阅读说明：本技术 一种基于降低牛乳致敏性的液体乳制备方法 (Liquid milk preparation method based on reduction of cow milk sensitization ) 是由 郑艳 梁肖娜 孙敬 杨慧 程娇 杨梅 曹雪妍 王宗洲 武俊瑞 岳喜庆 于 2020-06-08 设计创作，主要内容包括：本发明涉及一种基于降低牛乳致敏性的液体乳制备方法,包括以下步骤：(1)脱脂；(2)预热；(3)酶解：预热后,取脱脂牛乳,以点滴滴加方式依次添加上述三种蛋白酶溶液于脱脂乳中,并进行酶解；酶解时间为60-120 min,温度50-60℃,分时段调节pH,获得酶解液；(4)灭酶。所述低致敏性液体乳中分子量小于5000道尔顿的小肽约占99%。所述降低牛乳致敏性的液体乳制备方法用于降低牛乳中多个致敏原蛋白的致敏性,所述牛乳致敏原蛋白包括酪蛋白,αs<Sub>1</Sub>-酪蛋白,αs<Sub>2</Sub>-酪蛋白,β-酪蛋白,k-酪蛋白,α-乳白蛋白,β-乳球蛋白,免疫球蛋白,乳铁结合蛋白中的一种或几种。(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% 1 Casein, α s 2 -one or more of casein, β -casein, k-casein, α -lactalbumin, β -lactoglobulin, immunoglobulin, lactoferrin.)

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.

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