阅读说明：本技术 一种低致敏性蛋清粉的制备方法 (Preparation method of hypoallergenic egg white powder ) 是由 顾璐萍 陈杰 杨严俊 李俊华 苏宇杰 常翠华 于 2020-06-29 设计创作，主要内容包括：本发明公开了一种低致敏性蛋清粉的制备方法,包括以下步骤：(1)分离蛋清；(2)蛋清预处理；(3)树脂吸附；(4)酶解；(5)浓缩；(6)发酵；(7)灭菌、干燥。本发明采用等电点沉淀、树脂吸附、定向酶解等技术有针对性地去除蛋清中的主要过敏原,进一步采取发酵、浓缩工艺,最后采用低温喷雾干燥或电离喷雾干燥等手段制备低致敏性蛋清粉。相对于现有技术,本发明技术针对性去除蛋清中过敏原,致敏性降低至5％,并使得卵白蛋白发生适度水解,基本保留了蛋清原有的功能特性,同时去除了蛋腥味,可作为食品原料或者配料应用到固体饮料、保健食品、功能食品等产品的开发,具有广阔的市场应用前景。(The invention discloses a preparation method of hypoallergenic egg white powder, which comprises the following steps: (1) separating egg white; (2) egg white pretreatment; (3) resin adsorption; (4) carrying out enzymolysis; (5) concentrating; (6) fermenting; (7) sterilizing and drying. The invention specifically removes main allergens in egg white by adopting the technologies of isoelectric precipitation, resin adsorption, directional enzymolysis and the like, further adopts the processes of fermentation and concentration, and finally adopts the means of low-temperature spray drying or ionization spray drying and the like to prepare the hypoallergenic egg white powder. Compared with the prior art, the technology disclosed by the invention can specifically remove the allergen in the egg white, reduce the allergenicity to 5%, enable the egg white to be moderately hydrolyzed, basically keep the original functional characteristics of the egg white, remove the fishy smell of the egg, be used as a food raw material or an ingredient to be applied to the development of products such as solid beverages, health-care foods and functional foods, and have wide market application prospects.)

1. A method for removing ovotransferrin in egg white is characterized in that the method adopts protease and lipase to carry out compound enzymolysis on the egg white, and the ovotransferrin in the egg white liquid is specifically removed.

2. The method of claim 1, wherein the protease comprises papain, trypsin, alcalase, or a neutral protease.

3. The method according to claim 1 or 2, wherein the compounding ratio of the protease to the lipase is 1: 5-1: 10, and the total enzyme amount is 1000-8000U/g egg white.

4. The method according to any one of claims 1 to 3, wherein the enzymolysis is carried out under conditions of pH 6 to 10, temperature 30 to 60 ℃ and enzymolysis time 0.5 to 4.5 hours.

5. Use of the method according to any one of claims 1 to 4 for reducing the allergenicity of egg white.

6. A method for preparing hypoallergenic egg white powder, characterized in that the method is for treating egg white using the method according to any one of claims 1 to 4, the method comprising the steps of:

(1) egg white separation: cleaning fresh eggs, breaking the shells of the fresh eggs, separating egg white and yolk, removing the yolk, and keeping the egg white for later use;

(2) pretreatment of raw materials: stirring the egg white separated in the step (1) to be uniform; adjusting the pH value of the egg white to 3.5-4.5 by using a food acidity regulator, centrifuging or filtering to remove precipitated ovomucoid, and collecting supernatant;

(3) resin adsorption: enabling the supernatant obtained by the pretreatment in the step (2) to pass through an ion exchange resin chromatographic column, and adsorbing to remove lysozyme in the egg liquid;

(4) enzymolysis: adjusting the pH of the residual egg white obtained in the step (3), adding protease and lipase for enzymolysis, and hydrolyzing ovotransferrin and ovalbumin;

(5) concentration: concentrating the egg white enzymatic hydrolysate obtained in the step (4);

(6) fermentation: introducing the egg white enzymolysis concentrated solution obtained in the step (5) into a fermentation tank for fermentation;

(7) and (3) sterilization and drying: and (4) sterilizing the fermented egg white liquid obtained in the step (6), and drying to obtain hypoallergenic egg white powder.

7. The method as claimed in claim 6, wherein the resin used in step (3) is a weakly acidic cation exchange resin, the amount of the resin is 30-60% of the volume of the egg white liquid, and the stirring and adsorbing time is 4-9 h.

8. The method according to claim 6 or 7, wherein the fermentation strain in the step (6) is mixed with the yeast for fermentation by lactic acid bacteria, the inoculation amount is 0.1-0.5%, the fermentation time is 3-6 h, and the fermentation temperature is 40-45 ℃.

9. Egg white powder obtainable by the process according to any one of claims 6 to 8.

10. Use of the egg white powder of claim 9 for preparing a solid beverage, health food or functional food.

Technical Field

The invention relates to a preparation method of hypoallergenic egg white powder, and belongs to the technical field of food processing.

Background

The eggs have rich nutritive value, are rich in various essential amino acids and mineral substances for human bodies, and have the absorption rate of up to 98 percent for the protein of the eggs by human bodies. In addition, eggs are not only cheap and high in quality and become important food in daily life, but also are often added into various foods as a food ingredient, such as baked products, meat products, bean products and the like. Although eggs are rich in nutritional value and have excellent functional characteristics such as solubility, emulsibility, gelation and the like, eggs are one of eight food allergens recognized in the world, the allergens are mainly present in egg white, and the allergens in the egg white are mainly ovomucoid (11%, Mw:28kDa), ovalbumin (54%, Mw:45kDa), ovotransferrin (12% -13%, Mw:77.7kDa) and lysozyme (3.4% -3.5%, Mw: 14.3-14.6 kDa). According to research reports, the allergenicity of the 4 allergens is from high to low according to the in vitro IgG binding capacity as an index: lysozyme is greater than ovotransferrin is greater than ovalbumin is greater than ovomucoid, and the sensitization of lysozyme and ovotransferrin can be seen to be higher. At present, no specific treatment is available for egg allergy, and an egg allergy patient can avoid anaphylactic reaction only by strictly avoiding eating food containing eggs. For the egg allergy patients, not only an important nutrient source in daily life is lost, but also complete avoidance of contact with egg allergens in diet becomes a challenge.

The processing method for reducing the allergenicity of the egg white comprises three methods, namely a physical method, a chemical method and a biological method. Physical methods, such as heating, irradiation, high pressure, ultrasound, microwave and the like, are simple and effective to operate, but only destroy conformational epitopes of the protein, and can not completely eliminate the sensitization of the protein; chemical methods, such as glycosylation methods, can damage certain sensitization epitopes and reduce the sensitization of egg white, but the safety cannot be guaranteed; a biological method such as an enzymolysis method is a mild and safe processing mode, can destroy linear epitopes of proteins, further destroys conformational epitopes of the proteins, and is a processing method for effectively reducing the allergenicity of eggs, but the currently reported enzymolysis technology has low selectivity on substrates, and needs to carry out deep hydrolysis on egg white to obtain higher allergen removal rate, so that the protein structure, especially the main component of the egg white protein, can be greatly destroyed, the food texture and the mouthfeel are poor, the functional properties of the proteins are remarkably reduced and even lost, and simultaneously bitter taste is generated. Therefore, there is a need to develop a new process that can specifically remove allergens while retaining the functional properties and nutritional value of the original egg white.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of hypoallergenic egg white powder, which adopts protease and lipase to carry out compound enzymolysis on egg white, specifically and synergistically removes ovotransferrin in the egg white, and simultaneously properly hydrolyzes the egg white protein, so as to achieve the purposes of reducing allergens in the egg white powder and simultaneously retaining the nutritional value and functional characteristics of the original egg white.

The first purpose of the invention is to provide a method for removing ovotransferrin in egg white, which adopts protease and lipase to carry out compound enzymolysis on the egg white, and specifically removes the ovotransferrin in egg white.

In one embodiment of the invention, the protease comprises papain, trypsin, alcalase, or a neutral protease.

In one embodiment of the invention, the compounding ratio of the protease to the lipase is 1: 5-1: 10, and the total enzyme amount is 1000-8000U/g egg white.

In one embodiment of the invention, the enzymolysis condition is pH 6-10, temperature 30-60 ℃, and enzymolysis time 0.5-4.5 h.

The second purpose of the invention is to provide the application of the method in reducing the allergenicity of egg white.

A third object of the present invention is to provide a method for preparing hypoallergenic egg white powder, the method comprising the steps of:

(1) egg white separation: cleaning several fresh eggs, breaking the shells of the fresh eggs, separating egg white and yolk, removing the yolk and keeping the egg white for later use;

(2) pretreatment of raw materials: slowly stirring the egg white separated in the step (1) to be uniform by adopting a magnetic stirrer; adjusting the pH value of the egg white liquid by using a food acidity regulator, centrifuging or filtering to remove precipitated ovomucoid, collecting supernatant, and storing at 0-4 ℃ for later use;

(3) resin adsorption: enabling the egg white liquid obtained by the pretreatment in the step (2) to pass through an ion exchange resin chromatographic column, and adsorbing to remove lysozyme in the egg white liquid;

(4) enzymolysis: adjusting the pH of the residual egg white obtained in the step (3), adding protease and lipase for enzymolysis, hydrolyzing the ovotransferrin under specific conditions, and properly hydrolyzing the ovalbumin;

(5) concentration: concentrating the egg white enzymolysis liquid obtained in the step (4) by using ultrafiltration equipment;

(6) fermentation: introducing the egg white enzymolysis concentrated solution obtained in the step (5) into a fermentation tank for fermentation, removing egg fishy smell, removing monosaccharide and disaccharide, and preventing the egg white powder from browning in the subsequent drying process;

(7) and (3) sterilization and drying: and (4) pasteurizing the fermented egg white obtained in the step (6), and drying to obtain hypoallergenic egg white powder.

In one embodiment of the invention, the food acidity regulator in the step (2) can be acetic acid, lactic acid, citric acid, tartaric acid, sodium carbonate, sodium citrate, potassium citrate and the like, the pH of the egg white is regulated to 3.5-4.5, and the centrifugation is performed by adopting a butterfly centrifuge with the rotation speed of 5000-8000 g.

In one embodiment of the present invention, the resin used in step (3) is a weakly acidic cation exchange resin, the amount of the resin is 30-60% of the volume of the egg white liquid, the stirring and adsorption time is 4-9 h, and the eluent is 10-20% (NH)₄)₂SO₄The elution time of the solution is 0.5-2.5 h, and the elution times are 1-5 times.

In one embodiment of the invention, in the step (4), one of papain, trypsin, alkaline protease, neutral protease and the like is selected for enzymolysis, and the lipase is compounded with the selected one, wherein the pH is 6-10, the temperature is 35-55 ℃, the enzymolysis time is 0.5-4.5 h, the enzyme amount is 1000-8000U/g, and the compounding ratio is 1: 5-1: 10.

In one embodiment of the present invention, the concentration in step (5) is performed to a solid content of 20% to 25%.

In one embodiment of the invention, the fermentation strain in step (6) is mixed and fermented by lactic acid bacteria and yeast, the inoculation amount is 0.1-0.5%, the fermentation time is 3-6 h, and the fermentation temperature is 40-45 ℃.

In one embodiment of the present invention, the pasteurization condition in the step (7) is 65-68 ℃ for 3 min. The drying mode is low-temperature spray drying or ionization spray drying and the like, the air inlet temperature is 110-140 ℃, and the air outlet temperature is 55-65 ℃.

The fourth purpose of the invention is to provide the egg white powder prepared by the method.

The fifth purpose of the invention is to provide the application of the egg white powder in preparing solid beverage, health-care food or functional food.

The invention has the beneficial effects that:

(1) the invention adopts an ion exchange adsorption method and a directional hydrolysis method, and adopts a low-temperature spray drying or ionization spray drying mode to specifically remove main allergens in egg white, egg mucoid and lysozyme are completely removed, ovotransferrin is basically removed, ovoalbumin is moderately hydrolyzed, the original functional properties and nutritional value of egg white are reserved, and the allergenicity of the egg white is reduced to 5 percent at least. The product can be used as food raw material or ingredient for development of solid beverage, health food, functional food, etc.

(2) The process of the invention relates to a fermentation technology, and has the following beneficial effects: 1) removing the fishy smell of the eggs; 2) removing monosaccharide and disaccharide, and preventing the egg white powder from browning in the subsequent drying process; 3) further reducing the egg white sensitization.

(3) The by-product lysozyme can be used as a natural preservative and also a good auxiliary material for infant functional food. In addition, the method can also be applied to the pharmaceutical industry, such as the antibiotic production industry.

Drawings

FIG. 1 is a process flow diagram of a hypoallergenic egg white powder;

FIG. 2 gel electrophoresis images of samples prepared in examples 1-3 and comparative examples 1-2, wherein band 1: comparative example 1; strip 2: comparative example 2; the strip 3: example 1; the strip 4: example 2; the strip 5: example 3.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

1. Determination of IgG binding Capacity in vitro

Diluting zymolytic protein to 1 mu g/mL by using a coating buffer solution, taking 100 mu L of zymolytic protein to a 96-hole enzyme label plate A, and coating overnight at 4 ℃; removing buffer solution the next day, adding 300 μ L PBST lotion into each well, washing for 3 times, each time for 5 min; then 250 μ L of 3% skim milk was added to each well and incubated for 1h at 37 ℃. Preparing an enzyme label plate B, adding 250 mu L of 3% skim milk for blocking, and incubating for 1h at 37 ℃; washing as above, adding 60 μ L of different concentrations of egg albumin (competitive antigen) and 60 μ L of diluted rabbit serum (1:60000), incubating at 37 deg.C for 1 h; taking out the plate A, washing the plate A as above, sucking the reaction solution in a 100 mu L B plate to the plate A, and incubating the plate A for 1h at 37 ℃; washing the plate A, adding 100 mu L of HRP-labeled goat anti-rabbit IgG (1:5000) to each well, and incubating for 1h at 37 ℃; washing the plate A, adding 100 mu L of TMB color development solution into each hole, and incubating for 15min at 37 ℃ in a dark place; add 50. mu.L of 2M H per well₂SO₄The color reaction was terminated and the OD value was measured at 450 nm. The calculation formula is as follows:

IgG inhibitory Rate (%) - (1-B/B)₀)*100

B, the OD value corresponding to the competitive protein is obtained;

B₀OD value corresponding to the non-competitive protein.

2. Biological material

Papain with enzyme activity of 2 × 10⁵U/g, neutral proteinase with enzyme activity of 3 × 10⁵U/g, alkaline protease with the enzyme activity of 2 × 10⁵U/g, all from Angel Yeast GmbH.

Lipase with enzyme activity of 9 × 10⁴U/g, from Nippon Tianye enzyme preparations.