CN1257917C - Process for preparing collagen active polypeptide from avian animal tissue - Google Patents
Process for preparing collagen active polypeptide from avian animal tissue Download PDFInfo
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- CN1257917C CN1257917C CN 200410052758 CN200410052758A CN1257917C CN 1257917 C CN1257917 C CN 1257917C CN 200410052758 CN200410052758 CN 200410052758 CN 200410052758 A CN200410052758 A CN 200410052758A CN 1257917 C CN1257917 C CN 1257917C
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- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a method for preparing collagen activity polypeptide from poultry source animal tissues. Duck feet are used as raw materials, and the raw materials are soaked and pre-treated in 1.0 to 5.0% of acidic liquid; after the raw materials are washed in water, the raw materials are extracted for 4 to 10 hours in hot water at 60 to 85 DEG C; therefore, an extracting solution of collagen gelatin is obtained. The extracting solution is concentrated to a situation that the concentration of collagen protein is from 5 to 25%(w/w), a pH value is adjusted to 0.5 to 4.0 by adding acid, hydrolysis is carried out for 3 to 8 hours at 65 to 90 DEG C, a pH value is neutralized to 7.0 to 8.0 by adding an alkali solution, a water solution of the collagen activity polypeptide is obtained by centrifugation, organic solvent is added to the water solution of the polypeptide, and the proportion of the organic solvent and the water solution of the collagen polypeptide is 3 to 5:1(v/v); a mixture solution is thoroughly stirred and is statically stood for 5 to 8 hours, polypeptide precipitation is obtained by centrifugation, and a product of the collagen activity polypeptide is obtained by pressure reduction and dryness. The present invention has the advantages of wide raw material resources, easy acquirement, low cost, convenient operation, mild condition, short production period and high product yield.
Description
Technical field
The present invention relates to biological product manufacturing technology field, relate in particular to a kind of by the method for preparing the collagen active polypeptide in the avian animal tissues.
Background technology
Collagen protein extensively exists in tissues such as animal skin, cartilage, bone, tendon, is a histone, has found nearly 20 kinds.80~85% is collagen protein in the corium, mainly is I, III collagen type, and in the organism of bone 1/3rd, the overwhelming majority is a type i collagen albumen.The natural collagen protein molecule is twined by the triple helix polypeptide chain to be formed, and with hydrogen bonded, molecular weight is big between spiral.Glycine, proline(Pro) and hydroxyproline content are abundant unusually in the collagen protein.Collagen protein is hydrolyzed, can obtain collagen active polypeptide than small molecular weight, its water-soluble increase, and kept many advantageous properties of collagen protein, at numerous industrial circles such as medical cosmetology, makeup, tissue repair, surgical hemostasis, medical capsule, microcapsule, medicinal, foodstuffs industry important application is arranged.
Usually, extract in skin, tendon and the bone of collagen protein by domestic animals such as ox, pigs.In recent years, be subjected to the influence of epizootics such as mad cow disease, foot and mouth disease in the world wide, the proteic security of poultry derived collagen has caused generally worry.By extracting collagen protein in the tissues such as avian animal skin, bone, tendon, become a new way obtaining natural collagen protein and hydrolytic activity polypeptide thereof.At present to the extraction research of avian collagen protein seldom, the research to its hydrolyzed peptide goods does not appear in the newspapers.U.S. Pat 5138030 (1992) has proposed to extract from the bird tissue with enzyme process and organic acid dissolving method the method for collagen protein; U.S. Pat 0142368A1 (2002) discloses with poultry skins such as chicken, goose, bone etc. and has been organized as raw material, carries out the method that heat treated is directly extracted collagen protein in water medium; TaiWan, China D.C.Liu etc. are at Asian-Australasian Journal of Animal Sciences, and 14 (11): 1638-1644, reported the method for extracting the solubility in acid collagen protein with the acid solution leaching from the chicken pin in 2001.What existing avian collagen protein extractive technique obtained is collagen gelatin, and molecular weight is big, and the water at normal temperature dissolubility is poor, and the purity of gelatin is low, and yield is not high.
To the further hydrolysis of avian collagen gelatin, can obtain collagen polypeptide.Do not appear in the newspapers at the technology of preparing of avian collagen active polypeptide at present.To poultry derived collagen proteic hydrolysis has been known technology, mainly contains alkaline process, enzyme hydrolysis method (for example Chinese patent CN1403585A, 2003) and acid system (for example U.S. Pat 4130555,1978).These methods cut both ways: alkaline process easily makes protein molecular structure destruction and sex change and loses activity, and especially most of Gelucystine and halfcystine can be destroyed, has reduced the nutritive value of collagen hydrolysate polypeptide; Enzyme process operational condition gentleness, the product that obtains belong to the single product that molecular weight is concentrated, but the long reaction time that needs, condition control is strict, the enzyme that uses is disposable, and cost is often higher, and easily is difficult to the separation difficulty that purifying makes hydrolysate because of used biological enzyme; The acid system hydrolysis is rapid, can avoid racemization, reduce the loss of hydrolyzed peptide, but the peptide molecule weight range that obtains is bigger, and control condition is strict.Avian natural collagen protein and poultry derived collagen are proteic, and different to be mainly reflected in tightness degree crosslinked between its collagen different, and the avian collagen cross-linking is weak (U.S. Pat 0142368A1).Therefore, when the avian collagen hydrolysate was active polypeptide, the technology for hydrolyzing that needs was different with known poultry derived collagen proteolysis technology with condition, and the one side that has specific characteristics need be studied exploration.
Summary of the invention
The purpose of this invention is to provide a kind of by the method for preparing the collagen active polypeptide in the avian animal tissues.
With the duck pin is raw material, with 1.0~5.0% diluted acids raw material is soaked pre-treatment, the washing back obtained the collagen gelatin extracting solution in 4~10 hours with 60~85 ℃ of hot-water extraction, it is 5~25% (w/w) that extracting solution is concentrated into collagen concentration, adding acid for adjusting pH value is 0.5~4.0,65~90 ℃ of hydrolysis 3~8 hours, adding alkali lye, to be neutralized to pH be 7.0~8.0, the centrifugal aqueous solution that obtains the collagen active polypeptide adds organic solvent in polypeptid solution, the ratio of the organic solvent and the collagen polypeptide aqueous solution is 3~5: 1 (v/v), left standstill 5~8 hours after fully stirring, centrifugal that polypeptide precipitates, through drying under reduced pressure, make collagen active polypeptide product.
Advantage of the present invention: (1) adopts the acid system hydrolysis, and technology is simply rapid, and cost is low, and has reduced the loss of hydrolyzed peptide, good product quality; (2) adopt organic solvent precipitation method, both separated obtaining the hydrolyzed peptide product, realize impurity elimination again simultaneously, discolor and go flavor, improved the quality of product; (3) the aminoacids content height of product; (4) by the suitable hydrolysising condition of control, make the collagen protein macromole be fractured into active polypeptide, increased the water-soluble of collagen protein, be convenient in industrial circles such as makeup, medicine, food, use
Concrete embodiment
By the method for preparing the collagen active polypeptide in the avian animal tissues, its production stage is as follows:
(1) extraction of raw materials pretreatment and collagen gelatin liquid
Getting the duck pin is duck's foot, die Schwimmhaut, the duck pin obstructs is raw material, clean up, soak with diluted acid, washing, soak with deionized water then, used acid is one or more mixtures that are made in sulfuric acid, hydrochloric acid, acetate, the citric acid, concentration 1.0~5.0%, soak time is 400~480 hours, and washing back pH value is 4.0~6.0, and the deionized water soak time is 12~24 hours, the raw material that pre-treatment is good adds deionized water, carry out extracting under 60~85 ℃ of temperature, obtain collagen gelatin liquid, the extracting time is 4~10 hours;
(2) collagen gelatin liquid concentrates and the acid system hydrolysis
The collagen gelatin liquid of gained is filtered, concentrating under reduced pressure under 50~70 ℃, 0.095~0.099MPa then, the concentration that makes collagen protein in the concentrated solution is 5~25% (w/w), directly be hydrolyzed with acid system, used acid solution is one or more mixtures that are made in sulfuric acid, hydrochloric acid, acetate, citric acid, the oxalic acid, regulating the pH value is 0.5~4.0, and hydrolysis temperature is 65~90 ℃, and hydrolysis time is 3~8 hours;
(3) hydrolyzed solution neutralization and organic solvent deposit
It is 7.0~8.0 that hydrolyzed solution is neutralized to pH with alkali lye, left standstill 2~8 hours, centrifugal 15~20min discards precipitation under 6000~8000rpm, obtain the aqueous solution of collagen polypeptide, used alkali lye is a kind of in yellow soda ash, sodium bicarbonate, ammoniacal liquor, sodium hydroxide, the potassium hydroxide; Adding organic solvent in the collagen polypeptide aqueous solution of gained separates out the collagen polypeptide precipitation, centrifugal 15~20min under 6000~8000rpm, collecting precipitation, used organic solvent is one or more mixtures that are made in acetone, methyl alcohol, ethanol, the Virahol, and the ratio of the organic solvent and the collagen polypeptide aqueous solution is 3~5: 1 (v/v);
(4) drying
Gained is deposited in carried out drying under 50~70 ℃ 24~36 hours, pulverize, perhaps join, obtain powder and be collagen polypeptide products for solution carries out spraying drying.
The collagen active polypeptide yield that makes according to above method is 3.5~5.5% (dry products/raw material weight in wet base).
The index of product is as follows:
Outward appearance: white is pale yellow powder slightly
Molecular weight: 2kDa~35kDa
Nitrogen content:>16%
Total amino acid content:>90%
Heavy metal content:<10ppm
Water-soluble:>15g/100mL water (25 ℃)
1% pH value of water solution: be 6.5~7.0
Moisture content: 2.0~4.5%.
In the above preparation process, the control of acid system hydrolysis suitable condition and the separation and purification of collagen hydrolyzed peptide are gordian techniquies of the present invention.
Duck is the poultry that China extensively raises.Fresh duck pin raw material sources are wide, especially at China's southern area duck pin aboundresources, are easy to obtain low price.Secondly be natural collagen protein more than 80% in duck's foot and the die Schwimmhaut, be that compositions such as a spot of fat, other albumen and moisture, duck pin obstruct that the overwhelming majority also is a collagen protein in 1/3 organism, best in quality.The used duck pin raw material of the present invention is available from Zhejiang Hangzhou.
The invention will be further described by the following examples:
Example 1
Get duck pin (duck toe, die Schwimmhaut and duck pin stalk) 10000g, clean up, in 5.0% acetic acid solution, soaked 400 hours, washing pH value 6.0, deionized water soaked 24 hours.Then, extracting is 8 hours in 72 ℃ of hot water, filters, and gained filtrate is concentrating under reduced pressure under 50 ℃, 0.099MPa, makes that the content of collagen protein is 5% (w/w) in the concentrated solution, and adding citric acid in concentrated solution, to transfer pH be 3.5.In 90 ℃ of following hydrolysis 3 hours, be 7.0 with the yellow soda ash adjust pH, left standstill 2 hours, centrifugal 20min under 8000rpm discards precipitation then, gets the collagen polypeptide aqueous solution.To wherein adding ORGANIC SOLVENT MIXTURES (acetone and methyl alcohol 1: 1, v/v), with the ratio of the collagen polypeptide aqueous solution be 4: 1 (v/v), centrifugal 20min under the 8000rpm, abandoning supernatant liquor must precipitate, to be deposited in 65 ℃ of dryings 30 hours, and get collagen polypeptide powder 440g, the collagen polypeptide yield is 4.4% (dry product/raw material weight in wet base).
Example 2
Get duck pin stalk 2000g, clean up, in 3.0% citric acid solution, soaked 480 hours, washing pH value 5.0, deionized water soaked 16 hours.Then, extracting is 4 hours in 85 ℃ of hot water, filters, and gained filtrate is concentrating under reduced pressure under 55 ℃, 0.099MPa, makes that the content of collagen protein is 15% (w/w) in the concentrated solution, and adding sulfuric acid in concentrated solution, to transfer pH be 4.0.In 68 ℃ of following hydrolysis 7 hours, be 7.5 with the potassium hydroxide adjust pH, left standstill 8 hours, centrifugal 15min under 8000rpm discards precipitation then, gets the collagen polypeptide aqueous solution.To wherein adding ORGANIC SOLVENT MIXTURES (acetone and ethanol 2: 1, v/v), with the ratio of the collagen polypeptide aqueous solution be 4.5: 1 (v/v), centrifugal 15min under the 8000rpm, abandoning supernatant liquor must precipitate, to be deposited in 50 ℃ of dryings 30 hours, and get collagen polypeptide powder 90g, the collagen polypeptide yield is 4.5% (dry product/raw material weight in wet base).
Example 3
Get duck pin 750g, clean up, in 1.0% sulfuric acid liquid, soaked 450 hours, washing pH value 4.0, deionized water soaked 18 hours.Then, extracting is 8 hours in 65 ℃ of hot water, filters, gained filtrate is concentrating under reduced pressure under 65 ℃, 0.097MPa, makes that the content of collagen protein is 25% (w/w) in the concentrated solution, adds acetate and hydrochloric acid mixed solution (acetate and concentrated hydrochloric acid 6: 1 in concentrated solution, v/v), transferring pH is 0.5.In 65 ℃ of following hydrolysis 8 hours, be 7.0 with the sodium bicarbonate adjust pH, left standstill 8 hours, centrifugal 18min under 7000rpm discards precipitation then, gets the collagen polypeptide aqueous solution.To wherein adding ORGANIC SOLVENT MIXTURES (ethanol and Virahol 4: 1, v/v), with the ratio of the collagen polypeptide aqueous solution be 3: 1 (v/v), centrifugal 18min under the 7000rpm, abandoning supernatant liquor must precipitate, to be deposited in 61 ℃ of dryings 28 hours, and get collagen polypeptide powder 31g, the collagen polypeptide yield is 4.1% (dry product/raw material weight in wet base).
Example 4
Get the about 40g of die Schwimmhaut, clean up, 4.0% salt acid soak 420 hours, washing, deionized water soaked 12 hours, washed pH value 5.0 again.60 ℃ of hot-water extraction 10 hours, extracting solution concentrating under reduced pressure under 50 ℃, 0.095atm, make that the content of collagen protein is 10% (w/w) in the concentrated solution, in concentrated solution, add oxalic acid and citric acid mixed solution (oxalic acid and citric acid 2: 1, v/v), transfer pH about 2.5.72 ℃ of following hydrolysis 4 hours are 8.0 with the sodium hydroxide adjust pH.Left standstill 5 hours, centrifugal 20min under 6000rpm discards precipitation then, gets the collagen polypeptide aqueous solution.To wherein adding acetone, with the ratio of the collagen polypeptide aqueous solution be 4: 1 (v/v), centrifugal 20min under the 6000rpm, abandoning supernatant liquor must precipitate, to be deposited in 70 ℃ of dryings 24 hours, and get collagen polypeptide powder 2.1g, the collagen polypeptide yield is 5.2% (dry product/raw material weight in wet base).
Example 5
Get duck pin 4500g, clean up, in 3.5% hydrochloric acid and acetic acid solution (weight ratio 2: 1), soaked 480 hours, washing pH value 4.0, deionized water soaked 18 hours.Then, extracting is 8 hours in 65 ℃ of hot water, filters, and gained filtrate is concentrating under reduced pressure under 70 ℃, 0.099MPa, makes that the content of collagen protein is 20% (w/w) in the concentrated solution.(acetate and concentrated hydrochloric acid 6: 1, v/v), transferring pH is 1.5 to add acetate and hydrochloric acid mixed solution in concentrated solution.In 71 ℃ of following hydrolysis 8 hours, be 7.0 with the sodium bicarbonate adjust pH, left standstill 8 hours, centrifugal 15min under 8000rpm discards precipitation then, gets the collagen polypeptide aqueous solution.To wherein adding ethanol and Virahol (2: 1, v/v), with the ratio of the collagen polypeptide aqueous solution be 5: 1 (v/v), centrifugal 20min under the 7000rpm, abandoning supernatant liquor must precipitate, to be deposited in 55 ℃ of dryings 36 hours, and get collagen polypeptide powder 220g, the collagen polypeptide yield is 4.8% (dry product/raw material weight in wet base).
Claims (3)
1, a kind of by the method for preparing the collagen active polypeptide in the avian animal tissues, it is characterized in that: with the duck pin is raw material, with 1.0~5.0% diluted acids raw material is soaked pre-treatment, the washing back obtained the collagen gelatin extracting solution in 4~10 hours with 60~85 ℃ of hot-water extraction, it is 5~25% that extracting solution is concentrated into collagen protein quality percentage concentration, adding acid for adjusting pH value is 0.5~4.0,65~90 ℃ of hydrolysis 3~8 hours, adding alkali lye, to be neutralized to pH be 7.0~8.0, the centrifugal aqueous solution that obtains the collagen active polypeptide, in polypeptid solution, add organic solvent, the volume ratio of the organic solvent and the collagen polypeptide aqueous solution is 3~5: 1, leaves standstill 5~8 hours after fully stirring, and is centrifugal that polypeptide precipitates, drying makes collagen active polypeptide product.
2, according to claim 1 a kind of by the method for preparing the collagen active polypeptide in the avian animal tissues, it is characterized in that its production stage is as follows:
(1) extraction of raw materials pretreatment and collagen gelatin liquid
Getting the duck pin is duck's foot, die Schwimmhaut, the duck pin obstructs is raw material, clean up, soak with diluted acid, washing, soak with deionized water then, used acid is one or more mixtures that are made in sulfuric acid, hydrochloric acid, acetate, the citric acid, concentration 1.0~5.0%, soak time is 400~480 hours, and washing back pH value is 4.0~6.0, and the deionized water soak time is 12~24 hours, the raw material that pre-treatment is good adds deionized water, carry out extracting under 60~85 ℃ of temperature, obtain collagen gelatin liquid, the extracting time is 4~10 hours;
(2) collagen gelatin liquid concentrates and the acid system hydrolysis
The collagen gelatin liquid of gained is filtered, concentrating under reduced pressure under 50~70 ℃, 0.095~0.099MPa then, the mass percent concentration that makes collagen protein in the concentrated solution is 5~25%, directly be hydrolyzed with acid system, used acid solution is one or more mixtures that are made in sulfuric acid, hydrochloric acid, acetate, citric acid, the oxalic acid, regulating the pH value is 0.5~4.0, and hydrolysis temperature is 65~90 ℃, and hydrolysis time is 3~8 hours;
(3) hydrolyzed solution neutralization and organic solvent deposit
It is 7.0~8.0 that hydrolyzed solution is neutralized to pH with alkali lye, left standstill 2~8 hours, centrifugal 15~20min discards precipitation under 6000~8000rpm, obtain the aqueous solution of collagen polypeptide, used alkali lye is a kind of in yellow soda ash, sodium bicarbonate, ammoniacal liquor, sodium hydroxide, the potassium hydroxide; Adding organic solvent in the collagen polypeptide aqueous solution of gained separates out the collagen polypeptide precipitation, centrifugal 15~20min under 6000~8000rpm, collecting precipitation, used organic solvent is one or more mixtures that are made in acetone, methyl alcohol, ethanol, the Virahol, and the volume ratio of the organic solvent and the collagen polypeptide aqueous solution is 3~5: 1;
(4) drying
Gained is deposited in carried out drying under 50~70 ℃ 24~36 hours, pulverize, perhaps join, obtain powder and be collagen polypeptide products for solution carries out spraying drying.
3, according to claim 1 and 2 a kind of by the method for preparing the collagen active polypeptide in the avian animal tissues, it is characterized in that optimum preparating condition is: the mass percent concentration of collagen protein is 20% in the said collagen gelatin concentrated solution; The pH value that concentrates gelatin solution during the acid system hydrolysis is adjusted to 4.0,71 ℃ of hydrolysis temperatures, hydrolysis time 8 hours; In the organic solvent deposit step, the volume ratio of the organic solvent and the collagen polypeptide aqueous solution is 5: 1.
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CN102199192A (en) * | 2011-03-29 | 2011-09-28 | 中国科学院过程工程研究所 | Method for separating functional polypeptide from tortoise-shell glue |
CN102304172B (en) * | 2011-08-12 | 2013-06-05 | 郑州奇泓生物科技有限公司 | Nisin extraction method |
CN109810187A (en) * | 2017-11-20 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of extracting method and application of animal tendon tendon collagen |
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