CN104770574A - Method of preparing feather protein powder from keratinase - Google Patents
Method of preparing feather protein powder from keratinase Download PDFInfo
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- CN104770574A CN104770574A CN201510115893.9A CN201510115893A CN104770574A CN 104770574 A CN104770574 A CN 104770574A CN 201510115893 A CN201510115893 A CN 201510115893A CN 104770574 A CN104770574 A CN 104770574A
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Abstract
The invention relates to the technical field of animal fodder processing, and especially relates to a method of preparing feather protein powder from keratinase. The method takes poultry feathers as the raw material and is characterized by comprising the following steps: (1) preparing feather powder; (2) performing enzymatic hydrolysis; (3) filtering and drying. The technology is simple, the suitable field of the technology is enlarged, the temperature of enzymatic hydrolysis is low, the pH range is wide, the cost of enzymatic hydrolysis is low, and the efficiency of enzymatic hydrolysis is high. The content of soluble protein in the supernate of enzymatic hydrolysis is high, the supernate contains 8 amino acids with a high content, wherein phenylalanine, isoleucine, leucine and valine are essential amino acids; the protein powder can be used to produce amino acid products and added into the feed, thus the production cost is reduced, and the comprehensive economic benefit is increased.
Description
Technical field
The present invention relates to animal feed processing technique field, particularly a kind of method utilizing keratinase to prepare feather albumen powder.
Background technology
Feather is one of important resource of China, output is very abundant, annual production more than 1,000,000 tons, be by a kind of double bond structure very firmly keratin form, chemical composition is the same with general protein, but molecular shape is similar to fiber, close structure is solid, and density is large, itself is difficult to be digested and assimilated by animal, after physics or chemical method processing process, the protein that animal can digest and assimilate need be become.
Adopting gas explosion enzyme solution to prepare feather albumen powder, there is potential safety hazard in the method complex procedures, and large to the amino acid whose destruction in feather meal on the other hand, the crude protein content in the feather albumen powder prepared reaches 90%, and can only be used as forage protein.Applicant is the preparation method disclosing a kind of feather albumen powder in the patent of 2014104846137 at application number, keratinase/neutral proteinase is utilized to carry out complex enzyme hydrolysis to feather, its the most practical temperature is 40 degree, higher concerning energy consumption actual production; PH sphere of action due to keratinase is 7 ~ 10, the pH sphere of action of neutral proteinase is 5.5 ~ 8.5, after two kinds of enzyme compounds, research finds that the suitableeest action pH is 8, both overlapping zones of action are less, can drop into too much expense in production for regulating pH, and the addition of the enzyme adapted to most is keratinase 0. 0375/ neutral proteinase 0. 125 g, consumption is higher, these all can increase the production cost of the producer, reduce economic benefit.In addition, because the enzymolysis site of different protease is variant, therefore in enzymolysis supernatant, amino acid whose composition is not identical yet, keratinase/neutral proteinase is adopted to carry out complex enzyme hydrolysis to feather, and undeclaredly in enzymolysis liquid, which kind of amino acid detected, greatly reduce its range of application.
Summary of the invention
The present invention, in order to make up the deficiencies in the prior art, provides the keratinase that utilizes that a kind of technique is simple, production cost is low, enzymatic hydrolyzation is high and prepares the method for feather albumen powder.
The present invention is achieved through the following technical solutions:
Utilize keratinase to prepare a method for feather albumen powder, it is characterized in that: take poultry feather as raw material, adopt following steps:
(1) preparation of feather meal: by poultry feather through cleaning, removal of impurities, shred, dry loading beaker, insert 120 DEG C of sterilizing 40min in high-pressure steam sterilizing pan, then treated, drain, 80 DEG C of dry 12h in thermostatic drying chamber, after pulverizing, grind, crossing 100 mesh sieves, obtain feather meal, kept dry is stand-by;
(2) enzymolysis: feather meal is placed in container, 1g feather meal adds 100ml distilled water, keratinase 0.01-0.06g, and wherein the pH of enzymolysis liquid is 5-10, hydrolysis temperature 25-50 DEG C, enzymolysis time 4-46h, is placed in constant-temperature table and carries out enzymolysis;
(3) filtering drying: filtered by enzyme digestion reaction liquid, measures filtered fluid soluble protein and amino acid content, weighs stand-by after filtering residue drying.
Poultry feather described in step (1) is chicken feather or duck feather.
Preferably, the enzymolysis process described in step (2), the enzyme addition of every 1g feather meal is keratinase 0.03g, and hydrolysis temperature is 35 DEG C, and enzymolysis liquid pH is 7, enzymolysis time 24h.
Preferably, the mensuration of the content of the soluble protein described in step (3) adopts Coomassie Brilliant Blue to measure, and take bovine serum albumin as standard protein, mensuration wavelength is 595nm, calculate soluble protein content (μ g/g) and feather resolution ratio (%), curve plotting.
Further, Vitro Digestibility is measured in step (3), assay method is leach in residue to add pepsin and the prefabricated acid water to 45 DEG C, its mixture is placed in 45 DEG C of constant-temperature tables and digests 16h, by reacting liquid filtering, dry, weigh, wherein, every 1g leaches the pepsin that residue adds 3000 units, and the addition of acid water is 100ml.
The invention has the beneficial effects as follows: present invention process is simple, widened technique suitable application area, and its hydrolysis temperature is low, pH sphere of action is wide, enzymolysis cost is low, enzymolysis efficiency is high, and enzymolysis supernatant soluble protein content is higher, simultaneously containing the aminoacid ingredient that 8 kinds of content is higher, wherein phenylalanine, isoleucine, leucine, valine are essential amino acid, amino acid products and feed interpolation albumen can be processed as, reduce production cost, improve overall economic efficiency.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further illustrated.
Accompanying drawing 1 is protein content canonical plotting of the present invention;
Accompanying drawing 2 is hydrolysis temperature trial curve figure of the present invention;
Accompanying drawing 3 is enzymolysis liquid of the present invention initial pH trial curve figure;
Accompanying drawing 4 is keratinase addition trial curve figure of the present invention;
Accompanying drawing 5 is enzymolysis time trial curve figure of the present invention.
Detailed description of the invention
For a better understanding of the present invention, further illustrate below in conjunction with specific embodiment.
Embodiment:
one. material and method
Material
The feather (factory obtains from butchering fowl) of chicken and duck, keratinase: 100000U/g(Jinan Nuo Neng bioengineering Co., Ltd), 1mol/L NaOH solution, 1mol/L HCL solution, distilled water, (take 100mg Coomassie brilliant G-250 to be dissolved in 50ml95% ethanol, add 100mL85% phosphoric acid, adding distil water is diluted to 1000mL to Coomassie brilliant blue reagent.), (crystallization bovine serum albumin measures protein nitrogen content through micro-Kjeldahl, is mixed with the protein standard solution of 100 μ g/mL according to its purity protein standard solution in advance.), pepsin (protease is actual tires as 1:3000).
2. key instrument equipment
Key instrument
250mL triangular flask 18, pipette (10mL), test tube and rack for test tube, volumetric flask (50mL), mortar, 100 mesh standard sieves, dropper, funnel, filter paper, graduated cylinder, pipettor (100-1000 μ l; 5ml), large lancet head, cuvette, lens wiping paper, glass bar, accurate pH test paper.
Capital equipment
Vertical automatic electric heating pressure steam sterilizer (LDZX-40BI type) (Shenan Medical Appliances Factory, Shanghai); Constant-temperature shaking incubator (Shanghai Zhi Cheng analytical instrument Manufacturing Co., Ltd); Visible spectrophotometer (Shanghai Precision Scientific Apparatus Co., Ltd); Thermostatic drying chamber; Trace electronic balance (Beijing Sai Duolisi balance Co., Ltd); The full-automatic amino-acid analyzer of Biochrom 30+ (hundred health).
Test method and content
The pretreatment of feather and the preparation of sample of feather
The sample of feather obtained from poultry treatment plant generally contains the impurity such as many blood silt, so need before using to clean removal of impurities, otherwise can affect test effect and quality.In order to enzyme digestion reaction evenly need feather to make powder.
Feather pretreatment: feather → cleaning, removal of impurities → shred, dry → load beaker → insert high-pressure steam sterilizing pan (120 DEG C, 40min) → process → drain → thermostatic drying chamber (80 DEG C, 12h) → pulverize, grind, (100 order) → feather meal → kept dry of sieving is stand-by.
Multiple enzyme (keratinase) enzymolysis feather powder test
Single factor experiment determines multiple enzyme enzymolysis optimum condition
Get 18 250ml triangular flasks, 6 processed group, often organize 3 repeating groups, every bottle adds 100ml distilled water and 1g feather meal sample.
A. hydrolysis temperature test:, keratinase addition 0.06g, regulate pH to be 7, enzymolysis time 24h, hydrolysis temperature is respectively 25,30,35,40,45,50 DEG C, reacts under constant-temperature table.
B. the initial pH test of enzymolysis liquid: hydrolysis temperature is with test last time for reference, and enzyme addition keratinase 0.06g, enzymolysis time 24h, enzymolysis liquid pH is respectively 5,6,7,8,9,10, reacts under constant-temperature table.
C. enzyme addition test: hydrolysis temperature and pH are with test last time for reference, and keratinase addition is respectively 0.01 g, 0.015g, 0.02g, 0.03g, 0.045g, 0.06g, react under constant-temperature table.
D. enzymolysis time test: hydrolysis temperature, pH, enzyme addition with test last time for reference, enzymolysis time is respectively 4,8,14,23,38,46h, react under constant-temperature table.
After often organizing off-test, enzyme digestion reaction liquid is filtered, get filtered fluid and measure soluble protein content, weigh after filtering residue drying stand-by.
3.2.2 soluble protein content measures
Adopt Coomassie Brilliant Blue to measure protein content, take bovine serum albumin as standard protein, mensuration wavelength is 595nm, calculates soluble protein content (μ g/g) and feather resolution ratio (%), curve plotting, as shown in Figure 1.The computational methods of protein content are as follows:
Protein content
;
In formula: W-by quality standard protein curve checking in protein, μ g;
1.0-draws extracting liquid volume, mL;
V-extract cumulative volume, mL;
M-takes sample mass, g;
Feather resolution ratio (%)=(sample of feather dry mass-enzymolysis liquid residue dry mass)/sample of feather dry mass × 100.
3.2.3 orthogonal experiment confirmatory experiment result
Choosing hydrolysis temperature, the initial pH of enzymolysis liquid, enzyme addition and enzymolysis time is observation factor, and each factor gets five levels, designs the test of four factors, five levels, selects L25(5
4) orthogonally to test.3 250mL triangular flasks are got in each experiment, and all add 1g feather meal sample and 100mL distilled water, the same terms in triplicate.The best enzymolysis parameter determined according to orthogonal test carries out enzyme Degrading experiment.
3.2.4 the mensuration of Vitro Digestibility
According to GB/T 17811-1999 " the mensuration acidity pepsin method of animals ' protein forage digestibility ", the assay method of pepsin Vitro Digestibility in acid water is adopted to test.
Test method step: take acid water 100ml that 1g enzymolysis feather powder residue → interpolation pepsin (adding pepsin by sample 1:3000 unit) → add is preheated to 45 DEG C → by triangular flask and be placed in 45 DEG C of constant-temperature tables and digest 16h → reacting liquid filtering → dry → weigh.
The mensuration of aminoacid ingredient in 3.3 enzymolysis liquids
Using Biochrom(hundred health) the full-automatic amino-acid analyzer of 30+ analyzes each seed amino acid contained in enzymolysis.
two. experimental result and analysis
3.1 protein content calibration curves
Accompanying drawing 1 is protein content canonical plotting, and obtaining protein standard curve equation according to this calibration curve is y=0.0043x+0.0879, can obtain soluble protein content according to equation and the data recorded.According to data creating data form and curve map, analyze data acquisition experimental result.
3.2 Single factor experiment results and analysis
3.2.1 hydrolysis temperature test
Soluble protein content (unit mg/L)
Enzymolysis feather powder test is carried out under condition of different temperatures, curve map is obtained as shown in Figure 2 according to above table 1, when being 35 DEG C from the known hydrolysis temperature of curve map, in enzymolysis liquid, soluble protein content is the highest, with temperature rising or reduce protein content and all reduce, therefore again the optimum temperature of enzyme enzymolysis feather powder is 35 DEG C.
3.2.2 the initial pH test of enzymolysis
Soluble protein content (unit mg/L)
The temperature determined with previous test 35 DEG C is reference, enzymolysis feather powder test is carried out under condition of different pH, curve map is obtained as shown in Figure 3 according to above table 2, when the initial pH of enzymolysis liquid is 7 as we know from the figure, in enzymolysis liquid, soluble protein content is the highest, with pH rising or reduce protein content and all reduce, therefore again the suitableeest initial pH of enzyme enzymolysis feather powder is 7.
3.2.3 enzyme addition is tested
Soluble protein content (unit mg/L)
The temperature determined with previous test 35 DEG C and pH7 are reference, enzymolysis feather powder test is carried out under different multiple enzyme additions, curve map is obtained as shown in Figure 4 according to above table 3, from curve map, the known increase soluble protein content with enzyme addition also increases thereupon, when enzyme addition reaches keratinase 0.03g, soluble protein content reaches 165.4mg/L, along with the increase soluble protein content of enzyme addition rises slowly.Therefore the suitableeest enzyme addition of enzyme enzymolysis feather powder is keratinase 0.03g again.
3.2.4 enzymolysis time test
Soluble protein content measures (unit mg/L)
The temperature determined with previous test, pH and enzyme addition condition are reference, test under different enzymolysis times, curve map is obtained as shown in Figure 5 according to above table 4, can find out that from curve map the increase soluble protein content with enzymolysis time also increases thereupon, when enzymolysis time reaches 23h, soluble protein content is 141.9mg/L, and along with the increase soluble protein content of enzymolysis time reduces, therefore the peak enzymolysis-ability time of keratinase enzymolysis feather powder is 24h.
Tentatively show that optimum reaction conditions be hydrolysis temperature is 35 DEG C, the initial pH of enzymolysis liquid is 7 according to data analysis, enzyme addition be keratinase 0.03g and enzymolysis time is 24h.By design orthogonal experiment, result is verified.
3.3 orthogonal experiment confirmatory experiment results
3.3.1 Orthogonal experiment results statistics
Experimental factor level is determined according to above test
Orthogonal result and data statistics
According to upper table to Orthogonal experiment results process, obtain form 7, as follows:
Note: K1: each group of experimental result sum of corresponding is each factor level " 1 "; K2: each group of experimental result sum of corresponding is each factor level " 2 "; K3: each group of experimental result sum of corresponding is each factor level " 3 "; K4: each group of experimental result sum of corresponding is each factor level " 4 "; K5: each group of experimental result sum of corresponding is each factor level " 5 ".Which factor value that extreme difference R is corresponding is maximum, and impact is just maximum: otherwise, affect minimum.
3.3.2 Orthogonal experiment results analysis
As can be seen from the extreme difference R in form 7, in certain scope, to feather meal degraded, what have the greatest impact is enzymolysis time, influence power second be the initial pH of enzymolysis liquid, the 3rd is enzyme addition, and affecting minimum is hydrolysis temperature.K2 > k1 > k3 > k4 > k5 in hydrolysis temperature test group, thus hydrolysis temperature the suitableeest be 35 DEG C; Determine that the initial pH of the suitableeest enzymolysis liquid is 7 by identical Statistics, the suitableeest enzyme addition is keratinase 0.03 g, and the peak enzymolysis-ability time is 24 h.Enzymolysis liquid soluble protein content is 492.7 mg/L.Residue dry for standby after filtering.
3.4 feather resolution ratios
Enzymolysis feather powder under optimum reaction conditions, enzymolysis liquid filters, and residue is weighed after drying.Under best enzymolysis parameter, it is 645mg that feather decomposes quality, and namely the resolution ratio of feather reaches 64.5%.
3.5 enzymolysis feather powder Vitro Digestibilities
Adopt chloropeptic acid solution to carry out Vitro Digestibility experiment, experimental result result display enzymolysis feather albumen powder Vitro Digestibility reaches 64%.
Enzymolysis feather resolution ratio adds enzymolysis feather powder Vitro Digestibility, and total resolution ratio of feather is 87.22%.
enzymolysis liquid Analysis on amino acid components
The series of experiments of this research by carrying out keratinase degraded to chicken feather meal, and verify through orthogonal experiment, obtain the optimum process condition of keratinase degradation of feather powder, the optimum process conditions adopting HTHP pretreatment and keratinase to degrade are: chicken feather is through 120 DEG C of high temperature high pressure process 40min, dry, pulverize, sieve for 80 DEG C, every gram of feather meal adds keratinase 0.03g, and the initial pH of enzymolysis liquid is 7, enzymolysis 24 h at 35 DEG C.Feather resolution ratio reaches 64.5%, and enzymolysis liquid soluble protein content is 492.7mg/L, is rich in each seed amino acid in enzymolysis supernatant; Enzymolysis feather albumen powder Vitro Digestibility reaches 64%.Enzymolysis feather resolution ratio adds enzymolysis feather powder Vitro Digestibility, and total resolution ratio of feather is 87.22%.Show and there is good hydrolysis result.The Pepsin digestibility that in enzymolysis supernatant, soluble protein content is higher and enzymolysis feather albumen powder is higher, can be processed as amino acid products and feed adds albumen.
the experiment of enzymolysis drake feather
To take chicken feather as the enzymolysis that the peak enzymolysis-ability condition obtained after raw material carries out enzymolysis research is applied to drake feather, obtain result: duck feather resolution ratio 78.7%, soluble protein content 396.75mg L.
In sum, this enzyme solution can be widely used in comprising the process that the poultry feather such as chicken, duck produces feather meal.
Claims (5)
1. utilize keratinase to prepare a method for feather albumen powder, it is characterized in that: take poultry feather as raw material, adopt following steps:
(1) preparation of feather meal: by poultry feather through cleaning, removal of impurities, shred, dry loading beaker, insert 120 DEG C of sterilizing 40min in high-pressure steam sterilizing pan, then treated, drain, 80 DEG C of dry 12h in thermostatic drying chamber, after pulverizing, grind, crossing 100 mesh sieves, obtain feather meal, kept dry is stand-by;
(2) enzymolysis: feather meal is placed in container, 1g feather meal adds 100ml distilled water, keratinase 0.01-0.06g, and wherein the pH of enzymolysis liquid is 5-10, hydrolysis temperature 25-50 DEG C, enzymolysis time 4-46h, is placed in constant-temperature table and carries out enzymolysis;
(3) filtering drying: filtered by enzyme digestion reaction liquid, measures filtered fluid soluble protein and amino acid content, weighs stand-by after filtering residue drying.
2. a kind of method utilizing keratinase to prepare feather albumen powder according to claim 1, is characterized in that: the poultry feather described in step (1) is chicken feather or duck feather.
3. a kind of method utilizing keratinase to prepare feather albumen powder according to claim 1, it is characterized in that: the enzymolysis process described in step (2), the enzyme addition of every 1g feather meal is keratinase 0.03g, and hydrolysis temperature is 35 DEG C, enzymolysis liquid pH is 7, enzymolysis time 24h.
4. the method utilizing keratinase to prepare feather albumen powder according to claim 1, it is characterized in that: the mensuration of the content of the soluble protein described in step (3) adopts Coomassie Brilliant Blue to measure, take bovine serum albumin as standard protein, mensuration wavelength is 595nm.
5. the method utilizing keratinase to prepare feather albumen powder according to claim 1, it is characterized in that: in step (3), measure Vitro Digestibility, assay method is leach in residue to add pepsin and the prefabricated acid water to 45 DEG C, its mixture is placed in 45 DEG C of constant-temperature tables and digests 16h, by reacting liquid filtering, dry, weigh, wherein, every 1g leaches the pepsin that residue adds 3000 units, and the addition of acid water is 100ml.
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Cited By (6)
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| CN105707410A (en) * | 2016-03-01 | 2016-06-29 | 江西鸿吉生物科技有限公司 | Processing method of enzymolytic protein powder |
| CN106387320A (en) * | 2016-08-30 | 2017-02-15 | 济南诺能生物工程有限公司 | Processing and treating method of feathers |
| 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 |
| CN109965086A (en) * | 2019-05-14 | 2019-07-05 | 华中农业大学 | A kind of hydrolyzed feather meal additive and its application in animal feed |
| 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 |
| CN110710595A (en) * | 2019-11-25 | 2020-01-21 | 河南省科学院生物研究所有限责任公司 | Feed containing zymolytic broussonetia papyrifera protein and feather protein for livestock and poultry |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105707410A (en) * | 2016-03-01 | 2016-06-29 | 江西鸿吉生物科技有限公司 | Processing method of enzymolytic protein powder |
| CN106387320A (en) * | 2016-08-30 | 2017-02-15 | 济南诺能生物工程有限公司 | Processing and treating method of feathers |
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| 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 |
| CN109965086A (en) * | 2019-05-14 | 2019-07-05 | 华中农业大学 | A kind of hydrolyzed feather meal additive and its application in animal feed |
| 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 |
| CN110710595A (en) * | 2019-11-25 | 2020-01-21 | 河南省科学院生物研究所有限责任公司 | Feed containing zymolytic broussonetia papyrifera protein and feather protein for livestock and poultry |
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Application publication date: 20150715 |