CN108866132B - Process for extracting squid active small peptide at ultralow temperature - Google Patents
Process for extracting squid active small peptide at ultralow temperature Download PDFInfo
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- CN108866132B CN108866132B CN201810639101.1A CN201810639101A CN108866132B CN 108866132 B CN108866132 B CN 108866132B CN 201810639101 A CN201810639101 A CN 201810639101A CN 108866132 B CN108866132 B CN 108866132B
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- 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
Abstract
The invention relates to the technical field of aquatic product deep processing, in particular to a process for extracting squid active small peptide at ultralow temperature, which comprises the following steps: (1) pretreating fresh squid, and air-drying at low temperature to obtain squid carcass; (2) crushing to obtain squid meat paste; (3) adding marine low-temperature alkaline protein complex enzyme, and performing enzymolysis to obtain a crude enzyme solution; (4) adding a protective agent into the crude enzyme solution, performing ultrasonic enzymolysis, and cooling to obtain a final enzymolysis solution; (5) centrifuging, and collecting supernatant to obtain squid active small peptide solution. According to the invention, the squid is crushed at ultralow temperature, the compound enzyme is used as the suitable enzyme for preparing the small peptide from the squid meat paste, the enzymolysis efficiency is higher, and the antioxidant activity of the obtained small peptide is high; the process steps are simple, the reaction process is easy to control, the operability is strong, and the production period is short; the squid meat paste has high hydrolysis degree, and the obtained squid skin active small peptide has high antioxidant activity and high nutrient effective components, thereby providing feasible theoretical basis for industrial production.
Description
Technical Field
The invention relates to the technical field of aquatic product deep processing, in particular to a process for extracting squid active small peptide at ultralow temperature.
Background
In recent years, with the rapid development of the oceanic squid fishing industry in China, squids increasingly become the main marine economic mollusks and aquatic product processing raw materials in China.
The squid meat is delicious, is mainly used in food processing industry, has rich protein content and extremely high nutritional value, most squid products can not ensure the nutritional ingredients of the squid while keeping the flavor, and the protein needs to be hydrolyzed and absorbed by intestinal tracts before being absorbed by human bodies, and the nutritional ingredients absorbed by the human bodies, such as small peptides, amino acids and the like, only account for a small part of the whole nutrient bodies, so that the squid meat is easy to absorb while ensuring the nutrition for extraction process requirements in order to avoid nutrient waste.
Chinese patent literature discloses a preparation method of squid skin active polypeptide, the publication number of which is CN105112478A, the preparation method of the invention is easy to oxidize in the conventional treatment process, the nutrient content of the squid is easy to lose, the preparation process is rough, and the preparation method is only used for squid skin, and the application range is narrow. Therefore, in the technical field of aquatic product deep processing, a technical means for efficiently ensuring the nutritional effective components in the processing process of the squid aquatic product is needed.
Disclosure of Invention
The invention provides a process for extracting squid active small peptides at ultralow temperature, which has the advantages of simple process steps, easily controlled reaction process, strong operability, short production period, high squid meat paste hydrolysis degree, high antioxidant activity of the obtained squid skin active small peptides and high nutritional effective components, and aims to solve the problems of nutrient component loss and narrow application range in the traditional squid deep processing technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
a process for extracting squid active small peptide at ultralow temperature comprises the following steps:
(1) cleaning fresh squid with ice water, removing head, feet, viscera, fins, etc., peeling, soaking squid meat in the treatment solution, and air drying at low temperature to obtain squid carcass;
(2) freezing the obtained squid carcass at ultralow temperature by using liquid nitrogen, and crushing the squid carcass in a low-temperature crusher in a frozen state to obtain minced squid meat; adding a sodium hydroxide solution into the squid meat paste, soaking for 18-24 h, then washing with purified water until the pH value is neutral, then adding an ethylene glycol solution, stirring for 1-2 d, washing with water, and draining to obtain drained squid meat paste; ice water and ultra-low temperature crushing treatment are carried out to achieve the effect of removing oxygen and prevent the squid meat paste from going bad in the preparation process. The glycol is used for removing lipid substances, and the sodium hydroxide is used for removing impurity proteins and redundant squid skin cutin;
(3) adding water with the amount of 5-10 times that of the drained squid minced meat into the drained squid minced meat, uniformly stirring, keeping the temperature to 15-25 ℃, adjusting the pH to 7-9 to obtain a raw material solution, adding a marine low-temperature alkaline protein complex enzyme into the raw material solution, performing enzymolysis for 1-2 days, and continuously stirring to obtain a crude enzyme solution; the squid meat paste is treated at a lower temperature, so that the squid meat paste can be prevented from being oxidized, the compound enzyme can be collected and reused, and the economic cost is saved;
(4) adding a protective agent into the crude enzyme solution, performing ultrasonic enzymolysis for 20-40 min at 15-25 ℃ to achieve an enzyme deactivation activity effect, and cooling to obtain a final enzymolysis solution;
(5) and centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid active small peptide solution.
Preferably, in the step (2), 40-50 mL of sodium hydroxide solution is added into every gram of squid meat paste, and the mass percentage concentration of the sodium hydroxide solution is 0.3-0.6%.
Preferably, in the step (2), 20-50 mL of glycol solution is added into each gram of squid meat paste, and the mass percentage concentration of the glycol solution is 12-15%.
Preferably, in the step (3), the feed-to-liquid ratio of the marine low-temperature basic protein complex enzyme to the raw material liquid is 1 g: (800-1000 mL).
Preferably, in the step (3), 14000-15000 u of the marine low-temperature alkaline protein complex enzyme is added to each gram of the raw material solution, and the marine low-temperature alkaline protein complex enzyme comprises the following enzymes in percentage by mass: 60-80% of alkaline protease, 5-10% of trypsin and the balance of neutral protease. The invention screens enzyme, selects the compound enzyme consisting of alkaline protease, trypsin and neutral protease to carry out enzymolysis on the squid meat paste, and has the advantages of higher enzymolysis efficiency, better enzymolysis effect and high antioxidant activity of the obtained small peptide.
Preferably, in the step (4), the protective agent is guar gum, and the addition amount of the guar gum is 0.2-0.5% of the mass of the crude enzyme solution.
Guar gum is also called guar gum, guar gum has excellent characteristics of gelatinization, thickening, emulsification, stable dispersion and the like, is straight-chain macromolecule, hydroxyl on the chain can form hydrogen bonds with some hydrophilic colloids and starch, and a proper amount of guar gum is added into crude enzyme liquid, so that the crude enzyme liquid keeps a complex stable state with the guar gum in the ultrasonic enzymolysis process, the viscosity of the guar gum is reduced along with the increase of temperature, and the principle of the guar gum is that the guar gum molecules present a long-chain extending structure with short mannose residues under the low-temperature condition, and the guar gum is favorable for being combined with solvent water molecules to be close to physical and chemical bonds; when the temperature is higher, the approaching is weakened due to Brownian motion, and the ultrasonic enzymolysis is carried out at the temperature of 15-25 ℃, so that the guar gum can fully play a stabilizing role, and simultaneously, the guar gum also serves as a buffering agent to stabilize the pH value.
Preferably, in the step (4), the frequency of ultrasonic enzymolysis is 30-50 kHz, and the power is 100-200 w.
Preferably, in the step (5), the centrifugation process conditions are as follows: the centrifugal force is controlled to be 4000-5000 g/min, and the centrifugal time is controlled to be 10-20 min.
Preferably, in the step (4), nitrogen gas is continuously blown into the chelate solution formed in the ultrasonic enzymolysis step, and nitrogen gas is continuously blown into the chelate solution during the ultrasonic enzymolysis step, thereby removing oxygen contained in the solution and further performing a stirring action.
Preferably, the low-temperature pulverizer comprises a pulverizing box and a driving motor, a pulverizing blade is arranged in the pulverizing box, a cavity is arranged in the pulverizing blade, and an ultralow-temperature phase-change material is filled in the cavity.
The low-temperature pulverizer used by the invention adopts excellent ultralow-temperature phase-change materials to automatically cool, has the advantage of large solid-liquid phase-change latent heat through selection and adjustment of the use amount of various substances, can form eutectic through mixing of various substances to reduce the solid-liquid phase-change temperature, and simultaneously can improve the phase-change latent heat per unit volume and adjust the phase-change temperature of the ultralow-temperature phase-change materials. The ultralow-temperature phase change material is prepared by stirring and mixing acrylic acid, hexyl acetate and butyl valerate in a mass ratio of 1:2:2 at a high speed, is a composite organic phase change material, can obtain a lower phase change temperature, has the phase change temperature within-30 to-10 ℃, has low saturated steam pressure, is difficult to volatilize, is liquid at room temperature, is easy to mix, is non-flammable, non-toxic and harmless to human bodies and environments, basically has no corrosion to metal equipment, and ensures that the squid carcass is crushed at low temperature to obtain the squid minced meat.
Therefore, the invention has the following beneficial effects:
(1) the squid is crushed at ultralow temperature, and the squid is prepared at low temperature by utilizing cold brittleness, so that the effects of oxygen removal and oxidation resistance can be achieved;
(2) the compound enzyme is used as the suitable enzyme for preparing the small peptide from the squid meat paste, so that the enzymolysis efficiency is high, the enzymolysis effect is good, and the obtained small peptide has high antioxidant activity;
(3) the preparation process of the squid meat paste active small peptide is optimized, the process steps are simple, the reaction process is easy to control, the operability is strong, and the production period is short;
(4) the squid meat paste has high hydrolysis degree, and the obtained squid skin active small peptide has high antioxidant activity and high nutrient effective components, thereby providing feasible theoretical basis for industrial production.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples.
In the present invention, unless otherwise specified, all equipment and materials are commercially available or commonly used in the industry, and all percentages are by weight unless otherwise specified, and the methods in the following examples are conventional in the art unless otherwise specified.
The low-temperature pulverizer used in the following embodiment of the invention comprises a pulverizing box and a driving motor, wherein a pulverizing blade is arranged in the pulverizing box, a cavity is arranged in the pulverizing blade, and an ultralow-temperature phase-change material is filled in the cavity and is formed by stirring and mixing 100g of acrylic acid, 200g of hexyl acetate and 200g of butyl valerate at a high speed.
Example 1
(1) Cleaning fresh squid with ice water, removing head, feet, viscera, fins, etc., peeling, soaking squid meat in the treatment solution, and air drying at low temperature to obtain squid carcass;
(2) freezing the obtained squid carcass at ultralow temperature by using liquid nitrogen, and crushing the squid carcass in a low-temperature crusher in a frozen state to obtain minced squid meat; adding 40mL of sodium hydroxide solution with the mass percentage concentration of 0.6% into each gram of minced squid meat, soaking for 18h, washing with purified water until the pH value is neutral, adding 20mL of ethylene glycol solution with the mass percentage concentration of 15% into each gram of minced squid meat, stirring for 1d, washing with water, and draining;
(3) adding water which is 5 times of the amount of the squid minced meat into the drained squid minced meat, uniformly stirring, keeping the temperature to 15 ℃, adjusting the pH to 7 to obtain a raw material solution, adding 14000u of marine low-temperature alkaline protein complex enzyme into each gram of the raw material solution, wherein the marine low-temperature alkaline protein complex enzyme consists of the following enzymes in percentage by mass: 60% of alkaline protease, 5% of trypsin and the balance of neutral protease; the feed-liquid ratio of the marine low-temperature alkaline protein complex enzyme to the raw material liquid is 1 g: performing enzymolysis for 1d in 800mL, and continuously stirring to finally obtain a crude enzyme solution;
(4) adding guanidine gum accounting for 0.2% of the mass of the crude enzyme solution into the crude enzyme solution, performing ultrasonic enzymolysis at 15 ℃ for 40min, wherein the ultrasonic enzymolysis frequency is 30kHz and the power is 100w, so as to achieve the enzyme deactivation effect, cooling to obtain a final enzymolysis solution, and continuously blowing nitrogen into the chelating solution during the enzymolysis period to remove oxygen in the solution;
(5) and (4) centrifuging the final enzymolysis liquid for 20min under the centrifugal force of 4000g/min, and collecting supernatant to obtain the squid active small peptide solution.
Example 2
(1) Cleaning fresh squid with ice water, removing head, feet, viscera, fins, etc., peeling, soaking squid meat in the treatment solution, and air drying at low temperature to obtain squid carcass;
(2) freezing the obtained squid carcass at ultralow temperature by using liquid nitrogen, and crushing the squid carcass in a low-temperature crusher in a frozen state to obtain minced squid meat; adding 50mL of sodium hydroxide solution with the mass percentage concentration of 0.3% into each gram of minced squid meat, soaking for 24 hours, washing with purified water until the pH value is neutral, adding 50mL of ethylene glycol solution with the mass percentage concentration of 12% into each gram of minced squid meat, stirring for 2 days, washing with water, and draining;
(3) adding water which is 10 times of the amount of the squid minced meat into the drained squid minced meat, uniformly stirring, keeping the temperature to 25 ℃, adjusting the pH to 9 to obtain a raw material solution, adding 15000u of the marine low-temperature alkaline protein complex enzyme into each gram of the raw material solution, wherein the marine low-temperature alkaline protein complex enzyme comprises the following enzymes in percentage by mass: 80% of alkaline protease, 10% of trypsin and the balance of neutral protease; the feed-liquid ratio of the marine low-temperature alkaline protein complex enzyme to the raw material liquid is 1 g: performing enzymolysis for 2 days by 1000mL, and continuously stirring to obtain a crude enzyme solution;
(4) adding guanidine gum accounting for 0.5% of the mass of the crude enzyme solution into the crude enzyme solution, performing ultrasonic enzymolysis at 25 ℃ for 20min, wherein the ultrasonic enzymolysis frequency is 50kHz and the power is 200w, so as to achieve the enzyme deactivation effect, cooling to obtain a final enzymolysis solution, and continuously blowing nitrogen into the chelating solution during the enzymolysis period to remove oxygen in the solution;
(5) and centrifuging the final enzymolysis liquid for 20min under the centrifugal force of 5000g/min, and collecting supernatant to obtain the squid active small peptide solution.
Example 3
(1) Cleaning fresh squid with ice water, removing head, feet, viscera, fins, etc., peeling, soaking squid meat in the treatment solution, and air drying at low temperature to obtain squid carcass;
(2) freezing the obtained squid carcass at ultralow temperature by using liquid nitrogen, and crushing the squid carcass in a low-temperature crusher in a frozen state to obtain minced squid meat; adding 45mL of sodium hydroxide solution with the mass percentage concentration of 0.4% into each gram of minced squid meat, soaking for 20 hours, washing with purified water until the pH value is neutral, adding 35mL of ethylene glycol solution with the mass percentage concentration of 13% into each gram of minced squid meat, stirring for 1.5 days, and then washing with water and draining;
(3) adding water which is 8 times of the amount of the squid minced meat into the drained squid minced meat, uniformly stirring, keeping the temperature to 20 ℃, adjusting the pH to 8 to obtain a raw material solution, adding 14500u of marine low-temperature alkaline protein complex enzyme into each gram of the raw material solution, wherein the marine low-temperature alkaline protein complex enzyme comprises the following enzymes in percentage by mass: 75% of alkaline protease, 8% of trypsin and the balance of neutral protease; the feed-liquid ratio of the marine low-temperature alkaline protein complex enzyme to the raw material liquid is 1 g: 900mL, performing enzymolysis for 1.5 days, and continuously stirring to finally obtain a crude enzyme solution;
(4) adding guanidine gum accounting for 0.3% of the mass of the crude enzyme solution into the crude enzyme solution, performing ultrasonic enzymolysis for 35min at 20 ℃, wherein the ultrasonic enzymolysis frequency is 40kHz, the power is 150w, so as to achieve the enzyme deactivation effect, cooling to obtain a final enzymolysis solution, continuously blowing nitrogen into the chelating solution during the enzymolysis period, and removing oxygen in the solution;
(5) and centrifuging the final enzymolysis liquid for 15min under the centrifugal force of 4500g/min, and collecting supernatant to obtain the squid active small peptide solution.
The hydrolysis degree of the squid meat paste is over 32.5 percent, so that the preparation method disclosed by the invention has the advantages that the hydrolysis degree of the squid meat paste is high, the antioxidant activity of the obtained squid meat paste active small peptide is high, and a feasible theoretical basis is provided for industrial production.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (7)
1. A process for extracting squid active small peptide at ultralow temperature is characterized by comprising the following steps:
(1) cleaning fresh squid with ice water, removing head, feet, viscera, fins, etc., peeling, soaking squid meat in the treatment solution, and air drying at low temperature to obtain squid carcass;
(2) freezing the obtained squid carcass at ultralow temperature by using liquid nitrogen, and crushing the squid carcass in a low-temperature crusher in a frozen state to obtain minced squid meat; adding a sodium hydroxide solution into the squid meat paste for soaking, then washing with purified water until the pH value is neutral, then adding an ethylene glycol solution, stirring, washing with water, and draining to obtain drained squid meat paste; the low-temperature pulverizer comprises a pulverizing box and a driving motor, wherein a pulverizing blade is arranged in the pulverizing box, a cavity is arranged in the pulverizing blade, and an ultralow-temperature phase-change material is filled in the cavity; the ultralow-temperature phase-change material is formed by stirring and mixing acrylic acid, hexyl acetate and butyl valerate in a mass ratio of 1:2:2 at a high speed, and the phase-change temperature of the ultralow-temperature phase-change material is within-30 to-10 ℃;
(3) adding water with the amount of 5-10 times that of the drained squid meat paste into the drained squid meat paste, uniformly stirring, keeping the temperature to 15-25 ℃, adjusting the pH to 7-9 to obtain a raw material solution, adding a marine low-temperature alkaline protein complex enzyme into the raw material solution, performing enzymolysis, and continuously stirring to obtain a crude enzyme solution; the marine low-temperature alkaline protein complex enzyme consists of the following enzymes in percentage by mass: 60-80% of alkaline protease, 5-10% of trypsin and the balance of neutral protease; the feed-liquid ratio of the marine low-temperature alkaline protein complex enzyme to the raw material liquid is 1 g: (800-1000 mL);
(4) adding a protective agent into the crude enzyme solution, performing ultrasonic enzymolysis at 15-25 ℃, and cooling to obtain a final enzymolysis solution; the protective agent is guanidine gum, and the addition amount of the guanidine gum is 0.2-0.5% of the mass of the crude enzyme solution;
(5) and centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid active small peptide solution.
2. The process for extracting the squid active small peptide at the ultralow temperature as claimed in claim 1, wherein in the step (2), 40-50 mL of sodium hydroxide solution is added into every gram of squid minced meat, and the mass percentage concentration of the sodium hydroxide solution is 0.3-0.6%.
3. The process for extracting the squid active small peptide at the ultralow temperature as claimed in claim 1, wherein in the step (2), 20-50 mL of glycol solution is added into every gram of squid minced meat, and the mass percentage concentration of the glycol solution is 12-15%.
4. The process for extracting the squid active small peptide at the ultralow temperature as claimed in claim 1, wherein 14000-15000 u of marine low-temperature alkaline protein complex enzyme is added to each gram of raw material liquid in the step (3).
5. The process for extracting the squid active small peptide at the ultralow temperature according to claim 1, wherein in the step (4), the ultrasonic enzymolysis frequency is 30-50 kHz, and the power is 100-200 w.
6. The process for extracting the squid active small peptide at the ultralow temperature according to the claim 1, wherein in the step (5), the centrifugal process conditions are as follows: the centrifugal force is controlled to be 4000-5000 g/min, and the centrifugal time is controlled to be 10-20 min.
7. The process for extracting squid active small peptide at ultralow temperature according to the claim 1, 2, 3, 5 or 6, is characterized in that in the step (4), nitrogen is continuously blown into the chelating liquid formed in the ultrasonic enzymolysis process.
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