CN110684815A - Preparation method of squid ink active peptide - Google Patents
Preparation method of squid ink active peptide Download PDFInfo
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- CN110684815A CN110684815A CN201910754484.1A CN201910754484A CN110684815A CN 110684815 A CN110684815 A CN 110684815A CN 201910754484 A CN201910754484 A CN 201910754484A CN 110684815 A CN110684815 A CN 110684815A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 24
- 239000006228 supernatant Substances 0.000 claims abstract description 23
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- 108091005804 Peptidases Proteins 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 10
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- 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
Abstract
The invention relates to the technical field of biological extraction, and provides a preparation method of squid ink active peptide aiming at the problem that squid leftovers are discarded to pollute the environment, which comprises the following steps: (a) putting the squid in a squid processing device, taking out an ink sac of the squid and collecting squid ink; (b) mixing squid ink and water according to a volume ratio of 1 (10-20), adjusting pH value to 1-4, performing enzymolysis for 1-3 h at 30-40 ℃ by using 0.1% pepsin, then adjusting pH value to 7-9, performing enzymolysis for 1-2 h at 50-60 ℃ by using 0.1% compound protease to prepare enzymolysis liquid, and centrifuging to obtain supernatant; (c) adding 5-20% (m/v) trichloroacetic acid solution into the supernatant of the enzymolysis solution, mixing, standing, centrifuging to obtain supernatant, desalting, and freeze drying to obtain the squid ink active peptide. The method effectively utilizes the ink which is one of the squid leftovers to prepare the active peptide with rich physiological functions, and changes waste into valuable.
Description
Technical Field
The invention relates to the technical field of biological extraction, and particularly relates to a preparation method of squid ink active peptide.
Background
Squid is a common name of squid and soft fish in cephalopod marine animals. The squid is an important protein resource for human beings gradually because of delicious taste and rich nutrition, but inedible parts of the squid cannot be effectively utilized, the waste comprises ink sacs, viscera, epidermis and the like which account for about 30 percent of the mass of the squid, wherein the ink sacs account for 11.3 percent of the mass of the squid, and the waste is buried in the spot by a common treatment method, so that the waste of fishery resources is avoided, and the environmental pollution is also aggravated.
The squid ink sac contains ink, and the biomacromolecule polymer contained in the squid ink is a known unique natural endogenous biopolymer capable of protecting organisms from being damaged by radiation, contains a plurality of bioactive components such as polypeptide fragments, acidic polysaccharide, tyrosinase and the like, has the characteristics of removing free radicals and combining potential toxic ions, and has great application potential. Since scientists confirmed that squid ink is rich in anticancer substances, squid ink which has been regarded as waste is gradually valued. For example, a chinese patent publication No. CN 106174412A, entitled "a method for producing a cuttlefish juice sauce", discloses a method for producing a cuttlefish juice sauce, which belongs to the technical field of food processing, and includes the following process steps: 1) treating raw materials; 2) hot soup treatment; 3) cooling; 4) blending; 5) passing through a colloid mill; 6) homogenizing; 7) boiling; 8) hot filling; 9) sterilizing; 10) and (6) cooling. The natural cuttlefish ink and the resistant starch are used as raw materials, and the novel physical processing technology is adopted for production, so that the natural effects of melanin and proteoglycan complexes are retained to the greatest extent, and the sensory quality and the processing characteristics of the product are improved through scientific compatibility. The research on the biological activity of the squid ink is rare, and the squid ink sac is inconvenient to treat and brings some difficulties for the research. Accordingly, an ideal technical solution is needed to solve the above problems.
Disclosure of Invention
The invention provides a preparation method of squid ink active peptide in order to overcome the problem that the discarded squid leftovers pollute the environment, and the active peptide with rich physiological functions is prepared by effectively utilizing ink which is one of the squid leftovers, so that waste is changed into valuable. The invention also provides a device for conveniently extracting the ink, which prevents the squid ink from splashing in the extraction process.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of squid ink active peptide comprises the following steps:
(a) putting the squid in a squid processing device, taking out an ink sac of the squid and collecting squid ink;
(b) mixing squid ink and water according to a volume ratio of 1 (10-20), adjusting pH value to 1-4, performing enzymolysis for 1-3 h at 30-40 ℃ by using 0.1% pepsin, then adjusting pH value to 7-9, performing enzymolysis for 1-2 h at 50-60 ℃ by using 0.1% compound protease to prepare enzymolysis liquid, and centrifuging to obtain supernatant;
(c) adding 5-20% (m/v) trichloroacetic acid solution into the supernatant of the enzymolysis solution, mixing, standing, centrifuging to obtain supernatant, desalting, and freeze drying to obtain the squid ink active peptide.
The squid ink is fresh compared with the squid ink extracted from a refrigerated squid ink sac when being taken at present, and the squid ink sac is not worried about rupture in the transportation and storage processes. The squid ink is subjected to enzymolysis twice by pepsin and compound protease, so that the protein decomposition is more complete. And then, trichloroacetic acid is used for enabling the redundant protein to form insoluble salt to precipitate, and finally, desalting, freezing and drying are carried out to obtain the squid ink active peptide. The bioactive peptide is prepared from squid ink which is one of squid leftovers, waste is changed into valuable, and the prepared bioactive peptide has antibacterial and antioxidant activities, reduces the burden on the environment and is environment-friendly.
Preferably, the squid processing apparatus in the step (a) comprises a platform, support legs positioned below the platform and used for supporting, and a transparent cover covering the platform and used for preventing squid ink from splashing, wherein an operation hole is formed in the side surface of the cover and used for allowing a hand to pass through, a processing platform for placing squid, a water supply faucet, an ink bag pool for placing the ink bag and a stirrer are arranged on the platform, the stirrer acts on the ink bag in the ink bag pool, a filtering hole is formed in the bottom surface of the ink bag pool, a collecting barrel for collecting squid ink is arranged below the platform, and the bottom surface of the ink bag pool is communicated with the collecting barrel. The ink sac of the squid is positioned at the deeper part of the belly, the squid is not easy to take, and the squid is easy to break in the taking process, so that the ink of the squid is extremely difficult to clean when splashed onto clothes. Aiming at the problem, the invention designs the squid processing device which is specially used for taking squid ink, a transparent cover is covered above the platform, an operation hole is arranged on the cover, when squid is processed, both hands stretch into the cover, and the internal condition is observed through the cover. Firstly, putting the squid on a processing table, picking the head, taking the ink sac, simply washing the ink sac, putting the squid in an ink sac pool, processing all the squids, opening a stirrer, breaking the ink sac by the stirrer, enabling ink of the squid to flow out, and enabling the ink to flow into a collecting barrel through the bottom surface of the ink sac pool. The bottom surface of the ink sac pool is provided with a filtering hole, so that impurities such as ink sac fragments in squid ink can be filtered. So, pluck the ink sac from the squid and collect the processing procedure of squid ink and all go on in squid processing apparatus, squid ink can not spill squid processing apparatus, can keep the clean and tidy of whole process, and it is convenient to handle moreover, need not purchase the ink sac in advance and refrigerate, has avoided the metamorphism and the damage of ink sac in the storage process.
Preferably, the end of the stirrer is provided with a stirring paddle, the stirring paddle is provided with pointed cones facing the side surface and the bottom surface respectively, and the pointed cones are used for puncturing the ink bag. The side surface and the bottom surface of the stirring paddle are respectively provided with the pointed cones, so that the ink bag can be punctured from the side surface and can also be punctured right below the stirring paddle, and the squid ink can flow out conveniently.
Preferably, the aperture of the filtering hole on the bottom surface of the ink sac pool is 0.15-0.25 mm. The squid ink filtered out by the pore diameter can be directly used for the next operation without treatment, and is simple and convenient.
Preferably, the mass ratio of the compound protease in the step (b) is alkaline protease, papain, flavourzyme = (1-3): 2-4. The enzymolysis efficiency of the enzyme compounded by a plurality of proteases is higher than that of single enzyme, and the enzymolysis effect is better.
Preferably, the centrifugation in the step (b) is performed at 8000 rpm for 5-10 min.
Preferably, the volume ratio of the enzymolysis liquid supernatant to the trichloroacetic acid solution in the step (c) is 1: 1.
Therefore, the invention has the following beneficial effects: (1) the squid ink which is one of squid leftovers is effectively utilized to prepare the active peptide with rich physiological functions, so that waste is changed into valuable, and the burden on the environment is reduced; (2) the squid processing device is specially used for taking squid ink, the processing processes of taking the ink sac from the squid and collecting the squid ink are all carried out in the squid processing device, the squid ink cannot splash out of the squid processing device, the whole process can be kept clean and tidy, the processing is convenient, the ink sac is not required to be purchased in advance for refrigeration, and the ink sac is prevented from going bad and being damaged in the storage process; (3) the squid ink is subjected to two times of enzymolysis by pepsin and compound protease, so that the protein is decomposed more thoroughly; (4) the composite protease compounded by a plurality of proteases has higher enzymolysis efficiency and better enzymolysis effect than single enzyme.
Drawings
Fig. 1 is a schematic structural view of a squid processing apparatus.
Fig. 2 is a top view of the platform.
In the figure: 1. the device comprises a platform, 2, a processing platform, 21, anti-skid lines, 3, a water faucet, 4, an ink sac pool, 41, a filtering hole, 42, a funnel part, 43, a collecting barrel, 5, a stirrer, 51, a stirring paddle, 511, a pointed cone, 6, a squid pool, 7, a waste pool, 8, a cover, 81, an operation hole, 9 and supporting legs.
Detailed Description
As shown in fig. 1 and 2, the squid processing device comprises a platform 1, a support leg 9 positioned below the platform 1 for supporting and a transparent cover 8 covering the platform 1 for preventing squid ink from splashing, an operation hole 81 is arranged on the side surface of the cover 8 for allowing a hand to pass through, the operation hole 81 is separated from the platform 1 by a certain distance, the squid ink is prevented from splashing out of the operation hole 81, a layer of film which can be lifted from the lower part can be arranged on the operation hole 81 for blocking, two hands stretch into the operation hole 81 for processing the squid during operation, and the eyes observe the internal conditions through the transparent cover 8. Platform 1 is last from left to right to be equipped with waste material pond 7, processing platform 2, ink sac pond 4 and squid pond 6 in proper order, and the squid is put on processing platform 2, breaks open the process that the squid got the squid ink sac and goes on processing platform 2. In order to prevent the squid from slipping, the upper surface of the processing platform 2 is provided with uneven anti-slip lines 21. The middle of the treatment table 2 is provided with a rotatable water tap 3 which supplies water for the whole squid treatment device. And (4) taking out the ink sac of the squid, simply washing the dirty surface of the ink sac, and putting the squid into the ink sac pool 4. The squid can be further processed, the part which can not be eaten, such as the head, the viscera and the like, is taken out and put into a waste pool 7, and the squid meat is put into a squid pool 6. The waste pool 7 protrudes from the bottom of the platform 1, and can be taken down and cleaned during the treatment process and then is arranged at the bottom of the platform 1. Waste pool 7 and 6 bottoms in squid pond all are equipped with the outlet, with the water pipe intercommunication, discharge sewage. The ink bag pool 4 is provided with the stirrer 5 on the edge, the stirrer 5 comprises a stirring shaft and stirring paddles 51, the stirring shaft extends into the ink bag pool 4 and is close to the bottom of the ink bag pool 4, the stirring paddles 51 are arranged at the bottom end of the stirring shaft, the six stirring paddles 51 in the shape of a bent knife are uniformly distributed on the periphery of the bottom end of the stirring shaft, the stirring shaft rotates towards the bent direction of the bent knife of the stirring paddles 51 when working, the stirring paddles 51 can break and stir the ink bag to enable ink to flow out, the stirring paddles 51 are respectively provided with pointed cones 511 towards the side surface and the bottom surface at the inner side and the bottom of the bent knife, the ink bag can be punctured from the side surface, the ink bag under the stirring paddles 51 can also be punctured, and the outflow of squid. The bottom surface of the ink sac pool 4 is provided with a filtering hole 41 (only for illustration in the figure, not representing the actual size) with the aperture of 0.20 mm, so that the impurities such as ink sac fragments in the squid ink can be filtered, even the squid ink flowing out from the filtering hole 41 can be directly used for the next operation without treatment, and the method is simple and convenient. The outer side surface of the bottom surface of the ink sac pool 4 is provided with a funnel part 42 which is reduced from big to small, the bottom end of the funnel part 42 is communicated with a collecting barrel 43 arranged below the platform 1 through a water pipe, and the squid ink finally flows into the collecting barrel 43 to be collected. If the squid ink is not completely flowed out, the collecting barrel 43 can be set into a sealed container with two branch openings, one branch opening is connected with the bottom end of the funnel part 42 in a sealing way through a water pipe, and the other branch opening is connected with a vacuum pump to perform decompression and suction filtration on the squid ink.
The method for processing the squid in the squid processing device comprises the following steps: firstly, a squid to be processed is placed on a processing table 2, a cover 8 is covered, two hands extend into an operation hole 81 to carry out treatments such as head picking, ink sac taking, viscera taking and the like on the squid, the ink sac is simply washed and then placed in an ink sac pool 4, inedible heads, viscera and the like are placed in a waste pool 7 to wait for subsequent treatment, and the squid meat which is processed to be clean is placed in a squid pool 6. After all the squids are processed, the stirrer 5 is opened, the ink sac is broken by the stirrer 5, and the ink of the squids flows out and flows into the collecting barrel 43 through the filtering hole 41 on the bottom surface of the ink sac pool 4. The squid ink bag is picked from the squid and the squid ink is collected in the squid processing device, so that the squid ink cannot splash out of the squid processing device, the cleanness and tidiness of the whole process can be kept, the processing is convenient, the squid ink bag is not required to be purchased in advance for refrigeration, and the deterioration and the damage of the squid ink bag in the storage process are avoided; meanwhile, the squid meat and the inedible part are separated, and the whole squid processing process is convenient and tidy.
The technical solution of the present invention is further illustrated by the following specific examples.
In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.
Example 1
(a) Collecting squid ink in the squid processing device;
(b) mixing squid ink and water according to a volume ratio of 1:20, adjusting the pH value to 1, carrying out enzymolysis for 3 h at 30 ℃ by using 0.1% pepsin, then adjusting the pH value to 7, carrying out enzymolysis for 1 h at 50 ℃ by using 0.1% compound protease, preparing enzymolysis liquid by using the mass ratio of alkaline protease to papain to flavourzyme =1:2:4, and centrifuging at 6000 rpm for 15 min to obtain supernatant;
(c) adding 2 times volume of 5% (m/v) trichloroacetic acid solution into the supernatant of the enzymolysis solution, mixing uniformly, standing at room temperature for 60 min, centrifuging to obtain supernatant, dialyzing for desalting, and freeze-drying to obtain the squid ink active peptide.
Example 2
(a) Collecting squid ink in the squid processing device;
(b) mixing squid ink and water according to a volume ratio of 1:15, adjusting the pH value to 4, carrying out enzymolysis for 1 h at 40 ℃ by using 0.1% pepsin, then adjusting the pH value to 9, carrying out enzymolysis for 1 h at 60 ℃ by using 0.1% composite protease, preparing enzymolysis liquid by using 0.1% composite protease and alkaline protease, namely papain and flavourzyme, and centrifuging at 8000 rpm for 5 min to obtain supernatant;
(c) adding 20% (m/v) trichloroacetic acid solution with the same volume into the supernatant of the enzymolysis solution, mixing uniformly, standing at room temperature for 60 min, centrifuging to obtain supernatant, dialyzing for desalting, and freeze-drying to obtain the squid ink active peptide.
Example 3
(a) Collecting squid ink in the squid processing device;
(b) mixing squid ink and water according to a volume ratio of 1:10, adjusting the pH value to 3, carrying out enzymolysis for 2 h at 35 ℃ by using 0.1% pepsin, then adjusting the pH value to 8, carrying out enzymolysis for 2 h at 55 ℃ by using 0.1% compound protease, preparing enzymolysis liquid by using the mass ratio of alkaline protease to papain to flavourzyme =1:2:2, and centrifuging at 8000 rpm for 5 min to obtain supernatant;
(c) adding an isovolumetric 15% (m/v) trichloroacetic acid solution into the supernatant of the enzymatic hydrolysate, uniformly mixing, standing at room temperature for 60 min, centrifuging to obtain a supernatant, dialyzing for desalination, and freeze-drying to obtain the squid ink active peptide.
Example 4
(a) Collecting squid ink in the squid processing device;
(b) mixing squid ink and water according to a volume ratio of 1:10, adjusting the pH value to 3, carrying out enzymolysis for 2 h at 35 ℃ by using 0.1% pepsin, then adjusting the pH value to 8, carrying out enzymolysis for 2 h at 55 ℃ by using 0.1% compound protease, preparing enzymolysis liquid by using 0.1% compound protease in a mass ratio of alkaline protease to papain =1:2, centrifuging at 8000 rpm for 5 min, and taking supernatant;
(c) adding an isovolumetric 15% (m/v) trichloroacetic acid solution into the supernatant of the enzymatic hydrolysate, uniformly mixing, standing at room temperature for 60 min, centrifuging to obtain a supernatant, dialyzing for desalination, and freeze-drying to obtain the squid ink active peptide.
According to the invention, the squid ink which is one of squid leftovers is effectively utilized to prepare the bioactive peptide, waste is turned into wealth, and the prepared squid ink bioactive peptide has antibacterial and antioxidant activities, reduces the burden on the environment and is environment-friendly.
Claims (7)
1. A preparation method of squid ink active peptide is characterized by comprising the following steps:
(a) putting the squid in a squid processing device, taking out an ink sac of the squid and collecting squid ink;
(b) mixing squid ink and water according to a volume ratio of 1 (10-20), adjusting pH value to 1-4, performing enzymolysis for 1-3 h at 30-40 ℃ by using 0.1% pepsin, then adjusting pH value to 7-9, performing enzymolysis for 1-2 h at 50-60 ℃ by using 0.1% compound protease to prepare enzymolysis liquid, and centrifuging to obtain supernatant;
(c) adding 5-20% (m/v) trichloroacetic acid solution into the supernatant of the enzymolysis solution, mixing, standing, centrifuging to obtain supernatant, desalting, and freeze drying to obtain the squid ink active peptide.
2. The preparation method of the squid ink active peptide according to claim 1, wherein the squid processing device in the step (a) comprises a platform (1), support legs (9) which are positioned below the platform (1) and play a supporting role, and a transparent cover (8) which covers above the platform (1) and prevents squid ink from splashing, an operation hole (81) is formed in the side surface of the cover (8) for a hand to pass through, a processing platform (2) for placing squid, a water faucet (3) for supplying water, an ink sac pool (4) for placing the ink sac and a stirrer (5) are arranged on the platform (1), the stirrer (5) acts on the ink sac in the ink sac pool (4), a filtering hole (41) is formed in the bottom surface of the ink sac pool (4), a collecting barrel for collecting squid ink is arranged below the platform (1), and the bottom surface of the ink sac pool (4) is communicated with the collecting barrel (43).
3. The method for preparing the squid ink active peptide according to the claim 2, wherein the end of the stirrer (5) is provided with a stirring paddle (51), the stirring paddle (51) is provided with pointed cones (511) which respectively face to the side surface and the bottom surface, and the pointed cones (511) are used for puncturing ink sacs.
4. The method for preparing the squid ink active peptide according to the claim 2, wherein the aperture of the filtering hole (411) on the bottom surface of the ink sac pool (4) is 0.15-0.25 mm.
5. The method for preparing the squid ink active peptide according to claim 1, wherein the mass ratio of the compound protease in the step (b) is alkaline protease, papain, flavourzyme = (1-3), 2-4 and 2-4.
6. The method for preparing squid ink active peptide according to claim 1, wherein the centrifugation condition in the step (b) is 8000 rpm for 5-10 min.
7. The method for preparing a squid ink active peptide according to claim 1, 5 or 6, wherein the volume ratio of the supernatant of the enzymolysis liquid and the trichloroacetic acid solution in the step (c) is 1: 1.
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