CN105648009A - Preparation method of high-Fischer-ratio oligopeptide from shark meat - Google Patents
Preparation method of high-Fischer-ratio oligopeptide from shark meat Download PDFInfo
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Abstract
The invention discloses a preparation method of high-Fischer-ratio oligopeptide from shark meat. The shark meat is taken as a raw material, shark meat prolamin is prepared through ultrasonic extraction, alkaline protease and papain are used for enzymolysis, the high-Fischer-ratio oligopeptide from the shark meat is prepared through ultrafiltration classification, activated carbon adsorption and purification and macroporous resin refinement. The Fischer ratio ranges from 35.2 to 39.6 and the molecular weight ranges from 630 Da to 720 Da. The high-Fischer-ratio oligopeptide has a significant anti-fatigue effect and can be developed as anti-fatigue functional food or healthcare food.
Description
Technical field
The preparation method that the present invention relates to a kind of animal high F value oligopeptide, the preparation method being specifically related to a kind of shark flesh of fish high F value oligopeptide.
Background technology
Oligo-peptide mixture be one by mixed small peptide (or the claim oligopeptide) system being made up of 3��7 residual amino acid residues of aminoacid. F value (Fischerratio) refers to the molal quantity ratio of branched-chain amino acid (BCAA:Val, Ile, Leu) and aromatic amino acid (AAA:Trp, Tyr, Phe) content. In the blood of normal person, F value is 3.0-3.5, and the F value suffering from hepatic disease patient only has 1.0 or lower, and the F value of high F value oligopeptide should be greater than 20, far above the ratio of this two amino acid in human body. Owing to it has aminoacid composition and the physiological function of uniqueness, have been subjected to showing great attention to of food and medicine circle.
Summary of the invention
The preparation method that the technical problem to be solved is to provide a kind of shark flesh of fish high F value oligopeptide.
The present invention solves that the technical scheme that above-mentioned technical problem is taked is: the preparation method of a kind of shark flesh of fish high F value oligopeptide, it comprises the following steps:
1) preparation of shark flesh of fish alcohol soluble protein:Take the alcoholic solution that the shark flesh of fish adds 90%��95% by 1g:15mL��1g:20mL solid-to-liquid ratio, be made into homogenate with high-speed tissue mashing machine, at 25��30 DEG C, power 650��700W supersound extraction 150min ~ 180min; The pH value 1mol/LNaOH of extracting solution being adjusted to 9.5��10.0, with distilled water, concentration of alcohol is adjusted to 55%��65%, room temperature stands 45min��60min, in the centrifugal 10min��15min of 8000��10000rpm; Take supernatant, and to add NaCl to concentration be 5%��8%, after standing 8h��10h, in the centrifugal 10��15min of 8000��10000rpm, collect precipitation, lyophilizing, obtain shark flesh of fish alcohol soluble protein.
2) enzymolysis of shark flesh of fish alcohol soluble protein:Take shark flesh of fish alcohol soluble protein, by solid-to-liquid ratio 1g:5mL��1g:10mL, add glycine sodium hydrate buffer solution (0.05mol/L, pH9.5), regulate pH to 9.0��1.0 with 0.1mol/LHCl or 0.1mol/LNaOH, obtain mixed liquor; Mixture temperature is modulated 55��65 DEG C, adds alkaline protease (enzyme activity 1.95 �� 10 according to the 0.5%��1.0% of alcohol soluble protein quality5), after enzymolysis time 3h��4h, enzymolysis solution is incubated 10��15min in 90��95 DEG C, and enzyme denaturing is lived; Enzymolysis solution temperature being adjusted to 50��60 DEG C, adds papain, enzymolysis time 4h��5h according to the 0.6%��0.8% of alcohol soluble protein quality, enzymolysis solution is down to room temperature, in the centrifugal 10��15min of 8000��10000rpm, takes supernatant.
3) enzymolysis of shark flesh of fish high F value oligopeptide:Above-mentioned supernatant is refined through ultrafiltration, activated carbon adsorption purification and macroporous resin successively, obtains shark flesh of fish high F value oligopeptide.
As preferably, the described shark in step 1) be Mustelus griseus (pietschmann) (Mustelusgriseus).
As preferably, the detailed process that the ultrafiltration of described step 3), activated carbon adsorption purification and macroporous resin are refined is:
Ultrafiltration: above-mentioned enzymolysis supernatant adopts 3kDa ultrafilter membrane carry out hyperfiltration treatment under the operating pressure of 0.1��0.15MPa and the operating temperature of 20��30 DEG C, collects molecular weight less than 3kDa part, obtains ultrafiltration enzymolysis solution;
Activated carbon adsorption purification system: adjust pH value to 2.5 ~ 3.5 ultrafiltration enzymolysis solution, it is that ultrafiltration enzymolysis solution is slowly added into activated carbon granule with 0.3��0.5mL/min flow velocity and is loaded in glass column by 10��15:1 according to material and activated carbon granule ratio, after completion of the sample, adopt 1mol/L ammonia and the dehydrated alcohol mixed solution elution chromatography post of the 1:1 ratio of 2��3 column volumes, collect eluent, dry, obtain the refining polypeptide of bioactive peptide
Macroporous resin is refined: refining for activated carbon polypeptide is made into the solution of 10��20mg/mL, adjust pH value to 6.5��7.5, be slowly added into material and AB-8 macroporous resin than be 10��15:1 glass column in, impurity is removed with the distilled water eluting of 2��3 times of column volumes, again with 95% ethanol elution of 2��3 column volumes, collect 95% ethanol elution thing, spray drying, be shark flesh of fish high F value oligopeptide.
After measured, the protein content of gained shark flesh of fish high F value oligopeptide is 95.1��96.4%, total amino acid content is 83.7��85.4%, the content of branched-chain amino acid is 16.8��17.1%, the content of aromatic amino acid is 0.43��0.48%, and F value is 35.2��39.6, and molecular weight is 630��720Da, in branched-chain amino acid, Val:Ile:Leu is 1:1��1.5:3��4, and in aromatic amino acid, Trp:Tyr:Phe is 1:1��1.2:1��1.5.
Compared with prior art, the advantage of the preparation method of a kind of shark provided by the invention flesh of fish high F value oligopeptide is in that: present invention process is scientific and reasonable, simple to operate, selects alkaline protease and papain as enzymolysis enzyme, and enzymolysis process is easily monitored; The ultrafiltration classification, activated carbon adsorption purification and the macroporous resin process for purification that utilize are simple, it is easy to commercial application; Simultaneously, shark flesh of fish high F value oligopeptide prepared by the present invention is that the shark flesh of fish prepares through enzyme hydrolysis, safe and free of toxic and side effects, and resisting fatigue is notable, old people, valetudinarian and postoperative patient are had good health-care effect, and shark flesh of fish high F value oligopeptide can be developed as anti-fatigue effect food.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment:
Preparation method of a kind of shark flesh of fish high F value oligopeptide and its production and use, preparation technology flow process is as follows: the shark flesh of fish �� supersound extraction alcohol soluble protein �� alkaline protease and papain enzymolysis �� ultrafiltration �� bioactive peptide adsorption and purification �� macroporous resin refine �� shark flesh of fish high F value oligopeptide.
1) preparation of shark flesh of fish alcohol soluble protein:Take the alcoholic solution that the Mustelus griseus (pietschmann) shark flesh of fish adds 95% by 1g:18mL solid-to-liquid ratio, be made into homogenate with high-speed tissue mashing machine, at 30 DEG C, power 680W supersound extraction 150min; The pH value 1mol/LNaOH of extracting solution being adjusted to 9.5, with distilled water, concentration of alcohol is adjusted to 60%, room temperature stands 50min, in the centrifugal 12min of 9000rpm;Take supernatant, and to add NaCl to concentration be 6%, after standing 9h, in the centrifugal 15min of 9000rpm, collect precipitation, lyophilizing, obtain shark flesh of fish alcohol soluble protein.
2) enzymolysis of shark flesh of fish alcohol soluble protein:Take shark flesh of fish alcohol soluble protein, by solid-to-liquid ratio 1g:8mL, add glycine sodium hydrate buffer solution (0.05mol/L, pH9.5), regulate pH to 9.5 with 0.1mol/LHCl or 0.1mol/LNaOH, obtain mixed liquor; Mixture temperature is modulated 60 DEG C, adds alkaline protease (enzyme activity 1.95 �� 10 according to the 0.6% of alcohol soluble protein quality5), after enzymolysis time 3h, enzymolysis solution is incubated 15min in 95 DEG C, and enzyme denaturing is lived; Enzymolysis solution temperature being adjusted to 55 DEG C, adds papain, enzymolysis time 4h according to the 0.8% of alcohol soluble protein quality, enzymolysis solution is down to room temperature, in the centrifugal 12min of 9000rpm, takes supernatant.
3) enzymolysis of shark flesh of fish high F value oligopeptide:Above-mentioned supernatant is refined through ultrafiltration, activated carbon adsorption purification and macroporous resin successively, obtains shark flesh of fish high F value oligopeptide. Detailed process is:
1. ultrafiltration: above-mentioned enzymolysis supernatant adopts 3kDa ultrafilter membrane carry out hyperfiltration treatment under the operating pressure of 0.2MPa and the operating temperature of 25 DEG C, collects molecular weight less than 3kDa part, obtains ultrafiltration enzymolysis solution;
2. activated carbon adsorption purification system: adjust pH value to 2.8 ultrafiltration enzymolysis solution, than for 12:1, ultrafiltration enzymolysis solution is slowly added into activated carbon granule with 0.4mL/min flow velocity with activated carbon granule according to material and is loaded in glass column, after completion of the sample, adopt 1mol/L ammonia and the dehydrated alcohol mixed solution elution chromatography post of the 1:1 ratio of 3 column volumes, collect eluent, dry, obtain the refining polypeptide of bioactive peptide
3. macroporous resin is refined: refining for activated carbon polypeptide is made into the solution of 15mg/mL, adjust pH value to 7.0, it is slowly added into material with AB-8 macroporous resin ratio in the glass column of 15:1, impurity is removed with the distilled water eluting of 2 times of column volumes, again with 95% ethanol elution of 3 column volumes, collect 95% ethanol elution thing, spray drying, be shark flesh of fish high F value oligopeptide.
After measured, the protein content of gained shark flesh of fish high F value oligopeptide is 96.0%, total amino acid content is 85.1%, the content of branched-chain amino acid is 17.0%, the content of aromatic amino acid is 0.45%, and F value is 37.7, and molecular weight is 657Da, in branched-chain amino acid, Val:Ile:Leu is 1:1.2:3.3, and in aromatic amino acid, Trp:Tyr:Phe is 1:1.1:1��1.3.
Prepared shark flesh of fish high F value oligopeptide is done anti-fatigue active research (impact on indexs such as white mice swimming time and blood lactase acid, hepatic glycogen, blood urea nitrogen) of mice, evaluates its anti-fatigue active.
Test result indicate that: the shark flesh of fish high F value oligopeptide prepared can dramatically increase mice swimming time, improves exercise tolerance, improves the hepatic glycogen content in Mice Body, reduces blood lactase acid and blood urea nitrogen generation amount. .
The flesh of fish high F value oligopeptide impact (n=15, meansigma methods �� SD) on mice swimming time of table 1 shark
| Group | Matched group | Low dose group (20mg/mL) | Middle dosage group (40mg/mL) | High dose group (80mg/mL) |
| Swimming time | 338��15 | 403��14 | 476��21 | 531��32 |
Note: gavage volume 0.25mL/ days, the normal saline of blank group gavage every day same volume.
Table 2 shark the flesh of fish high F value oligopeptide on Mouse Blood lactic acid, hepatic glycogen, blood urea nitrogen content impact (n=15, meansigma methods �� SD)
| Group | Blood lactase acid | Hepatic glycogen | Blood urea nitrogen |
| Normal group | 6.54��1.06 | 3.23��0.29 | 9.27 �� 1.54 |
| Low dose group | 5.22��0.76 | 3.48��0.15 | 8.67 �� 0.71 |
| Middle dosage group | 4.38��0.95 | 3.69��0.24 | 8.31 �� 0.54 |
| High dose group | 3.53��0.01 | 4.75��0.23 | 7.66 �� 0.41 |
Note: gavage volume 0.25mL/ days, the normal saline of blank group gavage every day same volume.
Finally, in addition it is also necessary to be only the specific embodiment of the present invention it is noted that listed above. It is clear that the invention is not restricted to above example, it is also possible to there are many deformation. All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.
Claims (4)
1. the preparation method of a shark flesh of fish high F value oligopeptide, it is characterised in that comprise the following steps:
1) preparation of shark flesh of fish alcohol soluble protein:Take the alcoholic solution that the shark flesh of fish adds 90%��95% by 1g:15mL��1g:20mL solid-to-liquid ratio, be made into homogenate with high-speed tissue mashing machine, at 25��30 DEG C, power 650��700W supersound extraction 150min ~ 180min; The pH value 1mol/LNaOH of extracting solution being adjusted to 9.5��10.0, with distilled water, concentration of alcohol is adjusted to 55%��65%, room temperature stands 45min��60min, in the centrifugal 10min��15min of 8000��10000rpm; Take supernatant, and to add NaCl to concentration be 5%��8%, after standing 8h��10h, in the centrifugal 10��15min of 8000��10000rpm, collect precipitation, lyophilizing, obtain shark flesh of fish alcohol soluble protein;
2) enzymolysis of shark flesh of fish alcohol soluble protein:Take shark flesh of fish alcohol soluble protein, by solid-to-liquid ratio 1g:5mL��1g:10mL, add glycine sodium hydrate buffer solution, regulate pH to 9.0��1.0 with 0.1mol/LHCl or 0.1mol/LNaOH, obtain mixed liquor; Mixture temperature being modulated 55��65 DEG C, adds alkaline protease according to the 0.5%��1.0% of alcohol soluble protein quality, after enzymolysis time 3h��4h, enzymolysis solution is incubated 10��15min in 90��95 DEG C, and enzyme denaturing is lived; Enzymolysis solution temperature being adjusted to 50��60 DEG C, adds papain, enzymolysis time 4h��5h according to the 0.6%��0.8% of alcohol soluble protein quality, enzymolysis solution is down to room temperature, in the centrifugal 10��15min of 8000��10000rpm, takes supernatant;
3) enzymolysis of shark flesh of fish high F value oligopeptide:Above-mentioned supernatant is refined through ultrafiltration, activated carbon adsorption purification and macroporous resin successively, obtains shark flesh of fish high F value oligopeptide;
Wherein, described step 2) in glycine sodium hydrate buffer solution containing 0.05mol/L, pH value is 9.5; Alkaline protease enzyme activity is 1.95 �� 105��
2. preparation method according to claim 1, it is characterised in that the shark in described step 1) be preferably Mustelus griseus (pietschmann) (Mustelusgriseus).
3. preparation method according to claim 1, it is characterised in that the detailed process that the ultrafiltration of described step 3), activated carbon adsorption purification and macroporous resin are refined is:
Ultrafiltration: above-mentioned enzymolysis supernatant adopts 3kDa ultrafilter membrane carry out hyperfiltration treatment under the operating pressure of 0.1��0.15MPa and the operating temperature of 20��30 DEG C, collects molecular weight less than 3kDa part, obtains ultrafiltration enzymolysis solution;
Activated carbon adsorption purification system: adjust pH value to 2.5 ~ 3.5 ultrafiltration enzymolysis solution, it is that ultrafiltration enzymolysis solution is slowly added into activated carbon granule with 0.3��0.5mL/min flow velocity and is loaded in glass column by 10��15:1 according to material and activated carbon granule ratio, after completion of the sample, adopt 1mol/L ammonia and the dehydrated alcohol mixed solution elution chromatography post of the 1:1 ratio of 2��3 column volumes, collect eluent, dry, obtain the refining polypeptide of bioactive peptide
Macroporous resin is refined: refining for activated carbon polypeptide is made into the solution of 10��20mg/mL, adjust pH value to 6.5��7.5, be slowly added into material and AB-8 macroporous resin than be 10��15:1 glass column in, impurity is removed with the distilled water eluting of 2��3 times of column volumes, again with 95% ethanol elution of 2��3 column volumes, collect 95% ethanol elution thing, spray drying, be shark flesh of fish high F value oligopeptide.
4. preparation method according to claim 1, it is characterized in that the protein content of prepared high F value oligopeptide is 95.1��96.4%, total amino acid content is 83.7��85.4%, the content of branched-chain amino acid is 16.8��17.1%, the content of aromatic amino acid is 0.43��0.48%, and F value is 35.2��39.6, and molecular weight is 630��720Da, in branched-chain amino acid, Val:Ile:Leu is 1:1��1.5:3��4, and in aromatic amino acid, Trp:Tyr:Phe is 1:1��1.2:1��1.5.
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Cited By (3)
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| CN107348092A (en) * | 2017-07-11 | 2017-11-17 | 浙江丰宇海洋生物制品有限公司 | A kind of method using the molten slurry production of albumen powder product of fish |
| CN107494886A (en) * | 2017-07-11 | 2017-12-22 | 浙江丰宇海洋生物制品有限公司 | A kind of albumen powder product using the molten slurry production of fish |
| CN109078164A (en) * | 2018-08-22 | 2018-12-25 | 厦门肽王基因科技有限公司 | Application of the grey gummy shark fish-skin mixed type protein peptides as skeleton development promotor and micro element supplement agent |
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| CN107348092A (en) * | 2017-07-11 | 2017-11-17 | 浙江丰宇海洋生物制品有限公司 | A kind of method using the molten slurry production of albumen powder product of fish |
| CN107494886A (en) * | 2017-07-11 | 2017-12-22 | 浙江丰宇海洋生物制品有限公司 | A kind of albumen powder product using the molten slurry production of fish |
| CN109078164A (en) * | 2018-08-22 | 2018-12-25 | 厦门肽王基因科技有限公司 | Application of the grey gummy shark fish-skin mixed type protein peptides as skeleton development promotor and micro element supplement agent |
| CN109078164B (en) * | 2018-08-22 | 2022-04-01 | 厦门肽王基因科技有限公司 | Application of shark skin mixed protein peptide of Astrocaryum platypomum as skeletal development promoter and microelement supplement |
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