CN109965086B - Hydrolyzed feather meal additive and application thereof in animal feed - Google Patents
Hydrolyzed feather meal additive and application thereof in animal feed Download PDFInfo
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- CN109965086B CN109965086B CN201910399281.5A CN201910399281A CN109965086B CN 109965086 B CN109965086 B CN 109965086B CN 201910399281 A CN201910399281 A CN 201910399281A CN 109965086 B CN109965086 B CN 109965086B
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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
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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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 hydrolyzed feather meal additive and application thereof in animal feed, wherein the preparation method of the additive comprises the following steps: adding water into an enzymolysis base material, uniformly stirring, adjusting the pH to 7.5-9 according to the weight ratio of the enzymolysis base material to the water, adding high-temperature resistant keratinase at the temperature of 60-65 ℃, adding the dry enzymolysis base material with the addition of 1000-6500U/g, and carrying out enzymolysis for 30-36 h to obtain an amino acid compound, and filtering, sterilizing and freeze-drying the amino acid compound to prepare nano-scale particles to obtain an additive; the additive has good antioxidant effect when used in animal feed, and can increase the content of available protein in the feed.
Description
Technical Field
The invention belongs to the technical field of animal feed production, and particularly relates to a hydrolyzed feather meal additive and application thereof in animal feed.
Background
Biologically, aging is a spontaneous inevitable process of an organism over time, and is a complex natural phenomenon, which is manifested as degeneration of structure and functional decline, and decline of adaptability and resistance. Physiologically, aging is considered as a history of ontogeny proceeding from the fertilized egg to the elderly. Pathologically, aging is the result of stress and strain, injury and infection, decline in immune response, nutritional imbalance, metabolic disorders, and the accumulation of negligence and abused drugs. In animal husbandry, especially female animals, which are responsible for the reproduction of offspring of the whole population, the animals gradually age with age, which not only leads to a decline in the functions of their own organs and a decrease in immunity, but also, for female animals, more importantly, affects the health of the offspring.
However, animals cannot slow down aging like human beings by means of measures such as exercise, beauty treatment and operation, so that how to research a feed which can delay aging of animal body functions to a certain extent and can not increase or slightly increase breeding cost is urgent.
Disclosure of Invention
The invention aims to provide a hydrolyzed feather meal additive, which takes animal feathers or hair as raw materials and forms a compound amino acid additive with antioxidant effect after enzymolysis of keratinase; the invention also aims to provide the application of the hydrolyzed feather meal additive in animal feed, and provides a new application for the utilization of animal hair.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the first technical scheme is as follows:
a hydrolyzed feather powder additive is prepared by the following steps: adding water into an enzymolysis base material, uniformly stirring, adjusting the pH to 7.5-9 according to the weight ratio of the enzymolysis base material to the water, adding high-temperature resistant keratinase at the temperature of 60-65 ℃, adding the dry enzymolysis base material with the addition of 1000-6500U/g, and carrying out enzymolysis for 30-36 h to obtain an amino acid compound, and filtering, sterilizing and freeze-drying the amino acid compound to prepare nano particles to obtain an additive; the enzymolysis base material is animal feather or hair. Preferably, the pasteurization is adopted for sterilization, the mixed raw materials are heated to 68-70 ℃, and are rapidly cooled to 4-5 ℃ after being kept at the temperature for 30 min; the filtering process is carried out by adopting an ultrafiltration membrane, residues after hydrolysis and insufficiently hydrolyzed feathers are fully removed, and hydrolysis products are recovered to the maximum extent; preferably, the pretreatment process of the enzymatic hydrolysis base material comprises the following steps: pulverizing the enzymolysis base material, soaking with 5 wt% sodium hydroxide dilute solution for 20-30min, washing with deionized water, and drying at room temperature in sterile environment. The freeze-drying method can directly freeze-dry the enzymatic hydrolysate into powder, the obtained powder can reach the nanometer level, and the method is one of the common methods for preparing the nano particles, and the specific process for preparing the nano particles by the freeze-drying method is common knowledge in the field and is not repeated herein.
As a further improvement of the invention, the enzymolysis base material is white poultry feather. The white bird feather has very little melanin content, and the white bird feather, especially the abdomen feather, has the finest protein filaments and the least light irradiation, and has the optimal quality.
As a further improvement of the invention, the keratinase is produced by the following method: fermenting the Pichia pastoris MMpk-GS115 by methanol feed; wherein the addition amount of the methanol is 0.5-1 wt%; inducing at low temperature of 20-30 ℃ for 60 h. The activity of the keratinase is 50000U/mL-100000U/mL, and the specific activity is 3000U/mg-5000U/mg. The keratinase prepared by the method can ensure that the degradation rate of keratin in feather protein reaches more than 60 percent, and the content of small peptide in the product reaches 10-50 percent. And the generated small peptide is a peptide consisting of 2-5 amino acids.
As a further improvement of the invention, the methanol addition is 0.5 wt%.
As a further improvement of the invention, 0.5 wt% of Tween 80 is also added in the fermentation process.
As a further improvement of the invention, the low temperature induction temperature is 20 ℃.
As a further improvement of the present invention, the fermentation process is high density cell fermentation; the high density cell fermentation is also called high density culture. Generally refers to the growth state or culture technique of microorganisms in liquid culture when the cell population density exceeds the conventional culture by more than 10 times.
The second technical scheme is as follows:
use of a hydrolyzed feather meal additive in animal feed; the addition amount of the hydrolyzed feather meal additive in the animal feed is 8-15 wt%.
Compared with the prior art, the invention has the following technical effects:
the aging mechanism is very complicated, but in summary, the aging mechanism is mainly endogenous factors and exogenous factors, wherein the endogenous factors refer to natural aging controlled by genetic factors, and the exogenous factors are mainly non-natural aging caused by external damage such as illumination. For either reason, the underlying mechanism is the increase of peroxidized substances (LPO) in blood or skin tissue. The additive has certain inhibiting effect on the generation of a peroxide substance LPO in blood, and the reduction of the content of the peroxide substance LPO in the blood has important significance on the delaying of the aging of animals; in addition, the additive after freeze drying is nano-scale particles, active sites are increased after the particles are subjected to nanocrystallization, and the activity is greatly improved, so that the antioxidant effect of the additive can be fully exerted.
Poultry breeding is one of the underpinnings of livestock husbandry in China, and the feather yield of chickens, ducks and geese is very rich, and the annual yield is up to more than million tons. Therefore, the feather meal is a common protein feed raw material, the crude protein of the feather meal is more than 80 percent, the feather meal is rich in a plurality of essential amino acids of animals, the total amount of the amino acids is more than 70 percent, the content of sulfur-containing amino acids is high, and the feather meal has high utilization value in the feed, but the development and the utilization of the feather meal are seriously influenced because the disulfide bond protein structure of the feather is difficult to digest and absorb by the animals. The feather powder is hydrolyzed by keratinase, and finally the compound of low molecular peptide with different molecular weights obtained by the hydrolysis of the keratinase, a small amount of undegraded complete keratin, a trace amount of keratinase and the like is obtained, wherein the low molecular peptide does not contain disulfide bonds and can be well absorbed by animals, the incompletely degraded keratin still contains certain disulfide bonds, and the redox reaction can form or break the disulfide bonds in protein molecules, and the process is reversible, namely the disulfide bonds can be accompanied with the redox reaction in the breaking process, the disulfide bonds and the trace amount of keratinase act in the bodies of the animals, the process of degrading the disulfide bonds by the keratinase can initiate the reduction reaction of peroxide LPO, so that the content of the peroxide LPO in the bodies of the animals is reduced, and the compound has important significance for delaying the aging of the animals, and the cost is low, and simultaneously, the novel application of the waste animal feathers is expanded, and the reasonable application of resources is realized.
The additive of the present invention is used as a protein supplement and an antioxidant substance additive in animal feed, and the addition amount thereof is specifically determined according to the specific animal species. The additive has certain effect on delaying animal aging after being applied to animal feed. A large number of safety experiments prove that the additive has no toxic or side effect on animals.
Detailed Description
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, to the extent there is also a numerical limitation, also is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
In addition, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, instrumentalities, and/or procedures known to those skilled in the art have not been described in detail so as not to obscure the present invention. In the present invention, the units indicating the contents of the raw materials are based on parts by weight unless otherwise specified. In addition, the determination methods of the technical indexes of the invention are all standard methods used in the field, and specific reference can be made to the latest national standard unless otherwise stated.
The Pichia pastoris MMpk-GS115 used in the invention is purchased from China Center for Type Culture Collection (CCTCC), is classified and named as Pichia pastoris MMpk-GS115(pichia pastoris MMpk-GS115), and the preservation number of the preservation center is as follows: CCTCC NO: M2014171, preservation date is: in 2014, 28 days 4 and 28, the preservation unit is as follows: china Center for Type Culture Collection (CCTCC), address: wuhan university in Wuchang Lojia mountain; the Pichia pastoris MMpk-GS115 morphological and physiological and biochemical characteristics comprise: is milk white, has oval or round shape when being examined by a thallus microscope, and is a methanol nutrition type bacterium.
Example 1:
fermenting the Pichia pastoris MMpk-GS115 by methanol feed; wherein the addition amount of methanol is 0.5 wt%; inducing at 20 deg.C for 60 h. The activity of the keratinase is 80000U/mLU/mL, and the specific activity is 5000U/mg.
Crushing the enzymolysis base material-white duck abdomen feather, soaking in 5 wt% sodium hydroxide dilute solution for 20-30min, washing with deionized water, and drying at room temperature in sterile environment. Adding water into the dried enzymolysis base material, uniformly stirring, adjusting the pH value to 9 according to the weight ratio of the enzymolysis base material to the water, adding high-temperature resistant keratinase at the temperature of 60 ℃, adding 1000U/g of the dried enzymolysis base material, and carrying out enzymolysis for 36 hours to obtain an amino acid compound, carrying out ultrafiltration on the amino acid compound by using an ultrafiltration membrane, heating the mixed raw material to 68-70 ℃, keeping the temperature for 30min, rapidly cooling to 4-5 ℃, and carrying out pasteurization; the nano-scale additive is prepared by a freeze-drying method.
Example 2:
this example differs from example 1 in that methanol was added in an amount of 0.8 wt% during the keratinase preparation, and the remaining steps and parameters were the same as those of example 1.
Example 3:
this example differs from example 1 in that methanol was added in an amount of 1 wt% during the keratinase preparation process, and the remaining steps and parameters were the same as those of example 1.
Example 4:
the difference between this example and example 1 is that 0.5 wt% Tween 80 is also added during the preparation of keratinase, and the rest of the steps and parameters are the same as those in example 1.
Example 5:
the difference between this example and example 1 is that the low temperature induction temperature in the keratinase production process is 30 ℃, and the rest of the steps and parameters are the same as those in example 1.
Example 5:
this example differs from example 1 in that the low temperature induction temperature during the keratinase preparation process is 25 ℃, and the rest of the steps and parameters are the same as those of example 1.
Example 6:
this example differs from example 1 only in that: the pH of the mixture was adjusted to 8 during the enzymatic hydrolysis, and the rest of the steps and parameters were the same as in example 1.
Example 7:
this example differs from example 1 only in that: the pH was adjusted to 7.5 during the enzymatic hydrolysis, and the rest of the steps and parameters were the same as in example 1.
Example 8:
this example differs from example 1 only in that: the temperature in the enzymolysis process is 65 ℃, and the rest steps and parameters are the same as those in the example 1.
Example 9:
this example differs from example 1 only in that: the addition of keratinase in the enzymolysis process is 1500U/g dry enzymolysis base material, and the rest steps and parameters are the same as those in example 1.
Comparative example 1:
this comparative example differs from example 1 only in that: the non-nano additive is obtained by adopting a spray drying method.
Comparative example 2:
this comparative example differs from example 1 only in that: the keratinase is a commercial product purchased from Shenzhen Hensheng biologicals GmbH.
The additives of examples 1 to 9 and comparative examples 1 to 2 were tested for protein content and the results are shown in Table 1.
TABLE 1
The total protein content is detected by adopting a Kjeldahl method, the soluble protein is detected by adopting a Coomassie brilliant blue G-250 method, and the ammonia nitrogen content is detected by adopting a formaldehyde method according to GB/T3600-2000; the specific process is common knowledge and will not be described herein.
The acid soluble protein assay method is as follows:
the method comprises the following operation steps:
(1) accurately weighing 6g of a sample to be detected in a 100mL beaker, accurately adding 50mL of 15% trichloroacetic acid solution, uniformly mixing, and standing for 5 min;
(2) performing dry filtration by using medium-speed qualitative filter paper, discarding a little initial filtrate, and transferring the filtrate to a centrifuge tube;
(3) centrifuging at 4000r/min for 10min, accurately transferring 10mL of supernatant into a digestion tube, and measuring the protein content by a Kjeldahl method.
(4) And (3) measuring the content of the crude protein by a Kjeldahl method.
Calculating the formula:
wherein: v1- -volume of hydrochloric acid standard solution consumed by distillate (mL);
v0- -blank test consumes volume (mL) of HCl standard solution;
c- - -molar concentration of HCl standard solution (mol/L);
0.014- -molar mass of nitrogen (g/mmol);
6.25- -conversion of Nitrogen to protein;
m- -dry matter weight of solid sample (g);
cp-crude protein content of test sample (%)
To verify the removal effect of the lipid peroxide LPO in blood in examples 1-9 and comparative examples 1-2, an animal experiment model was constructed.
1.1 test drugs
The additives prepared in examples 1-9 and comparative examples 1-2 were named additives 1-9 and comparative additives 1-2, respectively.
1.2 test animals
For better verification effect, a blank control group is introduced, all rats have good health condition, and the weight difference is less than +/-0.1 kg, and the method is provided by Jinfeng test animals of Jinan, Co., Ltd.
1.3 feeding mode
Mixing the additive with conventional feed for rats, wherein the amount of the additive is 15% of the total amount of the feed, the feeding amount and daily management of each rat are performed according to a conventional method, the daily management and feed feeding amount of each rat are completely consistent, and the rats in the control group are fed with the conventional feed without the additive.
1. Test method
(1) Firstly, the content of lipid peroxide LPO in the blood of each group of rats is detected, and the average value is recorded as A0。
(2) The daily management was carried out according to the conventional method and the feeding method of 1.3.
(3) After 30 days, the blood of each group of rats was again examined for the content of lipid peroxide LPO, and the mean value was recorded as A1。
(4) The rate of change of lipid peroxide LPO was calculated. Calculation method (A)1-A0)/A0×100％。
The calculation results are recorded in table 2.
TABLE 2
Wherein: "+" represents increase and "-" represents decrease.
The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.
Claims (6)
1. The application of the hydrolyzed feather meal additive in preparing animal feed is characterized in that the preparation method comprises the following steps: adding water into an enzymolysis base material, uniformly stirring, adjusting the pH to 7.5-9 according to the weight ratio of the enzymolysis base material to the water, adding high-temperature resistant keratinase at the temperature of 60-65 ℃, adding the dry enzymolysis base material with the addition of 1000-6500U/g, and carrying out enzymolysis for 30-36 h to obtain an amino acid compound, and filtering, sterilizing and freeze-drying the amino acid compound to prepare nano particles to obtain an additive; the enzymolysis base material is animal feather or hair;
the production method of the keratinase comprises the following steps: fermenting the Pichia pastoris MMpk-GS115 by methanol feed; wherein the addition amount of the methanol is 0.5-1 wt%; inducing at low temperature of 20-30 ℃ for 60 h;
0.5 wt% of Tween 80 is also added in the fermentation process;
the addition amount of the hydrolyzed feather meal additive in the animal feed is 8-15%;
the animal feed has the function of delaying the aging of animals.
2. The use according to claim 1, wherein the filtration is performed using an ultrafiltration membrane.
3. The use of claim 1, wherein the enzymatically hydrolyzed base material is white poultry feathers.
4. Use according to claim 1, wherein the methanol is added in an amount of 0.5 wt%.
5. Use according to claim 1, wherein the low temperature induction temperature is 20 ℃.
6. Use according to claim 1, wherein the fermentation process is a high density cell fermentation.
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| CN110591931A (en) * | 2019-09-27 | 2019-12-20 | 华中农业大学 | Pichia pastoris, generated high-temperature-resistant enzyme and application |
| CN110643521A (en) * | 2019-09-27 | 2020-01-03 | 华中农业大学 | Pichia pastoris and keratinase fermented by same and application of keratinase |
| CN110506839A (en) * | 2019-09-27 | 2019-11-29 | 华中农业大学 | A kind of feather flour additive agent, preparation method and applications |
| CN110683882A (en) * | 2019-11-13 | 2020-01-14 | 湛江市绿海生物工程有限公司 | Process method for preparing amino acid water-soluble fertilizer from feather meal |
| CN113604529B (en) * | 2021-08-13 | 2023-10-10 | 广东完美生命健康科技研究院有限公司 | Chicken feather oligopeptide and extraction method and application thereof |
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| JP2011155932A (en) * | 2010-02-02 | 2011-08-18 | Toyo Univ | Alkali keratinase, dna encoding the same and method for using the same |
| JP6161190B2 (en) * | 2013-02-21 | 2017-07-12 | 学校法人 東洋大学 | Thermostable keratinase enzyme, method for producing the same, and DNA encoding the same |
| CN103981164A (en) * | 2014-05-30 | 2014-08-13 | 华中农业大学 | High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis |
| CN104770574A (en) * | 2015-03-17 | 2015-07-15 | 齐鲁工业大学 | Method of preparing feather protein powder from keratinase |
| CN106387320B (en) * | 2016-08-30 | 2020-02-07 | 济南诺能生物工程有限公司 | Feather processing method |
| CN108497157A (en) * | 2018-04-09 | 2018-09-07 | 广东希普生物科技股份有限公司 | High digestibility enzymolysis feather powder and preparation method thereof |
| CN108713640A (en) * | 2018-05-29 | 2018-10-30 | 芜湖乾凯材料科技有限公司 | Feather meal feed and preparation method thereof |
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