阅读说明：本技术 脱除腰果过敏蛋白的联合方法 (Combined method for removing cashew nut allergic protein ) 是由 张爱琳 迟越 梁筱研 苗颖 刘翠翠 于 2020-12-07 设计创作，主要内容包括：本发明公开了脱除腰果过敏蛋白的联合方法,包括步骤一,原料选取；步骤二,微波与超声波协同；步骤三,高温与高压协同；步骤四,酶处理；步骤五,存储放置；其中在上述步骤一中,人工根据加工条件,选择合适的原始样品蛋白；将选择的原始样品蛋白进行筛选；该脱除腰果过敏蛋白的联合方法,操作简单,利用微波与超声波协同、高温与高压协同、酶处理三种方法协同脱敏,通过每次脱敏后的蛋白浓度及SDS-PAGE电泳图以及WB免疫印迹结果判断其脱敏效果,由于微波与超声波破坏氢键和范德华力,改变了过敏原构象结构使得高温高压处理更好的将蛋白结构展开使酶切位点与疏水基暴露在表外,为酶法脱敏提供了前期条件,有利于脱除腰果过敏蛋白。(The invention discloses a combined method for removing cashew nut allergic protein, which comprises the following steps of firstly, selecting raw materials; step two, microwave and ultrasonic wave are cooperated; thirdly, high temperature and high pressure are cooperated; step four, enzyme treatment; step five, storing and placing; in the first step, selecting proper original sample protein manually according to processing conditions; screening the selected original sample protein; the combined method for removing the cashew nut allergic protein is simple to operate, three methods of microwave and ultrasonic wave synergy, high-temperature and high-pressure synergy and enzyme treatment are utilized for synergistic desensitization, the desensitization effect is judged according to the protein concentration after each desensitization, an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis diagram and a WB (white-bar-printing) immunoblotting result, the conformation structure of the allergen is changed due to the fact that the microwaves and the ultrasonic waves destroy hydrogen bonds and van der Waals force, the protein structure is better unfolded through high-temperature and high-pressure treatment, enzyme cutting sites and hydrophobic groups are exposed outside the surface, early-stage conditions are provided for enzyme desensitization, and.)

1. The combined method for removing the cashew nut allergic protein comprises the steps of firstly, selecting raw materials; step two, microwave and ultrasonic wave are cooperated; thirdly, high temperature and high pressure are cooperated; step four, enzyme treatment; step five, storing and placing; the method is characterized in that:

in the first step, the raw material selection comprises the following steps:

1) manually selecting proper original sample protein according to processing conditions;

2) screening the selected original sample protein, and determining the concentration of the original sample protein to be 2.5 mg/ml;

in the second step, the microwave and the ultrasonic wave cooperate to comprise the following steps:

1) manually pouring the original sample protein in the step one 2) into a microwave and ultrasonic synergetic machine, and adjusting the power of the microwave and ultrasonic synergetic machine to 600W;

2) starting a microwave and ultrasonic synergetic machine, adjusting the temperature to 60-80 ℃, treating for 240-: 1.4mg/ml, minimum concentration: 0.7 mg/ml; in the third step, the high temperature and high pressure are cooperated to comprise the following steps:

1) manually pouring the original sample protein treated in the step 2) into a reaction kettle, and uniformly mixing and stirring;

2) sealing the reaction kettle, raising the temperature to 121 ℃ and 0.1MPa, continuously reacting for 20min, standing for 10-20min, and performing high-temperature and high-pressure synergy to obtain the following maximum concentration: 0.8mg/ml, minimum concentration: 0.32 mg/ml;

wherein in the fourth step, the enzyme treatment comprises the following steps:

1) manually stirring the mixture in the reaction kettle in the step three 2) for 10-20min, then adding papain and trypsin according to an enzyme substrate ratio of 3000u/g, and stirring while adding;

2) after the addition is finished, the reaction kettle is sealed for 20-40min, and the concentration is concentrated into: 10mg/ml, the maximum concentration after treatment is: 1.1mg/ml, minimum concentration: 0.4mg/ml, namely obtaining desensitized cashew nut protein;

in the fifth step, the desensitized cashew proteins obtained in the fourth step 2) are manually taken out and filled into bottles, and then are placed in a shady and cool place for storage.

2. The combined method for removing cashew nut allergic protein as claimed in claim 1, wherein: the original sample protein selected in the step 1) needs to be used after being qualified.

3. The combined method for removing cashew nut allergic protein as claimed in claim 1, wherein: and in the step two 1), the microwave and ultrasonic synergetic machine needs to be cleaned before use.

4. The combined method for removing cashew nut allergic protein as claimed in claim 1, wherein: and in the third step 2), the reaction kettle is sealed, and then the internal air needs to be exhausted.

5. The combined method for removing cashew nut allergic protein as claimed in claim 1, wherein: in the step four 1), the ratio of the papain to the trypsin is 2: 1.

6. The combined method for removing cashew nut allergic protein as claimed in claim 1, wherein: and in the fifth step, the bottle body needs to be disinfected before bottling.

Technical Field

The invention relates to the technical field of almond allergic protein, in particular to a combined method for removing cashew nut allergic protein.

Background

Cashew nuts are also called mesocarp and dendrocalamus latiflorus, and three main allergens of cashew nuts, namely pea globulin, globulin and albumin, have been discovered and reported at present, have high water solubility, comprise main reaction antigens recognized by IgE of cashew nut allergy human serum, and are the main allergens of cashew nuts; according to the traditional method for removing the cashew nut allergic protein, enzyme treatment is directly adopted for desensitization, and as a protein structure of an allergen conformation structure is not easy to unfold, enzyme cutting sites and hydrophobic groups cannot be exposed outside the surface, early-stage conditions cannot be provided for enzyme desensitization, and the cashew nut allergic protein cannot be completely removed; in view of these defects, it is necessary to design a combined method for removing cashew nut allergic protein.

Disclosure of Invention

The invention aims to provide a combined method for removing cashew nut allergic protein, which aims to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme: the combined method for removing the cashew nut allergic protein comprises the steps of firstly, selecting raw materials; step two, microwave and ultrasonic wave are cooperated; thirdly, high temperature and high pressure are cooperated; step four, enzyme treatment; step five, storing and placing;

in the first step, the raw material selection comprises the following steps:

1) manually selecting proper original sample protein according to processing conditions;

2) screening the selected original sample protein, and determining the concentration of the original sample protein to be 2.5 mg/ml;

in the second step, the microwave and the ultrasonic wave cooperate to comprise the following steps:

1) manually pouring the original sample protein in the step one 2) into a microwave and ultrasonic synergetic machine, and adjusting the power of the microwave and ultrasonic synergetic machine to 600W;

2) starting a microwave and ultrasonic synergetic machine, adjusting the temperature to 60-80 ℃, treating for 240-: 1.4mg/ml, minimum concentration: 0.7 mg/ml;

in the third step, the high temperature and high pressure are cooperated to comprise the following steps:

1) manually pouring the original sample protein treated in the step 2) into a reaction kettle, and uniformly mixing and stirring;

2) sealing the reaction kettle, raising the temperature to 121 ℃ and 0.1MPa, continuously reacting for 20min, standing for 10-20min, and performing high-temperature and high-pressure synergy to obtain the following maximum concentration: 0.8mg/ml, minimum concentration: 0.32 mg/ml;

wherein in the fourth step, the enzyme treatment comprises the following steps:

1) manually stirring the mixture in the reaction kettle in the step three 2) for 10-20min, then adding papain and trypsin according to an enzyme substrate ratio of 3000u/g, and stirring while adding;

2) after the addition is finished, the reaction kettle is sealed for 20-40min, and the concentration is concentrated into: 10mg/ml, the maximum concentration after treatment is: 1.1mg/ml, minimum concentration: 0.4mg/ml, namely obtaining desensitized cashew nut protein;

in the fifth step, the desensitized cashew proteins obtained in the fourth step 2) are manually taken out and filled into bottles, and then are placed in a shady and cool place for storage.

According to the technical scheme, the original sample protein selected in the step 1) needs to be used after being checked to be qualified.

According to the technical scheme, the microwave and ultrasonic synergetic machine in the step two 1) needs to be cleaned before being used.

According to the technical scheme, the reaction kettle in the step three 2) is sealed, and then the internal air needs to be exhausted.

According to the technical scheme, the ratio of the papain to the trypsin in the step four 1) is 2: 1.

According to the technical scheme, in the fifth step, the bottle body needs to be disinfected before bottling.

Compared with the prior art, the invention has the following beneficial effects: the combined method for removing the cashew nut allergic protein is simple to operate, low in cost, safe and reliable, and has the advantages that three methods of microwave and ultrasonic wave synergy, high-temperature and high-pressure synergy and enzyme treatment synergy desensitization are utilized, and the desensitization effect is judged according to the protein concentration after each desensitization, an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis image and a WB (white line) immunoblotting result: the protein concentration of the original sample is 2.5mg/ml, and the highest concentration after microwave and ultrasonic wave synergistic desensitization is as follows: 1.4mg/ml, minimum concentration: 0.7 mg/ml; the highest concentration after high-temperature and high-pressure synergistic desensitization is as follows: 0.8mg/ml, minimum concentration: 0.32 mg/ml; the concentration was concentrated to 10mg/ml before the enzyme treatment, and the maximum concentration after the treatment was: 1.1mg/ml, minimum concentration: 0.4mg/ml, the protein content is gradually reduced from the concentration, which proves that the desensitization method is effective, and the microwave and the ultrasonic wave destroy hydrogen bonds and Van der Waals force, so that the conformation structure of the allergen is changed, the high-temperature and high-pressure treatment is better to unfold the protein structure, the enzyme cutting site and a hydrophobic group are exposed outside the surface, the early-stage condition is provided for enzyme desensitization, and the removal of cashew nut allergic protein is facilitated.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides a technical solution: the combined method for removing the cashew nut allergic protein comprises the steps of firstly, selecting raw materials; step two, microwave and ultrasonic wave are cooperated; thirdly, high temperature and high pressure are cooperated; step four, enzyme treatment; step five, storing and placing;

in the first step, the raw material selection comprises the following steps:

1) manually selecting proper original sample protein according to processing conditions, wherein the selected original sample protein is used after being qualified;

2) screening the selected original sample protein, and determining the concentration of the original sample protein to be 2.5 mg/ml;

in the second step, the microwave and the ultrasonic wave cooperate to comprise the following steps:

1) manually pouring the original sample protein in the step one 2) into a microwave and ultrasonic synergetic machine, wherein the microwave and ultrasonic synergetic machine needs to be cleaned before use, and the power of the microwave and ultrasonic synergetic machine is adjusted to 600W;

2) starting a microwave and ultrasonic synergetic machine, adjusting the temperature to 60-80 ℃, treating for 240-: 1.4mg/ml, minimum concentration: 0.7 mg/ml;

in the third step, the high temperature and high pressure are cooperated to comprise the following steps:

1) manually pouring the original sample protein treated in the step 2) into a reaction kettle, and uniformly mixing and stirring;

2) sealing the reaction kettle, exhausting the internal air after the reaction kettle is sealed, raising the temperature to 121 ℃ and 0.1MPa, continuously reacting for 20min, standing for 10-20min, and performing high-temperature and high-pressure synergy to obtain the following components in percentage by weight: 0.8mg/ml, minimum concentration: 0.32 mg/ml;

wherein in the fourth step, the enzyme treatment comprises the following steps:

1) manually stirring the mixture in the reaction kettle in the step three 2) for 10-20min, and then adding papain and trypsin according to an enzyme substrate ratio of 3000u/g, wherein the ratio of the papain to the trypsin is 2: 1, and stirring while adding;

2) after the addition is finished, the reaction kettle is sealed for 20-40min, and the concentration is concentrated into: 10mg/ml, the maximum concentration after treatment is: 1.1mg/ml, minimum concentration: 0.4mg/ml, namely obtaining desensitized cashew nut protein;

in the fifth step, the desensitized cashew proteins obtained in the fourth step 2) are manually taken out and filled into bottles, and the bottle bodies need to be disinfected before bottling, and then are stored in the shade.

Based on the above, the combined method for removing the cashew nut allergic protein has the advantages of simple operation, low cost, safety and reliability, and the desensitization effect is judged by utilizing three methods of microwave and ultrasonic wave synergy, high-temperature and high-pressure synergy and enzyme treatment synergy desensitization and through the protein concentration after each desensitization, an SDS-PAGE electrophoresis chart and a WB immunoblotting result: the protein concentration of the original sample is 2.5mg/ml, and the highest concentration after microwave and ultrasonic wave synergistic desensitization is as follows: 1.4mg/ml, minimum concentration: 0.7 mg/ml; the highest concentration after high-temperature and high-pressure synergistic desensitization is as follows: 0.8mg/ml, minimum concentration: 0.32 mg/ml; the concentration was concentrated to 10mg/ml before the enzyme treatment, and the maximum concentration after the treatment was: 1.1mg/ml, minimum concentration: 0.4mg/ml, the protein content is gradually reduced from the concentration, which proves that the desensitization method is effective, and the microwave and the ultrasonic wave destroy hydrogen bonds and Van der Waals force, so that the conformation structure of the allergen is changed, the high-temperature and high-pressure treatment is better to unfold the protein structure, the enzyme cutting site and a hydrophobic group are exposed outside the surface, the early-stage condition is provided for enzyme desensitization, and the removal of cashew nut allergic protein is facilitated.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.