CN102669401A - Production method of fish protein hydrolyzed chelate - Google Patents
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Abstract
The invention discloses a production method of fish protein hydrolyzed chelate. The production method includes: preprocessing residual waste of low-value marine fishes such as ribbonfish, adding complex enzymes to hydrolyze the waste, and subjecting polypeptides to chelate modification. The fish protein hydrolyzed chelate has effects of antibiosis, antioxidation and ferric strengthening. The fish protein hydrolyzed chelate is purely natural, has good antioxidation, and has evident antibacterial effects on escherichia coli, bacillus subtilis, staphylococcus aureus and salmonella. Absolute ethyl alcohol can be recycled in production. Production cost is low. High-value development and high utilization of aquatic products are achieved.
Description
Technical field
The present invention relates to a kind of preparation method of hydrolysis chelate, relate in particular to a kind of preparation method of fish proteolysis chelate.
Background technology
Micromolecule polypeptide in the protein hydrolysate can directly be assimilated by enteron aisle, and can be used as the enteral nutrition agent or offering with the spoon meat form needs the crowd, so protein hydrolysate is widely used in medicine, health food.Therefore, also always be the important topic of nutrient research.The product that protein obtains through method hydrolysis such as enzymatics is called protein hydrolysate or protolysate (Protein hydrolysate).
Previous period, the developmental research to protolysate mainly rested on kinds such as lactoprotein, soybean protein, albumen, and is wherein deep with the research and development of milk protein hydrolysate and active peptide.In recent years, about the product research of animal derived protein hydrolysate and active peptide and exploitation come into one's own day by day, report is constantly arranged like aspect articles such as poultry blood, aquatic product proteins.Because the Chinese Marine Biology Resources reserves are very abundant; Wherein marine protein resource kind is especially various; Because marine environment is different from terrestrial environment; For adapting to some extreme living environment, constituting halobiontic albumen is still all having a great difference with the terrestrial organism source protein on the amino acid composition on sequence of amino acid.Thereby march to the ocean, ask for marine drug food, functional protein and sp act material, become an important content of each coastal state's ocean development of the world.
For a long time, hairtail (output is more than 1,000,000 tons) since low, process technology level of the market price and recovering means fall behind, wherein greatly by directly chopping; The breeding bait fish meal that just is used for simple processing; Cause the value of the product utilization rate low, economic benefit is not high, phenomenons such as serious waste of resources; Simultaneously because a large amount of nutrient loss such as good protein, amino acid, active mineral matter have more caused the pollution of environment.In recent years, the scarcity of the stock of fish more and more causes people's attention, and a large amount of fish scraps is abandoned because of poor processability, has wasted a large amount of protein sources.Simultaneously marine fishes protein results from special geographical environment, and amino acid contained ratio and people's muscle composition is very approaching again, and its grade is high for the absorption of human body utilization, thereby develops fish protein hydrolyzate and separator has caused people's extensive interest.Therefore, must carry out the particularly intensive processing of hairtail of the stock of fish, seek new approach for improving its value and economic worth.
Chinese patent notification number CN 1883289A; Open day on December 27th, 2006; Denomination of invention is the preparation method of active polypeptide solution of fresh water fish protein; This application case discloses a kind of preparation method of active polypeptide solution of fresh water fish protein, is hydrolyzed through interpolation animal protein complex enzyme, food flavor enzyme, adds five technology for removing offensive smell flow processs such as saccharomycete and lactobacillus-fermented; Both can thoroughly dispel fishy smell, the bitter taste of hydrolyzate, and make this hydrolyzate possess peat-reek and mouthfeel that common fish albumen hydrolysis solution does not have again.Its weak point does, is applicable to fresh-water fishes, system the hydrolyzate non-oxidizability, antibiotic low.
Summary of the invention
The present invention is few for the utilization of seawater fish offal material in order to solve in the prior art, the hydrolyzate non-oxidizability, and antibiotic low defective is to provide a kind of preparation method who has the fish proteolysis chelate of non-oxidizability, antibiotic and iron invigoration effect simultaneously.
The present invention mainly is able to solve the problems of the technologies described above through following technical proposals:
A kind of preparation method of fish proteolysis chelate, described preparation method's step is following:
A) raw material is handled: the hairtail leftover bits and pieces, and decaptitate, truncate, bone, it is subsequent use to do to leave and take the flesh of fish;
B) pre-treatment: with clear water the flesh of fish of step a gained is cleaned, drained away the water, homogenate under the room temperature condition of chopping back, chilled storage;
C) complex enzyme for hydrolyzing: step b gained flesh of fish slurry is pressed 2-5 times of weight ratio add deionized water, adjusting pH is 6-7, adds complex enzyme; Temperature 40-50 ℃ of hydrolysis, the percentage by weight of complex enzyme is the 0.2%-0.8% of flesh of fish slurry, hydrolysis 1.5-6 hour; Hydrolysis finishes the back and in boiling water, keeps the 8-15min enzyme that goes out, cooling back suction filtration, centrifugal filtration again; Last ultrafiltration membrance filter hydrolyzate is removed the hydrolysate that molecular weight is lower than 10kDa, collects the ultrafiltration hydrolyzate and under 4 ℃ of conditions, stores;
D) polypeptide chelate thing preparation: with step c gained ultrafiltration hydrolyzate adjustment pH7-8.3; In 40-50 part ultrafiltration hydrolyzate, slowly add 1 part of FeCl2 solution that mass fraction is 1%-3% by weight, at temperature 20-30 ℃, chelatropic reaction 20-30 minute; After chelatropic reaction finishes; Chelate is filtered, obtain elementary chelating sediment and elementary filtrating, afterwards elementary filtrating is carried out absolute ethyl alcohol stepwise gradient deposition and obtain secondary chelating sediment; The chelating sediment of above absolute ethyl alcohol stepwise gradient deposition gained is merged 40-60 ℃ of dry 3-5h down with the elementary chelating sediment that precipitates for the first time gained, obtain fish proteolysis chelate.
As preferably, said complex enzyme by food flavor enzyme, papain, neutral proteinase is two or more forms, the enzyme activity of food flavor enzyme is 400,000 U/ grams, the enzyme activity of papain is 2,000,000 U/ grams, the enzyme activity of neutral proteinase is 100,000 U/ grams.
As preferably, food flavor enzyme, papain enzyme, neutral proteinase weight ratio are 1:3:2 in the said complex enzyme.
As preferably, the used instrument rotating speed of homogenate is 2000-3000r/min among the step b.
As preferably, absolute ethyl alcohol stepwise gradient deposition is divided into 3 grades in the said steps d, and the mass percentage concentration of ethanol is respectively 60%, 80%, 95%.
This preparation technology is:
Raw material → pre-treatment → enzymolysis → adding metal → stirring → adjusting pH → concussion in proportion synthesizes → centrifugation → absolute ethanol washing → fractional precipitation → suction filtration → forced air drying → polypeptide-Fe
2+The chelate finished product.
Wherein, suction filtration is in order to remove broken fish-bone and fishbone in the hydrolysate among the step c, and centrifugal filtration is in order to remove the fish oil and the unhydrolysed flesh of fish.
Steps d is with step c gained ultrafiltration hydrolyzate adjustment pH7-8.3, by weight in 45 parts of ultrafiltration hydrolyzates, slowly adding 1 part of FeCl2 solution that mass fraction is 1%-4%; At temperature 20-30 ℃, carried out chelatropic reaction in 20-30 minute, after chelatropic reaction finishes; Chelate is filtered; Obtain elementary chelating sediment A and elementary filtrating, afterwards elementary filtrating is carried out the alcohol grading gradient and sink to the bottom, at room temperature add absolute ethyl alcohol; Make its mass percentage concentration reach 60%, centrifugal 10 min of post precipitation obtain 2 grades of chelate precipitate B and secondary filtrating; In secondary filtrating, continue to add absolute ethyl alcohol, make its mass percentage concentration reach 80%, centrifugal 10 min obtain 3 grades of chelate precipitate C and three grades of filtratings; In three grades of filter liquors, continue at last to add absolute ethyl alcohol, make its mass percentage concentration reach more than 95%, leave standstill 20 min under the room temperature, produce suspension and deposition, this system is rotated the evaporation decompression concentrates, can get 4 grades of chelate deposit D; After above 1-4 grades of chelates were deposited under the vacuum condition (0.1 MPa) 45 ℃ of oven dry 4h, it was subsequent use to place silica gel drier to preserve.
Wherein, food flavor enzyme is an excision enzyme, has the effect of eliminating bitter taste, ability caseinhydrolysate, elastin laminin, collagen, mucinoid and mucoprotein etc.; Papain is a protein incision enzyme, and hydrolysis ability is strong, long action time, and price is low; Neutral proteinase is got through fermented extracted by bacillus subtilis; Belong to a kind of restriction endonuclease, can be used for the range protein hydrolysis process, under uniform temperature, pH value; Can macro-molecular protein be hydrolyzed to products such as amino acid; Can be widely used in the hydrolysis of animal/vegetable protein, moderate cost, degree of hydrolysis is high.
Wherein, measure each composition of raw material according to standard method:
Moisture: normal pressure direct drying method
Total ash: total ash assay method
Crude fat: Suo Shi extraction process
Crude protein: micro-Kjeldahl
Nonprotein nitrogen: micro-Kjeldahl
Total reducing sugar: anthrone colorimetric method
Iron content: the luxuriant and rich with fragrance method of adjacent two nitrogen
Calcium content: EDTA titration
AAN: potentiometric titration.
Anti-oxidant, the antibacterial activity of ferrous ion polypeptide chelate thing are measured:
The anti-oxidant mensuration of polypeptide chelate thing---TBA method:
With vitamin C, tocopherol is contrast, and the ferrous ion chelate has good oxidation resistance, and its antioxidation is suitable with it;
The antibacterial activity of polypeptide chelate thing is measured---Oxford cup double-layer plate method:
The ferrous ion chelate is to Escherichia coli, bacillus subtilis, and staphylococcus aureus, Pseudomonas aeruginosa experimentizes, and structure shows has better anti-bacterial effect to gram-positive bacteria.
The invention has the beneficial effects as follows:
(1) be raw material with marine low-value fish tape fish processing back leftover bits and pieces; Preparation has the fish proteolysis chelate of antibiotic, anti-oxidant and iron invigoration effect simultaneously; Absolute ethyl alcohol in the production process can be recycled, and production cost is low, can realize the high-valued exploitation of aquatic products and highly utilize;
(2) production Technology is not high to equipment requirements, and equipment investment is few, and general aquatic products processing enterprise all possesses the condition and the ability of exploitation;
(3) product has the characteristics of pure natural, has good oxidation resistance, to Escherichia coli, and bacillus subtilis, staphylococcus aureus, salmonella have significant antibacterial effect.
Description of drawings
Fig. 1 is an antioxidation activity sketch map of the present invention.
Among the figure, A: one-level chelate deposition; B: secondary chelate deposition; C: three grades of chelate depositions; D: level Four chelate deposition; E:Vit.E.
The specific embodiment
Embodiment one:
A) raw material is handled: the hairtail leftover bits and pieces, and decaptitate, truncate, bone, it is subsequent use to do to leave and take the flesh of fish;
B) pre-treatment: with clear water the flesh of fish of step a gained is cleaned, drained away the water, homogenate under the room temperature condition of chopping back, chilled storage;
C) complex enzyme for hydrolyzing: step b gained flesh of fish slurry is added deionized water by 3 times of weight ratios, and regulating pH is 6.8, adds complex enzyme; 45 ℃ of hydrolysis of temperature, the weight of complex enzyme is 0.5% of flesh of fish slurry, complex enzyme is made up of food flavor enzyme and papain; The weight ratio of food flavor enzyme and papain is 1:3, and hydrolysis time is 4 hours, and hydrolysis finishes the back and in boiling water, keeps the 10 min enzyme that goes out; Cooling back suction filtration; Centrifugal filtration again, last ultrafiltration membrance filter is removed the hydrolysate that molecular weight is lower than 10kDa, collects the ultrafiltration hydrolyzate and under 4 ℃ of conditions, stores;
D) polypeptide chelate thing preparation:, be 1% FeCl2 solution by weight in 45 parts of ultrafiltration hydrolyzates, slowly adding 1 part of mass fraction, 24 ℃ of temperature with step c gained ultrafiltration hydrolyzate adjustment pH7.3; Chelatropic reaction 30 minutes; Chelatropic reaction filters chelate after finishing, and obtains elementary chelating sediment A and elementary filtrating; Afterwards elementary filtrating being carried out the absolute ethyl alcohol stepwise gradient sinks to the bottom; At room temperature add absolute ethyl alcohol, make its mass percentage concentration reach 60%, centrifugal 10 min of post precipitation obtain 2 grades of chelate precipitate B and secondary filtrating; Add absolute ethyl alcohol to the continuation of secondary filtrating, make its mass percentage concentration reach 80%, centrifugal 10 min obtain 3 grades of chelate precipitate C and three grades of filtratings; In three grades of filter liquors, continue at last to add absolute ethyl alcohol, make its concentration reach more than 95%, leave standstill 20 min under the room temperature, produce suspension and deposition, this system is rotated the evaporation decompression concentrates, can get 4 grades of chelate deposit D; After above 1-4 grades of chelates were deposited under the vacuum condition (0.1 MPa) 45 ℃ of oven dry 4h, it was subsequent use to place silica gel drier to preserve.
Embodiment two:
A) raw material is handled: the hairtail leftover bits and pieces, and decaptitate, truncate, bone, it is subsequent use to do to leave and take the flesh of fish;
B) pre-treatment: with clear water the flesh of fish of step a gained is cleaned, drained away the water, homogenate under the room temperature condition of chopping back, chilled storage;
C) complex enzyme for hydrolyzing: step b gained flesh of fish slurry is added deionized water by 2 times of weight ratios, and regulating pH is 6.0, adds complex enzyme; 40 ℃ of hydrolysis of temperature, the weight of complex enzyme is 0.2% of flesh of fish slurry, complex enzyme is made up of food flavor enzyme, papain and neutral proteinase; The weight ratio of food flavor enzyme, papain, neutral proteinase is 1:3:2, and hydrolysis time is 1.5 hours, and hydrolysis finishes the back and in boiling water, keeps the 8 min enzyme that goes out; Cooling back suction filtration; Centrifugal filtration again, last ultrafiltration membrance filter is removed the hydrolysate that molecular weight is lower than 10kDa, collects the ultrafiltration hydrolyzate and under 4 ℃ of conditions, stores;
D) polypeptide chelate thing preparation:, be 2% FeCl2 solution by weight in 40 parts of ultrafiltration hydrolyzates, slowly adding 1 part of mass fraction, 20 ℃ of temperature with step c gained ultrafiltration hydrolyzate adjustment pH7.0; Chelatropic reaction 20 minutes; Chelatropic reaction filters chelate after finishing, and obtains elementary chelating sediment A and elementary filtrating; Afterwards elementary filtrating being carried out the absolute ethyl alcohol stepwise gradient sinks to the bottom; At room temperature add absolute ethyl alcohol, make its mass percentage concentration reach 60%, centrifugal 10 min of post precipitation obtain 2 grades of chelate precipitate B and secondary filtrating; Add absolute ethyl alcohol to the continuation of secondary filtrating, make its mass percentage concentration reach 80%, centrifugal 10 min obtain 3 grades of chelate precipitate C and three grades of filtratings; In three grades of filter liquors, continue at last to add absolute ethyl alcohol, make its concentration reach more than 95%, leave standstill 20 min under the room temperature, produce suspension and deposition, this system is rotated the evaporation decompression concentrates, can get 4 grades of chelate deposit D; After above 1-4 grades of chelates were deposited under the vacuum condition (0.1 MPa) 40 ℃ of oven dry 3h, it was subsequent use to place silica gel drier to preserve.
Embodiment three
A) raw material is handled: the hairtail leftover bits and pieces, and decaptitate, truncate, bone, it is subsequent use to do to leave and take the flesh of fish;
B) pre-treatment: with clear water the flesh of fish of step a gained is cleaned, drained away the water, homogenate under the room temperature condition of chopping back, chilled storage;
C) complex enzyme for hydrolyzing: step b gained flesh of fish slurry is added deionized water by 5 times of weight ratios, and regulating pH is 7.0, adds complex enzyme; 50 ℃ of hydrolysis of temperature, the weight of complex enzyme is 0.8% of flesh of fish slurry, complex enzyme is made up of papain and neutral proteinase; The weight ratio of papain, neutral proteinase is 3:2, and hydrolysis time is 6 hours, and hydrolysis finishes the back and in boiling water, keeps the 15 min enzyme that goes out; Cooling back suction filtration; Centrifugal filtration again, last ultrafiltration membrance filter is removed the hydrolysate that molecular weight is lower than 10kDa, collects the ultrafiltration hydrolyzate and under 4 ℃ of conditions, stores;
D) polypeptide chelate thing preparation:, be 3% FeCl2 solution by weight in 50 parts of ultrafiltration hydrolyzates, slowly adding 1 part of mass fraction, 30 ℃ of temperature with step c gained ultrafiltration hydrolyzate adjustment pH8.3; Chelatropic reaction 25 minutes; Chelatropic reaction filters chelate after finishing, and obtains elementary chelating sediment A and elementary filtrating; Afterwards elementary filtrating being carried out the absolute ethyl alcohol stepwise gradient sinks to the bottom; At room temperature add absolute ethyl alcohol, make its mass percentage concentration reach 60%, centrifugal 10 min of post precipitation obtain 2 grades of chelate precipitate B and secondary filtrating; Add absolute ethyl alcohol to the continuation of secondary filtrating, make its mass percentage concentration reach 80%, centrifugal 10 min obtain 3 grades of chelate precipitate C and three grades of filtratings; In three grades of filter liquors, continue at last to add absolute ethyl alcohol, make its concentration reach more than 95%, leave standstill 20 min under the room temperature, produce suspension and deposition, this system is rotated the evaporation decompression concentrates, can get 4 grades of chelate deposit D; After above 1-4 grades of chelates were deposited under the vacuum condition (0.1 MPa) 50 ℃ of oven dry 5h, it was subsequent use to place silica gel drier to preserve.
Embodiment four, through the product of the foregoing description system, measure its non-oxidizability, and adopting the TBA method is the thiobarbituricacid method, and its antioxygenic property effect is seen Fig. 1:
The antioxidation activity of B is the highest, 92% of its oxidation activity Vit.E antioxidation activity.A and B hydrophobicity are stronger, and reason is that they are rich in histidine, and their chelate ring strengthened the oxidation resistance of histidine imidazole ring, so A and B can be used as natural.Compare with B with A; C and D have hydrophily preferably; Solvable in water environment; These prompting C and D possibly mainly be made up of hydrophilic amino acid such as aspartic acid, glutamic acid, amion acetic acid, alanine, threonine and serine etc., only contain the small number of groups propylhomoserin, so C and D can be used as hydrophilic natural.
Through the product of the foregoing description, measure its antibiotic property, adopt Oxford cup double-layer plate method, its effect is following:
A does not have any antibacterial activity to Escherichia coli, staphylococcus aureus, salmonella and Bacillus subtillis; And B has more weak antibacterial activity to staphylococcus aureus; C also has faint antibacterial activity to above-mentioned 4 kinds of bacteriums; For the highest, this activity depends on the hydrophily of chelating component to D, so in food industry, D can be used as hydrophilic natural antibacterial agent to the antibacterial activities of 4 kinds of bacteriums.
Claims (5)
1. the preparation method of a fish proteolysis chelate is characterized in that, described preparation method's step is following:
A) raw material is handled: the hairtail leftover bits and pieces, and decaptitate, truncate, bone, it is subsequent use to do to leave and take the flesh of fish;
B) pre-treatment: with clear water the flesh of fish of step a gained is cleaned, drained away the water, homogenate under the room temperature condition of chopping back, chilled storage;
C) complex enzyme for hydrolyzing: step b gained flesh of fish slurry is pressed 2-5 times of weight ratio add deionized water, adjusting pH is 6-7, adds complex enzyme; Temperature 40-50 ℃ of hydrolysis, the percentage by weight of complex enzyme is the 0.2%-0.8% of flesh of fish slurry, hydrolysis 1.5-6 hour; Hydrolysis finishes the back and in boiling water, keeps the 8-15min enzyme that goes out, cooling back suction filtration, centrifugal filtration again; Last ultrafiltration membrance filter hydrolyzate is removed the hydrolysate that molecular weight is lower than 10kDa, collects the ultrafiltration hydrolyzate and under 4 ℃ of conditions, stores;
D) polypeptide chelate thing preparation: with step c gained ultrafiltration hydrolyzate adjustment pH7-8.3; In 40-50 part ultrafiltration hydrolyzate, slowly add 1 part of FeCl2 solution that mass fraction is 1%-3% by weight, at temperature 20-30 ℃, chelatropic reaction 20-30 minute; After chelatropic reaction finishes; Chelate is filtered, obtain elementary chelating sediment and elementary filtrating, afterwards elementary filtrating is carried out absolute ethyl alcohol stepwise gradient deposition and obtain secondary chelating sediment; The chelating sediment of above absolute ethyl alcohol stepwise gradient deposition gained is merged 40-60 ℃ of dry 3-5h down with the elementary chelating sediment that precipitates for the first time gained, obtain fish proteolysis chelate.
2. the preparation method of a kind of fish proteolysis chelate according to claim 1; It is characterized in that; Said complex enzyme by food flavor enzyme, papain, neutral proteinase is two or more forms; The enzyme activity of food flavor enzyme is 400,000 U/ grams, and the enzyme activity of papain is 2,000,000 U/ grams, and the enzyme activity of neutral proteinase is 100,000 U/ grams.
3. the preparation method of a kind of fish proteolysis chelate according to claim 2 is characterized in that, food flavor enzyme, papain, neutral proteinase weight ratio are 1:3:2 in the said complex enzyme.
4. the preparation method of a kind of fish proteolysis chelate according to claim 1 and 2 is characterized in that, the used instrument rotating speed of homogenate is 2000-3000r/min among the step b.
5. according to the preparation method of claim 1 or 2 or 3 described a kind of fish proteolysis chelates, it is characterized in that absolute ethyl alcohol stepwise gradient deposition is divided into 3 grades in the said steps d, the mass percentage concentration of ethanol is respectively 60%, 80%, 95%.
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| CN105601706B (en) * | 2015-12-11 | 2020-11-27 | 浙江海洋学院 | Antibacterial hairtail tetrapeptide and preparation method thereof |
| CN105876809A (en) * | 2016-04-18 | 2016-08-24 | 厦门三九生物科技有限公司 | Marine polypeptide-selenium chelate preparation method and application |
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| CN107893097A (en) * | 2017-12-28 | 2018-04-10 | 中国海洋大学 | A kind of cod row protein peptides ferrous chelate compound and preparation method thereof |
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Application publication date: 20120919 |