WO2016173222A1 - 一种低致敏低腥味鱼蛋白低聚肽及其工业化制备方法和应用 - Google Patents

一种低致敏低腥味鱼蛋白低聚肽及其工业化制备方法和应用 Download PDF

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WO2016173222A1
WO2016173222A1 PCT/CN2015/092932 CN2015092932W WO2016173222A1 WO 2016173222 A1 WO2016173222 A1 WO 2016173222A1 CN 2015092932 W CN2015092932 W CN 2015092932W WO 2016173222 A1 WO2016173222 A1 WO 2016173222A1
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Prior art keywords
fish
low
fish protein
oligopeptide
water
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PCT/CN2015/092932
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English (en)
French (fr)
Inventor
蔡木易
谷瑞增
鲁军
马涛
潘兴昌
董哲
马勇
徐亚光
马永庆
金振涛
陈亮
陆路
刘文颖
魏颖
张海欣
刘艳
曹珂璐
王憬
李国明
周明
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中国食品发酵工业研究院
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Priority to JP2017516108A priority Critical patent/JP6483252B2/ja
Publication of WO2016173222A1 publication Critical patent/WO2016173222A1/zh
Priority to US15/442,574 priority patent/US9974321B2/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • 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
    • A23L17/20—Fish extracts
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a fish protein oligopeptide, in particular to a low sensitization and low odor fish protein oligopeptide and an industrial preparation method and application thereof.
  • the fish protein rich in fish is a highly nutritious protein, which contains a large amount of nutrients necessary for the human body, and contributes to the reduction of blood lipids and cholesterol content, especially containing a large amount of DHA, which can promote brain development.
  • Nutrients that are indispensable for the growth of the nervous system therefore, more and more people choose to process fish products.
  • the odor of the fish product itself has a great influence on the taste of the processed product.
  • the molecular weight of the protein contained in the fish meat is too large, which is not conducive to the absorption of the human body. In some existing treatment technologies, it is usually used too much. Extreme pH and temperature environments hydrolyze macromolecular proteins, but adversely affect the properties of the protein.
  • fish allergy is considered to be one of the most common food allergies because fish protein contains a large amount of allergens, such as small albumin, which is confirmed by IgE antibodies. People have an allergic reaction. Heating and chemical treatment are common desensitization treatment methods, but it is impossible to remove all sensitizers of fish protein by denaturation of proteins by heating. Chemical treatment mainly uses chemical agents to reduce the activity of trypsin inhibitors. Avoid food safety problems such as chemical residues.
  • the publication No. CN102008004A discloses a method for preparing fish protein powder rich in fish skin protein.
  • the process mainly uses a neutral protease and bromelain to carry out two-step enzymatic hydrolysis of the fish skin raw material, although the enzymatic hydrolysis of the raw material is carried out.
  • the average molecular weight of the final product is below 6000 Da, but the allergens are not treated and detected, so it is impossible to determine whether the process can reduce the sensitizing activity, and the operation steps are too complicated.
  • Li Yang et al. used 2940 U/g papain in a phosphate buffer solution with a pH of 8.00 to hydrolyze the salmon fish protein at 40 ° C for 4 h, and the antigenicity of the final salmon protein was reduced by 58.33%, but not Antigen research on Xiaoqing protein, and it is also very likely to expose some linear epitopes hidden inside or in the hydrophobic region of the protein to make the enzymatic product new. Allergic concerns.
  • the invention provides a low-sensitization and low-smell fish protein oligopeptide and an industrial preparation method and application thereof, which are used for solving the problem that the fish protein sensitization, the product taste is not good and the protein molecular weight is too large in the prior art. Conducive to absorption and other technical defects.
  • the industrial preparation method of the low sensitization and low odor fish protein oligopeptide provided by the invention comprises the following steps:
  • step 3 the denatured protein solution is washed with water and then subjected to the centrifugation, and the water washing comprises:
  • the invention does not limit the fish species to be used, and may be a deep-sea fish or a fresh-water fish, and the fish waste refers to a substance remaining after removing the fish meat, such as fish skin or fish scale; after washing the fish meat material In order to make the subsequent heat denaturation effect better, the washed fish meat and/or fish waste can be crushed to 15-20 mesh.
  • the mass-to-volume ratio of the fish meat and/or fish scrap to water in the step 1) is 1: (1 to 5), that is, 1 kg of fish and/or fish waste is mixed with 1 to 5 L of water. A mixture was prepared.
  • the mixed liquid is prepared, when the water is added too little, the fluidity of the mixed liquid is poor, which tends to cause a decrease in the enzymatic hydrolysis efficiency; and when the water is too much, the reaction volume is too large, which may affect the subsequent treatment (for example, concentration, etc.), and the cost is also increased accordingly.
  • water may be pure water, distilled water, deionized water or the like, and deionized water is used in the present invention.
  • the mass to volume ratio of the precipitate to the water in the step 3) is 1: (1 to 5).
  • the thermal denaturation comprises: heating the mixture to 75-95 ° C, and maintaining the heat Stirring is continued for 10 to 60 minutes.
  • the heat denaturation treatment can destroy the spatial structure of the fish protein, thereby reducing the sensitization of the fish protein, and at the same time solving the problem of poor fluidity of the mixed solution and viscous solution, which is beneficial to the subsequent enzymatic hydrolysis.
  • the denatured protein solution is centrifuged, and the precipitate obtained by centrifugation is ground with water to prepare a slurry.
  • the rotation speed of the centrifuge is controlled to be 2000-8000 r/min, and the centrifugation time is 10 to 60 min. Since the precipitate obtained after centrifugation is a solid substance having a large volume, in order to thoroughly carry out the subsequent enzymatic hydrolysis process, further, Water is added to the precipitate, and the pulp is crushed to obtain a slurry.
  • the mass-to-volume ratio of the precipitate to water is controlled to be 1:1 to 5, and the slurry is crushed until the precipitate is 20 to 30 mesh.
  • step 3 the denatured protein solution is washed with water and then subjected to the centrifugation, the water washing comprising: After the denatured protein solution is centrifuged, water is added to the precipitate obtained by centrifugation for washing.
  • the mass to volume ratio of the precipitate to the water is 1: (1 to 5), wherein the centrifuge speed is controlled to 2000 to 8000 r/min, and the centrifuge is centrifuged.
  • the time is 10 to 60 minutes, and the water washing step can be repeated 2 to 3 times.
  • the inventors have done a great deal on how to completely eliminate the sensitization of fish protein by enzymatic method without losing the nutrient content of fish, while eliminating all the astringency of the fish product itself and inhibiting the production of bitter substances in the enzymatic hydrolysate.
  • Research As a result, it was found that most proteases could not completely eliminate the sensitization of fish protein and/or inhibit the production of bitter substances in the enzymatic product.
  • the treatment of fish protein with neutral protease can eliminate the sensitization of fish protein to a certain extent, but the desensitization effect is general.
  • the use of trypsin to treat fish protein eliminates the sensitization of fish protein.
  • the detected sensitized small albumin content has a certain increase.
  • the inventors unexpectedly discovered during the research that only by using neutral protease, papain, and alkaline protease for enzymatic hydrolysis, the sensitization of fish protein can be completely eliminated while inhibiting the bitter and astringent substances in the enzymatic hydrolysate. produce.
  • the amount of the neutral protease is 10 to 100 U/g
  • the amount of the papain is 10 to 100 U/g
  • the amount of the alkaline protease is 10 to 100 U/ g.
  • the ratio between the neutral protease, papain and alkaline protease is 1: (1 to 3): (1 to 3).
  • the pH can be adjusted to 6-9 using a sodium hydroxide aqueous solution having a mass concentration of 10 to 20%, the enzymatic hydrolysis is carried out at a temperature of 30 ° C to 60 ° C, and the enzymatic hydrolysis time is controlled.
  • too short enzymatic hydrolysis time ( ⁇ 1h) is not conducive to protein degradation, and too long (> 3h) may lead to the production of bitter and astringent substances.
  • the combination of neutral protease, papain and alkaline protease helps to control enzymatic hydrolysis while fully degrading fish protein to eliminate its sensitization
  • the bitter taste of the product can be further accepted by consumers.
  • the above enzymatic hydrolysis can reduce the main allergen albumin of fish protein by more than 99%, and at the same time, moderately hydrolyze fish protein to form a peptide with a smaller molecular weight (for example, a peptide having a molecular weight of less than 1000 Da), thereby facilitating the improvement of fish protein.
  • the rate of absorption of the human body is not conducive to protein degradation, and too long (> 3h) may lead to the production of bitter and astringent substances.
  • the combination of neutral protease, papain and alkaline protease helps to control
  • the amount of each enzyme is based on the weight of the fish and/or fish waste, that is, when 1 g of fish meat and/or fish waste is used, a neutral protease of 10 to 100 U is used. Further, the inactivation of the enzyme is carried out at a temperature of 110 to 120 ° C, and the time for controlling the inactivation of the enzyme is 8 to 12 seconds.
  • the rotation speed of the centrifugation in the step 5) can be controlled to be 2000 to 8000 r/min, and the centrifugation can be performed by a conventional apparatus such as a tube centrifuge or the like.
  • the membrane filtration can be carried out by using a membrane having a pore diameter of 1 to 100 nm, and the pore diameter can be further 1 to 50 nm (for example, ultrafiltration); when membrane filtration, the absolute pressure of membrane filtration can be controlled to 0.2 to 0.4 MPa, and the temperature is 30 to 80 ° C.
  • Membrane filtration of the centrifugation supernatant of the enzymatic hydrolysate further removes components having a large molecular weight, thereby maximally removing the macromolecular sensitizing protein component in the enzymatic hydrolysate.
  • the filtrate after filtration of the membrane, the filtrate can be decolored and concentrated. Specifically, it can be decolorized by a conventional decolorizing agent, for example, the decolorizing agent can be activated carbon powder, the mass ratio of the decolorizing agent to the filtrate can be (5-10):100, and the decoloring temperature can be controlled at 70-90 ° C, for example. At 80 ° C, the decolorization time can be 20 to 40 minutes, and the decolorization can be carried out under stirring. After decolorization, the decolorizing agent can be removed by conventional means such as filtration, such as plate and frame filtration.
  • the decolorizing agent can be activated carbon powder
  • the mass ratio of the decolorizing agent to the filtrate can be (5-10):100
  • the decoloring temperature can be controlled at 70-90 ° C, for example.
  • the decolorization time can be 20 to 40 minutes, and the decolorization can be carried out under stirring.
  • the decolorizing agent can be removed by conventional means such as filtration, such
  • the filtrate from which the decolorizing agent is removed may be evaporated to concentrate the filtrate, for example, a double effect falling film evaporator may be used for evaporation concentration, and the vapor pressure during evaporation may be controlled to be 0.1 ⁇ 0.02 MPa, and the evaporation temperature is 40 to 80° C.
  • concentration the volume of the concentrate can be reduced to 1/3 to 1/2 of the original volume.
  • sterilization and drying can be carried out to prepare a low-sensitization and low-odor fish protein oligopeptide powder, and drying can be, for example, spray drying.
  • the present invention also provides a hypoallergenic low-smell fish protein oligopeptide prepared according to any one of the above methods, wherein the low-sensitivity low-smell fish protein oligopeptide has a small content of small albumin ⁇ 200mg/kg.
  • the content of the small albumin in the hypoallergenic low-smell fish protein oligopeptide can be reduced by more than 99% compared with the fish protein.
  • the low-sensitivity low-smell fish protein oligopeptide has a molecular weight of less than 5000 Da and a mass content of >85%, and a peptide having a molecular weight of less than 1000 Da has a mass content of >60%. Further, the peptide having a molecular weight of less than 5000 Da in the hypoallergenic low-smell fish protein oligopeptide has a mass content of >95%, and the peptide having a molecular weight of less than 1000 Da has a mass content of >85%.
  • the invention also provides the use of the above-mentioned hypoallergenic low-odor fish protein oligopeptide in food or medicine, the food includes but not limited to infant food, sports function food and health care, wherein the infant food can include infant milk powder and baby Children's rice noodles, etc.
  • the method of the invention utilizes three specific proteases for enzymatic hydrolysis after pre-denaturation of fish protein, which not only overcomes the sensitization caused by various allergens, but also avoids new sensitization of enzymatic products.
  • the content of small albumin in fish protein is reduced by more than 99%; in addition, the method effectively removes the astringency of the fish product itself, and the fish meat material is processed into a mass content of molecular weight of less than 1000 Da by more than 90% by physical process.
  • the oligopeptide ensures the absorption and utilization rate of the human body, has simple process and saves cost, and is suitable for large-scale production, and the prepared low-sensitization and low-odor fish protein oligopeptide is widely used.
  • proteases of the examples of the present invention were purchased from Novozymes Biotech Co., Ltd., respectively.
  • the commercially available squid was descaled and boned to the internal organs, and after washing, it was broken to 18 mesh to obtain 5 kg of fish meat foam. 15 L of deionized water was added to the above fish flakes to prepare a mixture.
  • the mixture was warmed to 80 ° C, kept warm and stirred for 40 min, and then a denatured protein solution was prepared.
  • the above denatured protein solution was centrifuged, the centrifuge speed was controlled to 5000 r/min, and the centrifugation time was 30 min. After the end of the centrifugation, the upper layer liquid was discarded to obtain 4.9 kg of the lower layer solid.
  • the pH of the slurry was adjusted to 7 with a 20% aqueous solution of sodium hydroxide, and neutral protease, papain and alkaline protease were added to the slurry, wherein the amount of neutral protease, papain and alkaline protease was About 50 U/g of fish meat was subjected to enzymatic hydrolysis at a temperature of about 50 ° C for about 2 hours, and then heated to 110 ° C for 10 seconds to obtain an enzymatic hydrolyzate.
  • the enzymatic hydrolysate was centrifuged at a speed of 6000 r/min, and the supernatant was collected for use;
  • the ultrafiltration membrane with a pore diameter of about 50 nm is used for ultrafiltration of the supernatant, and the absolute pressure of the ultrafiltration is controlled to be about 0.3 MPa, and the temperature is about 50 ° C to obtain an ultrafiltrate.
  • the activated carbon powder is added to the ultrafiltrate, and the mass ratio of the activated carbon powder to the ultrafiltrate is 10:100, and the mixture is stirred at a temperature of about 80 ° C for about 30 minutes for decolorization, and after decolorization, the plate frame is filtered to remove the activated carbon powder.
  • Decolorizing solution
  • the decolorizing liquid is subjected to evaporation concentration, the vapor pressure is controlled to be about 0.1 MPa, the evaporation temperature is about 60 ° C, and the decolorizing liquid is concentrated to 1/2 of the original volume, and the concentrated liquid is sterilized and spray dried to obtain low sensitization.
  • Low astringent fish protein oligopeptide Low astringent fish protein oligopeptide.
  • the sensitizers of the low-sensitization and low-smell fish protein oligopeptides were detected by Bio-check's Fish-Check ELISA Kit, and the untreated fish and meat mixture was used as a control. The results are shown in Table 1. .
  • the low-sensitization and low-smell fish protein oligopeptide prepared above is dissolved in water to prepare a low-sensitization low-smell fish protein oligopeptide solution having a mass content of 10%; a tissue evaluation group of 20 people (half each) The bitterness and astringency of the low-sensitization and low-odor fish protein oligopeptide solution were evaluated.
  • bitterness is as follows: 1 mL of low-sensitization and low-smell fish protein oligopeptide solution is taken, and the dilution is diluted to just taste bitterness, and the dilution factor is taken as the bitterness value, and the average bitterness value of 20 people is calculated. table 3.
  • the evaluation method of astringency is as follows: the evaluation method is: 0-no astringency; 1- slightly astringent; 2-flavor Weak; 3- odor; 4- odor; 5- ⁇ ;; 6 ⁇ ⁇ ;; 6 ⁇ ⁇ ⁇ ; ; ; ; 7 7 7 7 7 7 7 7 7 7 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
  • the commercially available salmon fish skin scraps were collected, washed, and crushed to 20 mesh to obtain 5 kg of fish meat foam. 10 L of deionized water was added to the above fish flakes to prepare a mixture.
  • the mixture was warmed to 75 ° C, kept warm and stirred for 60 min, and then a denatured protein solution was prepared.
  • the above denatured protein solution was centrifuged, the centrifuge speed was controlled to 3500 r/min, and the centrifugation time was 45 min. After centrifugation, the upper layer liquid was discarded to obtain 4.7 kg of the lower layer solid.
  • the pH of the slurry was adjusted to 7.5 with a 15% aqueous solution of sodium hydroxide, and neutral protease, papain and alkaline protease were added to the slurry, wherein the amount of neutral protease, papain and alkaline protease was The 70 U/g fish waste was left and right, and the enzyme was hydrolyzed at a temperature of about 30 ° C for about 5 hours, and then heated to 110 ° C for 10 seconds to obtain an enzymatic hydrolyzate.
  • the enzymatic hydrolysate was centrifuged at 8000 r/min, and the supernatant was collected for use;
  • the ultrafiltration membrane with a pore diameter of about 20 nm is used for ultrafiltration of the supernatant, and the absolute pressure of the ultrafiltration is controlled to be about 0.4 MPa, and the temperature is about 80 ° C to obtain an ultrafiltrate.
  • the activated carbon powder is added to the ultrafiltrate, and the mass ratio of the activated carbon powder to the ultrafiltrate is 5:100, and the mixture is stirred for 30 minutes at a temperature of about 80 ° C for decolorization. After decolorization, the plate frame is filtered to remove the activated carbon powder.
  • Decolorizing solution
  • the marketed squid was descaled and boned, and the fish and internal organs were taken. After washing, it was broken to 20 mesh to obtain 5 kg of fish meat and internal organs. 20 L of deionized water was added to the above fish meat and fish gut to prepare a mixture.
  • the mixture was warmed to 90 ° C, kept warm and stirred for 20 min, and then a denatured protein solution was prepared.
  • the above denatured protein solution was centrifuged, the centrifuge speed was controlled to 4500 r/min, and the centrifugation time was 35 min. After centrifugation, the upper layer liquid was discarded to obtain 4.6 kg of the lower layer solid.
  • the pH of the slurry was adjusted to 8.5 with a 10% aqueous sodium hydroxide solution, and neutral protease, papain and alkaline protease were added to the slurry, wherein the amount of neutral protease, papain and alkaline protease was 20 U/g of fish meat and fish waste were left and right, and the enzyme was hydrolyzed at about 50 ° C for about 3.5 hours, and then heated to 110 ° C for 10 seconds to obtain an enzyme solution.
  • the second enzymatic hydrolysate was centrifuged at a speed of 7500 r/min, and the supernatant was collected for use;
  • the supernatant of the centrifuged membrane was subjected to membrane filtration using a membrane having a pore size of about 50 nm, and the absolute pressure of the membrane filtration was controlled to be about 0.2 MPa, and the temperature was about 30 ° C to obtain a filtrate.
  • the activated carbon powder is added to the filtrate, and the mass ratio of the activated carbon powder to the ultrafiltrate is 8:100, and the mixture is stirred at a temperature of about 80 ° C for about 30 minutes for decolorization. After decolorization, the plate is filtered to remove the activated carbon powder to obtain a decolorizing solution. ;
  • the decolorizing liquid is subjected to evaporation concentration, the vapor pressure is controlled to be about 0.1 MPa, the evaporation temperature is about 60 ° C, and the decolorizing liquid is concentrated to 1/3 of the original volume, and the concentrated liquid is sterilized and spray-dried to obtain low sensitization.
  • Low-smell fish protein oligopeptide; the quality test results, molecular weight distribution and taste evaluation results of the low-sensitized fish low-odor protein oligopeptide are shown in Tables 1 to 3, respectively.
  • Example 1 The slurry prepared in Example 1 was adjusted to a pH of about 7, and a neutral protease was added to the slurry according to the amount of 150 U/g of fish meat, and the enzyme was hydrolyzed at a temperature of about 40 ° C for about 2 hours.
  • the method of Example 1 was carried out by centrifugation (without membrane filtration decolorization treatment), concentration, and sterilization to obtain a low-sensitization and low-odor fish protein oligopeptide.
  • the quality test results are shown in Table 1 and Table 3.
  • Example 1 The slurry prepared in Example 1 was adjusted to a pH of about 8, and trypsin was added to the slurry according to the dosage of 200 U/g of fish meat, and the enzyme was hydrolyzed at a temperature of about 40 ° C for about 2 hours, and then the enzymatic hydrolyzate was as follows. 1 Method Centrifugal (without membrane filtration decolorization treatment), concentration, sterilization, to produce low sensitization fish low astringency protein oligopeptide, the quality test results are shown in Table 1 and Table 3.
  • Example 1 The slurry prepared in Example 1 was adjusted to a pH of about 7, and a neutral protease of 0.2% by weight of the fish raw material was added at 55 ° C, and the solution was hydrolyzed for 0.5 h; the temperature was lowered to 45 ° C, and the weight of the fish raw material was 0.1. % bromelain, after enzymatic hydrolysis for 0.5 h, the enzymatic hydrolysate was centrifuged, decolorized, concentrated, and sterilized according to the method of Example 1, to obtain a low-sensitization and low-odor fish protein oligopeptide, and the quality test results are shown in the table. 1 and Table 3.
  • Comparative Example 1 3.24*10 4 mg/kg Comparative Example 2 1.52*10 5 mg/kg Comparative Example 3 3.65*10 4 mg/kg
  • the low-sensitization and low-odor fish protein oligopeptide prepared by the invention can reduce the quality of the main sensitizing protein small albumin by more than 99%, and the effect is remarkable. It is indicated that the method of the invention can completely eliminate the sensitization of fish protein, and the desensitization effect is good.
  • proteases or combinations thereof can reduce or eliminate the sensitization of fish protein when the fish protein is treated, but only adopt a specific composition of protease and adopt a specific process (for example, pH environment, temperature, etc. of enzymatic hydrolysis) ) can more completely eliminate the sensitization of fish protein.
  • Example 1 Example 2
  • Example 3 More than 5000 0.09 0.15 1.78 3000-5000 0.04 0.29 0.71 1000-3000 3.78 3.69 8.23 500-1000 16.59 14.21 21.82 140-500 73.57 64.70 60.49 Below 140 5.82 16.79 6.83 Proportion of 1000 or less 95.98 95.70 89.14 Proportion of less than 5,000 99.80 99.67 98.07
  • the low sensitization and low odor fish protein oligopeptide prepared by the invention has a mass content of less than 5000 Da and a mass content of >85%, and a peptide having a molecular weight of less than 1000 Da has a mass content of >60%. Therefore, the low-sensitization and low-odor fish protein oligopeptide of the present invention has good absorption effect and can utilize a high protein content.
  • the low sensitization and low-smell fish protein oligopeptide prepared by the invention has less bitterness and astringency and good taste, indicating that the method of the invention can effectively inhibit the production of bitter substances in the enzymatic hydrolysate, and greatly removes the fish protein.
  • the composition of the taste substance; the use of protease to treat fish protein can not effectively avoid the release of bitter and astringent ingredients in fish protein and the removal of astringent substances, the specific composition of the protease and the use of specific processes (such as pre-denaturation, membrane filtration) Only in a more thorough way can remove the astringency and bitterness and ensure the taste of the product.

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Abstract

提供一种低致敏低腥味鱼蛋白低聚肽及其制备方法和应用。该方法包括如下步骤：1）将新鲜的鱼肉和/或鱼下脚料洗净、破碎，加水制成混合液；2）对混合液加热变性；3）对变性蛋白溶液离心水洗，所得沉淀加水磨制成浆料；4）调节浆液的pH值至6～9，依次加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶酶解，灭酶后得酶解液；5）酶解液离心，膜过滤上清液，制得低致敏低腥味鱼蛋白低聚肽。

Description

一种低致敏低腥味鱼蛋白低聚肽及其工业化制备方法和应用 技术领域
本发明涉及一种鱼蛋白低聚肽，特别是涉及一种低致敏低腥味鱼蛋白低聚肽及其工业化制备方法和应用。
背景技术
鱼肉中富含的鱼蛋白是一种营养价值极高的蛋白，其含有大量的人体所必须的营养物质，有助于血脂以及胆固醇含量的降低，尤其含有大量DHA，能够促进大脑的发育，是神经系统成长所不可或缺的养分，因此，越来越多的人选择对鱼产品进行加工食用。但是，鱼类产品本身携带的腥臭味对加工品的口味有极大的影响，同时鱼肉中含有的蛋白分子量过大，不利于人体的吸收，在一些现有的处理技术中，通常是采用过于极端的pH和温度环境对大分子蛋白进行水解，但是会对蛋白的性质造成不利的影响。
最更为重要的是，鱼类过敏被认为是最常见的食物过敏症之一，因为鱼蛋白含有大量的致敏原，例如小清蛋白，经IgE抗体证实，小清蛋白能使超过95％的人产生过敏反应。加热、化学处理是常见的脱敏处理方法，但是不可能通过采用加热使蛋白质变性而去除鱼蛋白的所有致敏原，化学处理主要是利用化学试剂来降低胰蛋白酶抑制剂的活性，然而其不可避免地会产生化学残留等食品安全问题。
公告号为CN102008004A的专利公开了一种富含鱼皮蛋白的鱼蛋白粉的制备方法，该工艺主要采用中性蛋白酶以及菠萝蛋白酶对鱼皮原料进行两步酶解，虽然其对原料进行酶解使终产品的平均分子量在6000Da以下，但是却未对其中的过敏原进行处理以及检测，因此无法确定该工艺是否能降低致敏活性，并且操作步骤过于繁杂。
李阳等人在体系pH值为8.00的磷酸盐缓冲液中，采用2940U/g的木瓜蛋白酶，在40℃对鳜鱼鱼肉蛋白水解4h，最终鳜鱼蛋白的抗原性降低58.33％，但也未针对小清蛋白做出抗原研究，同时也极有可能使一些隐藏在蛋白三维结构内部或疏水区的线性抗原表位暴露出来而使酶解产物具有新的 致敏性的担忧。
发明内容
本发明提供一种低致敏低腥味鱼蛋白低聚肽及其工业化制备方法和应用，用于解决现有技术中无法彻底消除鱼蛋白致敏性、产品口感不佳以及蛋白分子量过大不利于吸收等技术缺陷。
本发明提供的低致敏低腥味鱼蛋白低聚肽的工业化制备方法，包括如下步骤：
1)将新鲜的鱼肉和/或鱼下脚料洗净、破碎后，加水制成混合液；
2)对所述混合液进行热变性，制得变性蛋白溶液；
3)对所述变性蛋白溶液进行离心，将离心所得沉淀加水磨制成浆料；
4)调节所述浆液的pH值至6～9后，依次加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶进行酶解，灭酶后，制得酶解液；
5)将所述第二酶解液离心后，对离心上清液进行膜过滤，制得低致敏低腥味鱼蛋白低聚肽；
其中，步骤3)中，对所述变性蛋白溶液进行水洗后再进行所述离心，所述水洗包括：
将所述变性蛋白溶液离心后，向离心所得沉淀中加水进行洗涤，所述沉淀与所述水的质量体积比为1:1～5。
本发明对所采用的鱼类品种不做限制，可以是深海鱼，也可以是淡水鱼，并且鱼下脚料是指除去鱼肉以后所剩余的物质，例如鱼皮或者鱼鳞；将鱼肉料洗净后，为了使后续热变性效果更好，可以将洗净后的鱼肉和/或鱼下脚料破碎至15～20目。
进一步地，步骤1)中所述鱼肉和/或鱼下脚料与水的质量体积比为1:(1～5)，即：1kg的鱼和/或鱼下脚料与1～5L的水进行混合制备混合液。制备混合液时，加水过少时混合液流动性差，易导致酶解效率降低；而加水过多时反应体积过大，会影响后续处理(例如浓缩等)，此外成本也会相应增加。其中，水可以采用纯水、蒸馏水、去离子水等，本发明中采用去离子水。此外，步骤3)中所述沉淀与所述水的质量体积比为1:(1～5)。
进一步地，所述热变性包括：将所述混合液加热至75～95℃后，保温并 持续搅拌10～60min。该热变性处理能够破坏鱼蛋白的空间结构，从而降低鱼蛋白的致敏性，同时可解决混合液流动性差、溶液粘稠的问题，有利于后续酶解的进行。
进一步地，对所述变性蛋白溶液进行离心，将离心所得沉淀加水磨制成浆料。具体的，控制离心机转速为2000～8000r/min，离心时间为10～60min，由于离心后所得的沉淀为体积较大的固形物，为了使后续的酶解过程进行彻底，进一步地，可以向所述沉淀中加入水，破碎打浆，制得浆料。控制该沉淀与水的质量体积比为1:1～5，破碎打浆至该沉淀为20～30目即可。
此外，为了更好的去除腥味，并减少色素、脂肪等杂质提高蛋白含量，在步骤3)中，对所述变性蛋白溶液进行水洗后再进行所述离心，所述水洗包括：将所述变性蛋白溶液离心后，向离心所得沉淀中加水进行洗涤，所述沉淀与所述水的质量体积比为1:(1～5)，其中，，控制离心机转速为2000～8000r/min，离心时间为10～60min，该水洗步骤可重复2～3次。
本发明人对于如何在不损失鱼类营养成分的前提下，利用酶法彻底消除鱼蛋白的致敏性，同时消除鱼产品自身所有的腥味以及抑制酶解产物中苦涩物质的产生做了大量的研究。结果发现大多数的蛋白酶无法彻底消除鱼蛋白的致敏性和/或抑制酶解产物中苦涩物质的产生。例如，采用中性蛋白酶对鱼蛋白进行处理时能够在一定程度上能够消除鱼蛋白的致敏性，但脱敏效果一般，采用胰蛋白酶对鱼蛋白进行处理时消除鱼蛋白致敏性作用不明显，检测的致敏小清蛋白含量反而有一定升高。发明人在研究过程中意外地发现，只有同时采用中性蛋白酶、木瓜蛋白酶、碱性蛋白酶进行酶解，才能够较为彻底地消除鱼蛋白的致敏性同时抑制酶解产物中苦味涩味物质的产生。
特别是，在本发明的酶解中，所述中性蛋白酶的用量为10～100U/g，所述木瓜蛋白酶的用量为10～100U/g，所述碱性蛋白酶的用量为10～100U/g。并且，所述中性蛋白酶、木瓜蛋白酶和碱性蛋白酶之间的用量比为1：(1～3)：(1～3)。此外，在酶解前，可以采用质量浓度为10～20％的氢氧化钠水溶液调节pH值至6～9，所述酶解在30℃～60℃的温度下进行，并且控制酶解时间为2～6小时，酶解时间过短(＜1h)不利于蛋白的降解，而时间过长(＞3h)可能导致苦味涩味物质的产生。中性蛋白酶、木瓜蛋白酶以及碱性蛋白酶的结合，有利于在充分降解鱼蛋白以消除其致敏性的同时控制酶解 产物的苦涩口感，从而可以进一步的被广大消费者所接受食用。上述酶解可以将鱼蛋白的主要过敏原小清蛋白降低99％以上，同时，可对鱼蛋白适度水解，形成分子量较小的肽(例如分子量小于1000Da的肽)，从而有利于提高鱼蛋白的的人体吸收率。
在本发明中，各酶的用量是基于鱼肉和/或鱼下脚料的重量，即，采用1g鱼肉和/或鱼下脚料时，使用10～100U的中性蛋白酶。进一步地，在110～120℃的温度下进行所述灭酶，并且控制灭酶的时间为8～12秒。
进一步地，可控制步骤5)中所述离心的转速为2000～8000r/min，离心可采用常规设备进行，例如管式离心机等。此外，可采用孔径为1～100nm的滤膜进行所述膜过滤，孔径进一步可为1～50nm(例如超滤)；膜过滤时，可控制膜过滤的绝对压力为0.2～0.4MPa，温度为30～80℃。对酶解液的离心上清液进行膜过滤，可进一步地截留分子量较大的成分，从而最大限度地去除酶解液中的大分子致敏蛋白组分。
在本发明中，在膜过滤后，可对滤液进行脱色和浓缩。具体地，可以采用常规脱色剂进行脱色，脱色剂例如可以为活性碳粉，脱色剂与滤液的质量配比可以为(5～10)：100，脱色的温度可控制在70～90℃，例如80℃，脱色时间可以为20～40min，脱色可在搅拌下进行。在脱色后，可通过过滤等常规方式去除脱色剂，例如板框过滤。进一步地，可对去除脱色剂的滤液进行蒸发以浓缩滤液，例如可采用双效降膜蒸发器进行蒸发浓缩，并且可控制蒸发时的蒸汽压为0.1±0.02MPa，蒸发温度为40～80℃，经浓缩后，浓缩液的体积可降至原体积的1/3～1/2。进一步地，在浓缩后可进行灭菌和干燥，从而制得低致敏低腥味鱼蛋白低聚肽粉，干燥例如可以为喷雾干燥。
本发明还提供一种低致敏低腥味鱼蛋白低聚肽，按照上述任一所述制备方法制得，所述低致敏低腥味鱼蛋白低聚肽中小清蛋白的的质量含量≤200mg/kg。所述低致敏低腥味鱼蛋白低聚肽中小清蛋白的质量含量较鱼蛋白可以降低99％以上。
进一步地，所述低致敏低腥味鱼蛋白低聚肽中分子量小于5000Da的肽的质量含量＞85％，分子量小于1000Da的肽的质量含量＞60％。更进一步地，所述低致敏低腥味鱼蛋白低聚肽中分子量小于5000Da的肽的质量含量＞95％，分子量小于1000Da的肽的质量含量＞85％。
本发明还提供上述低致敏低腥味鱼蛋白低聚肽在食品或药品中的应用，食品包括但不限于婴幼儿食品、运动功能食品及保健，其中婴幼儿食品可以包括婴幼儿奶粉、婴幼儿米粉等。
本发明的方法在对鱼肉的鱼蛋白预变性后利用三种特定的蛋白酶进行酶解，不仅克服了多种致敏原所带来的致敏性，同时避免了酶解产物产生新的致敏性，鱼蛋白中小清蛋白含量降低了99％以上；此外，该方法有效去除了鱼类产品自身的腥味，通过物理工艺将鱼肉原料处理为分子量小于1000Da的肽的质量含量高达90％以上的低聚肽，从而保证了人体的吸收利用率，工艺简单、节省成本，适合大规模生产，所制得的低致敏低腥味鱼蛋白低聚肽应用范围广泛。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明中实施例的蛋白酶分别购自诺维信生物技术有限公司。
实施例1
1、混合液的制备
将市售鲫鱼去鳞去骨去内脏，清洗干净后，破碎至18目，制得5kg鱼肉沫。将15L去离子水加入上述鱼肉沫中制成混合液。
2、热变性
将混合液升温至80℃，保温并持续搅拌40min后，制成变性蛋白溶液。
3、浆料的制备
将上述变性蛋白溶液离心，控制离心机转速为5000r/min，离心时间为30min，离心结束后，弃上层液体，得4.9kg下层固体。
向上述4.9kg下层固体中加入15L去离子水，搅拌混匀，离心，控制离心机转速为4000r/min，离心时间为30min，取下层固体后再次重复该步骤2次，最终制得4.85kg沉淀。
向上述4.85kg沉淀中加入15L去离子水，破碎打浆至25目，制得浆料。
4、酶解
用质量浓度为20％的氢氧化钠水溶液调节上述浆料pH至7，向浆料中加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶，其中中性蛋白酶、木瓜蛋白酶和碱性蛋白酶的用量均为50U/g鱼肉左右，维持在50℃左右的温度下进行酶解约2h后，将其加热至110℃灭酶10秒，制得酶解液。
5、离心、膜过滤
在6000r/min的转速下对酶解液进行离心，收集离心上清液备用；
采用孔径为50nm左右的超滤膜对离心上清液进行超滤，控制超滤的绝对压力为0.3MPa左右，温度50℃左右，得到超滤液。
6、脱色、浓缩、灭菌
向超滤液中加入活性碳粉，活性碳粉与超滤液的质量配比为10：100，在80℃左右的温度下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为60℃左右，使脱色液浓缩至原体积的1/2后，浓缩液经灭菌、喷雾干燥后，制得低致敏低腥味鱼蛋白低聚肽。
7、质量检测及口感评价
采用Bio-check公司Fish-Check ELISA Kit对制得的低致敏低腥味鱼蛋白低聚肽中的多种致敏原进行检测，同时对未处理的鱼肉混合液作为对照，结果见表1。
采用国标海洋鱼低聚肽粉(GB/T 22729-2008)中所述方法对上述制备的低致敏低腥味鱼蛋白低聚肽中各组分的分子量分布进行检测，结果见表2。
将上述制备的低致敏低腥味鱼蛋白低聚肽溶于水中，制成质量含量为10％的低致敏低腥味鱼蛋白低聚肽溶液；组织20人评定小组(男女各半)对低致敏低腥味鱼蛋白低聚肽溶液进行苦味及腥味评价。
苦味评价方法为：取1mL低致敏低腥味鱼蛋白低聚肽溶液，对其进行梯度稀释至刚好尝出苦味为止，将稀释倍数计为苦味值，计算20人的平均苦味值，结果见表3。
腥味评价方法为：评价方法为：0-无腥味；1-略有腥味；2-腥味较 弱；3-有腥味；4-腥味一般；5-腥味偏重；6-腥味较重；7-腥味很重计算20人的平均腥味值，结果见表3。
实施例2
1、混合液的制备
将市售三文鱼鱼皮下脚料收集，清洗干净后，破碎至20目，制得5kg鱼肉沫。将10L去离子水加入上述鱼肉沫中制成混合液。
2、热变性
将混合液升温至75℃，保温并持续搅拌60min后，制成变性蛋白溶液。
3、浆料的制备
将上述变性蛋白溶液离心，控制离心机转速为3500r/min，离心时间为45min离心结束后，弃上层液体，制得4.7kg下层固体。
向上述4.7kg下层固体中加入10L去离子水，搅拌混匀，离心，控制离心机转速为4000r/min，离心时间为20min，取下层固体后再次重复该步骤2次，最终制得4.5kg沉淀。
向上述4.5kg沉淀中加入15L去离子水，破碎打浆至30目，制得浆料。
4、酶解
用质量浓度为15％的氢氧化钠水溶液调节上述浆料pH至7.5，向浆料中加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶，其中中性蛋白酶、木瓜蛋白酶和碱性蛋白酶的用量均为70U/g鱼下脚料左右，维持在30℃左右的温度下进行酶解约5h后，将其加热至110℃灭酶10秒，制得酶解液。
5、离心、膜过滤
在8000r/min的转速下对第酶解液进行离心，收集离心上清液备用；
采用孔径为20nm左右的超滤膜对离心上清液进行超滤，控制超滤的绝对压力为0.4MPa左右，温度80℃左右，得到超滤液。
6、脱色、浓缩、灭菌
向超滤液中加入活性碳粉，活性碳粉与超滤液的质量配比为5：100，在80℃左右的温度下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为80℃ 左右，使脱色液浓缩至原体积的1/3后，浓缩液经灭菌、喷雾干燥后，制得低致敏低腥味鱼蛋白低聚肽；该低致低腥味敏鱼蛋白低聚肽的质量检测结果、分子量分布和口感评价结果分别见表1至表3。
实施例3
1、混合液的制备
将市售鲫鱼去鳞去骨后取鱼肉与内脏，清洗干净后，破碎至20目，制得5kg鱼肉以及内脏。将20L去离子水加入上述鱼肉以及鱼内脏中制成混合液。
2、热变性
将混合液升温至90℃，保温并持续搅拌20min后，制成变性蛋白溶液。
3、浆料的制备
将上述变性蛋白溶液离心，控制离心机转速为4500r/min，离心时间为35min离心结束后，弃上层液体，制得4.6kg下层固体。
向上述4.6kg下层固体中加入20L去离子水，搅拌混匀，离心，控制离心机转速为4000r/min，离心时间为25min，取下层固体后再次重复该步骤2次，最终制得4.4kg沉淀。
向上述4.4kg沉淀中加入20L去离子水，破碎打浆至30目，制得浆料。
4、酶解
用质量浓度为10％的氢氧化钠水溶液调节上述浆料pH至8.5，向浆料中加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶，其中中性蛋白酶、木瓜蛋白酶和碱性蛋白酶的用量均为20U/g鱼肉以及鱼下脚料左右，维持在50℃左右的温度下进行酶解约3.5h后，将其加热至110℃灭酶10秒，制得酶解液。
5、离心、膜过滤
在7500r/min的转速下对第二酶解液进行离心，收集离心上清液备用；
采用孔径为50nm左右的滤膜对离心上清液进行膜过滤，控制膜过滤的绝对压力为0.2MPa左右，温度30℃左右，得到滤液。
6、脱色、浓缩、灭菌
向滤液中加入活性碳粉，活性碳粉与超滤液的质量配比为8：100，在80℃左右的温度下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为60℃左右，使脱色液浓缩至原体积的1/3后，浓缩液经灭菌、喷雾干燥后，制得低致敏低腥味鱼蛋白低聚肽；该低致敏鱼低腥味蛋白低聚肽的质量检测结果、分子量分布和口感评价结果分别见表1至表3。
对照例1
将实施例1制备的浆液，调节其pH值至7左右，按照用量150U/g鱼肉左右向浆液中加入中性蛋白酶，维持在40℃左右的温度下酶解约2h后，将酶解液按照实施例1方法进行离心(未经过膜过滤脱色处理)、浓缩、灭菌，制得低致敏低腥味鱼蛋白低聚肽，其质量检测结果见表1及表3。
对照例2
将实施例1制备的浆液，调节其pH值至8左右，按照用量200U/g鱼肉左右向浆液中加入胰蛋白酶，维持在40℃左右的温度下酶解约2h后，将酶解液按照实施例1方法进行离心(未经过膜过滤脱色处理)、浓缩、灭菌，制得低致敏鱼低腥味蛋白低聚肽，其质量检测结果见表1及表3。
对照例3
将实施例1制备的浆液，调节其pH值至7左右，在55℃下，加入鱼原料重量的0.2％的中性蛋白酶，酶解0.5h；降温至45℃，再加入鱼原料重量的0.1％的菠萝蛋白酶，酶解0.5h后，将酶解液按照实施例1方法进行离心、脱色、浓缩、灭菌，制得低致敏低腥味鱼蛋白低聚肽，其质量检测结果见表1及表3。
表1 低致敏低腥味鱼蛋白低聚肽质量检测结果
试验例 小清蛋白
鱼肉原料 1.37*105mg/kg
实施例1 121.37mg/kg
实施例2 56.49mg/kg
实施例3 88.63mg/kg
对照例1 3.24*104mg/kg
对照例2 1.52*105mg/kg
对照例3 3.65*104mg/kg
由表1结果可知：
1、本发明制备的低致敏低腥味鱼蛋白低聚肽中，主要致敏蛋白小清蛋白的质量含量可降低99％以上，效果显著。说明本发明方法能够较为彻底地消除鱼蛋白的致敏性，脱敏效果良好。
2、采用中性蛋白酶、菠萝蛋白酶对鱼蛋白进行处理时能够在一定程度上能够消除鱼蛋白的致敏性，但脱敏效果一般。采用胰蛋白酶对鱼蛋白进行处理时消除鱼蛋白致敏性作用不明显，检测的致敏小清蛋白含量反而有一定升高。
由此说明：并非任意的蛋白酶或其组合对鱼蛋白进行处理时均可降低或消除鱼蛋白的致敏性，而只有采用特定组成的蛋白酶并采用特定工艺(例如酶解的pH环境、温度等)才能够较为彻底地消除鱼蛋白的致敏性。
表2 低致敏低腥味鱼蛋白低聚肽的分子量分布
分子量范围 实施例1 实施例2 实施例3
5000以上 0.09 0.15 1.78
3000-5000 0.04 0.29 0.71
1000-3000 3.78 3.69 8.23
500-1000 16.59 14.21 21.82
140-500 73.57 64.70 60.49
140以下 5.82 16.79 6.83
1000以下所占比例 95.98 95.70 89.14
5000以下所占比例 99.80 99.67 98.07
由表2结果可知：
本发明制备的低致敏低腥味鱼蛋白低聚肽中分子量小于5000Da的肽的质量含量＞85％，分子量小于1000Da的肽的质量含量＞60％。因此本发明的低致敏低腥味鱼蛋白低聚肽人体吸收效果良好，可利用蛋白含量高。
表3 低致敏低腥味鱼蛋白低聚肽口感评价结果
试验例 平均苦味值 腥味值
实施例1 2 1
实施例2 3 2
实施例3 2 2
对照例1 8 6
对照例2 7 6.5
对照例3 5 4
由表3结果可知：
本发明制备的低致敏低腥味鱼蛋白低聚肽中苦味及腥味少，口感较好，说明本发明方法能够有效抑制酶解产物中苦味物质的产生，极大的去处鱼蛋白中腥味物质的成分；而只采用蛋白酶对鱼蛋白进行处理时无法有效避免鱼蛋白中的苦味涩味成分的释放及腥味物质去除，特定组成的蛋白酶并采用特定工艺(例如预变性、膜过滤)才能够较为彻底地去除腥味及苦味，保证产品口感。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. 一种低致敏低腥味鱼蛋白低聚肽的工业化制备方法，其特征在于，包括如下顺序进行的步骤：
    1)将新鲜的鱼肉和/或鱼下脚料洗净、破碎后，加水制成混合液；
    2)对所述混合液进行热变性，制得变性蛋白溶液；
    3)对所述变性蛋白溶液进行离心，将离心所得沉淀加水磨制成浆料；
    4)调节所述浆液的pH值至6～9后，依次加入中性蛋白酶、木瓜蛋白酶和碱性蛋白酶进行酶解，灭酶后，制得酶解液；
    5)将所述第二酶解液离心后，对离心上清液进行膜过滤，制得低致敏低腥味鱼蛋白低聚肽；
    其中，步骤3)中，对所述变性蛋白溶液进行水洗后再进行所述离心，所述水洗包括：将所述变性蛋白溶液离心后，向离心所得沉淀中加水进行洗涤，所述沉淀与所述水的质量体积比为1:(1～5)。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤1)中所述鱼肉和/或鱼下脚料与所述水的质量体积比为1:(1～5)，步骤3)中所述沉淀与所述水的质量体积比为1:(1～5)。
  3. 根据权利要求1所述的制备方法，其特征在于，所述热变性包括：将混合液加热至75～95℃后，保温并持续搅拌10～60min。
  4. 根据权利要求1所述的制备方法，其特征在于，所述中性蛋白酶的用量为10～100U/g，所述木瓜蛋白酶的用量为10～100U/g，所述碱性蛋白酶的用量为10～100U/g。
  5. 根据权利要求4所述的制备方法，其特征在于，所述中性蛋白酶、木瓜蛋白酶和碱性蛋白酶之间的用量比为1：(1～3)：(1～3)。
  6. 根据权利要求1所述的制备方法，其特征在于，所述酶解在30℃～60℃的温度下下进行，并且控制酶解时间为2～6小时。
  7. 根据权利要求1所述的制备方法，其特征在于，采用孔径为1～100nm的滤膜进行所述膜过滤。
  8. 一种低致敏低腥味鱼蛋白低聚肽，其特征在于，按照权利要求1至7中任一所述制备方法制得，所述低致敏低腥味鱼蛋白低聚肽中小清蛋白的的质量含量≤200mg/kg。
  9. 根据权利要求8所述的低致敏低腥味鱼蛋白低聚肽，其特征在于，分子量小于5000Da的肽的质量含量＞85％，分子量小于1000Da的肽的质量含量＞60％。
  10. 权利要求8或9所述的低致敏低腥味鱼蛋白低聚肽在食品或药品中的应用。
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