CN114176114A - Preparation method of shrimp meat antifreeze agent - Google Patents
Preparation method of shrimp meat antifreeze agent Download PDFInfo
- Publication number
- CN114176114A CN114176114A CN202111241862.XA CN202111241862A CN114176114A CN 114176114 A CN114176114 A CN 114176114A CN 202111241862 A CN202111241862 A CN 202111241862A CN 114176114 A CN114176114 A CN 114176114A
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- Prior art keywords
- enzymolysis
- freeze
- shrimp meat
- preparation
- antifreeze agent
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- 241000238557 Decapoda Species 0.000 title claims abstract description 89
- 235000013372 meat Nutrition 0.000 title claims abstract description 75
- 239000007798 antifreeze agent Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 62
- 241000251468 Actinopterygii Species 0.000 claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 claims abstract description 43
- 108010010803 Gelatin Proteins 0.000 claims abstract description 41
- 239000008273 gelatin Substances 0.000 claims abstract description 41
- 229920000159 gelatin Polymers 0.000 claims abstract description 41
- 235000019322 gelatine Nutrition 0.000 claims abstract description 41
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 41
- 239000000047 product Substances 0.000 claims abstract description 37
- 230000026731 phosphorylation Effects 0.000 claims abstract description 26
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 26
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 19
- 102000004142 Trypsin Human genes 0.000 claims abstract description 16
- 108090000631 Trypsin Proteins 0.000 claims abstract description 16
- 239000012588 trypsin Substances 0.000 claims abstract description 16
- 108010007119 flavourzyme Proteins 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 15
- 238000005374 membrane filtration Methods 0.000 claims abstract description 14
- 238000004108 freeze drying Methods 0.000 claims abstract description 13
- 239000012528 membrane Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 10
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims abstract description 8
- 235000011180 diphosphates Nutrition 0.000 claims abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 8
- 238000006011 modification reaction Methods 0.000 claims abstract description 7
- 230000000415 inactivating effect Effects 0.000 claims abstract description 6
- 230000004048 modification Effects 0.000 claims description 18
- 238000012986 modification Methods 0.000 claims description 18
- 239000000758 substrate Substances 0.000 claims description 15
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 9
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 9
- 238000002791 soaking Methods 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 4
- 230000009849 deactivation Effects 0.000 claims description 4
- 230000002255 enzymatic effect Effects 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 210000004347 intestinal mucosa Anatomy 0.000 claims description 3
- 238000009461 vacuum packaging Methods 0.000 claims description 3
- 239000000796 flavoring agent Substances 0.000 claims description 2
- 235000019634 flavors Nutrition 0.000 claims description 2
- 241000143060 Americamysis bahia Species 0.000 claims 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 230000002528 anti-freeze Effects 0.000 abstract description 19
- 238000007710 freezing Methods 0.000 abstract description 14
- 230000008014 freezing Effects 0.000 abstract description 13
- 238000003860 storage Methods 0.000 abstract description 5
- 238000005057 refrigeration Methods 0.000 abstract description 4
- 235000013305 food Nutrition 0.000 abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 66
- 239000000243 solution Substances 0.000 description 44
- 229940088598 enzyme Drugs 0.000 description 36
- 230000000052 comparative effect Effects 0.000 description 27
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 23
- 238000010438 heat treatment Methods 0.000 description 22
- 238000005119 centrifugation Methods 0.000 description 13
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 13
- 229940048086 sodium pyrophosphate Drugs 0.000 description 13
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 13
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 13
- 238000005303 weighing Methods 0.000 description 10
- 239000007864 aqueous solution Substances 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000013622 meat product Nutrition 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229940048084 pyrophosphate Drugs 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000003507 refrigerant Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 108010053481 Antifreeze Proteins Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- -1 compound phosphate Chemical class 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000533950 Leucojum Species 0.000 description 1
- 241000238553 Litopenaeus vannamei Species 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 235000019606 astringent taste Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 210000002468 fat body Anatomy 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000019692 hotdogs Nutrition 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/06—Freezing; Subsequent thawing; Cooling
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/24—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/40—Shell-fish
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Water Supply & Treatment (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Marine Sciences & Fisheries (AREA)
- Nutrition Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
The invention belongs to the technical field of food processing, and relates to a preparation method of a shrimp meat antifreeze agent, which comprises the following steps: s1, preparing a fish gelatin solution, and adjusting the pH value to 5.5-6.5; s2, adding flavourzyme into the fish gelatin solution for reaction, then inactivating enzyme, centrifuging, collecting supernatant and freeze-drying; s3, dissolving the freeze-dried product obtained by the first step of enzymolysis in water, adjusting the pH value to 7.5-8.5, adding trypsin for reaction, then inactivating enzyme, centrifuging, collecting supernatant, and freeze-drying; s4, dissolving the freeze-dried product obtained by the second step of enzymolysis in water, adjusting the pH value to 8.0-10.0, and adding pyrophosphate for reaction; s5, ultrafiltration membrane filtration: and separating and purifying the solution obtained by phosphorylation modification reaction by adopting an ultrafiltration membrane, collecting filtrate, and freeze-drying to obtain the shrimp meat antifreeze agent. The shrimp meat antifreeze agent disclosed by the invention is applied to shrimp meat, and can improve the refrigeration property of the shrimp meat, so that the shrimp meat achieves a better antifreeze effect in a low-temperature freezing storage process.
Description
Technical Field
The invention belongs to the technical field of food processing, and relates to a preparation method of an antifreeze agent for shrimp meat and application of the antifreeze agent in freezing of the shrimp meat.
Background
The penaeus vannamei boone is one of aquaculture shrimps with important commercial value, and is well received in domestic and foreign markets due to high nutritional value, thin shell, fat body and fresh and tender meat quality. However, fresh shrimp are susceptible to spoilage by biochemical, microbial, or physical changes during storage due to high water content, high free acid and protease activity. At present, freezing is one of the important methods for preserving shrimp meat. It is reported that freezing preservation at low temperature (-23 deg.C to-12 deg.C) can freeze more than 90% of water in shrimp meat, inhibit enzyme activity and microbial growth, and prolong shelf life. However, the quality of the shrimp meat can be reduced due to solute concentration and ice crystal formation in the freezing and freezing process of the shrimp meat; and the freeze denaturation can occur in the refrigeration process, so that the water retention property, the elasticity and the gel strength of the beverage are reduced, the juice is lost, and the nutritional value of the beverage is reduced.
In general, in order to delay the freezing denaturation of the protein of the frozen shrimp meat, an antifreeze such as saccharides, proteins, complex phosphates, amino acids, and the like needs to be added to the frozen shrimp meat. The compound phosphate is a good antifreeze, which can effectively reduce the loss of water and nutrient components of the shrimp meat in the processing, transportation and storage processes, maintain the tenderness of the product and improve the yield, however, the excessive addition of the compound phosphate in the shrimp meat can cause the shrimp product to generate unpleasant metallic astringency, rough tissue and the phenomena of snowflake and crystallization on the surface caused by the precipitation of the phosphate in the storage process; on the other hand, long-term consumption of foods containing high phosphate content can reduce the absorption of calcium by human body, and cause growth retardation, skeletal deformity, and poor quality of bones and teeth, thereby harming body health. Saccharides (mainly sucrose and sorbitol) are widely used in aquatic products as antifreeze agents, and the action principle is that the saccharides are mutually wound under the action of heat energy to form a three-dimensional network and are mutually combined with salt-soluble proteins in muscles to form a good network structure, but the saccharides antifreeze agents have the defects of higher sweetness and higher heat. The antifreeze peptide can reduce the formation of ice crystals and the damage to a network structure when shrimp meat is frozen, however, the preparation method of the antifreeze peptide is generally complex, for example, patent CN201310441149.9 discloses a fish-derived antifreeze polypeptide and a preparation method thereof, wherein fish skin collagen is used as a raw material, papain is used for enzymolysis, and a complex separation and purification is carried out to obtain the specific antifreeze polypeptide.
Disclosure of Invention
Aiming at the defects of the antifreeze agent in the prior art, the invention provides a preparation method of the antifreeze agent for shrimp meat, which prepares phosphorylated fish gelatin polypeptide by two-step enzymolysis, phosphorylation modification and ultrafiltration membrane filtration of fish gelatin, and applies the phosphorylated fish gelatin polypeptide to shrimp meat to improve the refrigeration characteristic of the shrimp meat, so that the shrimp meat achieves better antifreeze effect in the low-temperature freezing process.
One purpose of the invention is realized by the following technical scheme:
a preparation method of a shrimp meat antifreeze agent comprises the following steps:
s1, preparing a fish gelatin solution, and adjusting the pH value to 5.5-6.5;
s2, first-step enzymolysis: adding flavourzyme into the fish gelatin solution for enzymolysis reaction, then inactivating enzyme, centrifuging, collecting supernatant and freeze-drying;
s3, carrying out enzymolysis in the second step: dissolving the freeze-dried product obtained by the first-step enzymolysis in water, adjusting the pH value to 7.5-8.5, adding trypsin for enzymolysis reaction, then inactivating enzyme, centrifuging, collecting supernatant and freeze-drying;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to form a freeze-dried product solution, adjusting the pH to 8.0-10.0, and adding pyrophosphate to react;
s5, ultrafiltration membrane filtration: and separating and purifying the solution obtained by phosphorylation modification reaction by adopting an ultrafiltration membrane, collecting filtrate, and freeze-drying to obtain the shrimp meat antifreeze agent.
Preferably, a fish gelatin solution is prepared: adding fish gelatin into water, heating and dissolving in a water bath at 35-45 ℃ to form a fish gelatin water solution with the concentration of 1-6% (w/v).
Preferably, in the first step of enzymolysis, the added flavourzyme has the enzyme activity of 1X 104～1×106U/g, the mass ratio of the flavor protease to the substrate is 1: (50-200).
Preferably, in the second step of enzymolysis, the enzyme activity of the added trypsin is 1X 106～1×108U/g, the mass ratio of trypsin to the freeze-dried product is 1: (100-500).
Preferably, in the first step of enzymolysis and the second step of enzymolysis, the enzymolysis temperature is 40-60 ℃, and the enzymolysis time is 0.5-2 h.
Preferably, in the first step of enzymolysis and the second step of enzymolysis, the enzyme deactivation temperature is 85-95 ℃, and the enzyme deactivation time is 5-20 min.
Preferably, in the first step of enzymolysis and the second step of enzymolysis, the centrifugation is performed at 6000 to 10000rpm for 10 to 20 min.
Preferably, in the phosphorylation modification process, the ratio of the mass (g) of the added pyrophosphate to the volume (ml) of the freeze-dried product solution is (0.1-1): 100.
preferably, in the phosphorylation modification process, pyrophosphate is added and then the reaction is carried out for 0.5-2h at 45-55 ℃.
Preferably, the pyrophosphate is one or a mixture of sodium pyrophosphate and potassium pyrophosphate.
Preferably, the ultrafiltration membrane has a molecular weight cut-off of 1000-5000Da and a tangential flow rate of 4-8L (min. m)2) At a pressure of40～70pai。
In the preparation method of the shrimp meat antifreeze agent, the solution used for adjusting the pH is an acid solution or an alkali solution, the acid can be hydrochloric acid, sulfuric acid, nitric acid, acetic acid and the like, and the alkali can be sodium hydroxide, potassium hydroxide and the like. The concentration of the acid solution or the alkali solution is not limited, and is preferably 0.1-5 mol/L.
In the present invention, the enzyme activity is defined as: 1g of solid enzyme powder, under certain conditions of temperature and pH value, hydrolyzing casein for 1min to generate 1 activity unit of l mug tyrosine, which is expressed by U/g.
The other purpose of the invention is realized by the following technical scheme:
an antifreeze agent for shrimp meat, which is prepared by the preparation method.
The other purpose of the invention is realized by the following technical scheme:
the preparation method of the frozen peeled shrimp comprises the following steps:
removing heads, shells and intestinal glands of the shrimps, cleaning the shrimps, soaking the cleaned shrimps in a shrimp meat antifreeze agent solution prepared from the shrimp meat antifreeze agent at the temperature of less than or equal to 10 ℃ for 8-20 hours, taking out and standing for 2-5 min, then vacuum packaging, and storing in a refrigerator at the temperature of-15 to-25 ℃.
Preferably, the shrimp meat antifreeze solution is formed by dissolving the shrimp meat antifreeze in water, and the content of the shrimp meat antifreeze in the shrimp meat antifreeze solution is 1-5% (m/v).
Compared with the prior art, the invention has the following beneficial effects:
(1) the fish gelatin polypeptide is obtained through enzymolysis, and then the fish gelatin polypeptide is subjected to phosphorylation modification, so that the phosphorylation modified fish gelatin polypeptide has excellent anti-freezing performance, can be applied to the long-time low-temperature freezing or refrigerating process of shrimp meat products, reduces the loss of water and nutrient substances in the freezing and storing process, and better maintains the freshness and the tenderness of the frozen shrimp meat;
(2) according to the preparation method of the shrimp meat antifreeze agent, flavourzyme is adopted for carrying out first-step enzymolysis, trypsin is adopted for carrying out second-step enzymolysis, and the two-step enzymolysis of the flavourzyme and the trypsin improves the enzymolysis efficiency, so that fish polypeptide with appropriate molecular weight and molecular structure is obtained, and the antifreeze property is improved;
(3) the invention separates and purifies the phosphorylated fish polypeptide crude extract by using an ultrafiltration membrane technology to obtain the antifreeze agent with small molecular weight and high antifreeze efficiency;
(4) the invention successfully obtains the shrimp meat antifreeze agent through a novel preparation method, provides a novel method for preparing the antifreeze agent, and the obtained shrimp meat antifreeze agent can be used for freezing or refrigerating other aquatic products besides shrimp meat products.
(5) The preparation method of the shrimp meat antifreeze agent is simple and controllable to operate and can be applied to industrial production.
Detailed Description
The technical solutions of the present invention are further described and illustrated below by specific examples, it should be understood that the specific examples described herein are only for the purpose of facilitating understanding of the present invention, and are not intended to be specific limitations of the present invention. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.
Example 1
The preparation method of the shrimp meat antifreeze agent comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in water bath at 40 ℃ to prepare a 4% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 6.0 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:50(g/g)5U/g), the enzymolysis temperature is 40 ℃, the enzymolysis time is 0.5h, the enzyme is killed by heating in water bath at 95 ℃ for 10min, the centrifugation is carried out at 8000rpm for 15min, and the supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 4% (w/v) solution, and the pH was adjusted to 8.0 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:100(g/g) Trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 40 ℃, the enzymolysis time is 0.5h, the enzyme is inactivated by heating in water bath at 95 ℃ for 10min,centrifuging at 8000rpm for 15min, collecting supernatant, and lyophilizing;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to prepare a solution with the concentration of 5% (w/v), adjusting the pH value to 8.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the freeze-dried product solution is 0.1: reacting for 0.5h at 100 and 50 ℃;
s5, ultrafiltration membrane filtration: separating and purifying the solution obtained by phosphorylation modification reaction with ultrafiltration membrane with molecular weight of 1000Da at tangential flow rate of 4L (min. m)2) Collecting filtrate and freeze-drying to obtain the shrimp meat antifreeze agent under the pressure of 60 psi.
Example 2
The preparation method of the shrimp meat antifreeze agent comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 43 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 6.3 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:100(g/g)5U/g), the enzymolysis temperature is 50 ℃, the enzymolysis time is 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 5% (w/v) solution, and the pH was adjusted to 8.2 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:200(g/g) Trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 50 ℃, the enzymolysis is carried out for 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to prepare a solution with the concentration of 5% (w/v), adjusting the pH value to 9.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the freeze-dried product solution is 0.4: reacting for 1h at the temperature of 100 and 48 ℃;
s5, ultrafiltration membrane filtration: separating and purifying the solution obtained by phosphorylation modification reaction with ultrafiltration membrane with molecular weight of 3000Da at tangential flow rate of 6L (min. m)2) Pressure ofAnd the force is 65psi, and the filtrate is collected and freeze-dried to obtain the shrimp meat antifreeze agent.
Example 3
The preparation method of the shrimp meat antifreeze agent comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 38 ℃ to prepare a 3% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 5.8 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:200(g/g)5U/g), the enzymolysis temperature is 60 ℃, the enzymolysis time is 2h, the enzyme is killed by heating in water bath at 88 ℃ for 15min, centrifugation is carried out at 9000rpm for 13min, and supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 3% (w/v) solution, and the pH was adjusted to 7.8 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:500(g/g) Trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 60 ℃, the enzymolysis is carried out for 2h, the enzyme is killed by heating in water bath at 88 ℃ for 15min, centrifugation is carried out at 9000rpm for 13min, and supernatant is collected and freeze-dried;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to prepare a solution with 4% (w/v), adjusting the pH value to 10.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the freeze-dried product solution is 0.6: reacting for 2 hours at 100 and 50 ℃;
s5, ultrafiltration membrane filtration: separating and purifying the solution obtained by phosphorylation modification reaction with ultrafiltration membrane with molecular weight of 5000Da at tangential flow rate of 8L (min. m)2) Collecting filtrate and freeze-drying to obtain the shrimp meat antifreeze agent under the pressure of 55 psi.
Example 4
The preparation method of the shrimp meat antifreeze agent comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in water bath at 40 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 6.0 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:80(g/g)5U/g), the enzymolysis temperature is 50 ℃,the enzymolysis time is 0.5h, the enzyme is deactivated by heating in 90 ℃ water bath for 15min, the centrifugation is carried out for 18min at 8000rpm, and the supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 5% (w/v) solution, and the pH was adjusted to 8.0 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:300(g/g) Trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 50 ℃, the enzymolysis is carried out for 2h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out for 18min at 8000rpm, and the supernatant is collected and freeze-dried;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to prepare a solution with the concentration of 5% (w/v), adjusting the pH value to 9.0 by using 1M NaOH or 1M HCl, adding potassium pyrophosphate, wherein the ratio of the mass (g) of the added potassium pyrophosphate to the volume (ml) of the freeze-dried product solution is 1: reacting for 1h at 100 and 50 ℃;
s5, ultrafiltration membrane filtration: separating and purifying the solution obtained by phosphorylation modification reaction with ultrafiltration membrane with molecular weight of 1000Da at tangential flow rate of 7L (min. m)2) Collecting filtrate and freeze-drying to obtain the shrimp meat antifreeze agent under the pressure of 60 psi.
Comparative example 1
The difference between comparative example 1 and example 2 is that the fish gelatin of comparative example 1 is not subjected to enzymatic treatment and is directly modified by phosphorylation. The specific preparation method of comparative example 1 was:
weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 43 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, adjusting the pH to 9.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the fish gelatin aqueous solution is 0.4: reacting for 1h at the temperature of 100 and 48 ℃; the subsequent ultrafiltration membrane filtration step was the same as in example 2.
Comparative example 2
Comparative example 2 differs from example 2 in that the fish gelatin of comparative example 2 has not been modified by phosphorylation after two enzymatic steps. The specific preparation method of comparative example 2 was:
S1-S3 similar to example 2, the freeze-dried product obtained by the second step of enzymolysis is dissolved in water to prepare a solution with 5% (w/v), the pH value is adjusted to 9.0 by 1M NaOH or 1M HCl, and the reaction lasts for 1h at 48 ℃; the subsequent ultrafiltration membrane filtration step was the same as in example 2.
Comparative example 3
The preparation method of the shrimp meat antifreeze agent of the comparative example 3 comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 43 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 6.3 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:100(g/g)5U/g), the enzymolysis temperature is 50 ℃, the enzymolysis time is 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried;
s3, phosphorylation modification: dissolving the freeze-dried product obtained by the first step of enzymolysis in water to prepare a solution with the concentration of 5% (w/v), adjusting the pH value to 9.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the freeze-dried product solution is 0.4: reacting for 1h at the temperature of 100 and 48 ℃; the subsequent ultrafiltration membrane filtration step was the same as in example 2.
Comparative example 4
Comparative example 4 differs from example 2 in that the two-step enzymatic hydrolysis of comparative example 4 employs flavourzyme. The concrete preparation method of the shrimp meat antifreeze agent of the comparative example 4 comprises the following steps:
s1, weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 43 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 6.3 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: adding flavourzyme (1.83X 10) into fish gelatin substrate at a ratio of 1:100(g/g)5U/g), the enzymolysis temperature is 50 ℃, the enzymolysis time is 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 5% (w/v) solution, and the pH was adjusted to 6.3 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:100(g/g) flavourzyme (1.83X 10) was added5U/g), the enzymolysis temperature is 50 ℃, the enzymolysis time is 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried; subsequent phosphorylation modification and ultrafiltration membrane filtration steps were as in the examples2。
Comparative example 5
Comparative example 5 differs from example 2 in that trypsin is used for both enzymatic steps of comparative example 5. The concrete preparation method of the shrimp meat antifreeze agent of the comparative example 5 is as follows:
s1, weighing a proper amount of fish gelatin in distilled water, heating in a water bath at 43 ℃ to prepare a 5% (w/v) fish gelatin aqueous solution, and adjusting the pH value to 8.2 by using 1M NaOH or 1M HCl.
S2, first-step enzymolysis: according to the enzyme: 1:200(g/g) of fish gelatin substrate trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 50 ℃, the enzymolysis is carried out for 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried;
s3, carrying out enzymolysis in the second step: the lyophilized product obtained from the first step of enzymatic hydrolysis was dissolved in water to prepare a 5% (w/v) solution, and the pH was adjusted to 8.2 with 1M NaOH or 1M HCl, according to the enzyme: substrate 1:200(g/g) Trypsin (4.07X 10) was added7U/g), the enzymolysis temperature is 50 ℃, the enzymolysis is carried out for 1h, the enzyme is killed by heating in water bath at 90 ℃ for 15min, the centrifugation is carried out at 7000rpm for 18min, and the supernatant is collected and freeze-dried; the subsequent phosphorylation modification and ultrafiltration membrane filtration steps were the same as in example 2.
Comparative example 6
The preparation method of the shrimp meat antifreeze of the comparative example 6 comprises the following steps:
steps S1-S3 are the same as in example 2;
s4, dissolving the freeze-dried product obtained by the second step of enzymolysis in water to prepare a solution with 5% (w/v), adjusting the pH to 9.0 by using 1M NaOH or 1M HCl, adding sodium pyrophosphate, wherein the ratio of the mass (g) of the added sodium pyrophosphate to the volume (ml) of the freeze-dried product solution is 0.4: 100, respectively;
s5, ultrafiltration membrane filtration: separating and purifying the solution obtained in the step S4 with ultrafiltration membrane with molecular weight of 3000Da at tangential flow rate of 6L (min. m)2) Collecting filtrate and freeze-drying to obtain the shrimp meat antifreeze agent under the pressure of 65 psi.
The shrimp meat antifreeze agents prepared in examples 1 to 4 and comparative examples 1 to 6 were dissolved in water to prepare a 3% (w/v) shrimp meat antifreeze agent solution, and a commercial refrigerant (Convere chemical Co., Ltd., Guangzhou) was dissolved in water to prepare a 3% (w/v) shrimp meat antifreeze agent solution. Removing heads, shells and intestinal glands of the shrimps, cleaning the shrimp meat by using cold water (4 ℃), then respectively soaking the shrimp meat in shrimp meat antifreeze agent solutions prepared by the shrimp meat antifreeze agents of examples 1-4 and comparative examples 1-6 and commercial refrigerants, controlling the temperature below 10 ℃ in the whole operation process for 12 hours, taking out the shrimp meat after soaking, standing the shrimp meat for 5 minutes, then carrying out vacuum packaging and transferring the shrimp meat to a refrigerator with the temperature of-20 ℃ for storage for 14 days to obtain a frozen shrimp meat product.
Products S1-S11 correspond to frozen shrimp meat products obtained by soaking in shrimp meat antifreeze solutions prepared from the shrimp meat antifreeze of examples 1-4 and comparative examples 1-6 and a commercial refrigerant, respectively.
Products S1-S11 were tested for thaw loss and tenderness after cooking as follows:
(1) thawing loss rate: weighing appropriate amount of frozen shelled shrimp (m)1) Unfreezing at room temperature for 2h, wiping off water with gauze, and weighing and recording as m2. Thawing loss ratio (%) - (m)1-m2)/m1X 100. Higher values indicate a higher degree of deterioration in the frozen quality of the shrimp meat.
(2) And (3) tenderness determination: the smooth tenderness of the shrimp was tested using a TA-XT plus texture analyzer. The probe model is BSK, the test mode is hot dog, the speed before test is 2.0mm/s, the speed after test is 10.0mm/s, the descending distance is 20mm, and the thickness of the sample is 10 mm. The lower the shear, the higher the tenderness.
The test results for all samples are shown in table 1.
TABLE 1
| Sample (I) | Thawing loss Rate (%) | Shear force (N) |
| S1 | 1.92％ | 7.99 |
| S2 | 1.86％ | 7.21 |
| S3 | 1.91％ | 7.94 |
| S4 | 1.89％ | 7.33 |
| S5 | 2.87％ | 8.67 |
| S6 | 2.33％ | 8.22 |
| S7 | 2.68％ | 8.51 |
| S8 | 2.52％ | 8.43 |
| S9 | 2.43％ | 8.40 |
| S10 | 2.12％ | 8.05 |
| S11 | 2.53％ | 8.30 |
As can be seen from Table 1, the frozen shrimp meat, which was soaked in the shrimp meat antifreeze of examples 1-4, had a low freezing loss rate and high tenderness, exhibited good refrigeration characteristics and freeze resistance. The shrimp meat antifreeze of the comparative example 1 is not subjected to enzymolysis, the shrimp meat antifreeze of the comparative example 2 is not subjected to phosphorylation modification, the comparative example 3 adopts one-step enzymolysis, and the comparative examples 4-5 adopt single enzyme to carry out two-step enzymolysis, so that the freezing loss rate of the frozen shrimp meat is high, and the antifreeze effect is not as good as that of the example 2.
Finally, it should be noted that the specific examples described herein are merely illustrative of the spirit of the invention and do not limit the embodiments of the invention. Various modifications, additions and substitutions for the embodiments described herein will occur to those skilled in the art, and all such embodiments are neither required nor possible. While the invention has been described with respect to specific embodiments, it will be appreciated that various changes and modifications may be made without departing from the spirit and scope of the invention, as defined by the appended claims.
Claims (10)
1. The preparation method of the shrimp meat antifreeze agent is characterized by comprising the following steps:
s1, preparing a fish gelatin solution, and adjusting the pH value to 5.5-6.5;
s2, first-step enzymolysis: adding flavourzyme into the fish gelatin solution for enzymolysis reaction, then inactivating enzyme, centrifuging, collecting supernatant and freeze-drying;
s3, carrying out enzymolysis in the second step: dissolving the freeze-dried product obtained by the first-step enzymolysis in water, adjusting the pH value to 7.5-8.5, adding trypsin for enzymolysis reaction, then inactivating enzyme, centrifuging, collecting supernatant and freeze-drying;
s4, phosphorylation modification: dissolving the freeze-dried product obtained by the second step of enzymolysis in water to form a freeze-dried product solution, adjusting the pH to 8.0-10.0, and adding pyrophosphate to react;
s5, ultrafiltration membrane filtration: and separating and purifying the solution obtained by phosphorylation modification reaction by adopting an ultrafiltration membrane, collecting filtrate, and freeze-drying to obtain the shrimp meat antifreeze agent.
2. The process according to claim 1, wherein the enzyme activity of the flavourzyme added in the first enzymatic hydrolysis step is 1X 104～1×106U/g, the mass ratio of the flavor protease to the substrate is 1: (50-200).
3. The process according to claim 1, wherein in the second enzymatic hydrolysis, trypsin is added at an enzymatic activity of 1X 106～1×108U/g, the mass ratio of trypsin to the freeze-dried product is 1: (100-500).
4. The preparation method of claim 1, wherein the enzymolysis temperature is 40-60 ℃ and the enzymolysis time is 0.5-2h in the first step and the second step.
5. The preparation method of claim 1, wherein in the first step of enzymolysis and the second step of enzymolysis, the enzyme deactivation temperature is 85-95 ℃ and the enzyme deactivation time is 5-20 min.
6. The preparation method according to claim 1, wherein the ratio of the mass (g) of pyrophosphate added to the volume (ml) of the solution of the lyophilized product in the phosphorylation modification process is (0.1-1): 100.
7. the preparation method according to claim 1, wherein the reaction is carried out at 45-55 ℃ for 0.5-2h after pyrophosphate is added in the phosphorylation modification process.
8. The method of claim 1The preparation method is characterized in that the cutoff molecular weight of the ultrafiltration membrane is 1000-5000Da, and the tangential flow rate is 4-8L (min. m)2) The pressure is 40 to 70pa i.
9. An antifreeze agent for shrimp meat, which is prepared by the preparation method of claim 1.
10. The preparation method of the frozen peeled shrimp is characterized by comprising the following steps of:
removing heads, shells and intestinal glands of the shrimps, cleaning the shrimps, soaking the cleaned shrimps in the shrimp meat antifreeze agent solution prepared from the shrimp meat antifreeze agent in claim 9 at the temperature of less than or equal to 10 ℃ for 8-20 h, taking out and standing for 2-5 min, then vacuum packaging and storing in a refrigerator at the temperature of-15 to-25 ℃.
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