CN114668069A - Preparation method of soybean protein glycosylation modified product with different sugar contents - Google Patents

Preparation method of soybean protein glycosylation modified product with different sugar contents Download PDF

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CN114668069A
CN114668069A CN202210438352.XA CN202210438352A CN114668069A CN 114668069 A CN114668069 A CN 114668069A CN 202210438352 A CN202210438352 A CN 202210438352A CN 114668069 A CN114668069 A CN 114668069A
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soybean protein
protein
beta
maltodextrin
glycosylated
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CN114668069B (en
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宋春丽
任健
陈佳鹏
薛远
战思羽
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Qiqihar University
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23JPROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00Working-up of proteins for foodstuffs
    • A23J3/14Vegetable proteins
    • A23J3/16Vegetable proteins from soybean
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23JPROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00Working-up of proteins for foodstuffs
    • A23J3/30Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins

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Abstract

The invention relates to the technical field of food processing, in particular to a preparation method of a soybean protein glycosylation modified product with different sugar contents, which is prepared by catalyzing glycosylation soybean protein with beta-amylase or beta-glucosidase. The invention can change the sugar content of the glycosylated soybean protein on the basis of unchanged self-crosslinking degree of the protein, and obtain the soybean protein with different sugar contents. The invention mainly aims to degrade sugar chains based on glucoamylase (beta-amylase, beta-glucosidase) to change the side chain glycosyl content of a glycosylated product, and establish a soy protein glycosyl content regulation model, wherein the establishment of the regulation model is beneficial to analyzing the important role of the introduced side chain glycosyl in the regulation and control of the functional properties of the soy protein, and the establishment of the method provides technical support for expanding the application of the glycosylated protein in the food field.

Description

Preparation method of soybean protein glycosylation modified product with different sugar contents
Technical Field
The invention relates to the technical field of food processing, in particular to a preparation method of a soybean protein glycosylation modified product with different sugar contents.
Background
The soybean protein (including 7S glycinin, 11S glycinin and soybean protein isolate) has high nutritive value, complete types of essential amino acids, is close to animal protein, and is a high-quality plant protein. In addition, the protein also has functional properties such as good solubility and foamability, and the soybean protein has various properties such as solubility, emulsibility, gel property, foamability and the like, so that the soybean protein is widely applied to the food industry, for example, the soybean protein is used as an emulsifier to be added into the preparation of baked food, frozen food and soup food, so that the product state is stable; the soybean protein is used in the flour food, and can increase the gluten strength; the soybean protein can also be used as antibacterial fresh-keeping packaging material for food, etc. However, the solubility, foaming properties, etc. of natural soy proteins often do not meet the product requirements. Therefore, it is necessary to modify soy protein to obtain desired functional properties.
Maltodextrins, also known as water-soluble dextrins, are widely used in the food industry, for example, in food systems because they have better emulsifying and thickening properties instead of fats; the candy added with the sugar can reduce the risk of diabetes caused by the candy; because of being easily absorbed by human body, the functional beverage is often used as a formula component in functional beverages for athletes and formula milk powder for infants.
Under the condition of certain temperature and humidity, the Maillard reaction can introduce the hydrophilic carbonyl of the sugar molecule into the protein molecule by changing the molecular structure of the protein, and the reaction does not need to add chemical reagents and has high safety. The functional property of the soybean protein can be obviously improved by utilizing the Maillard reaction. Currently, protein glycosylation modification based on the maillard pathway generally adopts two ways to regulate the glycosyl introduction amount of the protein: one approach is to control the glycosylation reaction time, but this method has certain limitations, such as excessive production of melanoid-like harmful substances (Maillard L C, 1912), and the functional properties of proteins are destroyed; another method is to obtain proteins having different glycosyl transfer amounts by changing the molecular weight of the glycosyl group transferred, such as transferring monosaccharide, oligosaccharide, and polysaccharide to the protein separately. It is noted that the Maillard reaction, while introducing a glycosyl group, is accompanied by a phenomenon of self-crosslinking of the protein (Oliveira F C, Coimbra J S R, Oliveira E B, etc., 2016). Therefore, when analyzing the rule of the influence of the introduced glycosyl on the functional properties of the Maillard product, the method is difficult to define the potential influence caused by protein self-crosslinking.
By utilizing the different degrees of broken copolymerized sugar chains of highly specific glycosidases (such as beta-amylase and beta-glucosidase), different sugar chain length regulation modes (shortening 1 maltose unit or 1 glucose unit) can be implemented, so that the sugar content of the glycosylated soybean protein is regulated, the soybean protein glycosylated products with different sugar contents are obtained, and the structure-activity relationship of Maillard products is analyzed by taking the soybean protein glycosylated products as a model, and the method has remarkable advantages.
Disclosure of Invention
The invention aims to provide a method for regulating and controlling the functional properties of soybean protein by directionally breaking Maillard copolymerized sugar chains, which aims to solve the problems in the prior art. Adopting Maillard reaction to obtain soybean protein glycosylation product, changing side chain glycosyl content of glycosylation product based on hydrolysis (beta-amylase, beta-glucosidase), obtaining soybean protein isolate with different sugar content, and establishing soybean protein sugar content regulation model. The soybean protein glycosylation modification is realized by taking the soybean protein rich in amino and the maltodextrin rich in carbonyl or resistant dextrin as substrates to carry out Maillard reaction. The sugar content of the glycosylated soybean protein is changed on the basis of unchanged self-crosslinking degree of the protein, so that the soybean protein with different sugar contents is obtained. The establishment of the soybean protein sugar content regulation and control model is helpful for analyzing the important role of side chain glycosyl in the soybean protein functional property, the establishment of the method provides technical support for expanding the application of protein in the food field, and the invention provides the following scheme for realizing the purpose:
a method for preparing a soybean protein glycosylation modified product with different sugar contents is to prepare the glycosylation soybean protein under the catalysis of beta-amylase or beta-glucosidase.
Preferably, the glycosylation modified product of the soybean protein is prepared by Maillard reaction of the soybean protein and maltodextrin.
The preparation method further comprises the steps of enzyme deactivation, dialysis and drying, so that a glycosylation modification product which does not change the main chain of the soybean protein molecule and only changes the sugar content can be obtained, and the soybean protein sugar content regulation model is established.
By utilizing the method, the Maillard copolymerized sugar chain can be directionally broken so as to achieve the purpose of regulating and controlling the functional properties of the soybean protein.
Preferably, the preparation method of the soy protein glycosylation modification products with different sugar contents comprises the steps of adding glycosidase (beta-amylase and beta-glucosidase) into the glycosylation soy protein dispersion liquid at 50   -55 ℃, and shaking at constant temperature for 5   -80   min. And after the reaction is finished, inactivating enzyme, dialyzing to remove free sugar molecules, and freeze-drying to obtain the glycosylation modification products with different sugar contents.
In the invention, the optimum pH value range of the beta-amylase is 4.0-7.0, the optimum catalysis temperature is 50 ℃, and the beta-amylase is inactivated when the temperature reaches 70 ℃. The optimum pH value range of the beta-glucosidase is 5.0-7.0, the optimum catalysis temperature is 40-60 ℃, and the beta-glucosidase is inactivated when the temperature reaches 70 ℃. In the invention, the maltodextrin covalently cross-linked into the soybean protein is broken by beta-amylase or beta-glucosidase by utilizing a highly specific enzymatic reaction, the sugar chain can be cut off to different degrees by controlling the hydrolysis reaction time, and compared with a glycosylation product, the structure and functional properties of the modification product with different sugar contents are changed, thereby being beneficial to obtaining the glycosylation soybean protein with different functional properties.
Further, the enzyme deactivation conditions are as follows: and after the reaction is finished, immediately taking out, putting the mixture into a water bath kettle at the temperature of 85-95 ℃ for enzyme deactivation for 5-10 min, and then cooling to room temperature.
The enzyme deactivation is to stop the hydrolysis reaction and to deactivate the enzyme.
In a preferred embodiment, the present invention provides a method for preparing a glycosylated soybean protein product having different sugar contents, comprising the steps of:
taking soybean protein and maltodextrin as raw materials, and obtaining glycosylated soybean protein by the preparation of soybean protein dispersion liquid, the preparation of maltodextrin solution, the Maillard reaction of the soybean protein and the maltodextrin under the condition of damp heat, acid precipitation, centrifugal collection of precipitate, pH regulation, dialysis and drying; and then obtaining glycosylation modification products with different sugar contents under the catalysis of beta-amylase or beta-glucosidase, and finally inactivating enzyme, dialyzing and drying to obtain the soy protein glycosylation products with different sugar contents.
Preferably, the soy protein is selected from the group consisting of 7S glycinin, 11S glycinin, soy protein isolate; preferably, the soy protein is a soy protein dispersion, and the preparation conditions of the dispersion are as follows: adding distilled water with a certain volume into soybean protein serving as a raw material to prepare a soybean protein dispersion liquid, heating the soybean protein dispersion liquid in a water bath at 90-100   ℃ for 10   min, cooling to 37   ℃, and adjusting the pH value to 7.5-8.5, preferably, adjusting the pH value to 8.
The soybean protein has high nutritive value, complete types of essential amino acids, is close to animal protein, and is high-quality plant protein. Soy protein is widely used in many food industries because of its good nutritional quality and many functional properties. The soybean protein is called glycinin according to the part precipitated between pH4.5-4.8 by a high-speed centrifugation method, and the heat treatment can extend the globulin and is beneficial to promoting the Maillard reaction.
Preferably, the maltodextrin is a maltodextrin solution, and the preparation conditions of the solution are as follows: the method comprises the steps of taking maltodextrin as a raw material, adding a certain volume of distilled water to prepare a maltodextrin solution, and adjusting the pH value to 7.5-8.5, preferably, 8.
The maltodextrin has the characteristics of low sweetness, no peculiar smell, easy digestion, good solubility, strong thickening property, good carrier property, good stability and difficult deterioration, is widely applied to various foods, and can be used as a drying aid and an embedding wall material besides a filling agent and a thickening agent of the foods in recent years to improve the function of protein functional characteristics, thereby further expanding the application scene of the maltodextrin. Maltodextrin contains rich hydrophilic carbonyl groups and can perform Maillard reaction, so that the solubility and the emulsifying property of the whole molecule can be obviously improved. The maltodextrin is fully dissolved in distilled water, which is more beneficial to the Maillard reaction.
Preferably, the preparation method of the soy protein glycosylation product comprises the following steps: mixing the soybean protein dispersion liquid and the maltodextrin solution according to the mass ratio of 3: 1-1: 3 to enable the concentration of the final solution to be 2-6%, stirring and heating for 20-120 min at the temperature of 85-95 ℃ to obtain a soybean protein-maltodextrin mixed solution, removing free sugar molecules from the solution by alkali-soluble acid precipitation and a dialysis method, and freeze-drying the product to prepare the glycosylated soybean protein.
Preferably, the soybean protein dispersion liquid and the maltodextrin solution are mixed according to the mass ratio of 1:2, so that the concentration of the final solution is 4%, and the mixture is stirred and heated for 20min at the temperature of 95 ℃ to obtain the soybean protein-maltodextrin mixed solution.
Under the condition of certain temperature and humidity, the Maillard reaction can introduce the hydrophilic carbonyl of sugar molecules into protein molecules by changing the molecular structure of the protein, change the molecular volume, the charge and the amino acid composition, further improve the structure and the functional property of the protein and improve the processing characteristic of the protein. The maltodextrin is introduced into the soybean protein, compared with the original protein, the secondary structure of a glycosylation product is changed, the foamability, the foam stability, the emulsibility and the emulsion stability are obviously improved, and a theoretical basis is provided for functional ingredients for producing vegetable protein. Therefore, the method aims to realize glycosylation reaction between protein molecules and sugar molecules through Maillard reaction to improve the structure and functional characteristics of the protein and has wide prospect.
Preferably, the alkali-dissolving and acid-precipitating conditions are as follows: adjusting the pH value of the protein-sugar solution to 4.5, centrifuging at 3000-5000   r/min for 5-15   min, collecting precipitate, adding distilled water to dissolve, and adjusting the pH value to 7.0.
In the present invention, a precipitation phenomenon occurs at pH =4.5 in a soy protein-maltodextrin solution, the soy protein is precipitated, unreacted maltodextrin is in a supernatant, and then the precipitate is re-dissolved in distilled water to obtain a protein dispersion. The method can effectively remove the maltodextrin molecules which do not participate in the reaction, and is beneficial to the subsequent reaction.
Further, the dialysis condition is that the solution is dialyzed (8-14   kDa) for 24   -48 h at 4   ℃, preferably, the dialysis time is 24 h.
The dialysis is completed by 8-14   kDa dialysis bag, the sample solution is filled into the bag, the dialysis bag is immersed in water or buffer solution, the soybean protein with large molecular weight in the sample solution is trapped in the bag, and salt and maltodextrin molecules (less than or equal to 5   kDa) are separated out of the bag until the concentrations of two sides inside and outside the bag reach equilibrium. The method effectively removes salt molecules and free maltodextrin molecules in the dispersion liquid, and purifies the glycosylation product of the soybean protein.
Meanwhile, the invention also provides a soybean protein glycosylation modified product with different sugar contents, which is prepared by the method.
Finally, the invention also provides a soybean protein sugar content regulation model established by directionally breaking the copolymerized sugar chain, which is characterized in that the model provides theoretical support for explaining the important role of protein side chain glycosyl in the functional property of the protein.
Based on the Maillard reaction principle, the soybean protein rich in free amino and the maltodextrin rich in hydrophilic carbonyl are used as substrates to generate cross linking between protein molecules and sugar molecules, thereby realizing the glycosylation modification of the soybean protein. The soybean protein and maltodextrin are crosslinked to enhance the functional properties of the soybean protein such as solubility, emulsifying activity, foamability, foam stability, water and oil retention, apparent viscosity, viscoelasticity and the like, so that the food ingredient with ideal functional characteristics can be prepared by the Maillard reaction, and the soybean protein glycosylation product can be used as the functional ingredient to be applied to food, such as enhancing the foaming of cakes and being applied to food of special people. By utilizing highly specific hydrolysis reaction, beta-glucosidase or beta-amylase is used for breaking and covalently crosslinking maltodextrin in the soybean protein, and the hydrolysis reaction time is controlled to cut off sugar chains to different degrees, thereby providing guarantee for the research on the functional property characterization of the glycosylated soybean protein with different sugar contents; in addition, Maillard glycosylation can be used for preparing food ingredients with ideal functional characteristics, and the glycosylation modified product of the soybean protein can be used as a functional ingredient in food, such as enhancing the foaming of cakes and being applied to food of special people.
The invention discloses the following technical effects:
the research is based on the Maillard reaction principle, and maltodextrin rich in hydrophilic hydroxyl is added into soybean protein dispersion liquid rich in free amino groups so as to ensure that the soybean protein is fully crosslinked with the maltodextrin, thereby changing the characteristics of the soybean protein, such as relative molecular mass, amino acid composition, molecular volume, electric charge and the like, even the structure of the soybean protein. Then carrying out highly specific hydrolysis reaction by beta-glucosidase or beta-amylase to break the maltodextrin covalently cross-linked into the soybean protein, preparing and cutting off sugar chains in different degrees by controlling hydrolysis reaction time to obtain soybean protein isolate with different sugar contents, changing the sugar content of glycosylated soybean protein on the basis of unchanged self-crosslinking degree of protein, obtaining soybean protein with different sugar contents, establishing a soybean protein sugar content regulation model, evaluating a system and representing the property change of a modified product, aiming at clarifying the change rule of the soybean protein with different sugar contents in functional properties, thereby providing theoretical support for the important function of protein side chain glycosyl in the functional property of the protein, expanding the range of the protein as functional ingredients of food and meeting the special requirements of the protein in food processing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the grafting degree of modified SPI's for sample numbers 2, 3, 1, 4, 5 under different conditions.
FIG. 2 shows the grafting degree of modified SPI for sample numbers 9, 8, 6, 1, 7 under different conditions.
FIG. 3 shows the grafting degree of modified SPI's for sample numbers 10, 9, 1, 11, 12 under different conditions.
FIG. 4 is a bar graph of foaming and foam stability for samples 10, 14-16.
FIG. 5 is a graph showing fluorescence spectra in samples 10, 14 to 16.
FIG. 6 is the apparent viscosity of commercial Soy Protein (SPI), samples 10, 14-16.
FIG. 7 is a bar graph of foaming and foam stability for samples 10, 17-19.
FIG. 8 is a graph showing fluorescence spectra in samples 10, 17 to 19.
FIG. 9 is the apparent viscosity of commercial Soy Protein (SPI), samples 10, 17-19.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
A method for regulating and controlling the functional properties of soybean protein by directionally breaking Maillard copolymer sugar chains comprises the following steps:
(1) the preparation conditions of the soybean protein dispersion liquid are as follows: adding distilled water with a certain volume into soybean protein as raw material to obtain protein dispersion, heating SPI (soy protein isolate) dispersion in water bath at 95 deg.C for 10   min, cooling to 37   deg.C, and adjusting pH to 8.
(2) The preparation conditions of the maltodextrin solution are as follows: taking maltodextrin as a raw material, adding a certain volume of distilled water to prepare a maltodextrin solution, and adjusting the pH value to 8.
(3) The preparation conditions of the soybean protein glycosylation product are as follows: mixing the soybean protein dispersion liquid and the maltodextrin solution according to the mass ratio of 1:2 to ensure that the concentration of the final solution is 4 percent, stirring and heating for 40min at the temperature of 95 ℃ to obtain a soybean protein-maltodextrin mixed solution, removing free sugar molecules from the solution by alkali-soluble acid precipitation and dialysis, and freeze-drying the product to prepare the glycosylated soybean protein.
(4) The alkali dissolution and acid precipitation conditions are as follows: adjusting the pH value of the soybean protein-maltodextrin solution to 4.5, centrifuging at 3000   r/min for 10 min, collecting precipitate, adding distilled water to dissolve, and adjusting the pH value to 7.0.
(5) The dialysis conditions were that the solution was dialyzed (8-14   kDa) at 4   ℃ for 24   h.
Example 2
The only difference from example 1 was that the final concentration of the reaction solution in step (3) was changed to 2%.
Example 3
The only difference from example 1 was that the final concentration of the reaction solution in step (3) was changed to 3%.
Example 4
The only difference from example 1 was that the final concentration of the reaction solution in step (3) was changed to 5%.
Example 5
The only difference from example 1 was that the final concentration of the reaction solution in step (3) was changed to 6%.
Example 6
The only difference from example 1 was that the mass ratio of the soybean protein dispersion liquid and the maltodextrin solution of step (3) was changed to 1: 1.
Example 7
The only difference from example 1 was that the mass ratio of the soybean protein dispersion liquid and the maltodextrin solution of step (3) was changed to 1: 3.
Example 8
The only difference from example 1 was that the mass ratio of the soybean protein dispersion liquid and the maltodextrin solution of step (3) was changed to 2: 1.
Example 9
The only difference from example 1 was that the mass ratio of the soybean protein dispersion liquid and the maltodextrin solution of step (3) was changed to 3: 1.
Example 9
The only difference from example 1 was that the reaction time in step (3) was changed to 30 min.
Example 10
The only difference from example 1 was that the reaction time in step (3) was changed to 20 min.
Example 11
The only difference from example 1 was that the reaction time in step (3) was changed to 60 min.
Example 12
The only difference from example 1 was that the reaction time in step (3) was changed to 120 min.
Example 13
The only difference from example 1 is that the maltodextrin of step (2) was replaced by a resistant dextrin.
Experimental example 1
The free amino groups of examples 1 to 12 (labeled as samples 1 to 12) were measured, and the optimal conditions for the Maillard reaction were determined by comparing the free amino group contents; the specific process is as follows:
1.1 determination of free amino groups
The free amino group is the content of free amino group contained in protein molecules, and Maillard reaction can convert free amino group into binding state by using the reaction of free amino group of protein and sugar molecules, resulting in reduction of free amino group content.
The formula for calculating the Degree of Grafting (DG) of the glycosylated product is as follows:
Figure 35778DEST_PATH_IMAGE001
(C0is the free amino content before reaction; c1For the content of free amino groups after the reaction)
The results are shown in FIGS. 1 to 3.
Example 14
A method for regulating and controlling the functional properties of soybean protein by directionally breaking copolymerized sugar chains comprises the following steps:
(1) the glycosylation product was prepared under Maillard reaction conditions as determined in Experimental example 1, beta-amylase was added at 50   deg.C in an amount of 150   U/g protein, and shaking was carried out at constant temperature for 5   min. After the reaction is finished, inactivating enzyme, freeze-drying to obtain a modified product, dialyzing to remove free maltodextrin, and freeze-drying the glycosylation modified product for later use.
(2) Enzyme deactivation: after the reaction is finished, taking out the sample, putting the sample in a water bath kettle at the temperature of 90 ℃ for 5 minutes for enzyme deactivation, and cooling to room temperature.
Example 15
The only difference from example 14 was that the reaction time in step (1) was changed to 40 min.
Example 16
The only difference from example 14 was that the reaction time in step (1) was changed to 80 min.
Example 17
A method for regulating and controlling the functional properties of soybean protein by directionally breaking copolymerized sugar chains comprises the following steps:
(1) the glycosylation product is prepared under the Maillard reaction conditions determined in the experimental example 1, beta-glucosidase is added at 50   ℃ in an amount of 90   U/g protein, and the mixture is shaken at constant temperature for 5   min. After the reaction is finished, inactivating enzyme, freeze-drying to obtain a modified product, dialyzing to remove free maltodextrin, and freeze-drying the glycosylation modified product for later use.
(2) Enzyme deactivation: after the reaction is finished, taking out the sample, putting the sample in a water bath kettle at the temperature of 90 ℃ for 5 minutes for enzyme deactivation, and cooling to room temperature.
Example 18
The only difference from example 17 was that the reaction time in step (1) was changed to 20 min.
Example 19
The only difference from example 17 was that the reaction time in step (1) was changed to 80 min.
Example 20
A method for regulating and controlling the functional properties of soybean protein by directionally breaking copolymerized sugar chains comprises the following steps:
(1) the glycosylation product was prepared under Maillard reaction conditions as determined in Experimental example 1, beta-amylase was added at 50   deg.C in an amount of 90   U/g protein, and shaking was carried out at constant temperature for 5   min. After the reaction is finished, inactivating enzyme, freeze-drying to obtain a modified product, dialyzing to remove free resistant dextrin, and freeze-drying the glycosylation modified product for later use.
(2) Enzyme deactivation: after the reaction is finished, taking out the sample, putting the sample in a water bath kettle at the temperature of 90 ℃ for 5 minutes for enzyme deactivation, and cooling to room temperature.
Example 21
The only difference from example 20 was that the reaction time in step (1) was changed to 40 min.
Example 22
The only difference from example 20 was that the reaction time in step (1) was changed to 80 min.
Experimental example 2
Performance validation experiments were performed on examples 14-16 (labeled as samples 14-16), sample 10, and commercially available unmodified soy protein to compare technical effects; sugar content experiments were performed for examples 20-22 (labeled as samples 20-22) and for example 13 (labeled as sample 13) with the following results:
2.1 determination of sugar content in glycosylation modified products
Colorimetry is a method of determining the content of a component to be measured by comparing or measuring the color depth of a colored substance solution. The principle of determining the sugar content in the glycosylation modified product by using a colorimetric method is that polysaccharide is firstly hydrolyzed into monosaccharide under the action of sulfuric acid, and is quickly dehydrated to generate a furfural derivative, and finally generates an orange yellow compound with phenol, and the sugar content in the solution can be determined according to the intensity of light absorbed by a colored solution.
Drawing a glucose standard curve: preparing 0.1   mg/mL glucose standard solution, respectively taking 0, 0.2, 0.4, 0.6, 0.8 and 1.0   mL glucose standard solution in a test tube with a plug, supplementing 1.0   mL with distilled water, respectively adding 1.0   mL 5% phenol solution, then quickly adding 5.0   mL concentrated sulfuric acid, shaking uniformly, and standing for 10   min. Then treated in water bath at 30   deg.C for 20   min. The absorbance of the reaction solution was measured at 490   nm. The abscissa of the standard curve is the glucose mass concentration and the ordinate is the absorbance.
And (3) determination of a sample: the samples (4%, w/v) were diluted 100-fold, and the absorbance of the samples was measured in the same manner as in tables 1 and 2, except that 1   mL of the sample solution was used.
TABLE 1 sugar content (maltodextrin)
Figure DEST_PATH_IMAGE002
TABLE 2 sugar content (resistant dextrin)
Figure 860908DEST_PATH_IMAGE003
2.2 foamability and foam stability
Foam generally refers to a dispersion of gas bubbles dispersed in a continuous liquid phase or semi-solid containing a surfactant. Protein foaming characteristics include foaming capacity, which refers to the amount of foam produced under certain conditions (e.g., whipping), and foam stability, which is the stability (over time) of the foam formed.
The calculation method is as follows:
a protein sample was dissolved in a phosphate buffer (pH   7.0.0) to prepare a protein solution having a mass concentration of 10   g/L. Collecting 100   mL of the solution, whipping at 12000   r/min for 1   min, recording data, and measuring in parallel for 3 times[11]. Foaming Properties of protein: (F C ) And foam stability: (F S ) Are respectively expressed as formula (2) and formula (3)
Figure 843908DEST_PATH_IMAGE005
In the formula:V L is the volume of liquid before whipping;V 0 is the volume of foam immediately after whipping has stopped;V 30 the volume of the foam when left standing for 30   min after whipping is shown in FIG. 4.
2.3 solubility
The solubility of a substance in various solvents is often expressed in terms of solubility. Solubility is one of the major functional properties of proteins, and more importantly, solubility is a prerequisite and basis for the physiological and other processing functional properties of proteins. The measurement method is as follows:
the protein sample was dissolved in a buffer solution having a pH of 7.0 to prepare a 2   g/L protein solution. Storing at 4 deg.C after vortexing to make it fully hydrated. Centrifuging at 8000 r/min for 20   min the next day, collecting supernatant, and determining soluble protein content by Folin phenol method. The protein solubility was expressed as the ratio of the soluble protein content in the sample to be tested, and the results are shown in Table 3.
TABLE 3 solubility
Figure DEST_PATH_IMAGE006
2.4 hydrated particle size distribution and Zeta potential
The hydrated particle size reflects the aggregation behavior of the glycosylation modification product. Zeta potential is the potential of the shear plane of charged particles in a protein solution, and is generally used to measure the mutual attraction and repulsion between particles; and is also an important parameter for measuring the dispersion stability of the colloid system.
A concentrated protein dispersion was prepared and diluted to a concentration of 0.1   mg/mL (pH   7.0.0), filtered through a 0.45   μm water membrane, and then the hydrated particle size distribution and Zeta potential of the sample were measured in parallel 3 times using a particle size analyzer, with the results shown in Table 4.
TABLE 4 hydrated particle size distribution and zeta potential
Figure 380062DEST_PATH_IMAGE007
2.5 internal fluorescence Spectroscopy
The fluorescent effect is generated by fluorescent substances in the sample, which can be originated from the interior of the food sample, namely, the fluorescent effect is generated by the self components of the sample, and the fluorescent substances are called endogenous fluorescent substances.
Dissolving a protein sample in 0.01   mol/L phosphate buffer solution (pH   7.0.0) to prepare 1   mg/mL protein sample solution, centrifuging for 15   min under the condition of 10000   r/min, taking 4   mL supernatant, and scanning an emission spectrum in the range of 300-400   nm under the conditions that the emission wavelength is 290   nm and the luminosity of an excitation and emission slit is 5   nm. The wavelength is plotted on the abscissa and the relative fluorescence intensity is plotted on the ordinate, and the result is shown in FIG. 5.
2.6 determination of apparent viscosity
The apparent viscosity refers to the ratio of the corresponding shear stress to the shear rate under a certain velocity gradient. The apparent viscosity number can reflect the fluidity of the study object to a certain extent.
The apparent viscosity of the protein was measured using a malvern rheometer. A protein sample solution (pH   7.0.0) was prepared at a mass concentration of 4% (w/v). For the measurement, the sample dispersion was slowly poured into a filling jig (a conical plate having a diameter of 60   mm and a cone angle of 0.5   °). The measurement conditions are that the temperature is 25   ℃, and the frequency is 0.1-100   s-1The apparent viscosity of the sample was measured and the results are shown in FIG. 6.
Experimental example 3
Performance verification experiments were performed on examples 17-19 (labeled as samples 17-19), sample 10, and commercially available unmodified soy protein, comparing the technical effects, and the results were as follows:
3.1 determination of sugar content in glycosylation modified products
The results are shown in Table 5.
TABLE 5 sugar content
Figure DEST_PATH_IMAGE008
3.2 foamability and foam stability
Foam generally refers to a dispersion of gas bubbles dispersed in a continuous liquid phase or semi-solid containing a surfactant. Protein foaming characteristics include foaming capacity, which refers to the amount of foam produced under certain conditions (e.g., whipping), and foam stability, which is the stability (over time) of the foam formed.
The calculation method is as follows:
the protein sample was dissolved in a phosphate buffer (pH   7.5.5) to prepare a protein solution having a mass concentration of 10   g/L. Collecting 100   mL of the solution, whipping at 12000   r/min for 1   min, recording data, and measuring in parallel for 3 times[11]. Foaming Properties of protein: (F C ) And foam stability: (F S ) Are respectively expressed as formula (2) and formula (3)
Figure 833915DEST_PATH_IMAGE005
In the formula:V L is the volume of liquid before whipping;V 0 is the volume of foam immediately after whipping has stopped;V 30 the volume of the foam when left standing for 30   min after whipping is shown in FIG. 7.
3.3 solubility
The results are shown in Table 6.
TABLE 6 solubility
Figure 585DEST_PATH_IMAGE009
3.4 hydrated particle size distribution and Zeta potential
The hydrated particle size reflects the aggregation behavior of the glycosylation modification product. Zeta potential is the potential of the shear plane of charged particles in a protein solution, and is generally used to measure the mutual attraction and repulsion between particles; and is also an important parameter for measuring the dispersion stability of the colloid system.
A concentrated protein dispersion was prepared and diluted to a concentration of 0.1   mg/mL (pH   7.5.5), filtered with a 0.45   μm water film, and the hydrated particle size distribution and Zeta potential of the sample were measured in parallel 3 times using a particle size analyzer, the results of which are shown in Table 7.
TABLE 7 hydrated particle size distribution and zeta potential
Figure DEST_PATH_IMAGE010
3.5 internal fluorescence Spectroscopy
The fluorescent effect is generated by fluorescent substances in the sample, which can be originated from the interior of the food sample, namely, the fluorescent effect is generated by the self components of the sample, and the fluorescent substances are called endogenous fluorescent substances.
Dissolving a protein sample in 0.01   mol/L phosphate buffer solution (pH   7.5.5) to prepare 1   mg/mL protein sample solution, centrifuging for 15   min under the condition of 10000   r/min, taking 4   mL supernatant, and scanning an emission spectrum in the range of 300-400   nm under the conditions that the emission wavelength is 290   nm and the luminosity of an excitation and emission slit is 5   nm. The wavelength is plotted on the abscissa and the relative fluorescence intensity is plotted on the ordinate, and the result is shown in FIG. 8.
3.6 determination of apparent viscosity
The apparent viscosity of the protein was measured using a malvern rheometer. A protein sample solution (pH 7.5) was prepared at a mass concentration of 4% (w/v). For the measurement, the sample dispersion was slowly poured into a filling jig (a conical plate having a diameter of 60 mm and a cone angle of 0.5 °). The apparent viscosity of the sample is measured under the measurement conditions of 25 ℃ and 0.1-100 s < -1 > frequency, and the result is shown in figure 9.

Claims (14)

1. A process for preparing the glycosylated soybean protein with different contents of sugar includes such steps as preparing the glycosylated soybean protein by beta-amylase or beta-glucosidase.
2. The method of claim 1, further comprising the steps of inactivating enzyme, dialyzing, and drying.
3. The method according to claim 1, wherein the soybean protein is selected from the group consisting of 7S glycinin, 11S glycinin, isolated soybean protein; adding beta-amylase to the glycosylated soybean protein dispersion liquid at 50-50   -55 ℃ or adding beta-glucosidase to the glycosylated soybean protein dispersion liquid at 50-55 ℃, wherein the adding amount is 50-150U/g of protein, oscillating for 5   -80   min at constant temperature, inactivating enzyme after reaction is finished, dialyzing to remove free sugar molecules, and freeze-drying to prepare the glycosylated modified product with different sugar contents.
4. The method according to claim 3, wherein the pH value is 4.0-7.0 when beta-amylase is used as a catalyst; when beta-glucosidase is used as a catalyst, the pH value range is 5.0-7.0.
5. The preparation method according to claim 3, wherein the enzyme deactivation step is carried out under the conditions: and after the reaction is finished, immediately taking out, putting the mixture into a water bath kettle at the temperature of 85-95 ℃ for enzyme deactivation for 5-10 min, and then cooling to room temperature.
6. A method for preparing a soy protein glycosylation modification product with varying sugar content, the method comprising the steps of:
taking soybean protein and maltodextrin as raw materials, and obtaining glycosylated soybean protein by the preparation of soybean protein dispersion liquid, the preparation of maltodextrin solution, the Maillard reaction of the soybean protein and the maltodextrin under the condition of damp heat, acid precipitation, centrifugal collection of precipitate, pH regulation, dialysis and drying; and then, obtaining glycosylation modification products with different sugar contents by using beta-amylase or beta-glucosidase, specifically comprising enzymolysis, enzyme deactivation, dialysis and drying to obtain the glycosylation modification products which do not change the main chain of the soybean protein molecules and only change the sugar contents, and establishing the soybean protein sugar content regulation model.
7. The method according to claim 6, wherein the soy protein dispersion is prepared under the following conditions: the preparation method comprises the steps of taking soybean protein as a raw material, adding a certain volume of distilled water to prepare a soybean protein dispersion liquid, heating the soybean protein dispersion liquid in a water bath at 90-100   ℃ for 10   min, cooling to 37   ℃, and adjusting the pH value to 7.5-8.5.
8. The method according to claim 6, wherein the maltodextrin solution is prepared under the following conditions: taking maltodextrin as a raw material, adding a certain volume of distilled water to prepare a maltodextrin solution, and adjusting the pH value to 7.5-8.5.
9. The method of claim 6, wherein the Maillard reaction conditions are: mixing the soybean protein dispersion liquid and the maltodextrin solution according to the mass ratio of 3: 1-1: 3 to enable the concentration of the final solution to be 2-6%, and stirring and heating for reaction for 20-120 min at the temperature of 85-95 ℃ to obtain different soybean protein-maltodextrin mixed solutions.
10. The method according to claim 6, wherein the acid precipitation conditions are: the pH of the soy protein-maltodextrin solution was adjusted to 4.5.
11. The method of claim 6, wherein the centrifugation is carried out at 3000-5000   r/min for 5-15   min.
12. The method according to claim 6, wherein the pH adjustment is carried out by dissolving with distilled water and adjusting the pH to 7.0.
13. The preparation method of claim 6, wherein the dialysis conditions are dialysis (8-14   kDa) of the solution at 4   ℃ for 24   -48 h; the material in the dialysis bag is collected as a solution from which free sugar molecules are removed, and is dried to obtain glycosylated soybean protein.
14. A soy protein glycosylation modification product with different sugar content prepared according to any one of claims 1 to 13.
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