WO2019119998A1 - 一种适用于贻贝的蛋白源重金属脱除剂及其制备方法 - Google Patents

一种适用于贻贝的蛋白源重金属脱除剂及其制备方法 Download PDF

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WO2019119998A1
WO2019119998A1 PCT/CN2018/114324 CN2018114324W WO2019119998A1 WO 2019119998 A1 WO2019119998 A1 WO 2019119998A1 CN 2018114324 W CN2018114324 W CN 2018114324W WO 2019119998 A1 WO2019119998 A1 WO 2019119998A1
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enzymatic
minutes
mussels
meat
peptidase
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PCT/CN2018/114324
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English (en)
French (fr)
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杨会成
郑斌
廖妙飞
李瑞雪
洪瑶
周宇芳
相兴伟
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浙江省海洋开发研究院
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/22—Animal feeding-stuffs from material of animal origin from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/30—Oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the invention relates to a heavy metal removing agent, in particular to a protein source heavy metal removing agent suitable for mussels and a preparation method thereof, and belongs to the field of marine biological technology.
  • Mussel is a bivalve mollusc that lives on the rocks of the sea. It is a popular seafood product that can be steamed and cooked. It can also be sautéed with other vegetables and tasted delicious. According to analysis, each 100 grams of fresh shellfish contains 10.8 grams of protein, 2.4 grams of sugar, 2.4 grams of ash, 1.4 grams of fat, and the protein content of dried mussel meat is as high as 59.3%. Mussels also contain a variety of vitamins and essential trace elements such as manganese, zinc, selenium and iodine.
  • mussels have been extensively farmed and the yield is quite high.
  • Mussels are typical filter-feeding shellfish, such as jade mussels, purple mussels, thick-shelled mussels, and mussels. They use the cilia on the clams to filter suspended particles in the water, so it is necessary to filter a large amount of seawater. In addition, because the living environment of mussels is generally nearshore, mussels are more likely to be enriched in harmful heavy metals and organic compounds such as Cd, Pb and Hg than other marine organisms.
  • the invention also provides a preparation method of the above heavy metal removing agent.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are combined to enzymatically digest and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into a 2 to 3 times weight of sodium chloride solution, stirring and mixing to obtain a meat slurry, and then stirring to 8 ⁇
  • the 10kGy dose was irradiated for 5 to 10 minutes.
  • the fresh mussel meat is pretreated by removing the viscera from the fresh mussel meat, washing it with water for 2 to 3 times, and draining the water.
  • the comminution is to mash the fresh mussel meat using a high speed tissue masher.
  • the concentration of the sodium chloride solution is from 0.1 to 0.2 mol/L.
  • the enzymatic cosolvent is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1 to 2: 0.5 to 0.7.
  • the linear alcohol is selected from any one of n-hexadecanol or n-octadecanol.
  • the alkaline protease and trypsin complex enzymatic hydrolysis process conditions are: adjusting the pH to 7.5-8.5, adding a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and adding an enzymatic auxiliaries in the ultrasonic Under the shaking treatment, the enzyme is hydrolyzed at 50-60 ° C for 30-40 minutes, and the enzyme is deactivated to obtain the first enzymatic hydrolyzate; the mixed protease is added in an amount of 900-1200 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5-6, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 50-60 ° C for 10-20 minutes under ultrasonic vibration treatment, and inactivating the enzyme Live, get the second enzymatic hydrolysate; papain is added in an amount of 700-900 U per gram of meat slurry.
  • the specific method for separating and removing the macromolecular substance is: filtering the clear liquid through a ceramic membrane, and then separating the clear liquid through an ultrafiltration membrane having a molecular weight cut off of 5 to 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is: adjusting the pH of the permeate to 7-8, adding the complex peptidase, and enzymatically decomposing at 40-50 ° C for 10-20 minutes under ultrasonic vibration treatment. Enzyme activity, the third enzymatic solution is obtained, centrifuged to remove insoluble matter; then ferrous chloride and calcium chloride at a mass ratio of 1:1.2 are added, heated to 70-75 ° C with stirring, and chelate reaction is carried out by pH gradient adjustment method. After the reaction, it is concentrated to a paste, precipitated with absolute ethanol, and filtered by suction; the amount of the complex peptidase is 2000 to 2500 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.2 to 1.5.
  • the rotational speed of the centrifugal separation is 3,000 to 5,000 rpm, and the time is 20 to 30 minutes.
  • the specific method of the chelation reaction is to sequentially set four pH gradients of pH 4, pH 6, pH 8, and pH 10, and maintain the reaction at 70 to 75 ° C for 40 to 50 minutes under each pH gradient.
  • the ultrasonic oscillation process conditions are: a frequency of 40 to 60 kHz and a power of 300 to 320 W.
  • the enzyme is inactivated by a water bath at 85 to 100 ° C for 10 to 15 minutes.
  • the pH is adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • the invention provides a protein source heavy metal removing agent, which is prepared by using fresh mussel meat as a raw material, irradiating the meat slurry, and initially degrading the protein, and then sequentially performing the action of the enzymatic auxiliary agent.
  • It is a small molecular weight amino acid (rich in L-cysteine, containing a sulfhydryl group, especially for chelation of heavy metals), and can be chelated with ferrous ions and calcium ions.
  • the resulting chelate is particularly suitable for mussels. It can effectively remove harmful heavy metals such as lead and cadmium, and does not affect the calcium and zinc components contained in mussels.
  • Fresh mussel meat is made into a meat slurry by using sodium chloride solution, which helps the protein to dissolve or precipitate, which is beneficial to the degradation of protein under irradiation conditions.
  • the enzymatic auxiliary agent is a mixture of dimerized linoleic acid copolymer, alkylphenol ethoxylate and linear alcohol, and the three synergistically reduce the physical adsorption of the enzyme on the surface of the mussel meat raw material and promote the enzyme entry. Inside the mussel meat, the enzymatic efficiency is improved and the enzyme consumption is reduced.
  • the macromolecular substance is separated and removed, and then further enzymatic hydrolysis is carried out to obtain a third enzymatic solution which is basically degraded into a small molecular weight amino acid, and the third enzymatic solution
  • the liquid is rich in L-cysteine, which helps to chelate heavy metals. It chelate with ferrous ions and calcium ions, and has better removal of heavy metals than single ion chelate products.
  • the invention relates to alkaline protease, trypsin and papain, purchased from Guangzhou Dajiu Biotechnology Co., Ltd.; aminopeptidase, purchased from Nanjing Donghenghuadao Biotechnology Co., Ltd.; carboxypeptidase, purchased from Beijing Shengdong Technology Ltd. (Carboxypeptidase A, Bovine Pancreas).
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into a double weight sodium chloride solution (0.1 mol/L), stirring and mixing to obtain a meat slurry, and then stirring while stirring Irradiation was carried out for 5 minutes at a dose of 8 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1:0.5.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 7.5, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzyme digestion aid, under ultrasonic vibration treatment, 50 After enzymolysis for 30 minutes, the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 900 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 50 °C for 10 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 700 U per gram of the meat slurry.
  • a specific method for separating and removing the macromolecular substance is to obtain a clear solution by filtration through a ceramic membrane, and then separating the supernatant solution through an ultrafiltration membrane having a molecular weight cut off of 5 kDa to obtain a permeate.
  • Peptidase enzymatic hydrolysis and chelation reaction specific method is: adjust the pH of the permeate to 7, add the complex peptidase, under the ultrasonic vibration treatment, 40 °C enzymatic hydrolysis for 10 minutes, inactivate the enzyme to obtain the third enzymatic hydrolysate Centrifugation, remove insoluble matter; then add ferrous chloride and calcium chloride at a mass ratio of 1:1.2, stir and heat to 70 ° C, and carry out chelation reaction by pH gradient adjustment method. After the reaction is finished, concentrate to a paste, add or not The ethanol was precipitated by suction and filtered; the amount of the complex peptidase was 2000 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.2.
  • the centrifugal separation speed was 3000 rpm and the time was 20 minutes.
  • the ultrasonic vibration process conditions are: frequency 40 kHz, power 300 W.
  • the enzyme was inactivated for 10 minutes using a water bath at 85 °C.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into a three-fold weight of sodium chloride solution (0.2 mol/L), stirring and mixing to obtain a meat slurry, and then stirring while stirring Irradiation was carried out for 10 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and washed with water for 3 times to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:2:0.7.
  • the linear alcohol is n-octadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8.5, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 60 After enzymatic hydrolysis for 40 minutes, the enzyme was deactivated to obtain the first enzymatic hydrolysate; the mixed protease was added in an amount of 1200 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjust the pH to 6, add papain to the first enzymatic hydrolysate, enzymatically hydrolyze at 60 ° C for 20 minutes under ultrasonic shaking treatment, and inactivate the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 900 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • Peptidase enzymatic hydrolysis and chelation reaction specific method is: adjust the pH of the permeate to 8, add the complex peptidase, under the ultrasonic vibration treatment, enzymatic hydrolysis at 50 ° C for 20 minutes, inactivate the enzyme to obtain the third enzymatic hydrolysate , centrifugation, remove insoluble matter; then add ferrous chloride and calcium chloride at a mass ratio of 1:1.2, stir and heat to 75 ° C, and carry out chelation reaction by pH gradient adjustment method. After the reaction is finished, concentrate to a paste, add or not The ethanol was precipitated by suction and filtered; the amount of the complex peptidase was 2500 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.5.
  • the centrifugal separation speed was 5000 rpm and the time was 30 minutes.
  • the ultrasonic vibration process conditions are: frequency 60 kHz, power 320 W.
  • the enzyme was inactivated for 15 minutes using a 100 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into a double weight sodium chloride solution (0.2 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 10 minutes at a dose of 8 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:2:0.5.
  • the linear alcohol is n-octadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 7.5, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 60 After enzymolysis for 30 minutes, the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1200 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5, adding papain to the first enzymatic hydrolysate, enzymatically dissolving for 10 minutes at 60 ° C under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 900 U per gram of the meat slurry.
  • a specific method for separating and removing the macromolecular substance is to obtain a clear solution by filtration through a ceramic membrane, and then separating the supernatant solution through an ultrafiltration membrane having a molecular weight cut off of 5 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 8 and add the complex peptidase.
  • the enzyme Under ultrasonic vibration treatment, the enzyme is hydrolyzed at 40 ° C for 20 minutes to deactivate the enzyme to obtain the third enzymatic hydrolysate. Centrifugation, remove insoluble matter; then add ferrous chloride and calcium chloride at a mass ratio of 1:1.2, stir and heat to 70 ° C, and carry out chelation reaction by pH gradient adjustment method. After the reaction is finished, concentrate to a paste, add or not The ethanol was precipitated by suction and filtered; the amount of the complex peptidase was 2500 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.2.
  • the centrifugal separation speed was 5000 rpm and the time was 20 minutes.
  • the ultrasonic oscillation process conditions are: frequency 60 kHz, power 300 W.
  • the enzyme was inactivated for 10 minutes using a 100 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into a three-fold weight of sodium chloride solution (0.1 mol/L), stirring and mixing to obtain a meat slurry, and then stirring while stirring Irradiation was carried out for 5 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and washed with water for 3 times to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary agent is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1:0.7.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8.5, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 50 After enzymatic hydrolysis for 40 minutes, the enzyme was deactivated to obtain the first enzymatic hydrolysate; the mixed protease was added in an amount of 900 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are as follows: adjust the pH to 6, add papain to the first enzymatic hydrolysate, enzymatically hydrolyze at 50 °C for 20 minutes under ultrasonic vibration, and kill the enzyme to obtain a second enzymatic solution; papaya The protease was added in an amount of 700 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • Peptidase enzymatic hydrolysis and chelation reaction specific method is: adjust the pH of the permeate to 7, add the complex peptidase, under the ultrasonic vibration treatment, enzymatic hydrolysis at 50 ° C for 10 minutes, inactivate the enzyme to obtain the third enzymatic hydrolysate , centrifugation, remove insoluble matter; then add ferrous chloride and calcium chloride at a mass ratio of 1:1.2, stir and heat to 75 ° C, and carry out chelation reaction by pH gradient adjustment method. After the reaction is finished, concentrate to a paste, add or not The ethanol was precipitated by suction and filtered; the amount of the complex peptidase was 2000 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.5.
  • the centrifugal separation speed was 3000 rpm and the time was 30 minutes.
  • the ultrasonic oscillation process conditions are: frequency 40 kHz, power 320W.
  • the enzyme was inactivated for 15 minutes using a water bath at 85 °C.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into 2.5 times weight of sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 8 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1.5:0.6.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 55
  • the enzyme was decomposed for 35 minutes, and the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 7.5, add the complex peptidase, and digest the enzyme at 45 ° C for 15 minutes under ultrasonic vibration to deactivate the enzyme to obtain the third enzymatic hydrolysate. After centrifugation, the insoluble matter is removed; then, ferrous chloride and calcium chloride having a mass ratio of 1:1.2 are added, and the mixture is heated to 72 ° C with stirring, and the chelate reaction is carried out by a pH gradient adjustment method. After the reaction, the solution is concentrated to a paste, and the addition is completed. The ethanol was precipitated by ethanol and filtered by suction; the amount of the complex peptidase was 2200 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method for preparing the fresh mussel meat into the meat slurry is: pretreating the fresh mussel meat, pulverizing, pouring into a 2.5 times weight sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1.5:0.6.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 55
  • the enzyme was decomposed for 35 minutes, and the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 7.5, add the complex peptidase, and digest the enzyme at 45 ° C for 15 minutes under ultrasonic vibration to deactivate the enzyme to obtain the third enzymatic hydrolysate. After centrifugation, the insoluble matter is removed; then, ferrous chloride and calcium chloride having a mass ratio of 1:1.2 are added, and the mixture is heated to 72 ° C with stirring, and the chelate reaction is carried out by a pH gradient adjustment method. After the reaction, the solution is concentrated to a paste, and the addition is completed. The ethanol was precipitated by ethanol and filtered by suction; the amount of the complex peptidase was 2200 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into 2.5 times weight of sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 8 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, under ultrasonic vibration treatment, enzymatic hydrolysis at 55 ° C for 35 minutes, extinction Enzyme activity, the first enzymatic solution was obtained; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 7.5, add the complex peptidase, and digest the enzyme at 45 ° C for 15 minutes under ultrasonic vibration to deactivate the enzyme to obtain the third enzymatic hydrolysate. After centrifugation, the insoluble matter is removed; then, ferrous chloride and calcium chloride having a mass ratio of 1:1.2 are added, and the mixture is heated to 72 ° C with stirring, and the chelate reaction is carried out by a pH gradient adjustment method. After the reaction, the solution is concentrated to a paste, and the addition is completed. The ethanol was precipitated by ethanol and filtered by suction; the amount of the complex peptidase was 2200 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into 2.5 times weight of sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 8 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1.5:0.6.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 55
  • the enzyme was decomposed for 35 minutes, and the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of the chelation reaction is: adding ferrous chloride and calcium chloride to the permeate, heating to 72 ° C with stirring, and performing a chelate reaction by pH gradient adjustment, and then concentrating to a paste after the reaction, adding anhydrous Ethanol precipitation, suction filtration; the amount of complex peptidase was added to 2200 U per gram of meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further digested to carry out a chelation reaction with calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into 2.5 times weight of sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 8 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer, an alkylphenol ethoxylate and a linear alcohol, and the mass ratio of the three is 1:1.5:0.6.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 55
  • the enzyme was decomposed for 35 minutes, and the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 7.5, add the complex peptidase, and digest the enzyme at 45 ° C for 15 minutes under ultrasonic vibration to deactivate the enzyme to obtain the third enzymatic hydrolysate. , centrifuge, remove insoluble matter; then add calcium chloride, stir to 72 ° C, use the pH gradient method to carry out the chelation reaction, after the reaction is concentrated to a paste, add absolute ethanol precipitation, suction filtration; composite peptide The enzyme was added in an amount of 2200 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • a method for preparing a protein source heavy metal remover suitable for mussels comprising the steps of:
  • Fresh mussel meat is made into a meat slurry and irradiated
  • the alkaline protease and trypsin are mixed and enzymatically hydrolyzed, and then papain is hydrolyzed;
  • the macromolecular substance is separated and removed, and the peptidase is further enzymatically cleaved and reacted with ferrous ions and calcium ions.
  • the specific method of the irradiation treatment is: pretreating fresh mussel meat, pulverizing, pouring into 2.5 times weight of sodium chloride solution (0.15 mol/L), stirring and mixing to obtain a meat slurry, and then stirring. Irradiation was carried out for 8 minutes at a dose of 10 kGy.
  • the pretreatment method of fresh mussel meat is as follows: the fresh mussel meat is removed from the internal organs, and the water is washed twice with water to drain the water. Crush to crush the fresh mussel meat with a high speed tissue masher.
  • the enzymatic auxiliary is a mixture of a dimerized linoleic acid copolymer and a linear alcohol, and the mass ratio of the three is 1:0.6.
  • the linear alcohol is n-hexadecanol.
  • the alkaline protease and trypsin mixed enzymatic hydrolysis conditions are: adjust the pH to 8, add a mass ratio of 1:1 alkaline protease and trypsin mixed protease, and add enzymatic auxiliaries, under ultrasonic vibration treatment, 55
  • the enzyme was decomposed for 35 minutes, and the enzyme was deactivated to obtain the first enzymatic solution; the mixed protease was added in an amount of 1000 U per gram of the meat slurry.
  • the enzymatic hydrolysis conditions of papain are: adjusting the pH to 5.5, adding papain to the first enzymatic hydrolysate, enzymatically decomposing at 55 °C for 15 minutes under ultrasonic vibration, and inactivating the enzyme to obtain a second enzymatic hydrolyzate; papaya
  • the protease was added in an amount of 800 U per gram of the meat slurry.
  • the specific method for separating and removing the macromolecular substance is as follows: the clear liquid is filtered through a ceramic membrane, and then the supernatant is separated by an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to obtain a permeate.
  • the specific method of peptidase enzymatic hydrolysis and chelation reaction is to adjust the pH of the permeate to 7.5, add the complex peptidase, and digest the enzyme at 45 ° C for 15 minutes under ultrasonic vibration to deactivate the enzyme to obtain the third enzymatic hydrolysate. After centrifugation, the insoluble matter is removed; then, ferrous chloride and calcium chloride having a mass ratio of 1:1.2 are added, and the mixture is heated to 72 ° C with stirring, and the chelate reaction is carried out by a pH gradient adjustment method. After the reaction, the solution is concentrated to a paste, and the addition is completed. The ethanol was precipitated by ethanol and filtered by suction; the amount of the complex peptidase was 2200 U per gram of the meat slurry.
  • the complex peptidase is a mixture of aminopeptidase and carboxypeptidase, and the mass ratio of the two is 1:1.4.
  • the centrifugal separation speed was 4000 rpm and the time was 25 minutes.
  • the ultrasonic oscillation process conditions are: frequency 50 kHz, power 310 W.
  • the enzyme was deactivated for 12 minutes using a 90 ° C water bath.
  • the pH was adjusted using a 0.2 mol/L sodium hydroxide solution or a 0.2 mol/L hydrochloric acid solution.
  • a protein source heavy metal remover suitable for mussels is obtained by the above preparation method.
  • the heavy metal removing agents of Examples 1 to 5 effectively remove heavy metals such as lead and cadmium, and do not affect the calcium and zinc components which are beneficial to the human body contained in the mussels.
  • Comparative Example 1 was not subjected to irradiation treatment, Comparative Example 2 was not added with enzymatic auxiliaries, Comparative Example 3 omitted the peptidase treatment step, Comparative Example 4 only chelated calcium ions, and Comparative Example 5 of the enzymatic auxiliaries was omitted. Alkylphenol ethoxylates, lead, cadmium and other heavy metals removal effects are significantly worse, calcium and zinc content is also more or less affected.

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Abstract

一种适用于贻贝的蛋白源重金属脱除剂及其制备方法，以新鲜贻贝肉为原料，制成肉浆液后进行辐照处理，初步降解蛋白质，然后在酶解助剂的作用下，依次进行碱性蛋白酶和胰蛋白酶混合酶解、木瓜蛋白酶酶解，蛋白质基本降解为较小分子量的肽和氨基酸，分离除去大分子物质(多糖以及残留的少量蛋白质等)，肽酶进一步酶解，基本降解为小分子量的氨基酸(富含L-半胱氨酸，含有巯基，特别有助于螯合重金属)，与亚铁离子和钙离子螯合反应即可，所得螯合物适用于贻贝，可以有效进行铅、镉等重金属脱除，同时还不影响贻贝中含有的对人体有益的钙、锌成分。

Description

一种适用于贻贝的蛋白源重金属脱除剂及其制备方法 技术领域
本发明涉及一种重金属脱除剂，具体涉及一种适用于贻贝的蛋白源重金属脱除剂及其制备方法，属于海洋生物技术领域。
背景技术
近年来我国养殖区域污染问题日益严重，海产品食用安全问题日显突出，特别是重金属污染已经成为贻贝食品安全的极大隐患，重金属超标会对人体造成暂时或者持久损伤。
贻贝一种双壳类软体动物，生活在海滨岩石上，是一种广受人们喜爱的海鲜品，可以蒸、煮食之，也可剥壳后和其他青菜混炒，味均鲜美。据分析，每百克鲜贝肉含蛋白质10.8克，糖2.4克，灰分2.4克，脂肪1.4克，干制贻贝肉蛋白质含量高达59.3％。贻贝还含有多种维生素及人体必需的锰、锌、硒、碘等多种微量元素。值得一提的是，贻贝的营养价值高还由于它所含的蛋白质有人体需要的缬氨酸、亮氨酸等8种必需氨基酸，其含量大大高于鸡蛋以及鸡、鸭、鱼、虾和肉类等必需氨基酸的含量，被誉为海洋牛奶。另据研究，贻贝脂肪中还含有人体所必需的脂肪酸，其饱和脂肪酸的含量，较猪、牛、羊肉和牛奶等食品为低，不饱和脂肪酸的含量相对较高。被誉为海中鸡蛋。目前贻贝已经广泛人工养殖，产量颇高。
贻贝为典型滤食性贝类，如翡翠贻贝、紫贻贝、厚壳贻贝、隔贻贝等，其利用鳃上的纤毛过滤水中的悬浮颗粒为食，因此需要过滤大量的海水。此外，由于贻贝的生存环境一般是近岸，所以贻贝富集Cd、Pb和Hg等有害重金属和有机化合物的几率也高于其他海洋生物。
重金属污染导致贻贝产品不但无法出口，国内的销售也屡屡受挫，严重影响了贻贝产业的发展和人体食用有害重金属超标的贻贝引发的各种健康问题。因此，脱除贻贝中有害重金属的研究受到广泛关注。净水暂养或净化是一种重金属脱除的常用方法，但是一般耗时较长，而重金属脱除剂使用便捷，不失为一种替代选择。但是目前针对贻贝的重金属脱除剂产品较少，重金属脱除效果也有很大的改良空间。
发明内容
本发明的目的是为克服上述现有技术的不足，提供一种适用于贻贝的蛋白源重金属脱除剂。
本发明还提供了上述重金属脱除剂的制备方法。
为实现上述目的，本发明采用下述技术方案：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶复合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
优选的，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2～3倍重量的氯化钠溶液中，搅拌混匀得到肉浆液，然后边搅拌边以8～10kGy剂量辐照5～10分钟。
进一步优选的，新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2～3次，沥净水分即可。
进一步优选的，所述粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
进一步优选的，氯化钠溶液的浓度为0.1～0.2mol/L。
优选的，所述酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1～2：0.5～0.7。
进一步优选的，所述直链醇选自正十六醇或正十八醇中的任一种。
优选的，碱性蛋白酶和胰蛋白酶复合酶解的工艺条件为：调整pH至7.5～8.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，50～60℃酶解30～40分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入900～1200U。
进一步优选的，木瓜蛋白酶酶解的工艺条件为：调整pH至5～6，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，50～60℃酶解10～20分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入700～900U。
优选的，分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量5～10kDa的超滤膜膜进行分离，得到透过液。
优选的，肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7～8，加入复合肽酶，在超声波振荡处理下，40～50℃酶解10～20分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至70～75℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2000～2500U。
进一步优选的，所述复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.2～1.5。
进一步优选的，离心分离的转速为3000～5000转/分，时间为20～30分钟。
进一步优选的，螯合反应的具体方法是：依次设置pH4、pH6、pH8、pH10四个pH梯度，在每个pH梯度下70～75℃保温反应40～50分钟。
进一步优选的，超声波振荡工艺条件为：频率40～60kHz，功率300～320W。
进一步优选的，采用85～100℃水浴灭酶活10～15分钟。
进一步优选的，利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
本发明的有益效果：
1、本发明提供了一种蛋白源重金属脱除剂，是以新鲜贻贝肉为原料，制成肉浆液后进行辐照处理，初步降解蛋白质，然后在酶解助剂的作用下，依次进行碱性蛋白酶和胰蛋白酶复合酶解、木瓜蛋白酶酶解，蛋白质基本降解为较小分子量的肽和氨基酸，分离除去大分子物质(多糖以及残留的少量蛋白质等)，肽酶进一步酶解，基本降解为小分子量的氨基酸(富含L-半胱氨酸，含有巯基，特别有助于螯合重金属)，与亚铁离子和钙离子螯合反应即可，所得螯合物特别适用于贻贝，可以有效进行铅、镉等有害重金属脱除，同时还不影响贻贝中含有的对人体有益的钙、锌成分。
2、新鲜贻贝肉利用氯化钠溶液制成肉浆液，有助于蛋白质的溶解或析出，进而有 利于辐照条件下蛋白质的降解。
3、酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者协同作用，减少酶在贻贝肉原料表面的物理性吸附，促进酶进入贻贝肉内部，提高酶解效率，减少酶的消耗量。
4、当蛋白质基本降解为较小分子量的肽和氨基酸，先分离除去大分子物质，再利用肽酶进一步酶解，可以获得基本降解为小分子量的氨基酸的第三酶解液，第三酶解液中富含有助于螯合重金属的L-半胱氨酸，与亚铁离子和钙离子螯合反应，与单一离子螯合产品相比，具有更好的重金属脱除效果。
具体实施方式
下面结合实施例对本发明进行进一步的阐述，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
本发明涉及的碱性蛋白酶、胰蛋白酶和木瓜蛋白酶，购自广州大久生物科技有限公司；氨肽酶，购自南京东恒华道生物科技有限公司；羧肽酶，购自北京生东科技有限公司(羧肽酶A，牛胰)。
实施例1：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2倍重量的氯化钠溶液(0.1mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以8kGy剂量辐照5分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1：0.5。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至7.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，50℃酶解30分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入900U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，50℃酶解10分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入700U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量5kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7，加入复合肽酶，在超声波振荡处理下，40℃酶解10分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至70℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2000U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.2。
离心分离的转速为3000转/分，时间为20分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下70℃保温反应40分钟。
超声波振荡工艺条件为：频率40kHz，功率300W。
采用85℃水浴灭酶活10分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
实施例2：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入3倍重量的氯化钠溶液(0.2mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照10分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤3次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：2：0.7。直链醇为正十八醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，60℃酶解40分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1200U。
木瓜蛋白酶酶解的工艺条件为：调整pH至6，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，60℃酶解20分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入900U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至8，加入复合肽酶，在超声波振荡处理下，50℃酶解20分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至75℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2500U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.5。
离心分离的转速为5000转/分，时间为30分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下75℃保温反应50分钟。
超声波振荡工艺条件为：频率60kHz，功率320W。
采用100℃水浴灭酶活15分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
实施例3：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2倍重量的氯化钠溶液(0.2mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以8kGy剂量辐照10分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：2：0.5。直链醇为正十八醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至7.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，60℃酶解30分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1200U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，60℃酶解10分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入900U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量5kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至8，加入复合肽酶，在超声波振荡处理下，40℃酶解20分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至70℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2500U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.2。
离心分离的转速为5000转/分，时间为20分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下75℃保温反应40分钟。
超声波振荡工艺条件为：频率60kHz，功率300W。
采用100℃水浴灭酶活10分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
实施例4：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入3倍重量的氯 化钠溶液(0.1mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照5分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤3次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1：0.7。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，50℃酶解40分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入900U。
木瓜蛋白酶酶解的工艺条件为：调整pH至6，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，50℃酶解20分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入700U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7，加入复合肽酶，在超声波振荡处理下，50℃酶解10分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至75℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2000U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.5。
离心分离的转速为3000转/分，时间为30分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下70℃保温反应50分钟。
超声波振荡工艺条件为：频率40kHz，功率320W。
采用85℃水浴灭酶活15分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
实施例5：
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照8分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1.5：0.6。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，55℃酶解35分 钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7.5，加入复合肽酶，在超声波振荡处理下，45℃酶解15分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
对比例1
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，新鲜贻贝肉制成肉浆液的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1.5：0.6。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，55℃酶解35分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7.5，加入复合肽酶，在 超声波振荡处理下，45℃酶解15分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
对比例2
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照8分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，在超声波振荡处理下，55℃酶解35分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7.5，加入复合肽酶，在超声波振荡处理下，45℃酶解15分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
对比例3
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照8分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1.5：0.6。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，55℃酶解35分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
螯合反应的具体方法是：向透过液中加入氯化亚铁和氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
对比例4
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的 氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照8分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1.5：0.6。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，55℃酶解35分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7.5，加入复合肽酶，在超声波振荡处理下，45℃酶解15分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
对比例5
一种适用于贻贝的蛋白源重金属脱除剂的制备方法，包括步骤：
新鲜贻贝肉制成肉浆液后进行辐照处理；
在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
其中，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2.5倍重量的氯化钠溶液(0.15mol/L)中，搅拌混匀得到肉浆液，然后边搅拌边以10kGy剂量辐照8分钟。新鲜贻贝肉的预处理方法为：将新鲜贻贝肉除去内脏，利用清水洗涤2次，沥净水分即可。粉碎为利用高速组织捣碎机将新鲜贻贝肉捣碎。
酶解助剂为二聚亚油酸共聚物与直链醇的混合物，三者质量比为1：0.6。直链醇为正十六醇。
碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至8，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，55℃酶解35分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入1000U。
木瓜蛋白酶酶解的工艺条件为：调整pH至5.5，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，55℃酶解15分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入800U。
分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量10kDa的超滤膜膜进行分离，得到透过液。
肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7.5，加入复合肽酶，在超声波振荡处理下，45℃酶解15分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至72℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2200U。
复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.4。
离心分离的转速为4000转/分，时间为25分钟。
螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下72℃保温反应45分钟。
超声波振荡工艺条件为：频率50kHz，功率310W。
采用90℃水浴灭酶活12分钟。
利用0.2mol/L氢氧化钠溶液或0.2mol/L盐酸溶液调节pH。
一种适用于贻贝的蛋白源重金属脱除剂，是通过上述制备方法得到的。
试验例
1、将同一批养殖的贻贝，进行铅、镉染毒，随机分为10组，基础饲料喂食，分别将实施例1～5或对比例1～5的重金属脱除剂与基础饲料以2g/kg的比例混合进行日常饲养，饲养周期为10天，检测养殖前后的钙、锌、铅、镉平均含量，结果见表1。
表1.养殖前后重金属含量比较
Figure PCTCN2018114324-appb-000001
由表1可知，实施例1～5的重金属脱除剂有效进行铅、镉等重金属脱除，同时还不影响贻贝中含有的对人体有益的钙、锌成分。对比例1不进行辐照处理，对比例2未添加酶解助剂，对比例3略去了肽酶处理步骤，对比例4仅仅螯合钙离子，对比例5的酶解助剂略去了烷基酚聚氧乙烯醚，铅、镉等重金属脱除效果均明显变差，钙、锌含量也或多或少受到影响。
上述虽然对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。

Claims (10)

  1. 一种适用于贻贝的蛋白源重金属脱除剂的制备方法，其特征在于，包括步骤：
    新鲜贻贝肉制成肉浆液后进行辐照处理；
    在酶解助剂的作用下，先碱性蛋白酶和胰蛋白酶混合酶解，再木瓜蛋白酶酶解；
    分离除去大分子物质，肽酶进一步酶解，与亚铁离子和钙离子进行螯合反应。
  2. 根据权利要求1所述的制备方法，其特征在于，辐照处理的具体方法是：将预处理的新鲜贻贝肉，粉碎后倒入2～3倍重量的氯化钠溶液中，搅拌混匀得到肉浆液，然后边搅拌边以8～10kGy剂量辐照5～10分钟。
  3. 根据权利要求1所述的制备方法，其特征在于，所述酶解助剂为二聚亚油酸共聚物、烷基酚聚氧乙烯醚与直链醇的混合物，三者质量比为1：1～2：0.5～0.7。
  4. 根据权利要求1所述的制备方法，其特征在于，碱性蛋白酶和胰蛋白酶混合酶解的工艺条件为：调整pH至7.5～8.5，加入质量比1：1的碱性蛋白酶和胰蛋白酶的混合蛋白酶，并加入酶解助剂，在超声波振荡处理下，50～60℃酶解30～40分钟，灭酶活，得到第一酶解液；混合蛋白酶的加入量为每克肉浆液中加入900～1200U。
  5. 根据权利要求4所述的制备方法，其特征在于，木瓜蛋白酶酶解的工艺条件为：调整pH至5～6，向第一酶解液中加入木瓜蛋白酶，在超声波振荡处理下，50～60℃酶解10～20分钟，灭酶活，得到第二酶解液；木瓜蛋白酶的加入量为每克肉浆液中加入700～900U。
  6. 根据权利要求1所述的制备方法，其特征在于，分离除去大分子物质的具体方法是：经陶瓷膜滤清得清液，然后将清液经截留分子量5～10kDa的超滤膜膜进行分离，得到透过液。
  7. 根据权利要求1所述的制备方法，其特征在于，肽酶酶解以及螯合反应的具体方法是：调整透过液的pH至7～8，加入复合肽酶，在超声波振荡处理下，40～50℃酶解10～20分钟，灭酶活，得到第三酶解液，离心，除去不溶物；然后加入质量比1:1.2的氯化亚铁和氯化钙，搅拌加热至70～75℃，采用pH梯度调节法进行螯合反应，反应结束后浓缩至膏状，加无水乙醇沉淀，抽滤即得；复合肽酶的加入量为每克肉浆液中加入2000～2500U。
  8. 根据权利要求7所述的制备方法，其特征在于，所述复合肽酶为氨肽酶和羧肽酶的混合物，两者的质量比为1：1.2～1.5。
  9. 根据权利要求7所述的制备方法，其特征在于，螯合反应的具体方法是：依次设置pH＝4、pH＝6、pH＝8、pH＝10四个pH梯度，在每个pH梯度下70～75℃保温反应40～50分钟。
  10. 一种适用于贻贝的蛋白源重金属脱除剂，其特征在于，是通过权利要求1～9中任一项所述的制备方法得到的。
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