CN110585244B - Low-temperature crude extraction process of squid ink antioxidant substances - Google Patents
Low-temperature crude extraction process of squid ink antioxidant substances Download PDFInfo
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- 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
- A23L17/20—Fish extracts
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- 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
- A23L17/50—Molluscs
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- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- A61K35/56—Materials from animals other than mammals
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention provides a low-temperature crude extraction process of squid ink antioxidant substances, which comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities by using coarse filter cloth, adding squid viscera self-solution, inactivating enzyme, filtering, adjusting pH by alkali liquor, adding magnetic immobilized complex enzyme for enzymolysis under stirring, circularly stirring, enabling the enzyme to fully contact with a substrate, and stopping reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH with acid liquor, centrifuging, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging until the washing water is neutral, vacuum freeze drying, and preserving at-20deg.C. The raw materials used in the invention are abundant and easy to obtain, the enzyme method immobilization technology can be reused, the cost is reduced, the oxidation rate of the product is reduced by low-temperature treatment, the content of melanin and ink polysaccharide with oxidation resistance is extremely high, and the substance can be used for preparing oxidation-resistant health-care foods or medicines.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a low-temperature crude extraction process of squid ink antioxidant substances.
Background
In recent years, along with the development of the ocean water product fishing industry in China, the fishing amount of squid and the total amount of international squid feed processing reach hundreds of thousands of tons each year, wherein the squid ink sac of the processing waste is 1.28% of the weight of squid, and the squid ink sac is considerable in quantity. The document reports that the squid ink antioxidant substance in the ink sac has the functions of resisting oxidization and improving the immunity of patients and has stronger anti-radiation effect.
The Loligo chinensis Gray is marine animals of the family Equisetaceae of the phylum mollusca, class Cephalopoda. The annual fishing amount of squid worldwide is reported to reach 400 ten thousand tons, and China is one of the countries with the highest worldwide fishing amount. In the squid processing process, most processing factories discard squid ink sacs, which causes environmental pollution. The squid ink bag mainly comprises melanin and proteoglycan complex, the content of melanin in fresh ink is about 20%, and the squid ink bag belongs to true melanin and is a polymer of indoloquinone. Starting in the nineties, japanese students found that squid ink had potent antitumor activity (Sasaki J, et al J Nutr Sci Vitaminol,1997, 43 (4): 455) and isolated peptidoglycan as an active ingredient therefrom (Takaya Y, et al, biol Pharm,1994, 17 (6): 846). At present, a great deal of research on active substances such as squid ink proteoglycan, peptidoglycan and the like is carried out, but no report on the extraction process of the squid ink antioxidant substances is yet seen.
Disclosure of Invention
The invention provides a low-temperature crude extraction process of squid ink antioxidant substances, which adopts self-solution and magnetic immobilized enzyme for multistage enzymolysis, the self-solution enzymolysis is complete, the enzyme method immobilization technology can be repeatedly used, the cost is reduced, the oxidation rate of the product is reduced by low-temperature treatment, the content of melanin and ink polysaccharide with oxidation resistance is extremely high, the activity is strong, and the squid ink antioxidant substances can be used for preparing antioxidant foods, health foods or medicines.
The invention provides a low-temperature crude extraction process of squid ink antioxidant substances, which comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities by using coarse filter cloth, adding squid viscera self-solution, inactivating enzyme, filtering, adjusting pH to a first pH value by using alkali liquor, adding magnetic immobilized complex enzyme for enzymolysis under stirring, circularly stirring, enabling the enzyme to fully contact with a substrate, and stopping the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating the pH value to a second pH value by using acid liquor, centrifuging, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging until the washing water is neutral, vacuum freeze drying, and preserving at-20deg.C;
the preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with a stirrer to obtain squid viscera self-solution, wherein the enzyme deactivation condition is 105 ℃ for 10min, and the volume ratio of squid ink to squid viscera self-solution is (5-10): 1;
the preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: ferric chloride hexahydrate and ferrous chloride tetrahydrate are heated to the reaction temperature in nitrogen atmosphere, ammonia water is added dropwise, the constant temperature reaction is carried out for 2-5 hours under the protection of nitrogen, the temperature is reduced to the room temperature, the synthesized magnetic nano particles are washed by deionized water for a plurality of times, and the magnets are separated for standby;
s2, preparing immobilized complex enzyme: regulating concentrated solution with food grade lipase and food grade papain respectively, mixing with equal volume, adding sodium alginate to make sodium alginate 5wt%, sucking the mixture with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: respectively adding magnetic nano particles and ammonia water into deionized water, heating to a reaction temperature, then dropwise adding a silane coupling agent, reacting for 3-6 hours under the protection of nitrogen, adding immobilized complex enzyme, continuing to react for 2-3 hours, cooling to room temperature, washing the synthesized magnetic immobilized complex enzyme with deionized water for multiple times, and separating by a magnet to obtain the magnetic immobilized complex enzyme;
the reaction temperature is 50-60 ℃, and the mass ratio of the ferric chloride hexahydrate to the ferrous chloride tetrahydrate is 1: (2-3); the mass fraction of the ammonia water is 20-24%; the mass volume ratio of the ferric chloride hexahydrate to the ammonia water is 1: (10-30); the mass ratio of the magnetic nano particles to the silane coupling agent to the immobilized complex enzyme is 2 (0.01-0.03): 5, a step of; the mass volume ratio of the magnetic nano particles to the ammonia water is 1: (10-20);
the magnetic immobilized complex enzyme comprises 2000U/g food grade lipase 0.5-15wt% and 10 6 U/g food grade trichosanthin 1-10wt%.
As a further improvement of the invention, the first pH is 4.8-11.0; the second pH value is 3.0-5.0.
As a further improvement of the invention, the enzymolysis condition is that the enzymolysis time is 48-72h and the enzymolysis temperature is 15-20 ℃.
As a further improvement of the invention, the centrifugal condition is that the centrifugal speed is 10000-15000r/min, the centrifugal temperature is 4 ℃, and the centrifugal time is 10-20min.
As a further improvement of the invention, the freeze-drying condition is that the pre-cooling is carried out for 20-30min at the temperature of minus 10 ℃, and the freeze-drying is carried out for 10-15h at the temperature of minus 40 ℃.
The invention further protects the squid ink antioxidant substance prepared by the preparation method.
The invention further protects the application of the squid ink antioxidant substance prepared by the preparation method in preparing medicaments or auxiliary medicaments, health-care foods or foods for resisting oxidation related diseases.
The invention has the following beneficial effects: the magnetic nano particles and the immobilized complex enzyme are connected through the silane coupling agent, the prepared immobilized complex enzyme has magnetism, is convenient for magnetic separation, avoids complex steps such as enzyme deactivation, filtration, centrifugation and the like after enzymolysis reaction, simplifies operation, and can be reused without inactivating the enzyme after the immobilized enzyme is removed by the magnet, thereby reducing cost;
the invention has the advantages of abundant and easily obtained raw materials, high-value utilization of low-value products, repeated use of enzyme immobilization technology, cost reduction, low-temperature treatment to reduce the oxidation rate of the products, extremely high content of melanin and ink polysaccharide with oxidation resistance, strong activity, simple process steps, less equipment investment, no environmental pollution and high product yield. The material can be used for preparing antioxidant health food or medicine.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a graph showing the comparison of the ability to scavenge hydroxyl radicals in test example 1 of the present invention;
FIG. 2 is a graph showing the comparison of the ability of the present invention to scavenge superoxide anion radical in test example 2.
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: 10g of ferric chloride hexahydrate and 20g of ferrous chloride tetrahydrate are heated to the reaction temperature in nitrogen atmosphere, 100mL of 20wt% ammonia water is added dropwise, the temperature is kept constant for 50 ℃ for reaction for 2 hours under the protection of nitrogen, the temperature is reduced to the room temperature, the synthesized magnetic nano particles are washed for a plurality of times by deionized water, and the magnets are separated for later use;
s2, preparing immobilized complex enzyme: regulating concentrated solution with food grade lipase and food grade papain respectively, mixing with equal volume, adding sodium alginate to make sodium alginate 5wt%, sucking the mixture with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: 2g of magnetic nano particles and 20mL of 20wt% ammonia water are respectively added into deionized water, after the temperature is raised to the reaction temperature, 0.01g of silane coupling agent is dripped, the constant temperature reaction is carried out for 3 hours under the protection of nitrogen, 5g of immobilized complex enzyme is added, the reaction is continued for 2 hours, the temperature is reduced to the room temperature, the synthesized magnetic immobilized complex enzyme is washed for a plurality of times by the deionized water, and the magnet is separated, so that the magnetic immobilized complex enzyme is obtained, and the yield is 94%.
The magnetically immobilized complex enzyme comprises 2000U/g food grade lipase 0.5wt% and 10 6 U/g food grade papain 1wt%.
Example 2
The preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: 10g of ferric chloride hexahydrate and 30g of ferrous chloride tetrahydrate are heated to a reaction temperature in a nitrogen atmosphere, 300mL of 24wt% ammonia water is added dropwise, the temperature is kept constant at 60 ℃ for reaction for 2-5h under the protection of nitrogen, the temperature is reduced to room temperature, the synthesized magnetic nano particles are washed with deionized water for multiple times, and magnets are separated for later use;
s2, preparing immobilized complex enzyme: regulating concentrated solution with food grade lipase and food grade papain respectively, mixing with equal volume, adding sodium alginate to make sodium alginate 5wt%, sucking the mixture with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: 2g of magnetic nano particles and 40mL of 24wt% ammonia water are respectively added into deionized water, after the temperature is raised to the reaction temperature, 0.03g of silane coupling agent is dripped, the constant temperature reaction is carried out for 6 hours under the protection of nitrogen, 5g of immobilized complex enzyme is added, the reaction is continued for 3 hours, the temperature is reduced to the room temperature, the synthesized magnetic immobilized complex enzyme is washed for a plurality of times by the deionized water, and the magnet is separated, so that the magnetic immobilized complex enzyme is obtained, and the yield is 95%.
The magnetically immobilized complex enzyme comprises 2000U/g food grade lipase 15wt% and 10 6 U/g food grade trichosanthin 10wt%.
Example 3
The preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: 10g of ferric chloride hexahydrate and 25g of ferrous chloride tetrahydrate are heated to the reaction temperature in nitrogen atmosphere, 200mL of 22wt% ammonia water is added dropwise, the temperature is kept constant at 55 ℃ for reaction for 3.5 hours under the protection of nitrogen, the temperature is reduced to the room temperature, the synthesized magnetic nano particles are washed by deionized water for a plurality of times, and the magnets are separated for later use;
s2, preparing immobilized complex enzyme: regulating concentrated solution with food grade lipase and food grade papain respectively, mixing with equal volume, adding sodium alginate to make sodium alginate 5wt%, sucking the mixture with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: 2g of magnetic nano particles and 30mL of 22wt% ammonia water are respectively added into deionized water, after the temperature is raised to the reaction temperature, 0.02g of silane coupling agent is dripped, the constant temperature reaction is carried out for 4.5 hours under the protection of nitrogen, 5g of immobilized complex enzyme is added, the reaction is continued for 2.5 hours, the temperature is reduced to the room temperature, the synthesized magnetic immobilized complex enzyme is washed for a plurality of times by the deionized water, and the magnet is separated, so that the magnetic immobilized complex enzyme is obtained, and the yield is 97%.
The magnetically immobilized complex enzyme comprises 2000U/g food grade lipase 7wt% and 10 6 U/g food grade papain 5wt%.
Comparative example 1
The preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: 10g of ferric chloride hexahydrate and 2g of ferrous chloride tetrahydrate are heated to a reaction temperature in a nitrogen atmosphere, 50mL of 12wt% ammonia water is added dropwise, the temperature is kept constant for reaction for 1h under the protection of nitrogen, the temperature is reduced to room temperature, the synthesized magnetic nano particles are washed for a plurality of times by deionized water, and the magnets are separated for later use;
s2, preparing immobilized complex enzyme: regulating the concentrated solution with sterile water respectively, mixing with sodium alginate to make sodium alginate 1wt%, sucking the mixed solution with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: 2g of magnetic nano particles and 10mL of 12wt% ammonia water are respectively added into deionized water, after the temperature is raised to the reaction temperature, 0.02g of silane coupling agent is dripped, the constant temperature reaction is carried out for 1h under the protection of nitrogen, 1g of immobilized complex enzyme is added, the reaction is continued for 1h, the temperature is reduced to the room temperature, the synthesized magnetic immobilized complex enzyme is washed for a plurality of times by the deionized water, and the magnet is separated, so that the magnetic immobilized complex enzyme is obtained, and the yield is 51%.
The magnetically immobilized complex enzyme comprises 2000U/g food grade lipase 0.1wt% and 10 6 U/g food grade papain 0.5wt%.
Example 4
A low-temperature crude extraction process of squid ink antioxidant substances comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities with coarse filter cloth, adding squid viscera self-solution according to the volume ratio of squid ink to squid viscera self-solution of 5:1, self-dissolving for 1h, inactivating enzyme at 105 ℃ for 10min, filtering, adjusting pH to 4.8 with alkali liquor, adding the magnetic immobilized complex enzyme prepared in example 1 under stirring for enzymolysis, wherein the enzymolysis time is 48h, the enzymolysis temperature is 15 ℃, and circularly stirring to enable the enzyme to fully contact with a substrate, and terminating the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH to 3.0 with acid solution, centrifuging at 10000r/min for 10min at 4deg.C, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 20min, freeze-drying at-40deg.C for 10h, and preserving at-20deg.C.
The preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with stirring machine to obtain squid viscera self-solution.
Example 5
A low-temperature crude extraction process of squid ink antioxidant substances comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities with coarse filter cloth, adding squid viscera self-solution according to the volume ratio of squid ink to squid viscera self-solution of 10:1, self-dissolving for 2h, inactivating enzyme at 105 ℃ for 10min, filtering, regulating pH to 11.0 with alkali liquor, adding the magnetic immobilized complex enzyme prepared in example 2 under stirring for enzymolysis, wherein the enzymolysis time is 72h, the enzymolysis temperature is 20 ℃, and circularly stirring to enable the enzyme to fully contact with a substrate, and terminating the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH to 5.0 with acid liquor, centrifuging at 15000r/min at 4deg.C for 20min, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 30min, freeze-drying at-40deg.C for 15h, and preserving at-20deg.C.
The preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with stirring machine to obtain squid viscera self-solution.
Example 6
A low-temperature crude extraction process of squid ink antioxidant substances comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities with coarse filter cloth, adding squid viscera self-solution according to the volume ratio of squid ink to squid viscera self-solution of 7:1, self-dissolving for 1.5h, inactivating enzyme at 105 ℃ for 10min, filtering, adjusting pH to 6 with alkali liquor, adding the magnetic immobilized complex enzyme prepared in example 3 under stirring for enzymolysis, wherein the enzymolysis time is 64h, the enzymolysis temperature is 17 ℃, and circularly stirring to enable the enzyme to fully contact with a substrate, and terminating the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH to 4.0 with acid liquor, centrifuging at 12500r/min for 15min at 4deg.C, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 25min, freeze-drying at-40deg.C for 12 hr, and preserving at-20deg.C.
The preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with stirring machine to obtain squid viscera self-solution.
Comparative example 2
The preparation process parameters were different compared to example 6.
A low-temperature crude extraction process of squid ink antioxidant substances comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities with coarse filter cloth, adding squid viscera self-solution according to the volume ratio of squid ink to squid viscera self-solution of 7:1, self-dissolving for 1.5h, inactivating enzyme at 105 ℃ for 10min, filtering, adjusting pH to 6 with alkali liquor, adding the magnetic immobilized complex enzyme prepared in comparative example 1 under stirring for enzymolysis, wherein the enzymolysis time is 64h, the enzymolysis temperature is 17 ℃, and circulating stirring to enable the enzyme to fully contact with a substrate, and terminating the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH to 4.0 with acid liquor, centrifuging at 12500r/min for 15min at 4deg.C, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 25min, freeze-drying at-40deg.C for 12 hr, and preserving at-20deg.C.
The preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with stirring machine to obtain squid viscera self-solution.
Comparative example 3
Compared with example 6, the viscera-free solution was replaced by a papain solution, and the papain solution was added in an amount of 500U/kg.
A low-temperature crude extraction process of squid ink antioxidant substances comprises the steps of thawing squid ink, adding distilled water, stirring, washing, removing impurities by using coarse filter cloth, adding papain solution according to the volume ratio of the squid ink to the papain solution of 7:1, adjusting the pH to 6, carrying out enzymolysis for 1.5 hours, inactivating enzyme at 105 ℃ for 10min, filtering, adjusting the pH to 6 by using alkali liquor, adding the magnetic immobilized complex enzyme prepared in the embodiment 3 for enzymolysis under stirring, wherein the enzymolysis time is 64 hours, the enzymolysis temperature is 17 ℃, and carrying out circular stirring to enable the enzyme to be fully contacted with a substrate, and terminating the reaction when the magnetic immobilized complex enzyme is removed by using a magnet; regulating pH to 4.0 with acid liquor, centrifuging at 12500r/min for 15min at 4deg.C, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 25min, freeze-drying at-40deg.C for 12 hr, and preserving at-20deg.C.
Comparative example 4
In contrast to example 6, no magnetically immobilized complex enzyme was added.
A low-temperature crude extraction process of oxidation-resistant substances of squid ink comprises thawing squid ink, adding distilled water, stirring, washing, removing impurities with coarse filter cloth, adding squid viscera self-solution according to the volume ratio of squid ink to squid viscera self-solution of 7:1, self-dissolving for 1.5h, inactivating enzyme at 105 ℃ for 10min, centrifuging at 12500r/min, centrifuging at 4 ℃ for 15min, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging at 2000r/min for 10min until the washing water is neutral, vacuum freeze-drying, pre-cooling at-10deg.C for 25min, freeze-drying at-40deg.C for 12 hr, and preserving at-20deg.C.
The preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with stirring machine to obtain squid viscera self-solution.
Test example 1 test for scavenging hydroxyl radical
The test method comprises the following steps:
taking 8-branch test tubes with stopper, and adding 0.2mL of 10mmol/L FeSO respectively 4 EDTA mixture, 0.2mL of 20 mmol/L2-D-deoxyribose solution, 0.2mL of the test sample (distilled water, examples 4-6, comparative examples 2-4 and positive control vitamin C), PBS was used to supplement 1.8mL, finally 0.2mL of 10mmol/L hydrogen peroxide was added, the reaction was heated in a 37℃water bath for 1 hour, benzoic acid (1 mg/mL) and ascorbic acid (1 mg/mL) were used as controls, 1mL of 10% trichloroacetic acid was added to terminate the reaction, 1mL of 1% thiobarbituric acid was added, the mixture was heated in a boiling water bath for 10 minutes after mixing, the supernatant was centrifuged after cooling, and the absorbance was measured at 532 nm.
Scavenging rate of hydroxyl radical/% = (a) Control- A Sample of ）/A Control ×100
The results are shown in FIG. 1.
Test example 2 determination of the superoxide anion radical scavenging Capacity
The experimental method comprises the following steps: the method of pyrogallol autoxidation is adopted to measure the capability of scavenging superoxide anion free radicals.
Measurement of the reaction rate of the pyrogallol autoxidation: the measurement test is carried out at 25 ℃, 1.5mL of Tris-HCl-EDTA buffer (pH 8.2) and 0.1mL of 6mmol/L of pyrogallol (10 mmol/L hydrochloric acid is used for replacing pyrogallol in a control group) are added into a test tube with a plug, deionized water is used for supplementing to 3mL, after the mixture is quickly mixed uniformly, the control tube is used for zeroing, absorbance is measured every 0.5min at the wavelength of 420nm, the total absorbance is measured for 4min, the absorbance is plotted with time, and the self-oxidation reaction speed (delta OD 420/min) is obtained according to the slope of a linear change part; the dosage of the pyrogallol is properly changed to ensure that the autoxidation speed is 0.02 delta OD 420 And/or about min.
Influence of the sample on the reaction rate of pyrogallol autoxidation: the measurement process is the same as that of pyrogallol autoxidation, 0.1mL of sample (distilled water, examples 4-6, comparative examples 2-4 and positive control vitamin C) is added into the reaction system, and the concentration of the sample is properly regulated to ensure that the oxidation reaction speed under inhibition is 0.007-0.013 delta OD 420 /min。
Superoxide anion clearance/% = [ Δod 420 Min (autoxidation) - ΔOD 420 /min (sample tube)]/ΔOD 420 Per min (autoxidation) ×100
The results are shown in FIG. 2.
As can be seen from FIGS. 1 and 2, the squid ink antioxidant substances prepared in examples 1-3 of the invention have good effect of scavenging hydroxyl free radicals and capability of scavenging superoxide anion free radicals, and are obviously superior to vitamin C. The squid ink prepared by adopting different process parameters in comparative example 2 has greatly reduced antioxidant effect; in comparative example 3, visceral self-solution was not used and papain was substituted, the enzymolysis effect was inferior to the solubility of the self-solution, and a large amount of antioxidant substances were not obtained, and thus the antioxidant effect was reduced; in comparative example 4, no magnetic immobilized complex enzyme was added, and no effective enzymatic hydrolysis was performed to produce an antioxidant substance, so that the antioxidant property was poor. The combination of several processes can obtain peptide liquid with good antioxidation effect.
Compared with the prior art, the magnetic nano particles and the immobilized complex enzyme are connected through the silane coupling agent, the prepared immobilized complex enzyme has magnetism, is convenient for magnetic separation, avoids complex steps such as enzyme deactivation, filtration, centrifugation and the like after enzymolysis reaction, simplifies operation, and can be reused without inactivation after the immobilized enzyme is removed by the magnet, so that the cost is reduced;
the invention has the advantages of abundant and easily obtained raw materials, high-value utilization of low-value products, repeated use of enzyme immobilization technology, cost reduction, low-temperature treatment to reduce the oxidation rate of the products, extremely high content of melanin and ink polysaccharide with oxidation resistance, strong activity, simple process steps, less equipment investment, no environmental pollution and high product yield. The material can be used for preparing antioxidant health food or medicine.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. A low-temperature crude extraction process of squid ink antioxidant substances is characterized in that squid ink is taken to defrost, distilled water is added to stir and wash, impurities are removed by using coarse filter cloth, squid viscera are added to self-solution, enzyme is deactivated and filtered after self-dissolution is carried out for 1-2 hours, alkali liquor is used for regulating pH value to a first pH value, magnetic immobilized complex enzyme is added to carry out enzymolysis under stirring, cyclic stirring is carried out, enzyme and substrate are fully contacted, and reaction is stopped when the magnetic immobilized complex enzyme is removed by using a magnet; regulating the pH value to a second pH value by using acid liquor, centrifuging, and collecting precipitate; repeatedly washing the precipitate with distilled water, centrifuging until the washing water is neutral, vacuum freeze drying, and preserving at-20deg.C;
the preparation method of the viscera self-solution comprises the following steps: adding deionized water into squid viscera at ratio of 1:5, and mincing with a stirrer to obtain squid viscera self-solution, wherein the enzyme deactivation condition is 105 ℃ for 10min, and the volume ratio of squid ink to squid viscera self-solution is (5-10): 1;
the preparation method of the magnetic immobilized complex enzyme comprises the following steps:
s1, preparation of magnetic nano particles: ferric chloride hexahydrate and ferrous chloride tetrahydrate are heated to the reaction temperature in nitrogen atmosphere, ammonia water is added dropwise, the constant temperature reaction is carried out for 2-5 hours under the protection of nitrogen, the temperature is reduced to the room temperature, the synthesized magnetic nano particles are washed by deionized water for a plurality of times, and the magnets are separated for standby;
s2, preparing immobilized complex enzyme: regulating concentrated solution with food grade lipase and food grade papain respectively, mixing with equal volume, adding sodium alginate to make sodium alginate 5wt%, sucking the mixture with syringe, and dripping 3.5wt% CaCl dropwise 2 Preparing a solution, preparing immobilized microspheres, crosslinking for 12 hours, cleaning with sterile water, and grinding to 10-100nm for later use;
s3, preparing magnetic immobilized complex enzyme: respectively adding magnetic nano particles and ammonia water into deionized water, heating to a reaction temperature, then dropwise adding a silane coupling agent, reacting for 3-6 hours under the protection of nitrogen, adding immobilized complex enzyme, continuing to react for 2-3 hours, cooling to room temperature, washing the synthesized magnetic immobilized complex enzyme with deionized water for multiple times, and separating by a magnet to obtain the magnetic immobilized complex enzyme;
the reaction temperature is 50-60 ℃, and the mass ratio of the ferric chloride hexahydrate to the ferrous chloride tetrahydrate is 1: (2-3); the mass fraction of the ammonia water is 20-24%; the mass volume ratio of the ferric chloride hexahydrate to the ammonia water is 1: (10-30); the mass ratio of the magnetic nano particles to the silane coupling agent to the immobilized complex enzyme is 2 (0.01-0.03): 5, a step of; the mass volume ratio of the magnetic nano particles to the ammonia water is 1: (10-20);
the magnetic immobilized complex enzyme comprises 2000U/g food grade lipase 0.5-15wt% and 10 6 U/g food grade trichosanthin 1-10wt%.
2. The low temperature crude extraction process of antioxidant substances of squid ink according to claim 1, wherein the first pH value is 4.8-11.0; the second pH value is 3.0-5.0.
3. The low-temperature crude extraction process of the squid ink antioxidant substances according to claim 1, wherein the enzymolysis condition is 48-72h and the enzymolysis temperature is 15-20 ℃.
4. The low-temperature crude extraction process of the squid ink antioxidant substances according to claim 1, wherein the centrifugation condition is that the centrifugation speed is 10000-15000r/min, the centrifugation temperature is 4 ℃, and the centrifugation time is 10-20min.
5. The low temperature crude extraction process of antioxidant substances of squid ink according to claim 1, wherein the freeze drying condition is pre-cooling for 20-30min at-10 ℃, and freeze drying for 10-15h at-40 ℃.
6. An oxidation-resistant material of squid ink prepared by the low-temperature crude extraction process according to any one of claims 1 to 5.
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