CN111631261A - Liquid milk preparation method based on reduction of cow milk sensitization - Google Patents
Liquid milk preparation method based on reduction of cow milk sensitization Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1209—Proteolytic or milk coagulating enzymes, e.g. trypsine
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- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Dairy Products (AREA)
Abstract
The invention relates to a liquid milk preparation method based on cow milk allergenicity reduction, which comprises the following steps of (1) degreasing, (2) preheating, (3) enzymolysis, namely preheating, taking the skim milk, sequentially adding the three protease solutions into the skim milk in a dropping and dripping mode, and carrying out enzymolysis, wherein the enzymolysis time is 60-120 min, the temperature is 50-60 ℃, the pH is adjusted in different periods, an enzymolysis solution is obtained, and (4) enzyme is inactivated, small peptides with the molecular weight less than 5000 daltons in the hypoallergenic liquid milk account for about 99%1Casein, α s2-one or more of casein, β -casein, k-casein, α -lactalbumin, β -lactoglobulin, immunoglobulin, lactoferrin.
Description
Technical Field
The invention relates to the technical field of dairy product processing, in particular to a liquid milk preparation method based on reduction of cow milk sensitization.
Background
A large number of researches show that the milk allergy rate of infants is 2-6%, adults account for about 0.1-0.5%, milk contains more than 30 kinds of allergenic proteins, wherein α -lactalbumin, β -lactoglobulin and casein have a plurality of epitopes with high content, and the allergenic proteins are considered as main allergens in the milk1Casein, α s2Casein, β -casein, k-casein, immunoglobulin, bovine serum albumin, lactoferrin, and the like, have also become hot spots in recent years for reducing or eliminating the allergenicity of milk proteins.
At present, the desensitization method of cow milk mainly comprises high pressure, heat treatment, enzyme degradation and the like. Among them, the enzymatic hydrolysis method has received much attention because of its simple process and strong operability. The enzyme method is a common protein modification method, can break or reduce the sensitization epitope of the cow milk sensitization protein, and can also break the primary structure of the sensitization epitope by breaking some chemical bonds, thereby reducing cow milk sensitization. In order to eliminate or reduce the allergenicity of milk protein, Penas et al, adopt enzymolysis and high static pressure to process a whey protein solution, study the influence of the whey protein solution on the reduction of the allergenicity of beta-LG, and determine two enzymes with the best beta-LG allergenicity reduction and corresponding conditions thereof. The antigenicity of both beta-lactoglobulin and alpha-lactalbumin is reduced by more than 90% by the composite treatment of alkaline protease, trypsin and papain by Von Fengqin and the like at Zhejiang university; the whey protein enzymolysis product is used for a rat passive skin allergy test by Sunpilong and the like of Zhejiang industrial university, and the discovery shows that the blue spot of the rat can be obviously reduced.
At present, the research on cow milk sensitization at home and abroad is only limited to a single allergenic protein in cow milk, and the sensitization research of the whole cow milk system and the development of low-allergenic cow milk products are at the beginning stage, so that the invention provides a liquid milk preparation method based on cow milk sensitization reduction.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a liquid milk preparation method based on reduction of cow milk sensitization, which reduces or eliminates allergen proteins in cow milk by using an enzyme method, thereby achieving the purpose of simultaneously reducing the sensitization of a plurality of cow milk allergen proteins and preparing hypoallergenic liquid milk.
The invention aims to realize the technical scheme that a method for preparing liquid milk based on reduction of cow milk sensitization comprises the following steps:
(1) degreasing
Centrifuging fresh cow milk at 12000 r/min for 20-30min to obtain skimmed cow milk;
the fresh cow milk has the following indexes: protein content: 2.8 to 3.5 percent; fat content: 3.0 to 5.0 percent; lactose content: 4.0 to 5.6 percent; mineral content: 0.5-0.7%;
(2) preheating
Diluting papain, neutral protease and alkaline protease with distilled water to obtain protease solution with concentration of 1.0 g/mL; preheating skim milk and protease solution in 50-60 deg.C water bath for 20 min;
the papain, the neutral protease and the alkaline protease are food-grade raw materials;
(3) enzymolysis
After preheating, taking skim milk, and sequentially adding the three protease solutions into the skim milk in a drip dripping mode to obtain a skim milk and protease mixed solution for enzymolysis; continuously and uniformly stirring during enzymolysis, wherein the stirring speed is 600 r/min, the enzymolysis time is 60-120 min, the temperature is 50-60 ℃, and the pH is adjusted in different periods to obtain an enzymolysis liquid;
the three proteases are added according to the sequence of papain, neutral protease and alkaline protease;
the addition amounts of the three protease solutions are respectively added according to the enzyme activities of the three proteases and the substrate protein, and the E/S ratio is 2000:1-10000:1 (E: enzyme activity; S: protein);
the pH is adjusted according to the following steps:
a. adding 10% citric acid aqueous solution into the mixture of skimmed milk and protease, adjusting pH to 6.0-7.0, and performing enzymolysis for 30-60 min;
b. adding sodium bicarbonate water solution with mass percent concentration of 18% into the mixed solution of the skim milk and the protease, adjusting the pH value of the mixed solution of the skim milk and the protease to 7.0-8.0, and performing enzymolysis for 30-60 min;
(4) enzyme deactivation
And (3) after the enzymolysis reaction is finished, carrying out enzyme deactivation on the enzymolysis liquid in a water bath for 10-15min, wherein the water bath temperature is 90-100 ℃, cooling the enzymolysis liquid to room temperature after enzyme deactivation, then carrying out high-speed centrifugation at 5000 r/min for 10-20 min, separating and precipitating, and obtaining supernate which is hypoallergenic liquid emulsion after separation.
Further, the addition amounts of the papain, the neutral protease and the alkaline protease solution are respectively added according to the enzyme activities and the substrate protein of the three proteases, the E/S ratio is 6000:1 (E: enzyme activity; S: protein), the pH value is 6.0-7.0, the enzymolysis is 40min, the pH value is 7.0-8.0, the enzymolysis is 40min, and the total enzymolysis is 80 min.
Further, the addition amounts of the papain, the neutral protease and the alkaline protease solution are respectively added according to the enzyme activities and the substrate protein of the three proteases, the E/S ratio is 10000:1 (E: enzyme activity; S: protein), the pH value is 6.0-7.0, the enzymolysis is carried out for 40min, the pH value is 7.0-8.0, and the enzymolysis is carried out for 80 min.
Further, the small peptide with the molecular weight less than 5000 daltons in the hypoallergenic liquid milk accounts for about 99%.
The invention also provides an application of the liquid milk preparation method based on reduction of cow milk sensitization, and the technical key points are as follows: for simultaneous reduction ofAllergenicity of multiple allergenic proteins in bovine milk, including casein, α s1Casein, α s2-one or more of casein, β -casein, k-casein, α -lactalbumin, β -lactoglobulin, immunoglobulin, lactoferrin.
The invention has the beneficial effects that:
(1) the natural skim milk is used as a carrier, the original characteristics (natural pH, flavor and the like) of the natural skim milk are ensured, and the allergenic milk protein in the milk is reduced or broken by an enzyme method under mild conditions, so that the aims of reducing the antigenicity and the allergenicity of the milk are fulfilled; the method is simple to operate, easy to control, safe and economical; the prepared cow milk product has low allergenicity and high nutritional value, can meet the consumption requirements of cow milk allergy patients, and has good popularization and application prospects.
(2) Compared with the traditional milk product, the small peptide with the molecular weight less than 5000 daltons accounts for about 99 percent in the method, can promote the digestion and absorption of intestinal tracts, has higher absorption rate, does not stimulate intestines and stomach and the like, and can meet the basic requirements of human bodies on nutrients.
(3) Compared with the traditional milk product, the milk allergenic protein in the method is enzymolyzed into small peptides and amino acids, and the small peptides and the amino acids are beneficial to human digestion and absorption, have low allergenicity, and have biological functions of enhancing human immunity, promoting organism growth, reducing blood pressure and cholesterol and the like.
(4) Compared with hypoallergenic milk powder, the hypoallergenic liquid milk prepared by the method can meet the growth requirements of infants, can meet the nutritional requirements of various consumer groups except infants with milk allergy history, improves the biological utilization rate of the infants, improves the gastrointestinal environment, stimulates the immune response, improves the immunity of the infants and the like.
(5) Compared with hypoallergenic cow milk protein, the hypoallergenic liquid milk of the method makes up for the sensitization research of the predecessors only aiming at a single protein, and takes the whole cow milk as a research system, reduces or eliminates the allergenic protein in the cow milk on the premise of keeping the cow milk flavor, thus opening up a new path and providing a new idea for the further research and development of cow milk products.
Detailed Description
Example 1 a method for preparing liquid milk based on reduction of cow's milk sensitization, comprising the steps of:
(1) degreasing
Centrifuging fresh cow milk at 12000 r/min for 20-30min to completely defat to obtain defatted cow milk;
the fresh cow milk has the following indexes: protein content: 2.8 to 3.5 percent; fat content: 3.0 to 5.0 percent; lactose content: 4.0 to 5.6 percent; mineral content: 0.5 to 0.7 percent.
(2) Preheating
Diluting papain, neutral protease and alkaline protease with distilled water to obtain protease solution with concentration of 1.0 g/mL; preheating skim milk and protease solution in 50-60 deg.C water bath for 20 min;
the papain, the neutral protease and the alkaline protease are food-grade raw materials;
(3) enzymolysis
After preheating, taking skim milk, and sequentially adding the three protease solutions into the skim milk in a drip dripping mode to obtain a skim milk and protease mixed solution for enzymolysis; continuously and uniformly stirring during enzymolysis, wherein the stirring speed is 600 r/min, the enzymolysis time is 60-120 min, the temperature is 50-60 ℃, and the pH is adjusted in different periods to obtain an enzymolysis liquid;
the three proteases are added according to the sequence of papain, neutral protease and alkaline protease;
the addition amount of the three protease solutions is respectively added according to the enzyme activities of the three proteases and the substrate protein, and the E/S ratio is 2000:1-10000:1 (E: enzyme activity; S: protein);
the pH is adjusted according to the following steps:
a. adding 10 wt% citric acid aqueous solution into the mixture of skimmed milk and protease, adjusting pH to 6.0-7.0, and performing enzymolysis for 30-60 min;
b. adding sodium bicarbonate water solution with mass percentage concentration of (18%) into the mixed solution of the skim milk and the protease, adjusting the pH value of the mixed solution of the skim milk and the protease to 7.0-8.0, and performing enzymolysis for 30-60 min;
(4) enzyme deactivation
And after the enzymolysis reaction is finished, rapidly carrying out water bath on the enzymolysis liquid for inactivating enzyme for 10-15min, wherein the water bath temperature is 90-100 ℃, rapidly cooling the enzymolysis liquid to room temperature after inactivating enzyme, then carrying out high-speed centrifugation for 10-20 min at 5000 r/min, separating and precipitating, and separating to obtain supernate, namely the hypoallergenic liquid emulsion.
The small peptide with the molecular weight less than 5000 daltons in the hypoallergenic liquid milk accounts for about 99 percent.
Example 2 Effect of different treatments on the reduction of the sensitization of the major proteins in liquid milk
(1) Milk samples: the indexes of the fresh cow milk are as follows: protein content: 3.5 percent; fat content: 5.0 percent; lactose content: 5.6 percent; mineral content: 0.7 percent;
(2) the test method comprises the following steps:
treatment 1: bovine milk samples were assayed using an indirect ELISA method referred to as Elena, PRESTAMO, Guadaliup, BAEZA, Luisa M, MARTINEZMOLER, Isabel M, GOMEZ, Rosa, Effects of combined high pressure and enzymatic assays on the hydrolytics and immunoreactivity of dairy spiral proteins [ J ]. International Dairy Journal, 2006, 18(9): 918-22.
And (3) treatment 2:
centrifuging fresh cow milk at 12000 r/min for 20-30min to completely defat to obtain defatted cow milk; diluting papain with distilled water to obtain a protease solution with a concentration of 1.0 g/mL, adding the papain solution according to an enzyme-substrate protein ratio of 6000:1 (E/S), carrying out enzymolysis at 60 ℃ for 80min, and continuously stirring during the enzymolysis period so as to keep the pH value in an enzymolysis system relatively stable and carry out enzymolysis fully; enzyme deactivation method reference is made to example 1.
And (3) treatment:
centrifuging fresh cow milk at 12000 r/min for 20-30min to completely defat to obtain defatted cow milk; diluting papain into a protease solution with the concentration of 1.0 g/mL by using distilled water, adding the papain solution according to the ratio of the enzyme to the protein in the substrate of 6000:1 (E/S), wherein the enzymolysis temperature is 60 ℃, the pH value of an enzymolysis product is measured in real time by using an acidimeter, a, adding a citric acid aqueous solution with the mass percentage concentration of 10% into a mixed solution of skim milk and the protease, adjusting the pH value range to be 6.0-7.0, carrying out enzymolysis for 80min, and continuously stirring during the enzymolysis period so as to keep the pH value in an enzymolysis system relatively stable and carry out full enzymolysis; enzyme deactivation method reference is made to example 1.
And (4) treatment:
centrifuging fresh cow milk at 12000 r/min for 20-30min to completely defat to obtain defatted cow milk; diluting papain, neutral protease and alkaline protease with distilled water to obtain protease solution with concentration of 1.0 g/mL, sequentially adding the three protease solutions according to enzyme-substrate protein ratio of 6000:1 (E/S), enzymolysis temperature of 60 deg.C,
the pH value adjusting method in the enzymolysis process comprises the following steps:
step 1: adding 10 wt% citric acid aqueous solution into the mixture of skimmed milk and protease, adjusting pH to 6.0-7.0, and performing enzymolysis for 40 min;
step 2: adding sodium bicarbonate water solution with mass percentage concentration of (18%) into the mixed solution of the skim milk and the protease, adjusting the pH value of the mixed solution of the skim milk and the protease to 7.0-8.0, and performing enzymolysis for 40 min;
the pH value in the enzymolysis process is measured in real time by an acidimeter, and the continuous stirring during the enzymolysis period aims at keeping the pH value in an enzymolysis system relatively stable and fully performing enzymolysis; enzyme deactivation method reference is made to example 1.
(3) Results of measurement of decrease in sensitization of major proteins
After the fresh cow milk is treated by the four treatments, the sensitization reduction rate of ten proteins of the liquid milk after enzyme deactivation of each treatment is measured, the table 1 is a comparison table of the sensitization reduction rate of main proteins of the four treatments, and the results show that the sensitization reduction rate of 10 test proteins of the fresh cow milk obtained by the preparation method of the liquid milk for reducing cow milk sensitization of the treatment 4 is higher than that of the treatment 1-the treatment 3. Therefore, papain, neutral protease and alkaline protease are sequentially added into the skim milk for enzymolysis, and the pH value segmentation adjusting method in the enzymolysis process can reduce or break sensitized lactoprotein in the milk under mild conditions so as to achieve the purpose of reducing the antigenicity and the sensitization of the milk.
TABLE 1 comparison of the reduction rates of sensitization of major proteins in cow milk
EXAMPLE 3 Effect of different enzyme and substrate addition amounts on the reduction Rate of liquid milk protein sensitization
The method is used for simulating the digestion of the gastrointestinal tract of a human body in vitro and measuring the reduction rate of the protein allergenicity in the liquid milk by adopting the following method:
A. simulating gastric digestion: dissolving appropriate amount of liquid milk in 5% formic acid with concentration of 1 mg/mL and pepsin/substrate ratio of 1%, incubating at 37 deg.C for 1 h, heating in boiling water for 5min, and stopping reaction.
B. Simulating intestinal digestion: dissolving a proper amount of liquid milk in phosphate buffer (pH7.0), wherein the ratio of trypsin to substrate is 1%, the ratio of chymotrypsin to substrate is 1%, and the ratio of elastase to substrate is 0.2%, and incubating for 1 h at 37 ℃. The reaction was stopped by heating in boiling water for 5 min.
The method for measuring the hydrolysis degree of each treatment in the present example is as follows: the OPA method.
And (4) comparison treatment: the liquid milk adopts fresh cow milk, the fresh cow milk is fresh pasteurized skim milk provided by the keffiyan milk industry, 150mL skim milk is measured, an indirect ELISA method is adopted, the hydrolysis degree is measured to be 0.1%, and the allergenicity reduction rate of each allergenic protein is not detected.
The liquid milk in the treatment methods 1 to 7 is obtained by a liquid milk preparation method for reducing cow's milk sensitization, and the conditions of degreasing, preheating, enzymolysis pH value adjustment, enzyme deactivation and the like refer to the liquid milk preparation method for reducing cow's milk sensitization in the example 1; the papain, neutral protease and alkaline protease are food grade and purchased from Novoxin (China) Biotechnology, Inc. The fresh cow milk is fresh pasteurized skim milk provided by the kefir dairy industry.
Treatment 1:
weighing 150mL of skim milk, preparing three protease solutions at a ratio of 0.1 g/mL, preparing the enzyme and substrate at a ratio of 2000:1, performing enzymolysis at pH6.0-7.0 for 60min and pH 7.0-8.0 for 60min for 120min, wherein the hydrolysis degree is 5.6%, the reduction rate of α -lactalbumin allergenicity is 2.3%, the reduction rate of β -lactoglobulin allergenicity is 10.2%, the reduction rate of casein allergenicity is 15.5%, and α s1The reduction in casein allergenicity was 3.3%, α s2The reduction rate of casein sensitization was 4.8%, the reduction rate of β -casein sensitization was 5.1%, the reduction rate of k-casein sensitization was 2.2%, the reduction rate of immunoglobulin sensitization was 1.3%, the reduction rate of bovine serum albumin sensitization was 2.7% and the reduction rate of lactoferrin sensitization was 2.3%.
And (3) treatment 2:
weighing 150mL of skim milk, preparing three protease solutions into 0.1 g/mL, preparing the enzyme and substrate ratio adding amount of 4000:1, performing enzymolysis for 60min at pH6.0-7.0, performing enzymolysis for 60min at pH 7.0-8.0, performing enzymolysis for 120min altogether, wherein the hydrolysis degree is 8.9%, the sensitization reduction rate of α -lactalbumin is 4.6%, the sensitization reduction rate of β -lactoglobulin is 23.2%, the sensitization reduction rate of casein is 19.7%, α s1The reduction in casein allergenicity was 6.4%, α s2The reduction rate of casein sensitization was 5.9%, the reduction rate of β -casein sensitization was 4.1%, the reduction rate of k-casein sensitization was 2.3%, the reduction rate of immunoglobulin sensitization was 1.9%, the reduction rate of bovine serum albumin sensitization was 3.2% and the reduction rate of lactoferrin sensitization was 3.6%.
And (3) treatment:
weighing 150mL of skim milk, preparing three protease solutions at a ratio of 0.1 g/mL, adjusting the addition amount of the three protease solutions at a ratio of 6000:1, performing enzymolysis at pH6.0-7.0 for 60min, performing enzymolysis at pH 7.0-8.0 for 60min, performing enzymolysis for 120min altogether, wherein the hydrolysis degree is 11.5%, the sensitization reduction rate of α -lactalbumin is 6.4%, the sensitization reduction rate of β -lactoglobulin is 34.5%, the sensitization reduction rate of casein is 42.6%, and α s1The reduction in casein allergenicity was 12.4%, α s2Reduction of Casein sensitization to 9.9%, β Casein sensitizationThe decrease in the sensitivity was 10.1%, the decrease in the sensitivity to k-casein was 8.9%, the decrease in the sensitivity to immunoglobulin was 3.7%, the decrease in the sensitivity to bovine serum albumin was 4.6% and the decrease in the sensitivity to lactoferrin was 5.3%.
And (4) treatment:
weighing 150mL of skim milk, preparing three protease solutions into 0.1 g/mL, preparing the enzyme and substrate ratio adding amount into 8000:1, performing enzymolysis at pH6.0-7.0 for 60min, performing enzymolysis at pH 7.0-8.0 for 60min, performing enzymolysis for 120min altogether, wherein the hydrolysis degree is 15.3%, the sensitization reduction rate of α -lactalbumin is 9.4%, the sensitization reduction rate of β -lactoglobulin is 56.3%, the sensitization reduction rate of casein is 67.6%, α s1The reduction in casein allergenicity was 25.4%, α s2The reduction rate of casein sensitization was 13.9%, the reduction rate of β -casein sensitization was 18.7%, the reduction rate of k-casein sensitization was 17.2%, the reduction rate of immunoglobulin sensitization was 4.8%, the reduction rate of bovine serum albumin sensitization was 5.1%, and the reduction rate of lactoferrin sensitization was 5.9%.
And (4) treatment 5:
weighing 150mL of skim milk, preparing 0.1 g/mL of three protease solutions, preparing the addition amount of the protease and the substrate in a ratio of 10000:1, performing enzymolysis for 60min at pH6.0-7.0, performing enzymolysis for 60min at pH 7.0-8.0, performing enzymolysis for 120min altogether, wherein the hydrolysis degree is 19.9%, the reduction rate of α -lactalbumin allergenicity is 10.9%, the reduction rate of β -lactoglobulin allergenicity is 80.2%, the reduction rate of casein allergenicity is 89.7%, and α s1The reduction in casein allergenicity was 29.9%, α s2The reduction rate of casein sensitization was 21.3%, the reduction rate of β -casein sensitization was 19.6%, the reduction rate of k-casein sensitization was 19.0%, the reduction rate of immunoglobulin sensitization was 5.8%, the reduction rate of bovine serum albumin sensitization was 6.3%, and the reduction rate of lactoferrin sensitization was 7.2%.
And (6) treatment:
weighing 150mL of skim milk, preparing 0.1 g/mL of three protease solutions, preparing the enzyme and substrate ratio adding amount of 10000:1, performing enzymolysis at pH6.0-7.0 for 30min and at pH 7.0-8.0 for 30min, performing enzymolysis for 60min altogether, wherein the hydrolysis degree is 10.9%, the α -lactalbumin allergenicity reduction rate is 7.9%, the β -lactoglobulin allergenicity reduction rate is 34.2%, the casein allergenicity reduction rate is 69.7%, and α s1The reduction in casein allergenicity was 19.2%, α s2The reduction rate of casein sensitization was 17.7%, the reduction rate of β -casein sensitization was 14.7%, the reduction rate of k-casein sensitization was 12.8%, the reduction rate of immunoglobulin sensitization was 6.9%, the reduction rate of bovine serum albumin sensitization was 7.5% and the reduction rate of lactoferrin sensitization was 8.0%.
And (7) treatment:
weighing 150mL of skim milk, preparing 0.1 g/mL of three protease solutions, preparing the addition amount of the protease and the substrate in a ratio of 10000:1, performing enzymolysis for 40min at pH6.0-7.0, performing enzymolysis for 40min at pH 7.0-8.0, performing enzymolysis for 80min altogether, wherein the hydrolysis degree is 17.4%, the reduction rate of α -lactalbumin allergenicity is 11.9%, the reduction rate of β -lactoglobulin allergenicity is 84.8%, the reduction rate of casein allergenicity is 90.7%, and α s1The reduction in casein allergenicity was 29.3%, α s2The reduction rate of casein sensitization was 27.7%, the reduction rate of β -casein sensitization was 17.7%, the reduction rate of k-casein sensitization was 15.1%, the reduction rate of immunoglobulin sensitization was 6.9%, the reduction rate of bovine serum albumin sensitization was 8.5%, and the reduction rate of lactoferrin sensitization was 9.0%.
Claims (6)
1. A preparation method of liquid milk based on reduction of cow milk sensitization is characterized by comprising the following steps: the method comprises the following steps:
(1) degreasing
Centrifuging fresh cow milk at 12000 r/min for 20-30min to obtain skimmed cow milk;
(2) preheating
Diluting papain, neutral protease and alkaline protease with distilled water to obtain protease solution with concentration of 1.0 g/mL; preheating skim milk and the three protease solutions in 50-60 deg.C water bath for 20 min; the papain, the neutral protease and the alkaline protease are food-grade raw materials;
(3) enzymolysis
After preheating, taking skim milk, sequentially adding papain, neutral protease and alkaline protease solution into the skim milk to obtain skim milk and protease mixed solution, and performing enzymolysis; continuously and uniformly stirring during enzymolysis, wherein the stirring speed is 600 r/min, the enzymolysis time is 60-120 min, the temperature is 50-60 ℃, and the pH is adjusted in different periods to obtain an enzymolysis liquid; the addition amounts of the three protease solutions are respectively added according to the enzyme activities of the three proteases and the substrate protein, and the E/S ratio is 2000:1-10000:1 (E: enzyme activity; S: protein);
the method for adjusting the pH by time intervals comprises the following steps:
a. adding 10% citric acid aqueous solution into the mixture of skimmed milk and protease, adjusting pH to 6.0-7.0, and performing enzymolysis for 30-60 min;
b. adding sodium bicarbonate water solution with mass percent concentration of 18% into the mixed solution of the skim milk and the protease, adjusting the pH value of the mixed solution of the skim milk and the protease to 7.0-8.0, and performing enzymolysis for 30-60 min;
(4) enzyme deactivation
And (3) after the enzymolysis reaction is finished, carrying out enzyme deactivation on the enzymolysis liquid in a water bath for 10-15min, wherein the water bath temperature is 90-100 ℃, cooling the enzymolysis liquid to room temperature after enzyme deactivation, centrifuging at 5000 r/min for 10-20 min, and separating to obtain supernatant, namely the hypoallergenic liquid emulsion.
2. The method for preparing liquid milk based on reduction of cow's milk sensitization according to claim 1, wherein: the fresh cow milk has the following indexes: protein content: 2.8-3.5%, fat content: 3.0-5.0%, lactose content: 4.0-5.6%, mineral content: 0.5 to 0.7 percent.
3. The method for preparing liquid milk based on reduction of cow's milk sensitization according to claim 1, wherein: the addition amounts of the papain, the neutral protease and the alkaline protease solution are respectively added according to the enzyme activities and the substrate protein of the three proteases, and the enzymolysis is carried out for 40min at the pH of 6.0-7.0 and for 40min at the pH of 7.0-8.0 according to the E/S ratio of 6000:1 (E: enzyme activity and S: protein), and for 80 min.
4. The method for preparing liquid milk based on reduction of cow's milk sensitization according to claim 1, wherein: the addition amounts of the papain, the neutral protease and the alkaline protease solution are respectively added according to the enzyme activities and the substrate protein of the three proteases, and the enzymolysis is carried out for 40min at the pH of 6.0-7.0 and for 40min at the pH of 7.0-8.0 according to the E/S ratio of 10000:1 (E: enzyme activity and S: protein), and the enzymolysis is carried out for 80 min.
5. The method for preparing liquid milk based on reduction of cow's milk sensitization according to claim 1, wherein: the small peptide with the molecular weight less than 5000 daltons in the hypoallergenic liquid milk accounts for about 99 percent.
6. The use of the liquid milk preparation method based on reduction of bovine milk sensitization according to claim 1 is characterized in that the liquid milk preparation method based on reduction of bovine milk sensitization is used for reducing sensitization of a plurality of allergen proteins in bovine milk, wherein the bovine allergen proteins comprise casein, α s1Casein, α s2-one or more of casein, β -casein, k-casein, α -lactalbumin, β -lactoglobulin, immunoglobulin, lactoferrin.
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