Preparation method of squid skin active polypeptide
Technical Field
The invention relates to the technical field of aquatic product deep processing, in particular to a preparation method of squid skin active polypeptide.
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. At present, the processing and utilization technology content of the squid in China is low, and about 35% of squid heads, feet, skin, viscera and other byproducts are generated in the production, wherein the squid skin accounts for about 8% -13%.
The squid skin has a high collagen content, which is about 80% of the dry weight. The absorption rate of collagen polypeptide is nearly 100%, and the collagen polypeptide has the functions of resisting ulcer, reducing blood pressure and promoting Ca2+Absorption and the like. Therefore, the polypeptide substance with antioxidant activity is obtained from the squid skin, which is beneficial to greatly improving the added value of the squid skin.
Chinese patent application publication No. CN 102217699A, application publication No. 2011.10.19 discloses a preparation method of squid skin protein active peptide, which comprises the steps of cleaning squid skin, cutting into blocks, adding NaOH aqueous solution, soaking for 2-8 days for decolorization, and washing to neutral pH; addition of Ca (OH)2Soaking the mixture in the solution for 2-4 days for color fixation, and washing the mixture with water until the pH value is neutral; treating the fish skin in neutral water with the pH value of 55-75 ℃ for 0.5-1.5 h, filtering, removing insoluble protein, and extracting to obtain fish skin protein liquid; and finally, carrying out high-pressure treatment on the liquid at 110-121 ℃ for 30-120 min to obtain the squid skin protein active peptide. The preparation method has the following disadvantages: (1) the production period of the whole preparation method is long; (2) the preparation of the bioactive peptide by treating the squid skin protein with high-pressure hot water needs to be carried out under high-pressure conditions, the process conditions are strict, the operability is poor, and the squid skin waterInsufficient hydrolysis, low degree of hydrolysis and low antioxidant activity.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides the preparation method of the squid skin active polypeptide, which has the advantages of simple process steps, easily controlled reaction process, strong operability, short production period and high squid skin hydrolysis degree.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of squid skin active polypeptide comprises the following steps:
(1) washing and mashing squid skin, adding a sodium hydroxide solution, soaking for 24-30 h, washing with water until the pH value is neutral, adding an n-butyl alcohol solution, stirring for 16-24 h, washing with water, and draining. Soaking in sodium hydroxide to remove foreign proteins; soaking in n-butanol solution to remove lipid.
(2) Adding water which is 90-95 times of the weight of the drained squid skin into the drained squid skin, uniformly stirring, heating to 40-60 ℃, preserving heat for 3-5 hours, and adjusting the pH value to 8-9 to obtain a primary enzymolysis liquid. Heating squid skin under 40~60 ℃, utilizing the enzyme of squid skin self to carry out preliminary enzymolysis, not only be favorable to improving the degree of hydrolysis, can reduce the use amount of compound enzyme in the follow-up enzymolysis process moreover, be favorable to reduce cost.
(3) Adding complex enzyme and sodium bicarbonate into the primary enzymolysis liquid, performing ultrasonic enzymolysis for 20-30 min at 40-50 ℃, inactivating enzyme, and cooling to obtain final enzymolysis liquid, wherein nitrogen is continuously blown into the chelating liquid during the enzymolysis. The invention carries out enzymolysis under the ultrasonic condition to improve the enzymolysis efficiency and effect, but the ultrasonic wave has heat effect at the same time, and can change the temperature of the whole system, so that the whole enzymolysis process is unstable, therefore, the invention solves the problems by introducing nitrogen during enzymolysis, and the introduced nitrogen can play a role of stirring, so that the raw materials of the enzymolysis reaction can be mixed more uniformly, and the enzymolysis is more sufficient; secondly, the heat dissipation function is achieved, so that heat generated by enzymolysis reaction and ultrasonic waves can be dissipated in time, and the temperature is kept stable during enzymolysis; the introduction amount of nitrogen is based on that the primary enzymolysis liquid does not splash; NaHCO 23The solution plays a role of buffering,so as to stabilize the pH value of the whole reaction system.
(4) And centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid skin active polypeptide solution.
Preferably, in the step (1), 30-50 ml of sodium hydroxide solution is added to each gram of squid skin, and the mass percentage concentration of the sodium hydroxide solution is 0.2-0.5%.
Preferably, in the step (1), 30-50 ml of n-butyl alcohol solution is added to each gram of squid skin, and the mass percentage concentration of the n-butyl alcohol solution is 10-15%.
Preferably, in the step (3), 14000-15000 u of complex enzyme is added into each gram of the primary enzymolysis liquid, and the complex enzyme comprises the following enzymes in percentage by mass: 20-30% of alkaline protease, 10-20% of trypsin and the balance of neutral protease. According to the invention, the enzyme is screened, and the squid skin is subjected to enzymolysis by selecting the compound enzyme consisting of the alkaline protease, the trypsin and the neutral protease, so that the enzymolysis efficiency is high, the enzymolysis effect is good, and the antioxidant activity of the obtained polypeptide is high.
Preferably, in the step (3), the addition amount of the sodium bicarbonate is 0.5-1% of the mass of the primary enzymolysis liquid.
Preferably, in the step (3), the ultrasonic process conditions are as follows: the ultrasonic frequency is 25-40 kHz, and the power is 100-120W.
Therefore, the beneficial effects of the invention are as follows: the method has the advantages that the compound enzyme is used as the suitable enzyme for preparing the polypeptide from the squid skin, the preparation process of the squid skin active polypeptide is optimized, the process steps are simple, the reaction process is easy to control, the operability is strong, the production period is short, the hydrolysis degree of the squid skin is high, the obtained squid skin active polypeptide has high antioxidant activity, and the theoretical basis is provided for industrial production.
Detailed Description
The invention is further described below by means of specific embodiments.
In the present invention, all percentages are by weight unless otherwise specified, all equipment and materials are commercially available or commonly used in the industry, and the methods in the following examples are conventional in the art unless otherwise specified.
Example 1
(1) Cleaning squid skin, mashing, adding a sodium hydroxide solution with the mass percentage concentration of 0.2%, adding 30ml of the sodium hydroxide solution into each gram of the squid skin, soaking for 24 hours, washing with water until the pH value is neutral, adding a n-butyl alcohol solution with the mass percentage concentration of 10%, adding 30ml of the n-butyl alcohol solution into each gram of the squid skin, stirring for 16 hours, washing with water, and draining.
(2) Adding water which is 90 times of the weight of the squid skin into the drained squid skin, uniformly stirring, heating to 40 ℃, keeping the temperature for 3 hours, and adjusting the pH value to 8 to obtain a primary enzymolysis liquid.
(3) Adding complex enzyme and sodium bicarbonate into the primary enzymolysis liquid, performing ultrasonic enzymolysis for 20min at 40 ℃, inactivating enzyme, and cooling to obtain final enzymolysis liquid, continuously blowing nitrogen into chelating liquid during enzymolysis, adding 14000u of complex enzyme into each gram of primary enzymolysis liquid, wherein the complex enzyme comprises the following enzymes in percentage by mass: 20% of alkaline protease, 10% of trypsin and the balance of neutral protease; the adding amount of the sodium bicarbonate is 0.5 percent of the mass of the primary enzymolysis liquid; the ultrasonic process conditions are as follows: the ultrasonic frequency is 25kHz, and the power is 100W.
(4) And centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid skin active polypeptide solution.
Example 2
(1) Cleaning squid skin, mashing, adding a sodium hydroxide solution with the mass percentage concentration of 0.5%, adding 50ml of the sodium hydroxide solution into each gram of the squid skin, soaking for 30h, washing with water until the pH value is neutral, adding a n-butyl alcohol solution with the mass percentage concentration of 15%, adding 50ml of the n-butyl alcohol solution into each gram of the squid skin, stirring for 24h, washing with water, and draining.
(2) Adding water with the mass 95 times of that of the drained squid skin into the drained squid skin, uniformly stirring, heating to 60 ℃, keeping the temperature for 5 hours, and adjusting the pH to 9 to obtain a primary enzymolysis liquid.
(3) Adding complex enzyme and sodium bicarbonate into the primary enzymolysis liquid, performing ultrasonic enzymolysis for 30min at 50 ℃, inactivating enzyme, and cooling to obtain final enzymolysis liquid, continuously blowing nitrogen into chelating liquid during enzymolysis, adding 15000u of complex enzyme into each gram of primary enzymolysis liquid, wherein the complex enzyme comprises the following enzymes in percentage by mass: 30% of alkaline protease, 20% of trypsin and the balance of neutral protease; the adding amount of the sodium bicarbonate is 1 percent of the mass of the primary enzymolysis liquid; the ultrasonic process conditions are as follows: the ultrasonic frequency is 40kHz and the power is 120W.
(4) And centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid skin active polypeptide solution.
Example 3
(1) Cleaning squid skin, mashing, adding a sodium hydroxide solution with the mass percentage concentration of 0.3%, adding 40 ml of the sodium hydroxide solution into each gram of the squid skin, soaking for 25 h, washing with water until the pH value is neutral, adding a n-butanol solution with the mass percentage concentration of 12%, adding 40 ml of the n-butanol solution into each gram of the squid skin, stirring for 20h, washing with water, and draining.
(2) Adding water which is 92 times of the weight of the squid skin into the drained squid skin, uniformly stirring, heating to 45 ℃, keeping the temperature for 4 hours, and adjusting the pH value to 8.5 to obtain a primary enzymolysis liquid.
(3) Adding complex enzyme and sodium bicarbonate into the primary enzymolysis liquid, performing ultrasonic enzymolysis for 25min at 45 ℃, inactivating enzyme, and cooling to obtain final enzymolysis liquid, continuously blowing nitrogen into the chelating liquid during the enzymolysis, adding 14500u of complex enzyme into each gram of the primary enzymolysis liquid, wherein the complex enzyme comprises the following enzymes in percentage by mass: 25% of alkaline protease, 12% of trypsin and the balance of neutral protease; the adding amount of the sodium bicarbonate is 0.6 percent of the mass of the primary enzymolysis liquid; the ultrasonic process conditions are as follows: the ultrasonic frequency is 30kHz and the power is 110W.
(4) And centrifuging the final enzymolysis liquid, and collecting supernatant to obtain the squid skin active polypeptide solution.
The hydrolysis degree of the squid skin is more than 18.5%, and the DPPH free radical clearance rate of the obtained squid skin active polypeptide is more than 45%. Therefore, the preparation method disclosed by the invention has the advantages that the squid skin hydrolysis degree is high, the obtained squid skin active polypeptide has high antioxidant activity, and a theoretical basis is provided for industrial production.
The above-described embodiments are only preferred embodiments of the present invention, and are 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.