN102321553A (en) * | 2011-08-24 | 2012-01-18 | 农业部沼气科学研究所 | Feather keratin anaerobic degradation strain 18D-TA |
| CN102326668A (en) * | 2011-08-24 | 2012-01-25 | 农业部沼气科学研究所 | Method for anaerobically degrading feather keratin with microbial strain 18D-TA |
| CN106350558A (en) * | 2016-09-06 | 2017-01-25 | 生物源生物技术（深圳）有限公司 | Method for jointly degrading feathers by aid of enzyme bacteria |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285086A (en) * | 2007-12-29 | 2008-10-15 | 青岛康地恩生物科技有限公司 | Feather products, processing technology and use thereof |
| CN102321553A (en) * | 2011-08-24 | 2012-01-18 | 农业部沼气科学研究所 | Feather keratin anaerobic degradation strain 18D-TA |
| CN102326668A (en) * | 2011-08-24 | 2012-01-25 | 农业部沼气科学研究所 | Method for anaerobically degrading feather keratin with microbial strain 18D-TA |
| CN106350558A (en) * | 2016-09-06 | 2017-01-25 | 生物源生物技术（深圳）有限公司 | Method for jointly degrading feathers by aid of enzyme bacteria |
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