CN113637063A - Preparation process of dextran aldehyde glycosylated sodium caseinate - Google Patents
Preparation process of dextran aldehyde glycosylated sodium caseinate Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention discloses a preparation process of sodium glucuronosylated caseinate. Mixing a glucan solution and a sodium periodate solution; reacting under stirring to prepare dextran aldehyde, and dialyzing and freeze-drying a product after the reaction is finished; mixing a sodium caseinate solution and a dextran aldehyde solution; wet reaction at 70-90 deg.c for 30-120 min; and (5) freeze-drying the product to obtain the target product. The invention carries out oxidation modification on glucan to obtain glucan aldehyde with more reactive groups, and the contact probability of the reactive groups can be increased by increasing the number of aldehyde groups in polysaccharide molecules. And after the oxidation degree of glucan is increased, the molecular weight of polysaccharide is reduced, glucan aldehyde not only can be crosslinked among protein molecules, but also can play a crosslinking role in the protein molecules, so that the reaction time can be shortened, and the reaction efficiency can be improved. The product of the invention has higher emulsifying activity and better emulsifying stability when dealing with salt ions and heat treatment.
Description
Technical Field
The invention relates to sodium caseinate, in particular to a preparation process of sodium glucuronosylated caseinate, belonging to the field of protein modification.
Background
Protein is one of the major components of food and can alter the appearance, taste and texture of food under different processing conditions. Its functional properties in food include solubility, emulsifiability, foamability, gelling properties, etc. However, proteins are unstable in industrial processes (e.g., heat, shear treatment) and have difficulty in simultaneously achieving multiple functional properties, which limits their industrial applicability. Therefore, suitable modification techniques are often employed to extend the use of proteins in the food industry. Among protein modification methods, glycosylation modification has been a hot point of recent research.
The glycosylation modification of protein is a carbonyl-ammonia condensation reaction based on the Maillard reaction mechanism, wherein carbonyl groups of sugar molecules are covalently bonded with amino groups of protein molecules, and polysaccharide is grafted on the surface of the protein. The introduction of a large number of hydrophilic groups on the surface of the protein leads to an increase in the solubility of the protein. On the one hand, the increased solubility of the protein facilitates the rapid migration of hydrophobic and hydrophilic groups to the oil/water interface, thereby improving the emulsifying properties; on the other hand, the glycosylation modification increases the average particle size of the protein, and the glycosylated protein forms a thicker surface layer around the oil droplets than the native protein, thereby enhancing the stability of the emulsion. Therefore, glycosylation modification is an effective chemical modification means for improving the functional properties of food protein, and protein-sugar covalent complex is often used as a multifunctional additive with excellent properties.
In the current research, the glycosylation reaction is commonly carried out on macromolecular polysaccharide, but the reaction groups (aldehyde groups) in the macromolecular polysaccharide are limited and are often influenced by space resistance, so that the reaction of the macromolecular polysaccharide and macromolecular protein usually needs several hours to several days to complete the preparation of glycosylated protein. The glycosylation modification of protein has the defects of long time consumption, low efficiency and the like in both a dry heat method and a wet heat method. This is a problem to be solved in the process of modifying glycosylation of proteins.
It can be seen from the above background art that the problems of long time consumption, low efficiency and the like exist in the current glycosylation modification of protein, and the industrial and large-scale production and application of the protein are limited. If the aldehyde group content in polysaccharide molecules can be increased, the reaction time can be shortened, the reaction efficiency can be improved, and the protein glycosylation modification can be widely applied to the industry.
Studies on the antioxidant effect of glucan glycosylation on mung bean protein [ J ] studies on Chinese foods and nutrients, 2017,23(10): 63-67). The technology takes Mung Bean Protein Isolate (MBPI) and glucan as raw materials, and prepares an MBPI-Dextran covalent complex by a wet-heat method. The influence of glycosylation reaction on the oxidation resistance of MBPI is evaluated by measuring the reducing power of the product, the DPPH free radical scavenging capacity, superoxide anion free radical scavenging capacity and hydroxyl free radical scavenging capacity. The results show that: the MBPI-Dextran covalent complex has certain antioxidant capacity, particularly the product obtained by reaction at 90 ℃, the antioxidant capacity is obviously improved compared with the product at 80 ℃, which is related to the Maillard reaction degree, the Maillard reaction process is accelerated at higher temperature, and more substances with antioxidant capacity are promoted to be generated. Therefore, the mung bean protein after glycosylation modification has certain application value in the research of antioxidant capacity. In the technology, the MBPI-Dextran covalent complex prepared by a wet-heat method needs 6 hours of reaction to reach better grafting degree, and has the problems of slow glycosylation reaction rate and low efficiency. The main reason for this may be that the reactive groups (aldehyde groups) are few or the reactive contact sites (aldehyde or amino groups) are masked.
Chinese patent CN200810244118.3 discloses a method for improving functional properties of rice protein by protein-sugar graft coupling technology, which takes rice protein, a byproduct of rice starch and rice syrup, as a raw material, and adopts different glycosyl donors to carry out glycosylation modification on the rice protein by a wet method. The rice protein is grafted with sugar to effectively improve and fully utilize the functional properties of solubility, emulsibility, foamability and the like of the rice protein. Compared with chemical modification methods of vegetable protein such as alkylation, phosphorylation, deamidation and the like, the method is safe and environment-friendly, and the rice protein-polysaccharide graft coupling product can be used as a natural macromolecular emulsifier or a protein nutrition enhancer. However, the emulsifying activity of the rice protein-glucose graft prepared by the technology is 1.37 times of that of the protein, the emulsifying stability is 1.62 times of that of a protein control sample, and the reaction needs to be carried out in a strong alkali system with the pH value of 11, so that the defects of low efficiency and high energy consumption exist, and the industrial and large-scale production and application of the rice protein-glucose graft are limited.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an efficient glycosylation crosslinking method between glucan and sodium caseinate, which improves the glycosylation modification efficiency of protein, obviously improves the emulsion stability and the emulsion activity of emulsion, and improves the glycosylation modification efficiency of protein.
The purpose of the invention is realized by the following scheme:
a preparation process of dextran aldehyde glycosylated sodium caseinate comprises the following steps:
1) mixing a dextran solution and a sodium periodate solution; reacting under stirring to prepare dextran aldehyde, and dialyzing and freeze-drying a product after the reaction is finished;
2) mixing a sodium caseinate solution and a dextran aldehyde solution; wet reaction at 70-90 deg.c for 30-120 min;
3) freeze-drying the product obtained in the step 2) to obtain a sodium glucuronosylated caseinate product.
To further achieve the object of the present invention, preferably, the dextran has a molecular weight of 60-100 kDa.
Preferably, the molar ratio of the glucose units in periodate to glucan is 1: 1.4-10 (particularly preferably 1: 10).
Preferably, the stirring in step 1) is magnetic stirring.
Preferably, the magnetic stirring time is 13-17 h.
Preferably, the reaction under stirring in step 1) is carried out under magnetic stirring at pH 6-8 and at temperature 5-20 ℃ in the absence of light.
Preferably, it is characterized in that: in the step 2), the mass ratio of the dextran aldehyde to the sodium caseinate is 1: 1.3-2 (particularly preferably 1: 1.6).
Preferably, the wet reaction conditions are water bath reactions.
Preferably, the time of the wet reaction is 60-90 min.
Preferably, the dialysis is dialysis through a 3.5kD dialysis bag.
The prior protein glycosylation reaction mainly comprises the reaction of polysaccharide molecules with large volume and protein molecules, the reaction groups (aldehyde groups) in the macromolecular polysaccharide are few, the reactive contact sites can be shielded, and the space resistance can be influenced, so that the expected modification effect can be achieved in a long time (several days). The polysaccharide contains a large number of free hydroxyl groups, and the polysaccharide containing ortho-dihydroxy can be oxidized into the polysaccharide containing ortho-dialdehyde by periodate. The glucan is subjected to oxidation modification to obtain the glucan aldehyde with more reactive groups, and in the reaction process, the number of aldehyde groups in polysaccharide molecules is increased to increase the contact probability of the reactive groups; secondly, after the oxidation degree of glucan is increased, the molecular weight of polysaccharide is reduced, and glucan aldehyde not only can be crosslinked among protein molecules, but also can play a crosslinking role in the protein molecules; in addition, under the same reaction conditions, compared with unmodified glucan, oxidized glucan aldehyde can achieve better effect after reacting for 1 hour, and the reaction time is greatly shortened. Therefore, the invention can reduce the reaction time and improve the reaction efficiency for the glycosylation reaction of the protein.
The glucan aldehyde contains a large amount of hydroxyl groups, has good hydrophilic capacity, and the introduction of a large amount of hydrophilic groups on the surface of the protein increases the solubility of the protein. On the one hand, the increased solubility of the protein facilitates the rapid migration of hydrophobic and hydrophilic groups to the oil/water interface, thereby improving the emulsifying properties; on the other hand, the glycosylation modification increases the average particle size of the protein, and the glycosylated protein forms a thicker surface layer around the oil droplets than the native protein, thereby enhancing the stability of the emulsion.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) polysaccharide and protein are both macromolecules, and both have structural effects in chemical modification, namely, sugar chains of polysaccharide or peptide chains of protein can play a shielding effect on reactive active sites. The invention solves the problems of long reaction time and low efficiency in the traditional protein glycosylation modification. Starting from the raw material polysaccharide, the polysaccharide is modified, reactive groups are added, and the reaction time can be reduced.
(2) In the prior art, glucan is used as a raw material, and in a protein glycosylation reaction, one polysaccharide molecule is combined with at most one protein molecule; the glycosylation reaction of the invention takes the glucan aldehyde as a raw material, and in the glycosylation modification of the protein, one polysaccharide molecule can be combined with a plurality of protein molecules, namely the glucan aldehyde has the cross-linking effect among the protein molecules.
(3) The sodium dextran aldehyde glycosylated caseinate prepared by the invention is suitable for preparing emulsion, has higher emulsifying activity, and has better emulsifying stability in response to salt ions and heat treatment.
Drawings
FIG. 1 is a liquid gel chromatogram of dextran and dextran aldehyde.
FIG. 2 is a gel electrophoresis image of sodium caseinate and its glycosylation products.
Detailed Description
The present invention will be further described with reference to the following examples for better understanding of the present invention, but the embodiments of the present invention are not limited thereto.
Example 1
A preparation process of dextran aldehyde glycosylated sodium caseinate comprises the following steps:
(1) using distilled water as a solvent, preparing 30mg/mL dextran and 3.94mg/mL sodium periodate solutions respectively, and mixing so that the molar ratio of total glucose units in periodate to dextran is 1: 10, magnetically stirring for 15 hours at the pH of 7 and the low temperature of 10 ℃ in a dark place;
(2) dialyzing the product obtained in the step (1) by using a 3.5kD dialysis bag until the conductivity is stable, and freeze-drying the dialyzate to obtain the dextran aldehyde;
(3) preparing 6mg/mL dextran aldehyde solution and 10mg/mL sodium caseinate solution by using distilled water as a solvent, and mixing, wherein the mass ratio of dextran aldehyde to sodium caseinate is 1: 1.7, stirring uniformly;
(4) and (4) maintaining the mixed solution in the step (3) at 80 ℃, reacting for 60min, stopping the reaction, and freeze-drying the product to obtain the sodium glucuronosylated casein.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has proven to be a reliable method for detecting whether covalent bonding between proteins and polysaccharides occurs during the maillard reaction. The change in molecular weight of the protein can be judged by the position of the band by gel electrophoresis. As shown in fig. 2, the NaCas-Dex1 (example 1), NaCas-Dex4 (example 2) and NaCas-Dex7 (example 3) bands were shifted up in position and lighter in color compared to NaCas (sodium caseinate). Indicating that sodium caseinate does form a high molecular weight complex covalently bound to dextran aldehyde.
Example 2
A preparation process of dextran aldehyde glycosylated sodium caseinate comprises the following steps:
(1) using distilled water as a solvent, preparing 30mg/mL dextran and 15.78mg/mL sodium periodate solutions respectively, and mixing so that the molar ratio of total glucose units in periodate to dextran is 1: 2.5, magnetically stirring for 15 hours at the pH of 7 and the low temperature of 10 ℃ in a dark place;
(2) dialyzing the product obtained in the step (1) by using a 3.5kD dialysis bag until the conductivity is stable, and freeze-drying the dialyzate to obtain the dextran aldehyde;
(3) preparing 6mg/mL dextran aldehyde solution and 10mg/mL sodium caseinate solution by using distilled water as a solvent, and mixing, wherein the mass ratio of dextran aldehyde to sodium caseinate is 1: 1.7, stirring uniformly;
(4) and (4) maintaining the mixed solution in the step (3) at 80 ℃, reacting for 60min, stopping the reaction, and freeze-drying the product to obtain the sodium glucuronosylated casein.
Example 3
A preparation process of dextran aldehyde glycosylated sodium caseinate comprises the following steps:
(1) using distilled water as a solvent, preparing 30mg/mL dextran and 27.61mg/mL sodium periodate solutions respectively, and mixing so that the molar ratio of total glucose units in periodate and dextran is 1: 1.4, magnetically stirring for 15 hours at the pH of 7 and the low temperature of 10 ℃ in a dark place;
(2) dialyzing the product obtained in the step (1) by using a 3.5kD dialysis bag until the conductivity is stable, and freeze-drying the dialyzate to obtain the dextran aldehyde;
(3) preparing 6mg/mL dextran aldehyde solution and 10mg/mL sodium caseinate solution by using distilled water as a solvent, and mixing, wherein the mass ratio of dextran aldehyde to sodium caseinate is 1: 1.7, stirring uniformly;
(4) and (4) maintaining the mixed solution in the step (3) at 80 ℃, reacting for 60min, stopping the reaction, and freeze-drying the product to obtain the sodium glucuronosylated casein.
The sodium glucuronosylated casein obtained in the step has the emulsifying activity index of 52m2The emulsion stability was 326% per gram.
Comparative example 1
(1) Preparing 6mg/mL dextran solution and 10mg/mL sodium caseinate solution by using distilled water as a solvent, and mixing, wherein the mass ratio of dextran to sodium caseinate is 1: 1.7, stirring evenly
(2) And (2) maintaining the mixed solution in the step (1) at 80 ℃, reacting for 60min, stopping the reaction, and freeze-drying the product to obtain the dextran glycosylation sodium caseinate.
The polysaccharide aldosylated sodium caseinate prepared in the above examples was measured for emulsifying activity and emulsifying stability, and the test results are listed in table 1. Table 1 shows the comparison of the properties of sodium glucuronosylated caseinate prepared under the conditions of example 1, example 2, example 3 with those of sodium glucuronosylated caseinate.
The preparation process of the emulsion comprises the following steps: first, dextran-glycosylated sodium caseinate or dextran aldehyde-glycosylated sodium caseinate solutions (10mg/mL) were prepared, the pH was adjusted to 7, and these solutions were used as dispersants. Secondly, corn oil is added into the dispersing agent, and the volume content of the corn oil is 10 percent. Then, the mixture was dispersed with a high-speed disperser at 11000r/min for 1min, at which time an emulsion was obtained.
Determination of the emulsifying activity and the emulsifying stability: the prepared emulsion was analyzed for emulsifying activity and emulsifying stability by colorimetry. At 0min and 10min after the preparation of the crude emulsion, sucking 100 microliter of sample from the bottom of the emulsion and fully mixing with 5mL of sodium dodecyl sulfate solution with the mass fraction of 0.1 percent, immediately measuring the light absorption value of the turbid solution at 500nm, and taking the sodium dodecyl sulfate solution as a reference. According to the light absorption value of the turbid liquid, the emulsifying activity is calculated by the formula (1-1), and the emulsifying stability is calculated by the formula (1-2).
In the formula: a. the0Represents the absorbance measured at 0min after the emulsion is prepared; d represents dilution factor (51); c represents the concentration of sodium glucosylated dextran (10000 g/m)3) (ii) a L represents an optical length (0.01 m); θ represents the oil phase volume fraction (0.1); a. the10Represents the absorbance measured at 10min after emulsion preparation.
TABLE 1 results of the emulsion activity and emulsion stability tests in examples 1 to 3 and comparative example 1
| Example 1 | Example 2 | Example 3 | Comparative example 1 | |
| Emulsifying Activity m2/g | 44 | 51 | 52 | 39 |
| Emulsion stability% | 65 | 149 | 326 | 25 |
As can be seen from table 1, the emulsification activity and the emulsification stability of sodium glucuronosylated caseinate were significantly enhanced compared to the reaction product of dextran and sodium caseinate (comparative example 1), and the emulsification stability of sodium glucuronosylated caseinate (example 3) was improved by 301% compared to that of sodium caseinate (comparative example 1). Therefore, the invention provides a high-efficiency glycosylation crosslinking method between glucan and sodium caseinate, which obviously improves the emulsion stability and the emulsion activity of the emulsion and improves the glycosylation modification efficiency of protein.
FIG. 1 is a liquid gel chromatogram of dextran and dextran aldehyde, Dex0, Dex1, Dex4, Dex7 being dextran, dextran aldehyde prepared in example 1, dextran aldehyde prepared in example 2, dextran aldehyde prepared in example 3, respectively; the molecular weight of glucan is 68.39kDa, and the molecular weights of Dex1, Dex4 and Dex7 are 67.60kDa, 62.17kDa and 58.75kDa, respectively, which indicates that the molecular weight of glucan aldehyde decreases after glucan is oxidized, and the molecular weight of glucan aldehyde decreases with the increase of the degree of oxidation.
FIG. 2 is a gel electrophoresis diagram of sodium caseinate and its glycosylation products, NaCas-Dex0, NaCas-Dex1, NaCas-Dex4, NaCas-Dex7 are sodium caseinate, sodium dextran-glycosylated caseinate, sodium dextran-aldo-glycosylated caseinate prepared in example 1, sodium dextran-aldo-glycosylated caseinate prepared in example 2, and sodium dextran-aldo-glycosylated caseinate prepared in example 3, respectively. The position and color intensity of the NaCas-Dex0 and NaCas band are not obviously changed, which indicates that the glucan grafted on the sodium caseinate is trace under the condition of heating at 80 ℃ for 1 h. The positions of three glycosylated casein bands of NaCas-Dex1, NaCas-Dex4 and NaCas-Dex7 are sequentially arranged upwards, which shows that glucan aldehyde and sodium caseinate are connected together in a covalent bond mode, and polysaccharide and protein with small molecular weight are crosslinked to form a larger glycoprotein molecule. And the higher the oxidation degree of glucan (the more the aldehyde group content of glucan), the higher the molecular weight of glucan-glycosylated sodium caseinate, indicating that more glucan is grafted on the protein molecule. This is because dextran aldehyde having a high degree of oxidation can provide more reactive aldehyde groups; on the other hand, the steric effect of the low molecular weight dextran aldehyde is reduced, and the low molecular weight dextran aldehyde is more beneficial to contact and react with protein.
The prior protein glycosylation reaction mainly comprises the reaction of polysaccharide molecules with large volume and protein molecules, the reaction groups (aldehyde groups) in the macromolecular polysaccharide are few, the reactive contact sites can be shielded, and the space resistance can be influenced, so that the expected modification effect can be achieved in a long time (several days). As can be seen from Table 1, compared with unmodified dextran (comparative example 1), oxidized dextran aldehyde reacts for 1h (both products obtained by reacting dextran aldehyde as a raw material with sodium caseinate for 1 h) under the same reaction conditions, and the difference of the examples is that the oxidation degree of dextran aldehyde prepared by using dextran is different, namely the oxidation degree of dextran aldehyde obtained by performing glycosylation reaction on sodium caseinate is different, and the higher the oxidation degree of dextran aldehyde is (the more sodium periodate is used), the better the effect is, the reaction time is greatly shortened, and the important large-scale production advantage is achieved. Therefore, the invention can obviously reduce the reaction time and improve the reaction efficiency for the glycosylation reaction of the protein.
It should be noted that the embodiments of the present invention are not limited by the above-mentioned examples, and any other changes, modifications, substitutions, combinations, and simplifications which are made without departing from the spirit and principle of the present invention should be regarded as equivalent substitutions, and are included in the scope of the present invention.
Claims (10)
1. A preparation process of sodium dextran aldehyde glycosylated caseinate is characterized by comprising the following steps:
1) mixing a dextran solution and a sodium periodate solution; reacting under stirring to prepare dextran aldehyde, and dialyzing and freeze-drying a product after the reaction is finished;
2) mixing a sodium caseinate solution and a dextran aldehyde solution; wet reaction at 70-90 deg.c for 30-120 min;
3) freeze-drying the product obtained in the step 2) to obtain a sodium glucuronosylated caseinate product.
2. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: the molecular weight of the glucan is 60-100 kDa.
3. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: the molar ratio of glucose units in periodate to glucose units in glucan is 1: 1.4-10.
4. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: the stirring in the step 1) is magnetic stirring.
5. The process for preparing sodium dextran-aldose caseinate according to claim 4, wherein: the magnetic stirring time is 13-17 h.
6. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: the reaction under stirring in the step 1) refers to the reaction under magnetic stirring at the temperature of 5-20 ℃ and under the condition of light shielding and pH of 6-8.
7. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: in the step 2), the mass ratio of the dextran aldehyde to the sodium caseinate is 1: 1.3-2.
8. The process for preparing sodium dextran-aldose caseinate according to claim 1, wherein: the wet reaction condition is water bath reaction.
9. The process for preparing sodium dextran aldehyde glycosylated caseinate according to claim 8, wherein: the time of the wet reaction is 60-90 min.
10. The process for preparing sodium dextran aldehyde glycosylated caseinate according to claim 8, wherein: the dialysis is performed through a 3.5kD dialysis bag.
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