CN108728507A - The preparation method of fishskin collagen polypeptide - Google Patents
The preparation method of fishskin collagen polypeptide Download PDFInfo
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- CN108728507A CN108728507A CN201810408287.XA CN201810408287A CN108728507A CN 108728507 A CN108728507 A CN 108728507A CN 201810408287 A CN201810408287 A CN 201810408287A CN 108728507 A CN108728507 A CN 108728507A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 84
- 108010035532 Collagen Proteins 0.000 title claims abstract description 84
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 81
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 73
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 63
- 229920001436 collagen Polymers 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 72
- 239000007788 liquid Substances 0.000 claims abstract description 33
- 239000000843 powder Substances 0.000 claims abstract description 22
- 108091005804 Peptidases Proteins 0.000 claims abstract description 17
- 239000004365 Protease Substances 0.000 claims abstract description 17
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 17
- 238000001728 nano-filtration Methods 0.000 claims abstract description 17
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 17
- 238000003756 stirring Methods 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 239000000706 filtrate Substances 0.000 claims abstract description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000796 flavoring agent Substances 0.000 claims abstract description 12
- 235000019634 flavors Nutrition 0.000 claims abstract description 12
- 239000012528 membrane Substances 0.000 claims abstract description 12
- MNEIPOIFQCXXGM-UHFFFAOYSA-M O.O.O.[OH-].[Na+] Chemical compound O.O.O.[OH-].[Na+] MNEIPOIFQCXXGM-UHFFFAOYSA-M 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 238000005360 mashing Methods 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 13
- 235000005979 Citrus limon Nutrition 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- 244000248349 Citrus limon Species 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 9
- 239000004299 sodium benzoate Substances 0.000 claims description 9
- 235000010234 sodium benzoate Nutrition 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 8
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 8
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 8
- 238000004042 decolorization Methods 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000002386 leaching Methods 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- PWEBUXCTKOWPCW-UHFFFAOYSA-N squaric acid Chemical compound OC1=C(O)C(=O)C1=O PWEBUXCTKOWPCW-UHFFFAOYSA-N 0.000 claims description 5
- 238000007654 immersion Methods 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 239000004833 fish glue Substances 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 13
- 239000002253 acid Substances 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000017854 proteolysis Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000000284 extract Substances 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 241000251468 Actinopterygii Species 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 11
- 238000006460 hydrolysis reaction Methods 0.000 description 9
- 239000000049 pigment Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 230000007071 enzymatic hydrolysis Effects 0.000 description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 150000003384 small molecules Chemical class 0.000 description 6
- 238000009826 distribution Methods 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 238000004332 deodorization Methods 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- -1 sodium benzoate ethyl acetate Chemical compound 0.000 description 4
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 4
- 238000010792 warming Methods 0.000 description 4
- 230000010148 water-pollination Effects 0.000 description 4
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000001768 carboxy methyl cellulose Substances 0.000 description 3
- 239000003610 charcoal Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 241000238366 Cephalopoda Species 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000002144 chemical decomposition reaction Methods 0.000 description 2
- 230000000536 complexating effect Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 230000006837 decompression Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000005660 hydrophilic surface Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- KZBUYRJDOAKODT-UHFFFAOYSA-N Chlorine Chemical compound ClCl KZBUYRJDOAKODT-UHFFFAOYSA-N 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses the preparation method of fishskin collagen polypeptide, the specific steps are：Tissue mashing after fish-skin is shredded is pre-processed through oxygen sodium hydroxide solution and aqueous isopropanol successively；Pretreatment fish-skin acid extracts to obtain collagen protein powder；Collagen protein powder is digested through clostridiopetidase A and flavor protease again, with ultrafiltration membrane ultrafiltration to get ultrafiltrate in enzymolysis process；Modified activated carbon, ultrasonic wave stirring will be added in ultrafiltrate, filtering obtains clear filtrate；By clear filtrate through NF membrane nanofiltration, condensing peptide liquid is obtained, is freeze-dried up to fishskin collagen polypeptide.It has the beneficial effect that：Preparation method of the present invention is with albumen enzyme dosage is few, enzymolysis time is short, protein degradation effect is good, fishskin collagen polypeptide yield is high, collagen polypeptide can reduce the generation of bitter peptides and the loss of product compared with white, the higher advantage of sensory-acceptance.
Description
Technical field
The present invention relates to biotechnologies, the more particularly to preparation method of fishskin collagen polypeptide.
Background technology
Vast territory and abundant resources in China, and waters is vast, aquatic resources very abundant, in recent years, with the improvement of living standards, people
The diversity and practicability of aquatic resources demand are gradually increased, to promote the rapid development of China's fish processing industry.But
It is that will produce a large amount of fish pomace, such as fish-skin, fish scale, internal organ, fish head etc. while fish processing industry high speed development,
These leftover bits and pieces account for about the 40% ~ 50% of raw material fish.Relevant information show in fish pomace contain abundant nutrient substance,
The a large amount of wasting of resources is not only caused in this way, if dealt with improperly it is also possible to causing serious environmental pollution.Therefore, how
The great attention of domestic and international researcher can efficiently have been caused using the nutrient substance in fish pomace.In fish-skin
The content of collagen reaches as high as 80% of total protein or more, and fat content is extremely low, in order to make full use of in fish-skin
Protein resource, many researchers are explored generates collagen using fish-skin hydrolysis.Crude protein master in hydrolytic fish skin at present
It will be there are two types of method：Chemical degradation method and enzyme hydrolysis method.Chemical degradation method is to reach protein hydrolysate using strong acid and strong base peptide bond
Purpose, experimental method is easy to operate, but reacts acutely not easy to control, often results in the damage of amino acid.Enzyme hydrolysis method is anti-
Mild condition is answered, protein isolate low molecular polypeptide and amino acid can be obtained well, so obtaining the blueness of vast researcher
It looks at.
Existing zymolysis technique is all to lay particular emphasis on the initial reactions conditions such as control concentration of substrate, temperature, the dosage of pH, enzyme,
However as the progress of enzyme digestion reaction, concentration of substrate and pH are variations, cause collagen polypeptide enzymolysis process to exist complicated, numerous
Trivial, the problem of being difficult to control, therefore the prior art is difficult to control effectively to the variation of these conditions in reaction process, simultaneously
Control to bitter peptides is also to take method to remove after enzyme digestion reaction, without controlling the generation of bitter peptides during the reaction.Separately
Outside, existing collagen polypeptide product has that one common is：The irrational distribution of molecular weight, and collagen peptide
Physics and chemistry and functional character and its molecular weight distribution, amino acid composition are closely related.The polypeptide that Relative average molecular weight is 3000 produces
Molecular weight polypeptide in product between 3000-5000 dalton also accounts for the more of certain proportion or even the above molecular weight of 5000 dalton
There is also, 500 dalton low molecular weight polypeptide ratios below up to 20% for peptide.500 dalton part below is by oligopeptides and amino
Acid composition, the bioactivity of this constituent part is poor, and most of is bitter peptides.
The prior art such as Authorization Notice No. is the Chinese invention patent of 103805665 B of CN, discloses a kind of deep-sea fish skin
The preparation method of collagen polypeptide, this method are that compound protease is added at 55 DEG C of temperature in collagen liquid crude product to carry out enzyme
It solves, ultrafiltration membrance filter is repeatedly used in enzymolysis process, enzymolysis liquid composite activated carbon is then used into gradation absorption method decoloration deodorization
Nanofiltration afterwards, it is dry, obtain powdered collagen polypeptide.The preparation method is accomplished really using enzymolysis and ultra-filtration and separation combination technique
Artificial control reaction condition, makes that the molecular weight of product is controllable, molecular weight distribution is reasonable, to product various physicochemical properties more
It is good.But the composite activated carbon adsorption effect that the preparation method uses is not satisfactory.
Invention content
That the purpose of the present invention is to provide a kind of albumen enzyme dosages is few, enzymolysis time is short, and protein degradation effect is good, fish-skin
Collagen polypeptide yield is high, collagen polypeptide is relatively white, sensory-acceptance is higher, reduces the generation of bitter peptides and the loss of product
The preparation method of fishskin collagen polypeptide.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
The preparation method of fishskin collagen polypeptide, including pretreatment of raw material, collagen protein powder are prepared, digested, decolourizing and nanofiltration, tool
Body step is：
Feed pretreatment step is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:4-6g/mL fish-skin is added a concentration of
It in the oxygen sodium hydroxide solution of 0.08-0.13M, impregnates and constantly stirs, rinsed to neutrality with circulating water after 1.5-2.5h, then by material
Liquor ratio is 1:1.5-2.5g/mL is added the aqueous isopropanol of a concentration of 2-5%, impregnate cleaned repeatedly with distilled water after 10-15h to
Filtered through gauze after neutrality fully drains to get pretreatment fish-skin, and fishskin collagen polypeptide is many miscellaneous because wherein containing before preparation
Albumen and fat can reduce substance of this kind to fish skin collagen so first to carry out pre-treatment to remove these foreign proteins, fat etc.
The influence of polypeptide；
Collagen protein powder preparation process is：It is 1 by solid-liquid ratio:A concentration of 0.2-0.5M acetic acid is added into pretreatment fish-skin in 8-11
Solution, rotating speed are stirring and leaching 5-7h under 130-170r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, centrifugation
Supernatant is evaporated under reduced pressure, and is freeze-dried up to collagen protein powder, which makes collagen remain its unique supercoil knot
Structure, property are stablized, and the recovery rate of collagen is up to 23.46%, prepare for next enzymolysis；
Enzymolysis：It is that deionized water is added into collagen protein powder by 8-10% by concentration of substrate, pH to 7.4-8.6 is adjusted, by enzyme concentration
It is separately added into clostridiopetidase A and flavor protease for 2.4-3.5% and 1.6-2.0%, 10- is digested at being then 30-40 DEG C in temperature
15h, the ultrafiltration membrane ultrafiltration 10-20min that enzymolysis interval 1-2h is 8-12kDa with molecular cut off is to get ultrafiltrate, the step
It is digested using clostridiopetidase A and flavor protease, clostridiopetidase A can be opened exclusively under the premise of not destroying other protein
The distinctive superhelix of collagen, and flavor protease can carry out digestion from different amino acid sites, it is big to generate
The fishskin polypeptide of amount, while leading to reinfocing effect due to synergistic function between enzyme, the polypeptide fragment work(accordingly generated
It can further enhance, the content of polypeptide and the activity of fishskin polypeptide in the final enzymolysis efficiency for improving fish-skin albumen, enzymolysis liquid；
And small molecule product is removed with ultrafiltration during the reaction, and it avoids and continues to be digested into small molecule product as substrate, and it is small
Molecular product is the source of collagen bitter peptides, further reduces the generation of bitter peptides in this way, to reduce product
Loss, and the concentration of product in enzymatic hydrolysis system can be reduced so that enzyme digestion reaction rate can continue to keep, and greatly shorten enzymolysis
Time；
Decoloration：Ultrafiltrate is warming up to 50-70 DEG C, adjusting pH is 5-7, and the modified activated carbon of collagen weight 1-3% is added,
Ultrasonic wave stir 10-20min, filtering, obtain clear filtrate, the absorption property of the step modified activated carbon is good, ultrafiltrate can
It is uniformly dispersed, good decoloration deodorization effect can be reached under conditions of dosage is few, the time is short, and active ingredient is not lost,
Can be so that collagen polypeptide to be whiter, sensory-acceptance is higher；
Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 1-1.2kDa, the condensing peptide liquid of 1-1.2kDa or more, freeze-drying are obtained
Up to fishskin collagen polypeptide.
Preferably, enzymolysis step deionized water contains 0.2-0.4M potassium chloride and 3-5mM sodium carboxymethylcelluloses.Chlorine
Changing potassium and sodium carboxymethylcellulose may make in enzymatic hydrolysis system metal ion and acid ion by the interaction with protein,
Enable clostridiopetidase A and flavor protease that its structure is kept not to be destroyed, to play enzymolysis, and is conducive to protease
And the combination of substrate and catalytic hydrolysis reaction improve enzymolysis rate, and the presence of sodium carboxymethylcellulose is avoided that protein
It is intermolecular close, dispersing uniformity of the protein in enzymatic hydrolysis system is improved, the touch opportunity of protease and substrate is improved, into
And improve the content of polypeptide and the activity of fishskin polypeptide in enzymolysis rate enzymolysis efficiency, enzymolysis liquid.
Preferably, the preparation method of decolorization process modified activated carbon is：It is 1 by solid-liquid ratio:8-12g/mL will be active
Charcoal, which is put into the oxygen sodium hydroxide solution of a concentration of 0.8-1.2M, impregnates 45-50h, then uses deionized water quick wash 2-3 times, then
Activated carbon is put into the ethyl acetate containing lemon yellow and sodium benzoate, stirs 45-50h at 75-85 DEG C, it is dry to get
Modified activated carbon.By ethyl acetate and oxygen sodium oxide molybdena in activated carbon surface hydrolysis occurs for the step, then by hydrophily
Molecule sodium acetate is introduced into activated carbon surface, and internal structure is mainly based on micropore so that modified activated carbon can in ultrafiltrate
It is uniformly dispersed, improves contact of the modified activated carbon with ultrafiltrate, to quick adsorption pigment and fishy smell substance, while promoting space
Structure is converted to " Turbostratic " of disordering, expands the hole of activated carbon, is improved aperture ratio, is further increased activated carbon
Absorption property so that collagen polypeptide is whiter.
Further preferably, in ethyl acetate the lemon yellow containing 0.33-0.38wt% and 1.8-2.4wt% sodium benzoate.
The addition of lemon yellow and sodium benzoate is conducive to accelerate the hydrolysis and the organic salinity of hydrophily of ethyl acetate and oxygen sodium oxide molybdena
Son movement so that distribution of the organic molecules of salt of hydrophily inside activated carbon also more extensively with uniformly, have more pigments
It is combined in the form of oxygen key with hydrophilic surface groups inside modified activated carbon with fishy smell substance；Activated carbon surface can be improved simultaneously
Acid oxygen-containing functional group, and these functional groups can with the pigment in squid skin occur complexing, increase the suction to pigment
Attached amount, final modified activated carbon processing can be so that collagen polypeptide be whiter, and sensory-acceptance is higher.
Compared with the prior art, the advantages of the present invention are as follows：1）Preparation method of the present invention have put into it is low, easy to operate,
Protein degradation effect is good, fishskin collagen polypeptide yield is high, collagen polypeptide is compared with white, the higher advantage of sensory-acceptance, easily
In industrial-scale production, there is good application value and market potential；2）The absorption that the present invention is decolourized with modified activated carbon
Excellent performance can be uniformly dispersed in ultrafiltrate, and good decoloration deodorization effect can be reached under conditions of dosage is few, the time is short
Fruit, and active ingredient is not lost, it can be so that collagen polypeptide be whiter, sensory-acceptance is higher；3）The preparation method uses glue
Protoenzyme and flavor protease are digested, the content of polypeptide and the work of fishskin polypeptide in the enzymolysis efficiency of fish-skin albumen, enzymolysis liquid
Property is higher；4）The preparation method removes small molecule product in enzymolysis process with ultrafiltration, can reduce the generation of bitter peptides
With the loss of product, and the concentration of product in enzymatic hydrolysis system can be reduced so that enzyme digestion reaction rate can continue to keep, significantly
Shorten enzymolysis time.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
The preparation method of fishskin collagen polypeptide, including pretreatment of raw material, collagen protein powder are prepared, digested, decolourizing and nanofiltration, tool
Body step is：
1）Feed pretreatment step is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:4g/mL fish-skin is added a concentration of
It in the oxygen sodium hydroxide solution of 0.13M, impregnates and constantly stirs, rinsed to neutrality with circulating water after 1.5h, then it is 1 to press solid-liquid ratio:
A concentration of 2% aqueous isopropanol is added in 2.5g/mL, and filtered through gauze after being cleaned repeatedly to neutrality with distilled water after immersion 15h is filled
Divide and drain to get pretreatment fish-skin, fishskin collagen polypeptide is before preparation, because wherein containing many foreign proteins and fat, so wanting
Pre-treatment is first carried out to remove these foreign proteins, fat etc., influence of the substance of this kind to fishskin collagen polypeptide can be reduced；
2）Collagen protein powder preparation process is：It is 1 by solid-liquid ratio:8 are added a concentration of 0.5M acetic acid solutions into pretreatment fish-skin,
Rotating speed is stirring and leaching 7h under 130r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, and centrifuged supernatant decompression is steamed
It evaporates, is freeze-dried up to collagen protein powder, which makes collagen remain its unique superhelix, and property is stablized,
The recovery rate of collagen is up to 23.46%, prepares for next enzymolysis；
3）Enzymolysis：Deionized water is added into collagen protein powder for 8% by concentration of substrate, adjusts pH to 8.6, is by enzyme concentration
2.4% and 2.0% is separately added into clostridiopetidase A and flavor protease, and 15h is digested at being then 30 DEG C in temperature, and enzymolysis interval 1h, which is used, to be cut
Stay the ultrafiltration membrane ultrafiltration 10min that molecular weight is 12kDa to get ultrafiltrate, the step using clostridiopetidase A and flavor protease into
Row enzymolysis, clostridiopetidase A can exclusively open the distinctive superhelix of collagen under the premise of not destroying other protein,
And flavor protease can carry out digestion from different amino acid sites, to generate a large amount of fishskin polypeptide, at the same between enzyme because
Lead to reinfocing effect with synergistic function, the polypeptide fragment function of accordingly generating can further enhance, and finally improve fish
The content of polypeptide and the activity of fishskin polypeptide in the enzymolysis efficiency of hide collagen, enzymolysis liquid；And it is removed during the reaction with ultrafiltration
Small molecule product avoids and continues to be digested into small molecule product as substrate, and small molecule product is collagen bitter peptides
Source, further reduce the generation of bitter peptides in this way, to reduce the loss of product, and can reduce in enzymatic hydrolysis system
The concentration of product so that enzyme digestion reaction rate can continue to keep, and greatly shorten enzymolysis time；
4）Decoloration：Ultrafiltrate is warming up to 70 DEG C, it is 5 to adjust pH, and the modified activated carbon of collagen weight 3%, ultrasonic wave is added
10min is stirred, filtering obtains clear filtrate, and the absorption property of the step modified activated carbon is good, can be uniformly dispersed in ultrafiltrate,
Good decoloration deodorization effect can be reached under conditions of dosage is few, the time is short, and active ingredient is not lost, it can be so that collagen
Polypeptide is whiter, and sensory-acceptance is higher；
5）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 1kDa, the condensing peptide liquid of 1kDa or more is obtained, is freeze-dried up to fish
Collagen polypeptide.
Enzymolysis step deionized water contains 0.4M potassium chloride and 3mM sodium carboxymethylcelluloses.Potassium chloride and carboxymethyl are fine
The plain sodium of dimension may make that metal ion and acid ion pass through the interaction with protein in enzymatic hydrolysis system so that clostridiopetidase A and wind
Taste protease can keep its structure not to be destroyed, and to play enzymolysis, and be conducive to the combination of albumen enzyme-to-substrate simultaneously
Catalytic hydrolysis reaction improves enzymolysis rate, and the presence of sodium carboxymethylcellulose is avoided that approaching between protein molecule, carries
Dispersing uniformity of the high protein in enzymatic hydrolysis system improves the touch opportunity of protease and substrate, and then improves enzymolysis rate
The activity of the content of polypeptide and fishskin polypeptide in enzymolysis efficiency, enzymolysis liquid.
The preparation method of decolorization process modified activated carbon is：It is 1 by solid-liquid ratio:Activated carbon is put into a concentration of by 12g/mL
50h is impregnated in the oxygen sodium hydroxide solution of 0.8M, then uses deionized water quick wash 2 times, then activated carbon is put into containing lemon
In yellow and sodium benzoate ethyl acetate, 45h is stirred at 85 DEG C, it is dry to get modified activated carbon.The step passes through acetic acid second
With oxygen sodium oxide molybdena in activated carbon surface hydrolysis occurs for ester, and hydrophilic molecule sodium acetate is then introduced into activated carbon surface,
Internal structure is mainly based on micropore so that modified activated carbon can be uniformly dispersed in ultrafiltrate, improve modified activated carbon and surpass
The contact of filtrate to quick adsorption pigment and fishy smell substance, while promoting space structure to turn to " Turbostratic " of disordering
Change, expand the hole of activated carbon, improves aperture ratio, further increase the absorption property of activated carbon so that collagen polypeptide
It is relatively white.
The sodium benzoate of lemon yellow containing 0.38wt% and 1.8wt% in above-mentioned ethyl acetate.Lemon yellow and sodium benzoate
Addition be conducive to accelerate ethyl acetate and the hydrolysis of oxygen sodium oxide molybdena and the movement of the organic molecules of salt of hydrophily so that it is hydrophilic
Property the distribution inside activated carbon of organic molecules of salt also live more extensively modified with uniformly, having more pigments and fishy smell substance
Property charcoal inside combined in the form of oxygen key with hydrophilic surface groups；The acid oxygen-containing functional group of activated carbon surface can be improved simultaneously,
And complexing can occur with the pigment in squid skin for these functional groups, increase the adsorbance to pigment, final modified work
Property charcoal processing can make collagen polypeptide it is whiter, sensory-acceptance is higher.
Embodiment 2：
The preparation method of fishskin collagen polypeptide, including pretreatment of raw material, collagen protein powder are prepared, digested, decolourizing and nanofiltration, tool
Body step is：
1）Feed pretreatment step is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:5g/mL fish-skin is added a concentration of
It in the oxygen sodium hydroxide solution of 0.1M, impregnates and constantly stirs, rinsed to neutrality with circulating water after 2.0h, then it is 1 to press solid-liquid ratio:
A concentration of 4% aqueous isopropanol is added in 2.0g/mL, and filtered through gauze after being cleaned repeatedly to neutrality with distilled water after immersion 12h is filled
Divide and drains to get pretreatment fish-skin；
2）Collagen protein powder preparation process is：It is 1 by solid-liquid ratio:10 into pretreatment fish-skin, that a concentration of 0.3M acetic acid is added is molten
Liquid, rotating speed are stirring and leaching 6h under 150r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, and centrifuged supernatant subtracts
Pressure distillation, is freeze-dried up to collagen protein powder；
3）Enzymolysis：Deionized water is added into collagen protein powder for 8% by concentration of substrate, adjusts pH to 8.0, is by enzyme concentration
3.0% and 1.8% is separately added into clostridiopetidase A and flavor protease, and 12h is digested at being then 35 DEG C in temperature, and enzymolysis interval 1.5h is used
Molecular cut off is the ultrafiltration membrane ultrafiltration 15min of 10kDa to get ultrafiltrate；
4）Decoloration：Ultrafiltrate is warming up to 60 DEG C, it is 6 to adjust pH, and the modified activated carbon of collagen weight 2%, ultrasonic wave is added
15min is stirred, filtering obtains clear filtrate,；
5）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 1.1kDa, the condensing peptide liquid of 1.1kDa or more is obtained, freeze-drying is
Obtain fishskin collagen polypeptide.
Enzymolysis step deionized water contains 0.3M potassium chloride and 4mM sodium carboxymethylcelluloses.
The preparation method of decolorization process modified activated carbon is：It is 1 by solid-liquid ratio:Activated carbon is put into a concentration of by 10g/mL
48h is impregnated in the oxygen sodium hydroxide solution of 1.0M, then uses deionized water quick wash 2 times, then activated carbon is put into containing lemon
In yellow and sodium benzoate ethyl acetate, 48h is stirred at 80 DEG C, it is dry to get modified activated carbon.Contain in above-mentioned ethyl acetate
There are the lemon yellow of 0.35wt% and the sodium benzoate of 2.0wt%.
Embodiment 3：
The preparation method of fishskin collagen polypeptide, including pretreatment of raw material, collagen protein powder are prepared, digested, decolourizing and nanofiltration, tool
Body step is：
1）Feed pretreatment step is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:4g/mL fish-skin is added a concentration of
It in the oxygen sodium hydroxide solution of 0.13M, impregnates and constantly stirs, rinsed to neutrality with circulating water after 1.5h, then it is 1 to press solid-liquid ratio:
A concentration of 2% aqueous isopropanol is added in 2.5g/mL, and filtered through gauze after being cleaned repeatedly to neutrality with distilled water after immersion 15h is filled
Divide and drains to get pretreatment fish-skin；
2）Collagen protein powder preparation process is：It is 1 by solid-liquid ratio:8 are added a concentration of 0.5M acetic acid solutions into pretreatment fish-skin,
Rotating speed is stirring and leaching 7h under 130r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, and centrifuged supernatant decompression is steamed
It evaporates, is freeze-dried up to collagen protein powder；
3）Enzymolysis：Deionized water is added into collagen protein powder for 8% by concentration of substrate, adjusts pH to 8.6, is by enzyme concentration
2.4% and 2.0% is separately added into clostridiopetidase A and flavor protease, and 15h is digested at being then 30 DEG C in temperature, and enzymolysis interval 1h, which is used, to be cut
Stay the ultrafiltration membrane ultrafiltration 10min that molecular weight is 12kDa to get ultrafiltrate；
4）Decoloration：Ultrafiltrate is warming up to 70 DEG C, it is 5 to adjust pH, and the modified activated carbon of collagen weight 3%, ultrasonic wave is added
10min is stirred, filtering obtains clear filtrate；
5）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 1.2kDa, the condensing peptide liquid of 1.2kDa or more is obtained, freeze-drying is
Obtain fishskin collagen polypeptide.
Enzymolysis step deionized water contains 0.2M potassium chloride and 5mM sodium carboxymethylcelluloses.
The preparation method of decolorization process modified activated carbon is：It is 1 by solid-liquid ratio:Activated carbon is put into a concentration of by 8g/mL
45h is impregnated in the oxygen sodium hydroxide solution of 1.2M, then uses deionized water quick wash 3 times, then activated carbon is put into containing lemon
In yellow and sodium benzoate ethyl acetate, 50h is stirred at 75 DEG C, it is dry to get modified activated carbon.Contain in above-mentioned ethyl acetate
There are the lemon yellow of 0.33wt% and the sodium benzoate of 2.4wt%.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. the preparation method of fishskin collagen polypeptide, including pretreatment of raw material, collagen protein powder are prepared, digested, decolourizing and nanofiltration,
It is characterized in that：The decolorization process is：Modified activated carbon, ultrasonic wave stirring will be added in ultrafiltrate, filtering obtains clear filtrate.
2. the preparation method of fishskin collagen polypeptide according to claim 1, it is characterised in that：The system of the modified activated carbon
Preparation Method is：It is 1 by solid-liquid ratio:Activated carbon is put into the oxygen sodium hydroxide solution of a concentration of 0.8-1.2M by 8-12g/mL to be impregnated
45-50h, washing, then activated carbon is put into the ethyl acetate containing lemon yellow and sodium benzoate, stir 45- at 75-85 DEG C
50h is dry to get modified activated carbon.
3. the preparation method of fishskin collagen polypeptide according to claim 2, it is characterised in that：Contain in the ethyl acetate
The lemon yellow of 0.33-0.38wt% and the sodium benzoate of 1.8-2.4wt%.
4. the preparation method of fishskin collagen polypeptide according to claim 2, it is characterised in that：The decolorization condition is：Temperature
Degree is 50-70 DEG C, pH 5-7, and modified activated carbon is added to as the 1-3% of collagen weight, time 20-40min.
5. the preparation method of fishskin collagen polypeptide according to claim 1, it is characterised in that：The feed pretreatment step
For：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:The oxygen that a concentration of 0.08-0.13M is added in fish-skin by 4-6g/mL aoxidizes
It in sodium solution, impregnates and constantly stirs, rinsed to neutrality with circulating water after 1.5-2.5h, then it is 1 to press solid-liquid ratio:1.5-2.5g/
The aqueous isopropanol of a concentration of 2-5% is added in mL, and filtered through gauze after being cleaned repeatedly to neutrality with distilled water after immersion 10-15h is filled
Divide and drains to get pretreatment fish-skin.
6. the preparation method of fishskin collagen polypeptide according to claim 1, it is characterised in that：It is prepared by the collagen protein powder
Step is：It is 1 by solid-liquid ratio:A concentration of 0.2-0.5M acetic acid solutions, rotating speed 130- is added into pretreatment fish-skin in 8-11
Stirring and leaching 5-7h under 170r/min, centrifuging and taking supernatant, precipitation continue quadratic acid extraction, and centrifuged supernatant is evaporated under reduced pressure,
It is freeze-dried up to collagen protein powder.
7. the preparation method of fishskin collagen polypeptide according to claim 1, it is characterised in that：The enzymolysis step is：It presses
Concentration of substrate is that deionized water is added into collagen protein powder by 8-10%, adjusts pH to 7.4-8.6, is 2.4-3.5% by enzyme concentration
It is separately added into clostridiopetidase A and flavor protease with 1.6-2.0%, 10-15h, enzymolysis interval are digested at being then 30-40 DEG C in temperature
The ultrafiltration membrane ultrafiltration 10-20min that 1-2h is 8-12kDa with molecular cut off is to get ultrafiltrate.
8. the preparation method of fishskin collagen polypeptide according to claim 7, it is characterised in that：The deionized water contains
0.2-0.4M potassium chloride and 3-5mM sodium carboxymethylcelluloses.
9. the preparation method of fishskin collagen polypeptide according to claim 1, it is characterised in that：The nano-filtration step is：It will
NF membrane nanofiltration of the clear filtrate through 1-1.2kDa obtains the condensing peptide liquid of 1-1.2kDa or more, is freeze-dried up to fish glue from skin
Former polypeptide.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486891A (en) * | 2019-01-04 | 2019-03-19 | 广东海洋大学 | A kind of extraction preparation method of small molecule collagen peptide |
| CN110467668A (en) * | 2019-09-10 | 2019-11-19 | 河北考力森生物科技有限公司 | A kind of extracting method of type III collagen |
| CN112608379A (en) * | 2020-12-03 | 2021-04-06 | 青岛大学附属医院 | Preparation method of donkey skin collagen |
| CN112899335A (en) * | 2021-04-13 | 2021-06-04 | 德兰梅勒（北京）分离技术股份有限公司 | Preparation method of fish skin collagen peptide |
| CN117466993A (en) * | 2023-12-28 | 2024-01-30 | 山东鑫日海食品有限公司 | Membrane filtration purification method of deep sea fish collagen peptide |
-
2018
- 2018-05-02 CN CN201810408287.XA patent/CN108728507A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486891A (en) * | 2019-01-04 | 2019-03-19 | 广东海洋大学 | A kind of extraction preparation method of small molecule collagen peptide |
| CN110467668A (en) * | 2019-09-10 | 2019-11-19 | 河北考力森生物科技有限公司 | A kind of extracting method of type III collagen |
| CN112608379A (en) * | 2020-12-03 | 2021-04-06 | 青岛大学附属医院 | Preparation method of donkey skin collagen |
| CN112899335A (en) * | 2021-04-13 | 2021-06-04 | 德兰梅勒（北京）分离技术股份有限公司 | Preparation method of fish skin collagen peptide |
| CN117466993A (en) * | 2023-12-28 | 2024-01-30 | 山东鑫日海食品有限公司 | Membrane filtration purification method of deep sea fish collagen peptide |
| CN117466993B (en) * | 2023-12-28 | 2024-03-08 | 山东鑫日海食品有限公司 | Membrane filtration purification method of deep sea fish collagen peptide |
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Application publication date: 20181102 |