CN103409489B - Preparation method for fish collagen antioxidant peptide - Google Patents
Preparation method for fish collagen antioxidant peptide Download PDFInfo
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Abstract
The invention provides a preparation method for fish collagen antioxidant peptide, which relates to a collagen. The preparation method comprises the following steps: with a fish-skin or fish scale as a raw material, soaking the raw material with an alkaline solution containing absolute ethyl alcohol, removing impurity proteins and fat, rinsing the raw material with clear water until the raw material is neutral, then soaking the raw material with an acidic solution and rinsing the raw material with clear water until the raw material is neutral; adding composite protease into the pretreated fish-skin or fish scale for enzymatic hydrolysis so as to obtain fish collagen enzymatic hydrolysate; heating the fish collagen enzymatic hydrolysate, carrying out enzyme deactivation, then filtering the fish collagen enzymatic hydrolysate to remove insoluble substances and removing amino acids and salts by using a nanofiltration membrane so as to obtain a fish collagen peptide solution; preparing a fish collagen antioxidant peptide solution by adding a hydrophobic adsorbent into the fish collagen peptide solution, standing the solution for adsorption, adding an organic solvent into the hydrophobic adsorbent after filtration and then carrying out filtering so as to obtain the fish collagen antioxidant peptide solution; and removing the organic solvent in the fish collagen antioxidant peptide solution through vacuum heating evaporation and carrying out spray drying or freeze drying so as to prepare the fish collagen antioxidant peptide with an antioxidant function.
Description
Technical field
The present invention relates to a kind of collagen protein, especially relate to utilize that hydrophobic adsorbent adsorbs collagen protein enzymolysis liquid, the preparation of the method for wash-out, there is the preparation method of a kind of fish collagen antioxidant peptide of good anti-oxidant function.
Background technology
Collagen protein is dermis of skin important component part, but with advancing age, the renewal rate of skin collagen declines gradually, therefore needs timely replenishing collagen.Collagen peptide is a kind of hydrolytic collagen polypeptide of small-molecular-weight, and the collagen for skin losses can award timely reparation, and then improves the water retention capacity of skin, delay skin aging.On the other hand, research shows, free radical excessive in human body can destroy lipid, protein and DNA and cause various damage, accelerates the senescence process of body and brings out various disease.Although natural and synthetized oxidation preventive agent can play the effect of scavenging free radicals, can there is certain potential safety hazard in the use of synthetized oxidation preventive agent as butylated hydroxy anisole (BHA).Along with the raising of the development of Chinese society, the quickening of rhythm of life and health care consciousness, natural, safe antioxidant receives increasing concern.And, along with people deepen continuously to the understanding of effective constituent in oceanic resources, the interest of the biologically active substance being derived from aquatic animal is constantly increased.
Owing to containing a large amount of polybasic unsaturated fatty acids in human inner cell's film, the collagen peptide with good lipophilic hydrophobic structure will play better effect in vivo.
China Agricultural University reports anti-oxidation peptide being derived from collagen protein and uses thereof in Chinese patent CN101824071A, discloses a series of anti-oxidation peptide being derived from collagen protein, and it has one of following aminoacid sequence: (1) Hyl-Cys; (2) Gln-Gly-Ala-Arg; (3) Leu-Gln-Gly-Met; (4) Leu-Gln-Gly-Met-Hyp; The hydrolysate separation and purification obtained by the synthesis of polypeptide instrument chemical process, also by by collagen hydrolysate, then can obtain by this anti-oxidation peptide.The applicant provides a kind of extraction preparation method of small molecules collagen of fish skin peptide in Chinese patent CN102703555A, with the fish-skin of fresh-water fishes for raw material, removing noncollagen protein, through degreasing, de-raw meat, the step such as to grind, in acid condition to the process of collagen protein heating hydrolysis, then enzymolysis process is adopted to control the palliating degradation degree of collagen protein, membrane separation process is adopted to obtain target micromolecular collagen peptide to proteolytic enzyme recycling, then nanofiltration desalination is passed through, after gac binding resin decoloration deodorization, spraying dry is utilized to obtain the small molecules collagen of fish skin Gly-His-Lys of colorless and odorless.But foregoing invention all ignores the extraction preparation of lipophilic hydrophobic structure collagen peptide.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of fish collagen antioxidant peptide with good anti-oxidant function.
The present invention includes following steps:
1) pre-treatment of raw material: with fish-skin or fish scale for raw material, soaks with the basic solution containing dehydrated alcohol, uses clear water rinsing to neutral after removing foreign protein and fat, then after soaking with acidic solution, by clear water rinsing extremely neutrality;
2) enzymolysis: add compound protease in pretreated fish-skin or fish scale, after enzymolysis, obtains Isin glue collagen enzymolysis solution;
3) filter, go out enzyme: by the Isin glue collagen enzymolysis solution heating obtained, filter after the enzyme that carries out going out, remove insolubles, then utilize nanofiltration membrane to remove amino acid and salinity, obtain collagen peptide solution;
4) preparation of fish collagen antioxidant peptide solution: add hydrophobic adsorbent in collagen peptide solution, standing adsorption, after filtration, adds organic solvent in hydrophobic adsorbent, obtains fish collagen antioxidant peptide solution after filtration;
5) preparation of fish collagen antioxidant peptide: utilize the organic solvent in heating under vacuum evaporative removal fish collagen antioxidant peptide solution, be prepared into the fish collagen antioxidant peptide with anti-oxidant function by spraying dry or lyophilize, obtained fish collagen antioxidant peptide is Powdered.
In step 1) in, described fish-skin or fish scale can adopt a kind of fish-skin in fresh-water fishes, sea water fish or fish scale; The concentration expressed in percentage by volume of described dehydrated alcohol can be 20%; The add-on of the described basic solution containing dehydrated alcohol can be 2 ~ 5 times of raw material; The condition that described basic solution soaks can be: basic solution is 0.01 ~ 0.05M NaOH solution, and the temperature of immersion is 4 ~ 20 DEG C, and the time of immersion is 12 ~ 24h; The add-on of described acidic solution can be 2 ~ 5 times of raw material; The condition that described acidic solution soaks can be: acidic solution is 0.05 ~ 0.20M HCl solution, and the temperature of immersion is 4 ~ 20 DEG C, and the time of immersion is 1 ~ 3h.
In step 2) in, described compound protease can be selected from least two kinds in Collagenase, Sumizyme MP, trypsinase, papoid, bromeline, neutral protease etc.; The add-on of compound protease can be 0.1% ~ 0.5% of raw materials quality by mass percentage; The condition of described enzymolysis can be enzymolysis 2 ~ 6h at 40 ~ 60 DEG C.
In step 3) in, the temperature of described heating can be 90 ~ 100 DEG C, and the time of heating can be 5 ~ 15min; Described filtration can adopt Plate Filtration or centrifuging; The molecular weight cut-off of described nanofiltration membrane can be 200 dalton.
In step 4) in, described hydrophobic adsorbent can be selected from least one in gac, ion exchange resin, macroporous adsorbent resin etc.; The add-on of described hydrophobic adsorbent can be 1% ~ 5% of collagen peptide solution by mass percentage; The time of described standing adsorption can be 30 ~ 90min; The composition of described organic solvent can be: the volume ratio of dehydrated alcohol/acetic acid/water is 50/ (0.5 ~ 2.5)/(49.5 ~ 47.5); The add-on of described organic solvent can be 2 ~ 5 times of hydrophobic adsorbent in mass ratio.
In step 5) in, the temperature of described heating under vacuum evaporation can be 50 ~ 80 DEG C.
The present invention with fish-skin or fish scale for raw material, removing noncollagen protein, compound protease is adopted to carry out enzymolysis, after enzymolysis solution removes amino acid and salt by nanofiltration membrane, utilize hydrophobic adsorbent to carry out adsorbing, the preparation method such as organic solvent wash-out, then remove the means such as organic solvent, drying by heating evaporation and prepare collagen peptide.In collagen peptide prepared by the present invention, hydrophobic amino acid content is high, and molecular weight integrated distribution, at 300 ~ 2000Da, has good anti-oxidant function, can be widely used in the industry that protective foods, biological medicine, makeup etc. are relevant to HUMAN HEALTH.
Containing a large amount of polybasic unsaturated fatty acids in human inner cell's film, easily oxidized.Antioxidant may be used for the chain reaction of oil-control autoxidation.But the current research about collagen peptide mainly concentrates on the extraction of wetting ability collagen peptide, and the collagen peptide of preparation is not owing to being soluble in grease, and range of application is subject to certain restrictions.The present invention utilizes hydrophobic adsorbent to adsorb Isin glue collagen enzymolysis solution; the recycling organic solvent such as alcohol, acetic acid carries out the collagen peptide that wash-out is prepared into will have good lipophilic hydrophobic structure; can be inserted in the microbial film of unsaturated fatty acids existence, play anti-oxidation protection effect.Treatment condition of the present invention are gentle, can reclaim the hydrophobic collagen peptide in the decoloration process of collagen peptide, effectively utilize collagen protein enzymolysis thing, reduce production cost.Therefore the present invention has the following advantages:
1, the collagen peptide that prepared by the present invention has good anti-oxidant function.
2, the collagen peptide that prepared by the present invention contains a large amount of hydrophobic amino acids, will have good oleophylic function.
3, the main integrated distribution of molecular weight of collagen peptide prepared of the present invention is at 300 ~ 3000Da, is conducive to absorption of human body and utilizes.
4, preparation technology of the present invention may be used for reclaiming the hydrophobic collagen peptide run off in collagen peptide decolorization, reduces environmental pollution, reduces the production cost of collagen peptide.
5, the present invention has utilized fish-skin, fish scale etc. to prepare to have good anti-oxidant function, collagen peptide containing a large amount of hydrophobic grouping, not only may be used for the collagen of supplementary skin losses, can also interior free yl be removed, have a good application prospect in the industry that medicine, the cosmetics of super quality, protective foods etc. are relevant to HUMAN HEALTH.
Accompanying drawing explanation
Fig. 1 is the HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 1.
Fig. 2 is the HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 2.
Fig. 3 is the HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 3.
Fig. 4 is the HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 4.
Embodiment
Following examples will the present invention is further illustrated by reference to the accompanying drawings.
Embodiment 1
The 0.01M NaOH solution containing 20% (v/v) dehydrated alcohol of 20L is added in 10kg tilapia fish scale, 24h is soaked at 20 DEG C, after utilizing tap water rinse clean, the 0.05M HCl solution adding 20L soaks 3h at 20 DEG C, with tap water rinse to neutral, separate type high-density paste mill is utilized to be ground by the fish scale after process, add the compound protease (25g Collagenase and 25g papoid) of 50g, enzymolysis 2h at 40 DEG C, 100 DEG C of heating 15min go out enzyme, filter and remove insolubles, molecular weight cut-off is utilized to be after 200 daltonian nanofiltration membrane remove amino acid and salinity, obtain the collagen protein peptide solution 20L that Brix is 12%, through being cooled to room temperature, add 1000g macroporous adsorbent resin, filter after leaving standstill 30min, the machine solvent aqueous solution that 2000ml contains 50% (v/v) alcohol and 2.5% (v/v) acetic acid is added in the macroporous adsorbent resin obtained, at room temperature stir 15min, filter and obtain collagen peptide solution, Rotary Evaporators is utilized down except organic solvent 50 DEG C of heating under vacuum, spraying dry obtains the collagen peptide powder of about 220g.The HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 1 is see Fig. 1.
Embodiment 2
The 0.01M NaOH solution containing 20% (v/v) dehydrated alcohol of 30L is added in 10kg large yellow croaker fish scale, 18h is soaked at 20 DEG C, after utilizing tap water rinse clean, the 0.05M HCl solution adding 30L soaks 2h at 20 DEG C, with tap water rinse to neutral, separate type high-density paste mill is utilized to be ground by the fish scale after process, add the compound protease (20g Sumizyme MP and 20g neutral protease) of 40g, enzymolysis 4h at 45 DEG C, 100 DEG C of heating 15min go out enzyme, filter and remove insolubles, molecular weight cut-off is utilized to be after 200 daltonian nanofiltration membrane remove amino acid and salinity, obtain the collagen protein peptide solution 18L that Brix is 13%, through being cooled to room temperature, add 700g ion exchange resin, filter after leaving standstill 45min, the aqueous solutions of organic solvent that 2100ml contains 50% (v/v) alcohol and 1.5% (v/v) acetic acid is added in the gac obtained, at room temperature stir 20min, filter and obtain collagen peptide solution, Rotary Evaporators is utilized down except organic solvent 60 DEG C of heating under vacuum, spraying dry obtains the collagen peptide powder of about 230g.The HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 2 is see Fig. 2.
Embodiment 3
The 0.01M NaOH solution containing 20% (v/v) dehydrated alcohol of 40L is added in 10kg shark fish-skin, 15h is soaked at 20 DEG C, after utilizing tap water rinse clean, the 0.05M HCl solution adding 40L soaks 2h at 20 DEG C, with tap water rinse to neutral, separate type high-density paste mill is utilized to be ground by the fish-skin after process, add the compound protease (10g trypsinase and 10g papoid) of 20g, enzymolysis 5h at 50 DEG C, 100 DEG C of heating 10min go out enzyme, filter and remove insolubles, molecular weight cut-off is utilized to be after 200 daltonian nanofiltration membrane remove amino acid and salinity, obtain the collagen protein peptide solution 16L that Brix is 15%, through being cooled to room temperature, add 320g gac, filter after leaving standstill 60min, the aqueous solutions of organic solvent that 1000ml contains 50% (v/v) alcohol and 1.0% (v/v) acetic acid is added in the gac obtained, at room temperature stir 25min, filter and obtain collagen peptide solution, Rotary Evaporators is utilized down except organic solvent 70 DEG C of heating under vacuum, lyophilize obtains the collagen peptide powder of about 320g.The HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 3 is see Fig. 3.
Embodiment 4
The 0.01M NaOH solution containing 20% (v/v) dehydrated alcohol of 50L is added in 10kg tilapia fishskin, 12h is soaked at 20 DEG C, after utilizing tap water rinse clean, the 0.05M HCl solution adding 50L soaks 1h at 20 DEG C, with tap water rinse to neutral, separate type high-density paste mill is utilized to be ground by the fish-skin after process, add the compound protease (5g trypsinase and 5g bromeline) of 10g, enzymolysis 6h at 60 DEG C, 100 DEG C of heating 5min go out enzyme, filter and remove insolubles, molecular weight cut-off is utilized to be after 200 daltonian nanofiltration membrane remove amino acid and salinity, obtain the collagen protein peptide solution 15L that Brix is 15%, through being cooled to room temperature, add 150g gac, filter after leaving standstill 90min, the aqueous solutions of organic solvent that 750ml contains 50% (v/v) alcohol and 0.5% (v/v) acetic acid is added in the gac obtained, at room temperature stir 30min, filter and obtain collagen peptide solution, Rotary Evaporators is utilized down except organic solvent 80 DEG C of heating under vacuum, lyophilize obtains the collagen peptide powder of about 300g.The HPLC-UV detection of fish collagen antioxidant peptide molecular weight distribution prepared by embodiment 4 is see Fig. 4.
The present invention utilizes the molecular weight distribution of high performance liquid chromatograph to the collagen peptide that the present invention produces to measure, and result as shown in figures 1-4.From Fig. 1 ~ 4, the molecular weight of fish collagen antioxidant peptide prepared by the present invention is mainly distributed in 300 ~ 2000 dalton, and the molecular weight of the collagen peptide wherein utilizing macroporous resin (embodiment 1) to prepare concentrates on 470 dalton (Da) near zone, and (embodiment 2 is 1150Da and 390Da; Embodiment 3 is 1800Da and 460Da; Embodiment 4 be 2000Da, 1230 and 400Da).It is reported, compare with amino acid and protein polypeptide, be not only easy to absorption of human body metabolism lower than 2000 daltonian oligopeptides, and there is good biological activity.The DPPH free radical of fish collagen antioxidant peptide of preparation and Scavenging action to hydroxyl free radical are measured, if shown in result table 1.As shown in Table 1, although the DPPH radical scavenging activity of collagen peptide prepared by embodiment 1 ~ 4 also has a certain distance with gsh, but Hydroxyl radical-scavenging ability is close to gsh, show that the collagen peptide utilizing the present invention to prepare has good resistance of oxidation.The amino acid composition of amino acidanalyser to fish collagen antioxidant peptide is utilized to analyze (table 2), found that, hydrophobic amino acid ratio in the fish collagen antioxidant peptide of preparation can reach 40.3% ~ 58.6%, apparently higher than about 30% of Isin glue collagen enzymolysis solution.And some document think glycine between polarity and nonpolar between, generally also it is classified as nonpolar in biology.Therefore in the collagen anti-oxidation peptide prepared of the present invention, hydrophobic amino acid ratio can up to more than 70%, good lipophilic hydrophobic structure will be had, anti-oxidation peptide can be strengthened with the interaction with hydrophobicity polyunsaturated fatty acid in vivo, and then anti-lipid peroxidation chain reaction, therefore can be widely used in the industries such as protective foods, biological medicine, makeup.
In sum, the present invention utilizes compound protease to carry out enzymolysis to through pretreated fish-skin or fish scale, after enzymolysis solution removes amino acid and salt by nanofiltration membrane, utilize hydrophobic adsorbent to carry out adsorbing, the preparation method such as organic solvent wash-out, then remove the means such as organic solvent, drying by heating evaporation and prepare collagen peptide.Present invention process can combine with conventional collagen peptide production technique, decoloration process, the hydrophobic collagen peptide run off in the decoloration process of efficient recovery collagen peptide, reduces environmental pollution, saves production cost; The small molecules collagen peptide of preparation has good anti-oxidant function, expands the range of application of collagen peptide.
It should be noted that: the fish-skin that the present invention is used or fish scale can be the one in fresh-water fishes or sea water fish.
Table 1 is the antioxidant property (half-inhibition concentration, IC50) of fish collagen antioxidant peptide
The amino acid composition analysis (individual/1000 amino-acid residues) of table 2 fish collagen antioxidant peptide
Note: * is hydrophobic amino acid.
Claims (10)
1. a preparation method for fish collagen antioxidant peptide, is characterized in that comprising the following steps:
1) pre-treatment of raw material: with fish-skin or fish scale for raw material, soaks with the basic solution containing dehydrated alcohol, uses clear water rinsing to neutral after removing foreign protein and fat, then after soaking with acidic solution, by clear water rinsing extremely neutrality; The add-on of the described basic solution containing dehydrated alcohol is 2 ~ 5 times of raw material; The add-on of described acidic solution is 2 ~ 5 times of raw material;
2) enzymolysis: add compound protease in pretreated fish-skin or fish scale, after enzymolysis, obtains Isin glue collagen enzymolysis solution; Described compound protease is selected from least two kinds in Collagenase, Sumizyme MP, trypsinase, papoid, bromeline, neutral protease etc.; The add-on of compound protease is 0.1% ~ 0.5% of raw materials quality by mass percentage;
3) filter, go out enzyme: by the Isin glue collagen enzymolysis solution heating obtained, filter after the enzyme that carries out going out, remove insolubles, then utilize nanofiltration membrane to remove amino acid and salinity, obtain collagen peptide solution;
4) preparation of fish collagen antioxidant peptide solution: add hydrophobic adsorbent in collagen peptide solution, standing adsorption, after filtration, adds organic solvent in hydrophobic adsorbent, obtains fish collagen antioxidant peptide solution after filtration; The add-on of described hydrophobic adsorbent is 1% ~ 5% of collagen peptide solution by mass percentage; The add-on of described organic solvent is 2 ~ 5 times of hydrophobic adsorbent in mass ratio;
5) preparation of fish collagen antioxidant peptide: utilize the organic solvent in heating under vacuum evaporative removal fish collagen antioxidant peptide solution, be prepared into the fish collagen antioxidant peptide with anti-oxidant function by spraying dry or lyophilize, obtained fish collagen antioxidant peptide is Powdered.
2. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 1) in, described fish-skin or fish scale adopt a kind of fish-skin in fresh-water fishes, sea water fish or fish scale; The concentration expressed in percentage by volume of described dehydrated alcohol is 20%.
3. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, it is characterized in that in step 1) in, the condition that described basic solution soaks is: basic solution is 0.01 ~ 0.05M NaOH solution, and the temperature of immersion is 4 ~ 20 DEG C, and the time of immersion is 12 ~ 24h.
4. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, it is characterized in that in step 1) in, the condition that described acidic solution soaks is: acidic solution is 0.05 ~ 0.20M HCl solution, and the temperature of immersion is 4 ~ 20 DEG C, and the time of immersion is 1 ~ 3h.
5. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 2) in, the condition of described enzymolysis is enzymolysis 2 ~ 6h at 40 ~ 60 DEG C.
6. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 3) in, the temperature of described heating is 90 ~ 100 DEG C, and the time of heating is 5 ~ 15min.
7. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 3) in, described filtration adopts Plate Filtration or centrifuging; The molecular weight cut-off of described nanofiltration membrane is 200 dalton.
8. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 4) in, described hydrophobic adsorbent is selected from least one in gac, ion exchange resin, macroporous adsorbent resin.
9. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 4) in, the time of described standing adsorption is 30 ~ 90min; Consisting of of described organic solvent: the volume ratio of dehydrated alcohol/acetic acid/water is 50/0.5 ~ 2.5/49.5 ~ 47.5.
10. the preparation method of a kind of fish collagen antioxidant peptide as claimed in claim 1, is characterized in that in step 5) in, the temperature of described heating under vacuum evaporation is 50 ~ 80 DEG C.
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| CN111471733B (en) * | 2020-04-27 | 2023-03-07 | 山西大学 | Preparation method and application of shark skin polypeptide dry powder |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN114836505B (en) * | 2022-05-19 | 2024-04-12 | 泸州品创科技有限公司 | Small molecule anti-alcohol peptide and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
-
2013
- 2013-08-15 CN CN201310354944.4A patent/CN103409489B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
Non-Patent Citations (1)
| Title |
|---|
| 大豆7S、11S蛋白酶解模拟分析及疏水性对抗氧化活性的影响;孙国威;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20111231(第14期);摘要，表1-1,1.3,3.2.1,图3-5 * |
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