CN106520876A - Preparation method for Chinese forest frog antioxidative peptide - Google Patents
Preparation method for Chinese forest frog antioxidative peptide Download PDFInfo
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- CN106520876A CN106520876A CN201611021248.1A CN201611021248A CN106520876A CN 106520876 A CN106520876 A CN 106520876A CN 201611021248 A CN201611021248 A CN 201611021248A CN 106520876 A CN106520876 A CN 106520876A
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Abstract
The invention provides a preparation method for Chinese forest frog antioxidative peptide. The preparation method comprises the first step of pretreatment, the second step of degreasing, the third step of enzymolysis, the fourth step of enzyme deactivation and the fifth step of antioxidative peptide preparation. The preparation method has the beneficial effects that in the enzymolysis process, an enzymolytic agent comprises components including, by weight, 5-10 parts of papain, 12-20 parts of flavourzyme, 1-3 parts of magnesium sulfate, 2-5 parts of cysteine, 1-3 parts of monocalcium phosphate, 0.5-1.5 parts of vitamin A and 2-5 parts of sodium dodecyl sulfonate. As the enzymolytic agent is used for carrying out enzymolysis on Chinese forest frog protein, conditions are mild, and the polypeptide activity cannot be destroyed; and the enzymolysis efficiency is high, and the preparation time is shortened. The Chinese forest frog antioxidative peptide prepared through the method is high in extraction rate, purity, polypeptide and antioxidative performance and suitable for industrialized production, and preparation operation is easy.
Description
Technical field
The present invention relates to anti-oxidant titanium preparing technical field, specifically a kind of preparation method of wood frog's fallopian tube anti-oxidation peptide.
Background technology
Substantial amounts of research in recent years shows, the isolated polypeptide from various marine protein hydrolyzates, with latent
Oxidation resistance.Report is moved from oceans such as cuttlefish, oyster, blue mussel, long-tail cod, scad and cods both at home and abroad at present
Isolated antioxidation active peptides in thing.These oxidation resistant bioactive peptide of halobios can effectively in removing machine body free radical and
Active oxygen is damaged by blocking body of the free chain reaction during lipid peroxidation to prevent to cause because of oxidation
Wound.
The extracting method of oceanic biological active peptides mainly has 3 kinds at present：Solvent extraction, fermentable and enzyme hydrolysis.By
Do not have the residual of organic solvent and poisonous chemicals to become the prefered method of food and medicine industry in enzyme hydrolysis method.
Chinese invention patent of the prior art such as Authorization Notice No. for CN103540638B, is disclosed one kind and is added using stripped tuna
The method that work accessory substance prepares anti-oxidation peptide, its method is：Will be enriched in the accessory substances such as the section end to end of albumen and cooking liquor composition powder
Broken, then synchronized three phase separation, after obtaining the protein sources that directly can be digested, adds flavor protease, in pH=6.5,50 DEG C of bars
4h is digested under part, Jing ultrafiltration, charcoal absorption, desalination are purified enzymolysis liquid again, and it is spray-dried prepare Gly-His-Lys, detect which
Various antioxidation activities, the Gly-His-Lys obtained by above-mentioned technique are to DPPH free radicals, ultra-oxygen anion free radical and hydroxy radical
With good clearance rate, its IC50 is respectively 3.55mg/ml, 1.39mg/ml and 0.61mg/ml, is excellent anti-oxidant
Agent.The method prepare Gly-His-Lys for it is faint yellow, without bitter taste, micro- fishlike smell, molecular weight（>90.1%）≤ 10000Da is controllable to,
Polypeptide gross protein（In terms of butt）For 88.6%, ash content is 7.2%.Containing the impurity such as grease, antioxygen in Gly-His-Lys prepared by the method
Change peptide purity is undesirable, does not take the measure of protection polypeptide active, some polypeptide structure be destroyed, lose activity；
Authorization Notice No. is the Chinese invention patent of CN103497985B, discloses a kind of Walnut protein antioxidant peptides and preparation method thereof,
The method utilizes walnut cake protein, by enzymatic hydrolysis, obtains protein enzymatic hydrolyzate, recycles gel filtration chromatography and reversed phase high efficiency
Liquid chromatogram is carried out isolating and purifying to which and obtains 14 kinds of new anti-oxidation peptide products, and determines its amino acid sequence.The method
Obtained anti-oxidation peptide has very strong antioxidation, and oxygen radical absorbability may each be about 4 times of thick peptide, and peptide
With molecular weight it is little, can be directly absorbed by the body the characteristics of, therefore can be widely applied to food, health products and cosmetics etc. neck
Domain.The method is cumbersome, and preparation cost is high, is not suitable for factorial praluction.
The content of the invention
The present invention provides a kind of recovery rate height, and simple to operate, the wood frog's fallopian tube anti-oxidation peptide purity of extraction is high, and polypeptide active is strong,
The strong preparation method of non-oxidizability.
The present invention is by solving the problems, such as to propose in above-mentioned background technology that the technical scheme taken is：
1）Pretreatment：Wood frog's fallopian tube is cleaned with frozen water 3 ~ 5 times, will be the spot cleaning on silt and wood frog's fallopian tube surface clean, do in an oven
Dry, baking temperature is 70 ~ 90 DEG C, and drying time is 40 ~ 60min, then is crushed with pulverizer, crosses 200 ~ 300 mesh sieves.By wood frog's fallopian tube powder
It is broken into powder and increases specific surface area, degreasing and enzyme digestion reaction can be made more abundant, also can shorten the reaction time；
2）Degreasing：According to solid-to-liquid ratio 1 in wood frog's fallopian tube powder:2~1:5 add petroleum ethers, and petroleum ether in containing 2 ~ 4% acetone and
1 ~ 5% secondary octyl phenol polyethenoxy ether, skimming temp be 60 ~ 80 DEG C, stir speed (S.S.) be 300 ~ 400r/min, the reaction time be 20 ~
40min, obtains solid-liquid separating, carries out suction filtration with Buchner funnel, obtains the wood frog's fallopian tube powder after degreasing.If not carrying out degreasing
Words, the grease in wood frog's fallopian tube powder can be extracted together with anti-oxidant titanium, affect the purity of anti-oxidant titanium, reduce the anti-oxidant of product
Property；Petroleum ether only molten grease insoluble protein, adds acetone and secondary octyl phenol polyethenoxy ether improve wood frog's fallopian tube in petroleum ether
Solubility of the grease in powder in petroleum ether, so as to improve the degreasing rate of wood frog's fallopian tube powder；
3）Enzymolysis：Solid-to-liquid ratio 1 is pressed in the wood frog's fallopian tube powder after degreasing:3~1:5 add distilled water, be stirring evenly and then adding into enzymolysis agent and
Additive, heating water bath, hydrolysis temperature be 40 ~ 50 DEG C, pH be 6.0 ~ 7.1, stir speed (S.S.) be 300 ~ 400r/min, the reaction time
For 1.5 ~ 3h.The characteristics of having mildness, selectivity and high efficiency using enzyme hydrolysis wood frog's fallopian tube albumen, obtains after enzymolysis agent enzymolysis
Polypeptide, space structure will not be destroyed, with obvious non-oxidizability；Additive can protect the activity of the polypeptide of hydrolysis generation,
In avoiding the aqueous solution, micro heavy metal ion is damaged to its structure；
4）Go out enzyme：After enzymolysis terminates, boiling water bath is carried out to enzymolysis liquid and heats the enzyme that goes out, the enzyme time of going out is 10 ~ 15min, natural cooling
Enzymolysis reaction, filters；
5）It is prepared by anti-oxidation peptide：Product Jing after reverse high performance liquid chromatography obtains various wood frog's fallopian tube proteolysis, by removing
The experiment of DPPH free radicals, hydroxyl radical free radical, ABTS free radicals and ultra-oxygen anion free radical, picks out strong several of non-oxidizability
Polypeptide is planted, its molecular weight is determined.The milipore filter being adapted to is selected to carry out ultrafiltration according to the component size for determining.Vacuum and low temperature concentration filter
It is spray dried to polypeptide powder after liquid, liquid concentration temperature is 20 ~ 30 DEG C, vacuum is -5 ~ 5pa, the reaction time is 30 ~
50min.In the polypeptide powder for obtaining, anti-oxidation peptide content is high, and non-oxidizability is strong, free radical that can effectively in removing machine body and
Active oxygen is damaged by blocking body of the free chain reaction during lipid peroxidation to prevent to cause because of oxidation
Wound, high financial profit.
Enzymolysis agent composition and its weight portion are：5 ~ 10 parts of papains, 12 ~ 20 parts of flavor proteases, 1 ~ 3 part of sulfuric acid
Magnesium, 2 ~ 5 parts of cysteines, 1 ~ 3 part of calcium dihydrogen phosphate, 0.5 ~ 1.5 part of vitamin A, 2 ~ 5 parts of dodecyl sodium sulfates and and
0.001 ~ 0.05 part of cetostearyl alcohol isononoate.Above-mentioned enzymolysis agent digests wood frog's fallopian tube albumen mild condition, will not destroy polypeptide work
Property；Enzymolysis efficiency is high, shortens preparation time.
Additive component and its weight portion are 2 ~ 5 parts of APEOs, 1 ~ 3 part of polyacrylamide, 0.5 ~ 2 part of fatty alcohol sulphur
Sour sodium and 3 ~ 6 parts of sorbic acids.Above-mentioned additive can protect the activity of the polypeptide of enzyme hydrolysis generation, it is to avoid micro weight in the aqueous solution
Metal ion is damaged to its structure.
Compared with prior art, it is an advantage of the current invention that：Using petroleum ether as wood frog's fallopian tube powder degreasing agent, petroleum ether is only molten
Grease insoluble protein, adds acetone and secondary octyl phenol polyethenoxy ether improve the grease in wood frog's fallopian tube powder in stone in petroleum ether
Solubility in oily ether, so as to improve the degreasing rate of wood frog's fallopian tube powder；Enzymolysis agent composition and its weight portion are：5 ~ 10 parts of Papains
Enzyme, 12 ~ 20 parts of flavor proteases, 1 ~ 3 part of magnesium sulfate, 2 ~ 5 parts of cysteines, 1 ~ 3 part of calcium dihydrogen phosphate, 0.5 ~ 1.5 part of dimension life
Plain A and 2 ~ 5 part of dodecyl sodium sulfate, above-mentioned enzymolysis agent digest wood frog's fallopian tube albumen mild condition, will not destroy polypeptide active；Enzymolysis
Efficiency high, shortens preparation time；Additive component and its weight portion are 2 ~ 5 parts of APEOs, 1 ~ 3 part of polyacrylamide,
0.5 ~ 2 part of sodium alkyl sulfate and 3 ~ 6 parts of sorbic acids, above-mentioned additive can protect the activity of the polypeptide of enzyme hydrolysis generation, it is to avoid
In the aqueous solution, micro heavy metal ion is damaged to its structure.Wood frog's fallopian tube anti-oxidation peptide recovery rate prepared by said method is high,
Purity is high, and polypeptide active is strong, and non-oxidizability is strong, simple to operate, is adapted to factorial praluction.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of preparation method of wood frog's fallopian tube anti-oxidation peptide, concretely comprises the following steps：
1）Pretreatment：Wood frog's fallopian tube is cleaned with frozen water 3 ~ 5 times, will be the spot cleaning on silt and wood frog's fallopian tube surface clean, do in an oven
Dry, baking temperature is 70 ~ 90 DEG C, and drying time is 40 ~ 60min, then is crushed with pulverizer, crosses 200 ~ 300 mesh sieves.By wood frog's fallopian tube powder
It is broken into powder and increases specific surface area, degreasing and enzyme digestion reaction can be made more abundant, also can shorten the reaction time；
2）Degreasing：According to solid-to-liquid ratio 1 in wood frog's fallopian tube powder:2~1:5 add petroleum ethers, and petroleum ether in containing 2 ~ 4% acetone and
1 ~ 5% secondary octyl phenol polyethenoxy ether, skimming temp be 60 ~ 80 DEG C, stir speed (S.S.) be 300 ~ 400r/min, the reaction time be 20 ~
40min, obtains solid-liquid separating, carries out suction filtration with Buchner funnel, obtains the wood frog's fallopian tube powder after degreasing.If not carrying out degreasing
Words, the grease in wood frog's fallopian tube powder can be extracted together with anti-oxidant titanium, affect the purity of anti-oxidant titanium, reduce the anti-oxidant of product
Property；Petroleum ether only molten grease insoluble protein, adds acetone and secondary octyl phenol polyethenoxy ether improve wood frog's fallopian tube in petroleum ether
Solubility of the grease in powder in petroleum ether, so as to improve the degreasing rate of wood frog's fallopian tube powder；
3）Enzymolysis：Solid-to-liquid ratio 1 is pressed in the wood frog's fallopian tube powder after degreasing:3~1:5 add distilled water, are stirring evenly and then adding into enzymolysis agent
And additive, heating water bath, hydrolysis temperature are 40 ~ 50 DEG C, and pH is 6.0 ~ 7.1, and stir speed (S.S.) is 300 ~ 400r/min, during reaction
Between be 1.5 ~ 3h.The characteristics of having mildness, selectivity and high efficiency using enzyme hydrolysis wood frog's fallopian tube albumen, obtains after enzymolysis agent enzymolysis
Polypeptide, space structure will not be destroyed, with obvious non-oxidizability；Additive can protect the work of the polypeptide of hydrolysis generation
Property, it is to avoid in the aqueous solution, micro heavy metal ion is damaged to its structure；
4）Go out enzyme：After enzymolysis terminates, boiling water bath is carried out to enzymolysis liquid and heats the enzyme that goes out, the enzyme time of going out is 10 ~ 15min, natural cooling
Enzymolysis reaction, filters；
5）It is prepared by anti-oxidation peptide：Product Jing after reverse high performance liquid chromatography obtains various wood frog's fallopian tube proteolysis, by removing
The experiment of DPPH free radicals, hydroxyl radical free radical, ABTS free radicals and ultra-oxygen anion free radical, picks out strong several of non-oxidizability
Polypeptide is planted, its molecular weight is determined.Select the milipore filter being adapted to carry out ultrafiltration according to the component size for determining, filtrate is first used
The film ultrafiltration of 10000 ~ 15000Da, its permeate with the film ultrafiltration of 1000 ~ 2000Da, take stagnant liquid again.Vacuum and low temperature is concentrated
After be spray dried to polypeptide powder, liquid concentration temperature is 20 ~ 30 DEG C, and vacuum is -5 ~ 5pa, and the reaction time is 30 ~ 50min.
In the polypeptide powder for obtaining, anti-oxidation peptide content is high, and non-oxidizability is strong, free radical and active oxygen that can effectively in removing machine body
Or by the free chain reaction during blocking lipid peroxidation come the body injury for preventing to cause because of oxidation, economy
High efficiency.
Enzymolysis agent composition and its weight portion are：5 ~ 10 parts of papains, 12 ~ 20 parts of flavor proteases, 1 ~ 3 part of sulfuric acid
Magnesium, 2 ~ 5 parts of cysteines, 1 ~ 3 part of calcium dihydrogen phosphate, 0.5 ~ 1.5 part of vitamin A and 2 ~ 5 parts of dodecyl sodium sulfates.Above-mentioned enzyme
Solution agent enzymolysis wood frog's fallopian tube albumen mild condition, will not destroy polypeptide active；Enzymolysis efficiency is high, shortens preparation time.
Additive component and its weight portion are 2 ~ 5 parts of APEOs, 1 ~ 3 part of polyacrylamide, 0.5 ~ 2 part of fatty alcohol sulphur
Sour sodium and 3 ~ 6 parts of sorbic acids.Above-mentioned additive can protect the activity of the polypeptide of enzyme hydrolysis generation, it is to avoid micro weight in the aqueous solution
Metal ion is damaged to its structure.
Embodiment 2：
A kind of preparation method of wood frog's fallopian tube anti-oxidation peptide, most preferably step is：
1）Pretreatment：Wood frog's fallopian tube is cleaned with frozen water 4 times, will be the spot cleaning on silt and wood frog's fallopian tube surface clean, it is dried in an oven,
Baking temperature is 80 DEG C, and drying time is 50min, then is crushed with pulverizer, crosses 250 mesh sieves.Wood frog's fallopian tube is ground into into powder increase
Specific surface area, can make degreasing and enzyme digestion reaction more abundant, also can shorten the reaction time；
2）Degreasing：According to solid-to-liquid ratio 1 in wood frog's fallopian tube powder:Containing 3% acetone and 4% Zhong Xin in 4 addition petroleum ethers, and petroleum ether
Base phenol polyethenoxy ether, skimming temp are 80 DEG C, and stir speed (S.S.) is 300r/min, and the reaction time is 30min, obtains separation of solid and liquid
Liquid, carries out suction filtration with Buchner funnel, obtains the wood frog's fallopian tube powder after degreasing.Grease meeting if not carrying out degreasing, in wood frog's fallopian tube powder
Extracted with anti-oxidant titanium together, affect the purity of anti-oxidant titanium, reduce the non-oxidizability of product；Only molten grease is not or not petroleum ether
Molten protein, adds acetone and secondary octyl phenol polyethenoxy ether improve the grease in wood frog's fallopian tube powder in petroleum ether in petroleum ether
Solubility, so as to improve the degreasing rate of wood frog's fallopian tube powder；
3）Enzymolysis：Solid-to-liquid ratio 1 is pressed in the wood frog's fallopian tube powder after degreasing:3 add distilled water, are stirring evenly and then adding into enzymolysis agent and addition
Agent, heating water bath, hydrolysis temperature are 45 DEG C, and pH is 6.5, and stir speed (S.S.) is 300r/min, and the reaction time is 2h.Using enzyme hydrolysis
The characteristics of wood frog's fallopian tube albumen has mildness, selectivity and high efficiency, the polypeptide that obtains after enzymolysis agent enzymolysis, space structure will not be by
Destruction, with obvious non-oxidizability；Additive can protect the activity of the polypeptide of hydrolysis generation, it is to avoid micro weight in the aqueous solution
Metal ion is damaged to its structure；
4）Go out enzyme：After enzymolysis terminates, boiling water bath is carried out to enzymolysis liquid and heats the enzyme that goes out, the enzyme time of going out is 15min, natural cooling end
Only enzyme digestion reaction, filters；
5）It is prepared by anti-oxidation peptide：Product Jing after reverse high performance liquid chromatography obtains various wood frog's fallopian tube proteolysis, by removing
The experiment of DPPH free radicals, hydroxyl radical free radical, ABTS free radicals and ultra-oxygen anion free radical, picks out strong several of non-oxidizability
Polypeptide is planted, its molecular weight is determined.Select the milipore filter being adapted to carry out ultrafiltration according to the component size for determining, filtrate is first used
The film ultrafiltration of 13000Da, its permeate with the film ultrafiltration of 1200Da, take stagnant liquid again.It is spray-dried after vacuum and low temperature concentration
Into polypeptide powder, liquid concentration temperature is 26 DEG C, and vacuum is -2pa, and the reaction time is 40min.Resist in the polypeptide powder for obtaining
Oxidation peptide content is high, and non-oxidizability is strong, free radical and active oxygen that can effectively in removing machine body or by blocking lipid mistake
Free chain reaction in oxidizing process is come the body injury that prevents to cause because of oxidation, high financial profit.
Enzymolysis agent composition and its weight portion are：5 ~ 10 parts of papains, 12 ~ 20 parts of flavor proteases, 1 ~ 3 part of sulfuric acid
Magnesium, 2 ~ 5 parts of cysteines, 1 ~ 3 part of calcium dihydrogen phosphate, 0.5 ~ 1.5 part of vitamin A, 2 ~ 5 parts of dodecyl sodium sulfates and 0.001 ~
0.05 part of cetostearyl alcohol isononoate.Above-mentioned enzymolysis agent digests wood frog's fallopian tube albumen mild condition, will not destroy polypeptide active；Enzymolysis
Efficiency high, shortens preparation time, it may be possible to which effect of the cetostearyl alcohol isononoate to above-mentioned composition generates coupling, improves
The activity of end-product, its mechanism are still not clear.Additive component and its weight portion are 2 ~ 5 parts of APEOs, 1 ~ 3 part poly- third
Acrylamide, 0.5 ~ 2 part of sodium alkyl sulfate and 3 ~ 6 parts of sorbic acids.Above-mentioned additive can protect the work of the polypeptide of enzyme hydrolysis generation
Property, it is to avoid in the aqueous solution, micro heavy metal ion is damaged to its structure.
Embodiment of above is merely to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this area
Member, without departing from the spirit and scope of the present invention, can also make a variety of changes and modification.Therefore, all equivalents
Technical scheme fall within scope of the invention, the scope of patent protection of the present invention should be defined by the claims.
Claims (8)
1. a kind of preparation method of wood frog's fallopian tube anti-oxidation peptide, it is characterised in that following steps：
1）Pretreatment：Wood frog's fallopian tube is cleaned, be dry, pulverize；
2）Degreasing：Organic solvent is added in wood frog's fallopian tube powder, is stirred, filtered；
3）Enzymolysis：Distilled water is added in the wood frog's fallopian tube powder after degreasing, enzymolysis agent and additive, heating water bath is added after stirring, is stirred
Mix；
4）Go out enzyme：After enzymolysis terminates, boiling water bath is carried out to enzymolysis liquid and heats the enzyme that goes out, filtered；
5）It is prepared by anti-oxidation peptide：Product Jing after reverse high performance liquid chromatography obtains various wood frog's fallopian tube proteolysis, by removing certainly
Tested by base, pick out the strong several polypeptides of non-oxidizability, determine its molecular weight, select what is be adapted to according to the component size for determining
Milipore filter carries out ultrafiltration, is spray dried to polypeptide powder after vacuum and low temperature concentration filtrate.
2. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 2
Organic solvent is containing 2 ~ 4% acetone and 1 ~ 5% secondary octyl phenol polyethenoxy ether in petroleum ether, and petroleum ether.
3. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：Described step 2 takes off
Fat temperature is 60 ~ 80 DEG C, and stir speed (S.S.) is 300 ~ 400r/min, and the reaction time is 20 ~ 40min.
4. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 3
Enzymolysis agent composition and its weight portion are：5 ~ 10 parts of papains, 12 ~ 20 parts of flavor proteases, 1 ~ 3 part of magnesium sulfate, 2 ~ 5 part half
Cystine, 1 ~ 3 part of calcium dihydrogen phosphate, 0.5 ~ 1.5 part of vitamin A, 2 ~ 5 parts of dodecyl sodium sulfates and 0.001 ~ 0.05 part of spermaceti
Stearyl alcohol isononoate.
5. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 3
Additive component and its weight portion are 2 ~ 5 parts of APEOs, 1 ~ 3 part of polyacrylamide, 0.5 ~ 2 part of sodium alkyl sulfate and 3 ~
6 parts of sorbic acids.
6. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 3
Hydrolysis temperature is 40 ~ 50 DEG C, and pH is 6.0 ~ 7.1, and stir speed (S.S.) is 300 ~ 400r/min, and the reaction time is 20 ~ 40min.
7. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 4
Boiling water bath goes out 10 ~ 15min of enzyme.
8. the preparation method of a kind of wood frog's fallopian tube anti-oxidation peptide according to claim 1, it is characterised in that：In described step 5
Liquid concentration condition is：20 ~ 30 DEG C, vacuum is -5 ~ 5pa, and the reaction time is 30 ~ 50min.
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| CN110438185A (en) * | 2019-07-31 | 2019-11-12 | 杭州妇帮医疗科技有限公司 | A method of extracting the polypeptide moiety in wood frog ovum |
| CN111296849A (en) * | 2020-03-24 | 2020-06-19 | 中科花鹿农业发展有限公司 | Anti-aging bird nest snow clam deer blood collagen compound peptide beverage and preparation method thereof |
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