CN111802508A - Preparation method of feather peptide powder - Google Patents
Preparation method of feather peptide powder Download PDFInfo
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- CN111802508A CN111802508A CN202010776866.7A CN202010776866A CN111802508A CN 111802508 A CN111802508 A CN 111802508A CN 202010776866 A CN202010776866 A CN 202010776866A CN 111802508 A CN111802508 A CN 111802508A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Animal Husbandry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention relates to the technical field of poultry slaughter waste resource utilization, and particularly discloses a preparation method of feather peptide powder. The method takes poultry feathers as raw materials and is characterized in that: pulverizing feather, placing into a container, injecting water, adding keratinase 50-400U and alkaline protease 100-360U per 1g feather, adjusting pH to 10-11.5, reacting at 40-50 deg.C for 4-8h, and drying filtrate to obtain peptide powder product. The process is simple to operate, safe and efficient, and has great significance for recycling the feather slaughter waste. The soluble protein content in the filtrate of the product is higher than 680ug/ml, the peptide content in the peptide powder is higher than 95%, the relative molecular mass of the peptide is below 2000 daltons, the product has high purity, can be processed into animal protein feed raw materials, has wide application range, and is suitable for wide popularization and application.
Description
Technical Field
The invention relates to the technical field of poultry slaughter waste resource utilization, and mainly relates to a preparation method of feather peptide powder.
Background
With the rapid development of the poultry industry, the yield of feathers as one of the byproducts of poultry processing is huge every year, and by 2019, the yield of feathers is over 500 million tons every year in the world, and the yield of feathers is over 100 million tons every year in China, so that improper treatment is not only a waste of resources, but also pollutes the environment. The protein content in the feather is up to more than 80 percent (mainly keratin), and the sulfur-containing amino acid content is the first of all natural feeds, so the feather is an excellent protein raw material. However, since keratin contains a large number of disulfide bonds, it has a crosslinking effect in the peptide chain of the protein, is extremely stable, and is not easily digested and absorbed by animals, and therefore, it must be subjected to special treatment to exert a positive effect in animal feed.
The processing method of feather powder at present mainly adopts high-temperature high-pressure hydrolysis method, acid-base treatment method and enzymolysis method. The high-temperature high-pressure hydrolysis method further destroys the space structure of the keratin by controlling pressure, temperature and time, the pressure during the reaction is 0.3MPa to 0.7MPa, the temperature is 150 ℃ to 200 ℃, the reaction time is 0.5 to 3 hours, and the conditions can cause the loss of heat-sensitive amino acid, the product quality is unstable, and the energy consumption is large. The acid-base treatment method is to treat the feathers by acid-base solution, which can seriously damage amino acid, lead a large amount of amino acid to be modified or degraded, lead L-amino acid to be racemized into D-amino acid during alkaline hydrolysis, be not easy to be digested by animals, lead the produced feather powder to have poor palatability, lead improper operation to corrode machines and pollute the environment. The enzymolysis method protects thermosensitive amino acids, increases the content of digestible amino acids and further improves the digestibility by using the advantages of high efficiency, low reaction temperature and the like of enzymes under proper types of enzymes and conditions.
In the prior art, patent publication No. CN104798981A discloses a method for hydrolyzing feather with keratinase and alkaline protease in a complex manner, which uses an orthogonal test when optimizing the hydrolysis conditions, so that the soluble protein content is about 494.18 mg/L. Therefore, there is a further need for improvement, but it is difficult to further improve with the conventional orthogonal test.
Disclosure of Invention
The invention aims to provide a preparation method of feather peptide powder which has high peptide content and is easy to digest and absorb by animals aiming at the defects of the prior art. The method has the characteristics of high efficiency, safety and simple process.
The invention is realized by the following technical scheme: a preparation method of feather peptide powder mainly comprises the following steps:
(1) feather pretreatment: cleaning poultry feather, removing impurities, pulverizing, sterilizing with high pressure steam at 121 deg.C for 20min, drying at 85 deg.C for 10 hr, drying and storing.
(2) Enzymolysis: adding keratinase of 50-400U and alkaline protease of 100-360 ten thousand U into the feather pretreated in the step (1) according to 1g of feather, adding 100ml of water solution, adjusting the pH value to 10-11.5, and reacting for 4-8h at the temperature of 40-50 ℃.
(3) And (3) filtering and drying: and (3) determining the content of soluble protein and the relative molecular mass distribution of the peptide in the enzymolysis solution, and drying the filtrate into powder, namely the peptide powder.
In a preferred embodiment, 0.0035g of keratinase with the enzyme activity of 7 ten thousand U/g, 0.0018g of alkaline protease with the enzyme activity of 200 ten thousand U/mg are added into 1g of feather, the pH value of an aqueous solution is 11.4, the reaction temperature is 44.4 ℃, and the reaction time is 5.4 hours.
In a preferred embodiment, when the optimal pH value of the reaction is explored, in order to eliminate the influence of alkaline solution on the alkaline hydrolysis of the feather, feather groups without enzyme are set for comparison.
In a preferred embodiment, Design-expert.V8.0.6.1 software is adopted for optimizing the enzymolysis conditions, and a response surface analysis test is carried out.
In a preferred embodiment, the determination of the soluble protein content is performed by total protein assay (BCA method). Under alkaline conditions, BCA binds to proteins that bind Cu2+Reduction to Cu+One of Cu+Chelating two BCA molecules, wherein the working reagent forms a purple complex from the original apple green, and the maximum light absorption intensity is in direct proportion to the protein concentration.
In a preferred embodiment, the peptides in the enzymatic hydrolysate have a relative molecular mass distribution below 2000 daltons.
The invention has the beneficial effect that the disulfide bond of keratin in the feather can be destroyed, so that the feather is decomposed into small peptide which is easy to be digested and absorbed by animals. The process is safe and efficient, the reaction temperature is low, the process plays a role in protecting sulfur-containing amino acids such as cystine and methionine, the utilization rate of the amino acids is improved, the content of soluble protein in the enzymolysis liquid is higher than 680ug/ml, the content of peptide in the peptide powder is higher than 95%, and the relative molecular mass of the peptide is below 2000 daltons. In addition, the method has the advantages of easily controlled process parameters, simple operation and safe production, plays a role in recycling feather resources, can replace conventional protein resources, and has important significance in saving resources and protecting the environment.
Drawings
FIG. 1 shows the results of a single-factor test of the parameters of the renalase digestion;
FIG. 2 shows the response surface (upper) and contour line (lower) of the interaction of the initial pH value and enzymolysis temperature of the enzymolysis solution on the content of soluble protein;
FIG. 3 shows the response surface (upper) and contour line (lower) of the interactive effect of the initial pH value and enzyme addition amount of the enzymolysis solution on the content of soluble protein;
FIG. 4 shows the response surface (upper) and contour line (lower) of the interaction of the initial pH value and enzymolysis time of the enzymolysis solution on the content of soluble protein;
FIG. 5 is a response surface (upper) and contour lines (lower) of the interactive influence of the enzymolysis temperature and the enzyme addition amount on the soluble protein content;
FIG. 6 is a response surface (upper) and a contour line (lower) of the interactive influence of enzymolysis temperature and enzymolysis time on the content of soluble protein;
FIG. 7 is a graph showing the response surface (upper) and contour line (lower) of the interactive effect of enzyme addition and enzymolysis time on soluble protein content;
FIG. 8 is a gel chromatogram of peptide in the renalase hydrolysate after optimization.
The specific implementation mode is as follows:
the present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not to be construed as limiting the invention, and any modifications and variations of the present invention are intended to fall within the spirit and scope of the appended claims.
Example (b):
firstly, test materials:
chicken feathers; alkaline protease (200U/mg), keratinase (7 ten thousand U/g), sodium hydroxide (analytically pure), hydrochloric acid (analytically pure), BCA total protein concentration determination kit, standard peptide marker, acetonitrile (chromatographic grade).
II, testing the instrument:
a pH desk-top acidimeter, a water bath, a high-speed centrifuge, a high-pressure steam sterilization pot, an enzyme-labeling instrument, an ultra-pure water device, a micro-porous plate rapid oscillator, a desk-top air constant-temperature oscillator, a dryer and a gel chromatograph.
Thirdly, a test method and contents:
1 feather pretreatment: cleaning feather, removing impurities, pulverizing with pulverizer to obtain feather with length of about 1-2cm, sterilizing in high pressure steam sterilizing pot (121 deg.C, 20min), drying and storing.
2, soluble protein content determination: and (4) determining by using a BCA kit.
3 single factor test to determine the optimal enzymolysis condition of the compound enzyme
(1) And (3) compound enzyme proportioning test: taking 1g of feather (the weight of the feather is between 1.0000g and 1.0500 g), heating in a water bath at 55 ℃, reacting for 8h, controlling the pH to be 11, fixing the total addition amount of keratinase and alkaline protease to be 0.003g, carrying out tests under the conditions that the ratio of the two enzymes is 3:1, 2:1, 1:2, 1:3 and 5 gradients in sequence, setting 5 times for each group for repetition, heating in boiling water for 10min after the reaction is finished, inactivating the enzymes, centrifuging at 7000r/min for 10min, taking supernatant, measuring BCA, and taking an average value.
(2) Initial pH test of enzymatic hydrolysate: accurately weighing 0.002g keratinase and 0.001g alkaline protease, heating in water bath at 55 deg.C for 8h, performing test under gradient of pH 10, 10.5, 11, 11.5, 12, 5, each group is repeated, and the rest steps are the same as above.
(3) And (3) enzymolysis temperature test: the reaction time is 8h, the pH is 11.5, the water bath heating is respectively carried out at the temperature of 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 5 gradients, each group is repeated for 5 times, and the rest steps are the same as the above steps.
(4) Enzyme addition amount test: the pH value is 11.5, the mixture is heated in a water bath at 45 ℃, the reaction time is 8 hours, the total amount of the added enzyme is 0g, 0.0012g, 0.0024g, 0.0036g, 0.0048g and 0.006g in sequence, 5 gradients are used for carrying out the test, 5 groups are repeated, and the other steps are the same as the above steps.
(5) And (3) enzymolysis time test: accurately weighing 0.0032g keratinase and 0.0016g alkaline protease, adjusting pH to 11.5, heating in water bath at 45 deg.C, performing test under gradient of 1h, 2h, 3h, 4h, 5h, 6h, 7, each group is set to 5 times, and the rest steps are the same as above.
Response surface optimization test of 4-complex enzyme enzymolysis condition
According to the result of the single-factor test of the renalase, a Box-Behnken test design is adopted to carry out the initial pH (X) of the enzymolysis solution1) Temperature of enzymolysis (X)2) Enzyme addition amount (X)3) Time of enzymolysis (X)4) For 4 variables, 3 replicates were set for each group, and 3 replicates for each group were set for the enzyme-free control, with the difference (Y) in soluble protein content measured for the enzyme-added and enzyme-free groups as the response. The response surface test factors and level are designed as shown in the table below.
TABLE 1 renalase response surface design factors and levels
5 gel chromatography detection test of optimized compound enzyme enzymolysis liquid
And (4) according to the test result of the response surface, preparing the optimized complex enzyme supernatant into freeze-dried powder, and performing gel chromatography detection.
Fourth, test results
1-compound enzyme optimum enzymolysis condition single factor test result
The optimal compounding ratio of the keratinase to the alkaline protease is 2:1, and the optimal reaction conditions for the complex enzyme to carry out enzymolysis on the feathers are as follows: the initial pH value of the enzymolysis liquid is 11.5, the enzymolysis temperature is 45 ℃, the total addition amount of the enzyme is 0.0048g, 0.0032g of keratinase is added, 0.0012g of alkaline protease is added, the enzymolysis time is 5h, and the result of a single-factor test of the double-enzyme enzymolysis parameter is shown in figure 1.
2-optimization test result of response surface of complex enzyme enzymolysis condition
TABLE 2 test protocol and results
TABLE 3 analysis of variance results of regression equation
Note: "x" indicates p <0.01, the difference was very significant; "+" indicates p <0.05, the difference was significant.
P of model<0.0001, is extremely significant; mismatching term p is 0.1333>0.05, is not significant, and shows that experimental points can be explained by using a model; r20.9992, the model has high reliability. Performing polynomial fitting regression on the test data to obtain a regression equation:
soluble protein content ═ 381.39-99.41X1-0.77X2+24.87X3+12.11X4+2.81X1X2-3.14X1X3-13.12X1X4-21.45X2X3+0.020X2X4+6.39X3X4-268.91X12-39.67X22-39.98X32-21.63X42
The results of the renalase enzymolysis condition response surface optimization test are shown in FIGS. 2-7.
3 response surface test verification test
And (3) obtaining a result by verifying the response surface test result: under the reaction conditions that the initial pH value of the enzymolysis liquid is 11.40, the temperature is 44.4 ℃, the addition amount of keratinase is 0.0035g (245U), the addition amount of alkaline protease is 0.0018g (360U) and the enzymolysis time is 5.4h, the soluble protein content of the enzymolysis liquid is 681.954 ug/ml.
The soluble protein content value input into the software for analysis is the actual measured content of the enzyme-added histone-no content of the enzyme-added histone. And when analyzed by Design-expert.V8.0.6.1 software, the theoretical predicted value is 398.398 ug/ml. The result of the verification test is 400.397ug/ml, and has no significant difference (p is more than 0.05) from the theoretical predicted value, which indicates that the model is reliable. Meanwhile, a control group without enzyme is prepared, the test result is 281.557ug/ml, and the addition of the two results is the final soluble protein content 681.954 ug/ml.
4 gel chromatography detection test result of the optimized renalase hydrolysate, as shown in FIG. 8
TABLE 4 optimization of the relative molecular mass distribution of peptides in the post-renalase hydrolysate
From the results, the relative molecular mass of the peptide in the optimized renalase enzymolysis liquid is mostly below 2000 daltons. Therefore, the peptide content of the obtained product can reach more than 95 percent after the enzymolysis condition of the compound enzyme is optimized.
Claims (8)
1. A preparation method of feather peptide powder is characterized by comprising the following steps: taking poultry feather as a raw material, pretreating the feather, putting the feather into a container, adding keratinase of 50-400U and alkaline protease of 100-360 ten thousand U into each 1g of the feather, adjusting the pH value to 10-11.5, reacting for 4-8h at 40-50 ℃, and filtering.
2. The production method according to claim 1, characterized in that: the pretreatment is to clean, remove impurities and crush the feathers, sterilize the feathers by high-pressure steam, dry and store the feathers for later use.
3. The method according to claim 1, further comprising determining the soluble protein content after filtration, centrifuging the enzymolysis solution at 6000-8000r/min for 8-12min, and collecting the supernatant by total protein assay (BCA).
4. The production method according to claim 3, further comprising determining a relative molecular mass distribution of the peptide in the enzymatic hydrolysate, characterized by: and (4) preparing the enzymolysis liquid into freeze-dried powder, and measuring by adopting gel chromatography.
5. The method of claim 1, wherein the poultry feathers comprise one or more of chicken, duck, and goose feathers.
6. The preparation method of claim 1, wherein the soluble protein content in the feather peptide powder is higher than 650ug/ml, the peptide content in the peptide powder is higher than 95%, and the relative molecular mass of the peptide is below 2000 daltons.
7. The method as claimed in claim 1, wherein the initial pH of the enzymolysis solution is 11.20-11.6, the addition amount of keratinase is 240-255U, the addition amount of alkaline protease is 350-370U, and the enzymolysis time is 5-6h at 44-45 ℃.
8. The preparation method of claim 7, wherein the soluble protein content in the feather peptide powder is higher than 680ug/ml, the peptide content in the peptide powder is higher than 95%, and the relative molecular mass of the peptide is below 2000 daltons.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746092A (en) * | 2020-12-31 | 2021-05-04 | 安徽希普生物科技有限公司 | Method for preparing metal chelating peptide from poultry feather |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101653188A (en) * | 2009-09-03 | 2010-02-24 | 江南大学 | Feather albumen powder and preparation method thereof |
| CN104798981A (en) * | 2015-03-17 | 2015-07-29 | 齐鲁工业大学 | Method for preparing feather protein powder from alkaline protease/keratinase |
| 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 |
-
2020
- 2020-08-05 CN CN202010776866.7A patent/CN111802508A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101653188A (en) * | 2009-09-03 | 2010-02-24 | 江南大学 | Feather albumen powder and preparation method thereof |
| CN104798981A (en) * | 2015-03-17 | 2015-07-29 | 齐鲁工业大学 | Method for preparing feather protein powder from alkaline protease/keratinase |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 郝鲁江: "角蛋白酶/碱性蛋白酶制备羽毛蛋白粉的工艺研究", 《中国家禽》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746092A (en) * | 2020-12-31 | 2021-05-04 | 安徽希普生物科技有限公司 | Method for preparing metal chelating peptide from poultry feather |
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