WO2020134772A1 - β-LACTOGLOBULIN-VITAMIN CONJUGATE COMPRISING SUGAR ALCOHOLS AND PREPARATION METHOD THEREOF - Google Patents

β-LACTOGLOBULIN-VITAMIN CONJUGATE COMPRISING SUGAR ALCOHOLS AND PREPARATION METHOD THEREOF Download PDF

Info

Publication number
WO2020134772A1
WO2020134772A1 PCT/CN2019/120410 CN2019120410W WO2020134772A1 WO 2020134772 A1 WO2020134772 A1 WO 2020134772A1 CN 2019120410 W CN2019120410 W CN 2019120410W WO 2020134772 A1 WO2020134772 A1 WO 2020134772A1
Authority
WO
WIPO (PCT)
Prior art keywords
lactoglobulin
solution
electric field
place
glycosylation
Prior art date
Application number
PCT/CN2019/120410
Other languages
French (fr)
Chinese (zh)
Inventor
左齐乐
韩菲菲
毛宝兴
李勉
朱炫
陈杰
陈跃文
石丽华
张文瑶
Original Assignee
浙江华康药业股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江华康药业股份有限公司 filed Critical 浙江华康药业股份有限公司
Publication of WO2020134772A1 publication Critical patent/WO2020134772A1/en

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a ⁇ -lactoglobulin-vitamin conjugate containing sugar alcohol and a preparation method thereof.
  • VE is a fat-soluble vitamin. It has good antioxidant capacity in and outside the body, as well as certain anti-aging, anti-cancer functions, and functions such as prolonging shelf life.
  • VE is a fat-soluble substance, it is insoluble in water.
  • ultraviolet rays, alkali and oxygen can quickly destroy VE, making it difficult to be well used in the food industry. Therefore, it is necessary to use special methods to treat it in order to maintain the unique antioxidant properties of VE itself while enhancing its water solubility, stability and the effect of addition in food.
  • ⁇ -lactoglobulin is a kind of milk protein and a member of the lipid carrier protein family. It has strong ligand binding ability and can be combined with fat-soluble vitamins and fatty acids. It is used as a carrier for fat-soluble nutrients To strengthen the production of fat foods, fat-soluble vitamins and fatty acids can self-assemble into the hydrophobic holes and hydrophobic cracks on the surface of ⁇ -lactoglobulin molecules, so that ⁇ -lactoglobulin can not only bind these small fat-soluble molecules, but also Can promote the absorption of these substances.
  • the same ⁇ -lactoglobulin is also one of the main allergens in cow milk, so while using it in combination with vitamins to exert its effects, we also need to take certain measures for its adverse effects in order to better play its Function and broaden its application level.
  • Publication No. CN103783169A discloses a method for making fresh vitamin E fortified cheese. Although this method uses ⁇ -lactoglobulin to self-assemble with VE, it plays a certain role in protecting VE, but there are also some problems. : 1) The sensitization of ⁇ -lactoglobulin has not been eliminated, which limits its application. 2) The emulsifier is not used in combination with self-assembly, and its assembly effect is naturally reduced.
  • the present invention attempts to modify ⁇ -lactoglobulin to reduce its sensitization, so as to enhance the stabilizer during the binding process with VE.
  • the technical problem to be solved by the present invention is to provide a ⁇ -lactoglobulin-vitamin conjugate containing sugar alcohols and a preparation method thereof, and to provide a ⁇ -lactoglobulin with reduced sensitization.
  • sugar alcohol is used as a natural emulsifier to further improve the assembly effect of ⁇ -lactoglobulin-vitamin after assembly.
  • the process is simple and the operation is convenient.
  • the present invention is achieved in this way, providing a sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate, including ⁇ -lactoglobulin, VE, sorbitol, and maltitol after high-pressure pulse glycosylation treatment
  • High-pressure pulse glycosylation refers to the treatment of 80 ⁇ s by placing ⁇ -lactoglobulin prepared with 0.01 mg/L phosphate buffer solution of pH 7.4 into 1 mg/mL in a high-voltage electric field, where the electric field strength of the high-voltage electric field is 30kV/cm, the electric field waveform is a unipolar square wave.
  • the high-pressure pulse glycosylation treatment further includes first-level high-voltage electric field glycosylation-treated ⁇ -lactoglobulin and L-arabinose obtained by using ⁇ -lactoglobulin as previously obtained by the high-voltage electric field treatment method.
  • Galactose is mixed in a mass ratio of 1:0.01:2 and lyophilized.
  • the lyophilized powder is then placed in a centrifuge tube and placed in an incubator at a temperature of 65°C and a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide. After 3 hours of reaction in the solution, place the centrifuge tube in an ice bath to terminate the reaction immediately.
  • the high-pressure pulse glycosylation treatment further includes the following process: combining the ⁇ -lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment in the following manner: Take ⁇ -lactoglobulin and VE with a mass ratio of 25mg ⁇ 40mg: 2.5mg ⁇ 4mg, add 25ml ⁇ 40ml of purified water, 5g ⁇ 15g of sorbitol, 10g ⁇ 20g of maltitol, and vortex to mix for 40s ⁇ 60s.
  • the mixed solution was magnetically acted at 50°C ⁇ 60°C for 70min ⁇ 100min, put the solution into a dialysis bag, placed in 30 times the volume of pure water for 24h to remove uncoated VE to obtain the composition , Using a vacuum freeze dryer at -45 °C for 20h to obtain a dried ⁇ -lactoglobulin-vitamin conjugate product containing sugar alcohol.
  • the present invention is achieved in this way, and provides a method for preparing a sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate as described above, including the following steps:
  • Step 1 ⁇ -lactoglobulin is prepared into a 1 mg/mL ⁇ -lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4;
  • Step 2 Place the ⁇ -lactoglobulin solution in a high-voltage electric field for high-voltage pulse processing
  • Step 3 Combine ⁇ -lactoglobulin after high-pressure pulse glycosylation treatment with VE, sorbitol, and maltitol, and freeze-dry the composition to obtain dried ⁇ -lactoglobulin containing sugar alcohol- Vitamin conjugate products.
  • step two the ⁇ -lactoglobulin solution is placed in a high-voltage electric field for a treatment time of 80 ⁇ s, wherein the electric field strength of the high-voltage electric field is 30 kV/cm, and the electric field waveform is a unipolar square wave.
  • the combination method includes: weighing ⁇ -lactoglobulin and VE with a mass ratio of 25 mg to 40 mg: 2.5 mg to 4 mg, adding 25 ml to 40 ml of purified water, and 5 g to 15 g of sorbitol, 10g ⁇ 20g maltitol, vortex to mix for 40s ⁇ 60s, apply the magnetic force at 50°C ⁇ 60°C for 70min ⁇ 100min, put the solution into the dialysis bag and put it in 30 times volume of pure water Dialysis was performed for 24 hours to remove uncoated VE to obtain a ⁇ -lactoglobulin-vitamin composition containing sugar alcohol.
  • the present invention is achieved in this way, and provides a method for preparing a sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate as described above, including the following steps:
  • ⁇ -lactoglobulin is made into 1mg/mL ⁇ -lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH7.4;
  • the first-level high-pressure pulse glycosylation-treated ⁇ -lactoglobulin obtained in step (2) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction.
  • the sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate and its preparation method have the following characteristics:
  • ⁇ -lactoglobulin can be modified to force the secondary and tertiary structure of ⁇ -lactoglobulin to change, thereby effectively reducing protein sensitization;
  • Figure 1 is a comparison diagram of the measurement data of ⁇ -lactoglobulin free amino content of three groups of untreated, glycosylated and high-pressure pulse glycosylated test samples;
  • Figure 2 is a comparison diagram of the measured data of ⁇ -lactoglobulin antigenicity reduction rate of the three groups of untreated, glycosylated and high-pressure pulse glycosylated test samples;
  • FIG. 3 is a comparison diagram of the binding rate of ⁇ -lactoglobulin and VE between the formula without sugar alcohol group and the formula group of the present invention.
  • the preferred embodiment of the sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate of the present invention includes ⁇ -lactoglobulin, VE, sorbitol, and maltitol after high-pressure pulse glycosylation treatment, and the high-pressure pulse sugar
  • the basification treatment means that ⁇ -lactoglobulin prepared with 0.01 mol/L phosphate buffer solution of pH 7.4 and 1 mg/mL is placed in a high-voltage electric field for 80 ⁇ s, in which the electric field strength of the high-voltage electric field is 30 kV/cm ,
  • the electric field waveform is a unipolar square wave.
  • the high-pressure pulse glycosylation treatment also includes the first-level high-voltage electric field glycosylation-treated ⁇ -lactoglobulin, L-arabinose, and semi Lactose is mixed at a mass ratio of 1:0.01:2 and lyophilized.
  • the lyophilized powder is then placed in a centrifuge tube and placed in an incubator at a temperature of 65°C and a relative humidity of 65% by mass and a saturated potassium iodide solution of 59% by mass After 3 hours of reaction, place the centrifuge tube in an ice bath to terminate the reaction immediately.
  • the high-pressure pulse glycosylation treatment also includes the following processing procedure: the ⁇ -lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment are combined by: weighing The mass ratio is 25mg ⁇ 40mg: 2.5mg ⁇ 4mg of ⁇ -lactoglobulin and VE, add 25ml ⁇ 40ml of pure water, 5g ⁇ 15g sorbitol, 10g ⁇ 20g maltitol, vortex to mix for 40s ⁇ 60s, the obtained
  • the mixed solution is magnetically operated at 50°C ⁇ 60°C for 70min ⁇ 100min, put the solution into the dialysis bag, put it in 30 times the volume of pure water to avoid light dialysis for 24h, remove the uncoated VE, and get the combination
  • the product was frozen in a vacuum freeze dryer at -45°C for 20 hours to obtain a dried sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate product.
  • the invention also discloses a method for preparing the ⁇ -lactoglobulin-vitamin conjugate containing sugar alcohol as mentioned above, which includes the following steps:
  • Step 1 ⁇ -lactoglobulin is prepared into a 1 mg/mL ⁇ -lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
  • Step 2 Place the ⁇ -lactoglobulin solution in a high-voltage electric field under high-voltage pulse processing.
  • Step 3 Combine ⁇ -lactoglobulin after high-pressure pulse glycosylation treatment with VE, sorbitol, and maltitol, and freeze-dry the composition to obtain dried ⁇ -lactoglobulin containing sugar alcohol- Vitamin conjugate products.
  • step two the ⁇ -lactoglobulin solution is placed in a high-voltage electric field for a treatment time of 80 ⁇ s, wherein the electric field strength of the high-voltage electric field is 30 kV/cm, and the electric field waveform is a unipolar square wave.
  • the combination method includes: weighing ⁇ -lactoglobulin and VE with a mass ratio of 25 mg to 40 mg: 2.5 mg to 4 mg, adding 25 ml to 40 ml of purified water, 5 g to 15 g of sorbitol, and 10 g to 20 g Maltitol, vortex to mix for 40s ⁇ 60s, apply the magnetic force at 50°C ⁇ 60°C for 70min ⁇ 100min, put the solution into the dialysis bag, put it in 30 times volume of pure water to avoid light After 24 hours of dialysis, the uncoated VE was removed to obtain a ⁇ -lactoglobulin-vitamin composition containing sugar alcohol.
  • the invention also discloses a method for preparing the ⁇ -lactoglobulin-vitamin conjugate containing sugar alcohol as mentioned above, which includes the following steps:
  • the ⁇ -lactoglobulin is prepared into a 1 mg/mL ⁇ -lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
  • the first-level high-pressure pulse glycosylation-treated ⁇ -lactoglobulin obtained in step (2) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction.
  • the preparation method of the first ⁇ -lactoglobulin-vitamin conjugate containing sugar alcohol of the present invention includes the following steps:
  • ⁇ -lactoglobulin is prepared into a 1 mg/mL ⁇ -lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
  • the first-level high-pressure pulse glycosylation-treated ⁇ -lactoglobulin obtained in step (12) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction.
  • the second method for preparing a sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate of the present invention includes the following steps:
  • ⁇ -lactoglobulin is made into 1mg/mL ⁇ -lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH7.4.
  • the first-level high-pressure pulse glycosylation-treated ⁇ -lactoglobulin obtained in step (22) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction.
  • the third method for preparing a sugar alcohol-containing ⁇ -lactoglobulin-vitamin conjugate of the present invention includes the following steps:
  • ⁇ -lactoglobulin is prepared into a 1 mg/mL ⁇ -lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
  • the first-level high-pressure pulse glycosylation-treated ⁇ -lactoglobulin obtained in step (32) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction.
  • the characteristics of the ⁇ -lactoglobulin-vitamin conjugate product containing sugar alcohol prepared by the method of the present invention will be evaluated by the following test.
  • the glycosylation reaction is a chemical reaction in which a carbonyl group in a monosaccharide, a reducing sugar, or a carbonyl compound is covalently bonded to an amino group in a protein, so the degree of glycosylation reaction can be reflected by the free amino content of the protein.
  • the content of free amino group is determined by o-phthalaldehyde (OPA) method.
  • Figure 1 shows the comparison data of the determination of the free amino content of the three groups of untreated, glycosylated and high-pressure pulsed glycosylated test samples, where untreated refers to the beta sample without any treatment.
  • the glycosylation treatment means that the test sample is only glycosylated, and the high-pressure pulse glycosylation treatment refers to the test sample of Example 1 obtained by the method of the present invention.
  • ⁇ -lactoglobulin undergoes PEF (high-voltage pulsed electric field, Pulsed Electric Fields) After combined with glycosylation, the content of free amino groups is significantly reduced, indicating that ⁇ -lactoglobulin and sugar molecules are covalently bonded after glycosylation, and there is a downward trend with the increase of pulse field strength, possible reasons It is processed by high-intensity pulse intensity, and the spatial structure of ⁇ -lactoglobulin is expanded to a greater extent.
  • the amino groups in ⁇ -lactoglobulin molecules are exposed on the surface of the protein molecules, and the sugar groups are more easily covalently bound to the amino groups, making ⁇ -lactoglobulin
  • the free amino content of globulin is significantly reduced.
  • the determination of antigenicity adopts indirect competitive ELISA method (enzyme-linked immunosorbent assay, Enzyme Linked Immune Sorbent Assay).
  • ⁇ -lactoglobulin standard concentration 0.5 ⁇ g/mL ⁇ 64 ⁇ g/mL
  • absorbance value as the ordinate to draw a standard curve.
  • ⁇ -lactoglobulin can be calculated under different treatment conditions
  • the antigenicity of the sample ( ⁇ g/mL) to calculate the rate of decrease in antigenicity. Calculated as follows:
  • Antigen reduction rate (%) (C as- C to be tested ) ⁇ C as is *100,
  • Figure 2 shows the comparison data of the determination of the antigenicity reduction rate of the three groups of untreated, glycosylated and high-pressure pulsed glycosylated test samples, where untreated refers to the beta samples without any treatment Protein, glycosylation treatment means that the test sample is only subjected to glycosylation treatment, and high-pressure pulse glycosylation treatment refers to the test sample of Example 1 obtained by the method of the present invention.
  • the high-pressure pulse glycosylation treatment method and the glycosylation treatment method used in the present invention have reduced the antigenicity of ⁇ -lactoglobulin, and the high-pressure pulse glycosylation treatment in the invention
  • the way the antigenicity is reduced the most is 74%, indicating that the method of the invention can effectively reduce the sensitization of ⁇ -lactoglobulin.
  • Determination of the combination rate of ⁇ -lactoglobulin and VE Accurately weigh 0.2g of ⁇ -lactoglobulin and VE conjugate, add 1mL of pure water to fully dissolve, then add 10mL of ethanol into the shaker and shake to extract at 200r/min for 20min. Anhydrous sodium sulfate removes water. The solution was centrifuged at 3000r/min for 10min, and then the supernatant was sucked, diluted with ethanol to an appropriate concentration, and the absorbance was measured at 285nm with ethanol as a blank.
  • C is the sample concentration (mg/mL)
  • A is the absorbance of the sample
  • B is the dilution factor
  • V is the volume of the extraction solution (10mL)
  • m1 is the mass of the conjugate (mg)
  • m2 is the total amount of VE (mg ).
  • the binding rate of the formula-free sugar alcohol group and the invention formula group-that is, the sugar alcohol-added ⁇ -lactoglobulin-VE conjugate (Example 1 test sample) of the present invention is obtained as follows Figure 3 shows (the test sample is repeated 5 times).
  • the combination rate of the formula group of the present invention is more than 50% higher than that of the formula-free sugar alcohol group, and the effect of the present invention is significantly better than the formula-free sugar alcohol group.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Disclosed are a β-lactoglobulin-vitamin conjugate comprising sugar alcohols and a preparation method thereof. The β-lactoglobulin-vitamin conjugate comprising sugar alcohols comprises β-lactoglobulin that has been subjected to glycosylation treatment with high voltage pulses, VE, sorbitol and maltitol. The glycosylation treatment with high voltage pulses involves treating a 1 mg/mL β-lactoglobulin solution prepared with a 0.01 mol/L, Ph 7.4 phosphate buffer solution in a high voltage electric field for 80 μs, the field intensity of the high voltage electric field being 30 kV/cm, and the electric field waveform being a unipolar square wave. Modification of β-lactoglobulin by the glycosylation treatment with high voltage pulses alters the secondary and tertiary structures of β-lactoglobulin, thus effectively reducing protein sensitization. Further, addition of the sugar alcohols as a natural emulsifier enhances the rate of conjugation of the β-lactoglobulin-vitamin conjugate.

Description

一种含有糖醇的β-乳球蛋白-维生素结合物及其制备方法Beta-lactoglobulin-vitamin conjugate containing sugar alcohol and preparation method thereof 技术领域Technical field
本发明属于生物技术领域，特别涉及一种含有糖醇的β-乳球蛋白-维生素结合物及其制备方法。The invention belongs to the field of biotechnology, and in particular relates to a β-lactoglobulin-vitamin conjugate containing sugar alcohol and a preparation method thereof.
背景技术Background technique
VE是脂溶性维生素，在体内外都有较好的抗氧化能力，还有一定的抗衰老、抗癌功能作用以及有延长保存期等功能。然而由于VE是脂溶性物质不溶于水，同时紫外线、碱以及氧都能快速破坏VE，使其难以很好的应用于食品工业中。因此，要采用特殊的方法对其进行处理，以期达到维持VE本身特有抗氧化性的同时增强其水溶性、稳定性与在食品中的添加效果。VE is a fat-soluble vitamin. It has good antioxidant capacity in and outside the body, as well as certain anti-aging, anti-cancer functions, and functions such as prolonging shelf life. However, because VE is a fat-soluble substance, it is insoluble in water. At the same time, ultraviolet rays, alkali and oxygen can quickly destroy VE, making it difficult to be well used in the food industry. Therefore, it is necessary to use special methods to treat it in order to maintain the unique antioxidant properties of VE itself while enhancing its water solubility, stability and the effect of addition in food.
β-乳球蛋白是牛奶蛋白的一种，是脂质载体蛋白家族中的一员，具有较强的配体结合能力，可以与脂溶性维生素及脂肪酸结合，作为脂溶性营养物质的载体应用于强化脂类食品的生产，脂溶性维生素及脂肪酸可自组装于β-乳球蛋白分子内部的疏水空穴和表面的疏水裂缝，使得β-乳球蛋白不仅可以结合这些脂溶性小分子，而且还能促进这些物质的吸收。但同样的β-乳球蛋白也是牛乳中的主要过敏原之一，所以在利用它结合维生素发挥其效果的同时，我们对于其不利影响也需要采取一定的措施，才可以更好的发挥它的作用，拓宽其应用层次。β-lactoglobulin is a kind of milk protein and a member of the lipid carrier protein family. It has strong ligand binding ability and can be combined with fat-soluble vitamins and fatty acids. It is used as a carrier for fat-soluble nutrients To strengthen the production of fat foods, fat-soluble vitamins and fatty acids can self-assemble into the hydrophobic holes and hydrophobic cracks on the surface of β-lactoglobulin molecules, so that β-lactoglobulin can not only bind these small fat-soluble molecules, but also Can promote the absorption of these substances. However, the same β-lactoglobulin is also one of the main allergens in cow milk, so while using it in combination with vitamins to exert its effects, we also need to take certain measures for its adverse effects in order to better play its Function and broaden its application level.
公开号CN103783169A中公开了一种强化维生素E鲜奶酪的制作方法，该方法中虽然利用β-乳球蛋白与VE进行了自组装，对VE的保护起到了一定的保护作用，但是也存在一些问题：1）β-乳球蛋白的致敏性并未消除，使得其应用面受到了限制，2）结合自组装中未用到乳化剂，其组装效果自然有所降低。Publication No. CN103783169A discloses a method for making fresh vitamin E fortified cheese. Although this method uses β-lactoglobulin to self-assemble with VE, it plays a certain role in protecting VE, but there are also some problems. : 1) The sensitization of β-lactoglobulin has not been eliminated, which limits its application. 2) The emulsifier is not used in combination with self-assembly, and its assembly effect is naturally reduced.
本发明试图对β-乳球蛋白进行改性降低其致敏性，使其在与VE进行结合过程中增强稳定剂。The present invention attempts to modify β-lactoglobulin to reduce its sensitization, so as to enhance the stabilizer during the binding process with VE.
技术问题technical problem
本发明所要解决的技术问题在于，提供一种含有糖醇的β-乳球蛋白-维生素结合物及其制备方法，提供一种降低致敏性的β-乳球蛋白，通过这种蛋白对维生素进行组合，再以糖醇作为天然乳化剂，进一步提高组装后β-乳球蛋白-维生素的组装效果，工艺简单，操作方便。The technical problem to be solved by the present invention is to provide a β-lactoglobulin-vitamin conjugate containing sugar alcohols and a preparation method thereof, and to provide a β-lactoglobulin with reduced sensitization. After combination, sugar alcohol is used as a natural emulsifier to further improve the assembly effect of β-lactoglobulin-vitamin after assembly. The process is simple and the operation is convenient.
技术解决方案Technical solution
本发明是这样实现的，提供一种含有糖醇的β-乳球蛋白-维生素结合物，包括高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇，所述高压脉冲糖基化处理是指将用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白放置在高压电场中处理80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。The present invention is achieved in this way, providing a sugar alcohol-containing β-lactoglobulin-vitamin conjugate, including β-lactoglobulin, VE, sorbitol, and maltitol after high-pressure pulse glycosylation treatment, High-pressure pulse glycosylation refers to the treatment of 80 μs by placing β-lactoglobulin prepared with 0.01 mg/L phosphate buffer solution of pH 7.4 into 1 mg/mL in a high-voltage electric field, where the electric field strength of the high-voltage electric field is 30kV/cm, the electric field waveform is a unipolar square wave.
进一步地，所述高压脉冲糖基化处理还包括将β-乳球蛋白采用如前的高压电场处理方式得到的一级高压电场糖基化处理过的β-乳球蛋白与L-阿拉伯糖、半乳糖按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。Further, the high-pressure pulse glycosylation treatment further includes first-level high-voltage electric field glycosylation-treated β-lactoglobulin and L-arabinose obtained by using β-lactoglobulin as previously obtained by the high-voltage electric field treatment method. Galactose is mixed in a mass ratio of 1:0.01:2 and lyophilized. The lyophilized powder is then placed in a centrifuge tube and placed in an incubator at a temperature of 65°C and a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide. After 3 hours of reaction in the solution, place the centrifuge tube in an ice bath to terminate the reaction immediately. After the reaction is completed, add pre-cooled ultrapure water to reconstitute, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the solution Salt and excess sugar, the final concentration of protein in the solution was adjusted to 1mg/mL, and the resulting second high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
进一步地，所述高压脉冲糖基化处理还包括以下处理过程：将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。Further, the high-pressure pulse glycosylation treatment further includes the following process: combining the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment in the following manner: Take β-lactoglobulin and VE with a mass ratio of 25mg~40mg: 2.5mg~4mg, add 25ml~40ml of purified water, 5g~15g of sorbitol, 10g~20g of maltitol, and vortex to mix for 40s~60s. The mixed solution was magnetically acted at 50℃~60℃ for 70min~100min, put the solution into a dialysis bag, placed in 30 times the volume of pure water for 24h to remove uncoated VE to obtain the composition , Using a vacuum freeze dryer at -45 ℃ for 20h to obtain a dried β-lactoglobulin-vitamin conjugate product containing sugar alcohol.
本发明是这样实现的，提供一种如前所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The present invention is achieved in this way, and provides a method for preparing a sugar alcohol-containing β-lactoglobulin-vitamin conjugate as described above, including the following steps:
步骤一、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；Step 1: β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4;
步骤二、将β－乳球蛋白溶液放置在高压电场中高压脉冲处理；Step 2: Place the β-lactoglobulin solution in a high-voltage electric field for high-voltage pulse processing;
步骤三、将经过高压脉冲糖基化处理后的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇进行组合，再组合物进行冷冻干燥，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。Step 3: Combine β-lactoglobulin after high-pressure pulse glycosylation treatment with VE, sorbitol, and maltitol, and freeze-dry the composition to obtain dried β-lactoglobulin containing sugar alcohol- Vitamin conjugate products.
进一步地，在步骤二中，所述β－乳球蛋白溶液放置在高压电场中处理时间为80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。Further, in step two, the β-lactoglobulin solution is placed in a high-voltage electric field for a treatment time of 80 μs, wherein the electric field strength of the high-voltage electric field is 30 kV/cm, and the electric field waveform is a unipolar square wave.
进一步地，在步骤三中，所述组合方法包括：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到含有糖醇的β-乳球蛋白-维生素的组合物。Further, in step three, the combination method includes: weighing β-lactoglobulin and VE with a mass ratio of 25 mg to 40 mg: 2.5 mg to 4 mg, adding 25 ml to 40 ml of purified water, and 5 g to 15 g of sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, apply the magnetic force at 50℃~60℃ for 70min~100min, put the solution into the dialysis bag and put it in 30 times volume of pure water Dialysis was performed for 24 hours to remove uncoated VE to obtain a β-lactoglobulin-vitamin composition containing sugar alcohol.
本发明是这样实现的，提供一种如前所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The present invention is achieved in this way, and provides a method for preparing a sugar alcohol-containing β-lactoglobulin-vitamin conjugate as described above, including the following steps:
（1）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；(1), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH7.4;
（2）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs；(2) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs;
（3）、将步骤（2）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用；(3), the first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (2) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1mg/mL, place the obtained secondary high-pressure pulse glycosylation-treated β-lactoglobulin at -20℃ for use;
（4）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(4) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the secondary high-pressure pulse glycosylation treatment in the following way: Weigh the mass ratio of 25mg~40mg: 2.5mg~4mg of β -Lactoglobulin and VE, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, and apply the resulting mixture at 50℃~60℃ for 70min~ After 100min, put the solution into a dialysis bag, place it in 30 times the volume of pure water for 24h, remove the uncoated VE, get the composition, and freeze it at -45°C for 20h using a vacuum freeze dryer. A dry β-lactoglobulin-vitamin conjugate product containing sugar alcohol is obtained.
有益效果Beneficial effect
与现有技术相比，本发明的含有糖醇的β-乳球蛋白-维生素结合物及其制备方法，具有以下特点：Compared with the prior art, the sugar alcohol-containing β-lactoglobulin-vitamin conjugate and its preparation method have the following characteristics:
1）通过高压脉冲糖基化处理后可以对于β-乳球蛋白进行改性，迫使β-乳球蛋白的二、三级结构发生改变，从而有效降低蛋白的致敏性；1) After high-pressure pulse glycosylation, β-lactoglobulin can be modified to force the secondary and tertiary structure of β-lactoglobulin to change, thereby effectively reducing protein sensitization;
2）通过添加天然乳化剂糖醇后，提高了其组合的结合率。2) After adding the natural emulsifier sugar alcohol, the combination rate of the combination is improved.
附图说明BRIEF DESCRIPTION
图1为未处理、糖基化处理和高压脉冲糖基化处理三组试验样品的β-乳球蛋白自由氨基含量的测定数据对比示意图；Figure 1 is a comparison diagram of the measurement data of β-lactoglobulin free amino content of three groups of untreated, glycosylated and high-pressure pulse glycosylated test samples;
图2为未处理、糖基化处理和高压脉冲糖基化处理三组试验样品的β-乳球蛋白抗原性降低率的测定数据对比示意图；Figure 2 is a comparison diagram of the measured data of β-lactoglobulin antigenicity reduction rate of the three groups of untreated, glycosylated and high-pressure pulse glycosylated test samples;
图3为配方不含糖醇组与本发明配方组的β-乳球蛋白与VE的结合率的对比示意图。FIG. 3 is a comparison diagram of the binding rate of β-lactoglobulin and VE between the formula without sugar alcohol group and the formula group of the present invention.
本发明的最佳实施方式Best Mode of the Invention
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention.
本发明含有糖醇的β-乳球蛋白-维生素结合物的较佳实施例，包括高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇，所述高压脉冲糖基化处理是指将用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白放置在高压电场中处理80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。The preferred embodiment of the sugar alcohol-containing β-lactoglobulin-vitamin conjugate of the present invention includes β-lactoglobulin, VE, sorbitol, and maltitol after high-pressure pulse glycosylation treatment, and the high-pressure pulse sugar The basification treatment means that β-lactoglobulin prepared with 0.01 mol/L phosphate buffer solution of pH 7.4 and 1 mg/mL is placed in a high-voltage electric field for 80 μs, in which the electric field strength of the high-voltage electric field is 30 kV/cm , The electric field waveform is a unipolar square wave.
其中，所述高压脉冲糖基化处理还包括将β-乳球蛋白采用如前的高压电场处理方式得到的一级高压电场糖基化处理过的β-乳球蛋白与L-阿拉伯糖、半乳糖按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。Wherein, the high-pressure pulse glycosylation treatment also includes the first-level high-voltage electric field glycosylation-treated β-lactoglobulin, L-arabinose, and semi Lactose is mixed at a mass ratio of 1:0.01:2 and lyophilized. The lyophilized powder is then placed in a centrifuge tube and placed in an incubator at a temperature of 65°C and a relative humidity of 65% by mass and a saturated potassium iodide solution of 59% by mass After 3 hours of reaction, place the centrifuge tube in an ice bath to terminate the reaction immediately. After the reaction is completed, add pre-cooled ultrapure water to reconstitute, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the solution. For salt and excess sugar, the protein concentration in the solution was finally adjusted to 1 mg/mL, and the obtained second-stage high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
其中，所述高压脉冲糖基化处理还包括以下处理过程：将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。Wherein, the high-pressure pulse glycosylation treatment also includes the following processing procedure: the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment are combined by: weighing The mass ratio is 25mg~40mg: 2.5mg~4mg of β-lactoglobulin and VE, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, the obtained The mixed solution is magnetically operated at 50℃~60℃ for 70min~100min, put the solution into the dialysis bag, put it in 30 times the volume of pure water to avoid light dialysis for 24h, remove the uncoated VE, and get the combination The product was frozen in a vacuum freeze dryer at -45°C for 20 hours to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin conjugate product.
本发明还公开一种如前所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The invention also discloses a method for preparing the β-lactoglobulin-vitamin conjugate containing sugar alcohol as mentioned above, which includes the following steps:
步骤一、将β－乳球蛋白用0.01mol/L pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液。Step 1: β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
步骤二、将β－乳球蛋白溶液放置在高压电场中高压脉冲处理。Step 2: Place the β-lactoglobulin solution in a high-voltage electric field under high-voltage pulse processing.
步骤三、将经过高压脉冲糖基化处理后的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇进行组合，再组合物进行冷冻干燥，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。Step 3: Combine β-lactoglobulin after high-pressure pulse glycosylation treatment with VE, sorbitol, and maltitol, and freeze-dry the composition to obtain dried β-lactoglobulin containing sugar alcohol- Vitamin conjugate products.
在步骤二中，所述β－乳球蛋白溶液放置在高压电场中处理时间为80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。In step two, the β-lactoglobulin solution is placed in a high-voltage electric field for a treatment time of 80 μs, wherein the electric field strength of the high-voltage electric field is 30 kV/cm, and the electric field waveform is a unipolar square wave.
在步骤三中，所述组合方法包括：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到含有糖醇的β-乳球蛋白-维生素的组合物。In step three, the combination method includes: weighing β-lactoglobulin and VE with a mass ratio of 25 mg to 40 mg: 2.5 mg to 4 mg, adding 25 ml to 40 ml of purified water, 5 g to 15 g of sorbitol, and 10 g to 20 g Maltitol, vortex to mix for 40s~60s, apply the magnetic force at 50℃~60℃ for 70min~100min, put the solution into the dialysis bag, put it in 30 times volume of pure water to avoid light After 24 hours of dialysis, the uncoated VE was removed to obtain a β-lactoglobulin-vitamin composition containing sugar alcohol.
本发明还公开一种如前所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The invention also discloses a method for preparing the β-lactoglobulin-vitamin conjugate containing sugar alcohol as mentioned above, which includes the following steps:
（1）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液。(1) The β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
（2）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs。(2) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs.
（3）、将步骤（2）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。(3), the first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (2) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1 mg/mL, and the obtained second-stage high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
（4）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(4) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the secondary high-pressure pulse glycosylation treatment in the following way: Weigh the mass ratio of 25mg~40mg: 2.5mg~4mg of β -Lactoglobulin and VE, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, and apply the resulting mixture at 50℃~60℃ for 70min~ After 100min, put the solution into a dialysis bag, place it in 30 times volume of pure water and avoid dialysis for 24h, remove the uncoated VE, get the composition, and freeze it at -45℃ using a vacuum freeze dryer After 20 hours, a dry β-lactoglobulin-vitamin conjugate product containing sugar alcohol was obtained.
本发明的实施方式Embodiments of the invention
下面结合具体实施例进一步说明本发明的方法。The method of the present invention is further described below with reference to specific embodiments.
实施例1Example 1
本发明第一种含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The preparation method of the first β-lactoglobulin-vitamin conjugate containing sugar alcohol of the present invention includes the following steps:
（11）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液。(11) β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
（12）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs。(12) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs.
（13）、将步骤（12）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。(13). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (12) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1 mg/mL, and the obtained second-stage high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
（14）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为30mg:3mg的β-乳球蛋白和VE，加入30ml纯水，5g山梨糖醇，10g麦芽糖醇，涡旋混合60s，将得到的混合液在50℃下磁力作用90min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(14) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment by combining the following methods: Weigh the β-lactoglobulin with a mass ratio of 30mg: 3mg and VE, add 30ml of pure water, 5g sorbitol, 10g maltitol, vortex to mix for 60s, put the resulting mixture under magnetic force at 50 ℃ for 90min, put the solution into the dialysis bag and put it in 30 times volume Dialysis in pure water for 24h to remove uncoated VE to obtain a composition and freeze it at -45°C for 20h using a vacuum freeze dryer to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin conjugate product.
实施例2Example 2
本发明第二种含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The second method for preparing a sugar alcohol-containing β-lactoglobulin-vitamin conjugate of the present invention includes the following steps:
（21）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液。(21), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH7.4.
（22）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs。(22) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in a high-voltage pulse process for 80 μs.
（23）、将步骤（22）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。(23). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (22) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1 mg/mL, and the obtained second-stage high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
（24）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为40mg:4mg的β-乳球蛋白和VE，加入40ml纯水，10g山梨糖醇，20g麦芽糖醇，涡旋混合40s，将得到的混合液在50℃下磁力作用100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(24) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment by combining the following methods: Weigh the β-lactoglobulin with a mass ratio of 40mg: 4mg and VE, add 40ml pure water, 10g sorbitol, 20g maltitol, vortex to mix for 40s, put the resulting mixture under magnetic force at 50 ℃ for 100min, put the solution into the dialysis bag and place it in 30 times volume Dialysis in pure water for 24h to remove uncoated VE to obtain a composition and freeze it at -45°C for 20h using a vacuum freeze dryer to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin conjugate product.
实施例3Example 3
本发明第三种含有糖醇的β-乳球蛋白-维生素结合物的制备方法，包括如下步骤：The third method for preparing a sugar alcohol-containing β-lactoglobulin-vitamin conjugate of the present invention includes the following steps:
（31）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液。(31), β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4.
（32）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs。(32) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs.
（33）、将步骤（32）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。(33). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (32) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1 mg/mL, and the obtained second-stage high-pressure pulse glycosylation-treated β-lactoglobulin was placed at -20°C until use.
（34）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比25mg:2.5mg的β-乳球蛋白和VE，加入25ml纯水，15g山梨糖醇，15g麦芽糖醇，涡旋混合50s，将得到的混合液在55℃下磁力作用70min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中避光透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(34) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second-stage high-pressure pulse glycosylation treatment by combining the following methods: Weigh the β-lactoglobulin with a mass ratio of 25mg: 2.5mg and VE, add 25ml of pure water, 15g of sorbitol, 15g of maltitol, vortex to mix for 50s, put the resulting mixture under magnetic force at 55℃ for 70min, put the solution into the dialysis bag and place it in 30 times volume Dialysis in pure water for 24h to remove uncoated VE to obtain a composition and freeze it at -45°C for 20h using a vacuum freeze dryer to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin conjugate product.
下面通过以下试验来评价采用本发明的方法制备含有糖醇的β-乳球蛋白-维生素结合物产品的特性。The characteristics of the β-lactoglobulin-vitamin conjugate product containing sugar alcohol prepared by the method of the present invention will be evaluated by the following test.
A、致敏性评价。A. Evaluation of sensitization.
糖基化反应是单糖、还原糖中的羰基或羰基化合物与蛋白质中的氨基共价结合的化学反应，因此可以通过蛋白质的自由氨基含量来反映糖基化反应程度。自由氨基含量的测定采用邻苯二甲醛（o-phthalaldehyde， OPA）法测定自由氨基含量。The glycosylation reaction is a chemical reaction in which a carbonyl group in a monosaccharide, a reducing sugar, or a carbonyl compound is covalently bonded to an amino group in a protein, so the degree of glycosylation reaction can be reflected by the free amino content of the protein. The content of free amino group is determined by o-phthalaldehyde (OPA) method.
图1显示的是未处理、糖基化处理和高压脉冲糖基化处理三组试验样品的自由氨基含量测定的对比数据，其中，未处理是指试验样品不做任何处理的β-乳球蛋白，糖基化处理是指试验样品仅进行糖基化处理，高压脉冲糖基化处理是指采用本发明方法得到的实施例1的试验样品。Figure 1 shows the comparison data of the determination of the free amino content of the three groups of untreated, glycosylated and high-pressure pulsed glycosylated test samples, where untreated refers to the beta sample without any treatment. The glycosylation treatment means that the test sample is only glycosylated, and the high-pressure pulse glycosylation treatment refers to the test sample of Example 1 obtained by the method of the present invention.
从图1可以看出，β-乳球蛋白经PEF（高压脉冲电场，Pulsed Electric Fields）结合糖基化处理后，自由氨基含量显著降低，说明糖基化后β-乳球蛋白与糖分子发生了共价结合，而且随着脉冲场强的增大有下降趋势，可能的原因是经高强度脉冲强度处理，β-乳球蛋白的空间结构展开程度更大，β-乳球蛋白分子中的氨基暴露在蛋白分子表面，糖基更易与氨基发生共价结合，使得β-乳球蛋白的自由氨基含量显著降低。As can be seen from Figure 1, β-lactoglobulin undergoes PEF (high-voltage pulsed electric field, Pulsed Electric Fields) After combined with glycosylation, the content of free amino groups is significantly reduced, indicating that β-lactoglobulin and sugar molecules are covalently bonded after glycosylation, and there is a downward trend with the increase of pulse field strength, possible reasons It is processed by high-intensity pulse intensity, and the spatial structure of β-lactoglobulin is expanded to a greater extent. The amino groups in β-lactoglobulin molecules are exposed on the surface of the protein molecules, and the sugar groups are more easily covalently bound to the amino groups, making β-lactoglobulin The free amino content of globulin is significantly reduced.
B、抗原性评价。B. Antigenicity evaluation.
抗原性的测定采用间接竞争ELISA法（酶联免疫吸附法，Enzyme Linked Immune Sorbent Assay）。以β-乳球蛋白标准品浓度（0.5μg/mL~64μg/mL）的对数为横坐标，吸光值为纵坐标绘制标准曲线，根据标准曲线即可算出不同处理条件下β-乳球蛋白样品的抗原性（μg/mL），从而算出抗原性降低率。计算公式如下：The determination of antigenicity adopts indirect competitive ELISA method (enzyme-linked immunosorbent assay, Enzyme Linked Immune Sorbent Assay). Use the logarithm of β-lactoglobulin standard concentration (0.5μg/mL~64μg/mL) as the abscissa and the absorbance value as the ordinate to draw a standard curve. According to the standard curve, β-lactoglobulin can be calculated under different treatment conditions The antigenicity of the sample (μg/mL) to calculate the rate of decrease in antigenicity. Calculated as follows:
抗原性降低率（%）=（C 原样 -C 待测 ）÷C 原样 *100， Antigen reduction rate (%) = (C as- C to be tested ) ÷ C as is *100,
式中，C原样：根据标准曲线计算出的原样的抗原性（μg/mL）；C待测：根据标准曲线计算出的待测样品的抗原性（μg/mL）。In the formula, C as it is: the antigenicity of the original sample calculated according to the standard curve (μg/mL); C to be tested: the antigenicity of the sample to be tested calculated according to the standard curve (μg/mL).
图2显示的是未处理、糖基化处理和高压脉冲糖基化处理三组试验样品的抗原性降低率的测定对比数据，其中，未处理是指试验样品不做任何处理的β-乳球蛋白，糖基化处理是指试验样品仅进行糖基化处理，高压脉冲糖基化处理是指采用本发明方法得到的实施例1的试验样品。Figure 2 shows the comparison data of the determination of the antigenicity reduction rate of the three groups of untreated, glycosylated and high-pressure pulsed glycosylated test samples, where untreated refers to the beta samples without any treatment Protein, glycosylation treatment means that the test sample is only subjected to glycosylation treatment, and high-pressure pulse glycosylation treatment refers to the test sample of Example 1 obtained by the method of the present invention.
从图2可以看出，本发明中所运用的高压脉冲糖基化处理方法与糖基化处理方法对于β-乳球蛋白的抗原性均有所降低，而发明中的高压脉冲糖基化处理方式抗原性降低最多，达到74%，说明该发明方法可有效较低β-乳球蛋白的致敏性。It can be seen from FIG. 2 that the high-pressure pulse glycosylation treatment method and the glycosylation treatment method used in the present invention have reduced the antigenicity of β-lactoglobulin, and the high-pressure pulse glycosylation treatment in the invention The way the antigenicity is reduced the most is 74%, indicating that the method of the invention can effectively reduce the sensitization of β-lactoglobulin.
C、组合率测定。C. Determination of combination rate.
β-乳球蛋白与VE组合率测定：准确称取β-乳球蛋白与VE结合物0.2g，加入1mL纯水充分溶解，然后加入10mL乙醇放入振荡器中200r/min振荡提取20min，用无水硫酸钠除去水分。再将溶液3000r/min离心10min，而后吸取上清液，用乙醇稀释到合适浓度，并以乙醇作空白在285nm处测定吸光值。Determination of the combination rate of β-lactoglobulin and VE: Accurately weigh 0.2g of β-lactoglobulin and VE conjugate, add 1mL of pure water to fully dissolve, then add 10mL of ethanol into the shaker and shake to extract at 200r/min for 20min. Anhydrous sodium sulfate removes water. The solution was centrifuged at 3000r/min for 10min, and then the supernatant was sucked, diluted with ethanol to an appropriate concentration, and the absorbance was measured at 285nm with ethanol as a blank.
VE标准曲线的建立得到标曲公式：The VE standard curve is established to obtain the calibration formula:
VE含量计算：C=（A-0.0182）/1.4508*B，Calculation of VE content: C=(A-0.0182)/1.4508*B,
结合率计算：E=[(C*V*m 1/0.2)/m 2]*100， Calculation of binding rate: E=[(C*V*m 1 /0.2)/m 2 ]*100,
其中：C为样本浓度(mg/mL)，A为样本的吸光值，B为稀释倍数，V为提取液体积(10mL)，m1为结合物的质量(mg)，m2为VE总量(mg)。Where: C is the sample concentration (mg/mL), A is the absorbance of the sample, B is the dilution factor, V is the volume of the extraction solution (10mL), m1 is the mass of the conjugate (mg), and m2 is the total amount of VE (mg ).
根据以上结合率的计算方法比较配方不含糖醇组与发明配方组——即本发明的加入糖醇的β-乳球蛋白-VE结合物（实施例1试验样品）的结合率得到如下图3所示图形（试验样品5次重复）。According to the calculation method of the binding rate above, the binding rate of the formula-free sugar alcohol group and the invention formula group-that is, the sugar alcohol-added β-lactoglobulin-VE conjugate (Example 1 test sample) of the present invention is obtained as follows Figure 3 shows (the test sample is repeated 5 times).
从图3中可以看出，本发明配方组比配方不含糖醇组的结合率提高了50%以上，本发明的效果明显好于配方不含糖醇组。It can be seen from FIG. 3 that the combination rate of the formula group of the present invention is more than 50% higher than that of the formula-free sugar alcohol group, and the effect of the present invention is significantly better than the formula-free sugar alcohol group.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention should be included in the protection of the present invention Within range.
工业实用性Industrial applicability
在此处键入工业实用性描述段落。Type the paragraph describing the industrial applicability here.
序列表自由内容Sequence listing free content
在此处键入序列表自由内容描述段落。Type the sequence listing free content description paragraph here.

Claims (10)

  1. 一种含有糖醇的β-乳球蛋白-维生素结合物，其特征在于，包括高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇，所述高压脉冲糖基化处理是指将用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白放置在高压电场中处理80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。A β-lactoglobulin-vitamin conjugate containing sugar alcohols, characterized by comprising β-lactoglobulin, VE, sorbitol, maltitol after high-pressure pulse glycosylation treatment, the high-pressure pulse glycosyl group Chemical treatment means that β-lactoglobulin formulated with 0.01 mol/L phosphate buffer solution at pH 7.4 into 1 mg/mL is placed in a high-voltage electric field for 80 μs, where the electric field strength of the high-voltage electric field is 30 kV/cm, The electric field waveform is a unipolar square wave.
  2. 如权利要求1所述的含有糖醇的β-乳球蛋白-维生素结合物，其特征在于，所述高压脉冲糖基化处理还包括将β-乳球蛋白采用如权利要求1中的高压电场处理方式得到的一级高压电场糖基化处理过的β-乳球蛋白与L-阿拉伯糖、半乳糖按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用。The β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 1, wherein the high-pressure pulse glycosylation treatment further comprises applying β-lactoglobulin to the high-voltage electric field according to claim 1 The first-level high-voltage electric field glycosylation-treated β-lactoglobulin, L-arabinose, and galactose obtained by the treatment method are mixed in a mass ratio of 1:0.01:2, lyophilized, and then the lyophilized powder is packed in The centrifuge tube was placed in an incubator, and reacted in a saturated potassium iodide solution at a temperature of 65°C and a relative humidity of 65% by mass for 59% for 3 hours. After the reaction was completed, the centrifuge tube was immediately placed in an ice bath to terminate the reaction. Then add pre-cooled ultrapure water to reconstitute, then use ultrafiltration centrifuge tube with molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution, and finally adjust the protein concentration in the solution to 1mg/mL Β-lactoglobulin after high-pressure pulse glycosylation treatment was placed at -20℃ for use.
  3. 如权利要求2所述的含有糖醇的β-乳球蛋白-维生素结合物，其特征在于，所述高压脉冲糖基化处理还包括以下处理过程：将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。The β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 2, wherein the high-pressure pulse glycosylation treatment further includes the following process: after the second high-pressure pulse glycosylation treatment β-lactoglobulin, VE, sorbitol, maltitol are combined by the following methods: Weigh β-lactoglobulin and VE with a mass ratio of 25mg~40mg: 2.5mg~4mg, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, apply the magnetic force at 50℃~60℃ for 70min~100min, put the solution into the dialysis bag. Dialysis was performed in 30 volumes of pure water for 24 hours to remove uncoated VE to obtain a composition, which was frozen at -45°C for 20 hours using a vacuum freeze dryer to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin Conjugate products.
  4. 一种如权利要求1所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，包括如下步骤：A method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 1, comprising the following steps:
    步骤一、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；Step 1: β-lactoglobulin is prepared into a 1 mg/mL β-lactoglobulin solution with 0.01 mol/L phosphate buffer solution at pH 7.4;
    步骤二、将β－乳球蛋白溶液放置在高压电场中高压脉冲处理；Step 2: Place the β-lactoglobulin solution in a high-voltage electric field for high-voltage pulse processing;
    步骤三、将经过高压脉冲糖基化处理后的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇进行组合，再组合物进行冷冻干燥，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。Step 3: Combine β-lactoglobulin after high-pressure pulse glycosylation treatment with VE, sorbitol, and maltitol, and freeze-dry the composition to obtain dried β-lactoglobulin containing sugar alcohol- Vitamin conjugate products.
  5. 如权利要求4所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，在步骤二中，所述β－乳球蛋白溶液放置在高压电场中处理时间为80μs，其中，高压电场的电场强度为30kV/cm，其电场波形为单极性方波。The method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 4, wherein in step two, the β-lactoglobulin solution is placed in a high-voltage electric field for a treatment time of 80 μs , Where the electric field strength of the high-voltage electric field is 30kV/cm, and the electric field waveform is a unipolar square wave.
  6. 如权利要求4所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，在步骤三中，所述组合方法包括：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到含有糖醇的β-乳球蛋白-维生素的组合物。The method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 4, wherein in step three, the combination method comprises: weighing a mass ratio of 25 mg to 40 mg: 2.5 mg ~4mg of β-lactoglobulin and VE, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, and the resulting mixture at 50℃~60℃ Magnetic action 70min~100min, put the solution into the dialysis bag, placed in 30 times the volume of pure water for dialysis for 24h, to remove the uncoated VE, to obtain β-lactoglobulin-vitamin containing sugar alcohol combination.
  7. 一种如权利要求3所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，包括如下步骤：A method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 3, characterized in that it comprises the following steps:
    （1）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；(1), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH7.4;
    （2）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs；(2) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs;
    （3）、将步骤（2）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用；(3), the first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (2) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1mg/mL, place the obtained secondary high-pressure pulse glycosylation-treated β-lactoglobulin at -20℃ for use;
    （4）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为25mg~40mg:2.5mg~4mg的β-乳球蛋白和VE，加入25ml~40ml纯水，5g~15g山梨糖醇，10g~20g麦芽糖醇，涡旋混合40s~60s，将得到的混合液在50℃~60℃下磁力作用70min~100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(4) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the secondary high-pressure pulse glycosylation treatment in the following way: Weigh the mass ratio of 25mg~40mg: 2.5mg~4mg of β -Lactoglobulin and VE, add 25ml~40ml of pure water, 5g~15g sorbitol, 10g~20g maltitol, vortex to mix for 40s~60s, and apply the resulting mixture at 50℃~60℃ for 70min~ After 100min, put the solution into a dialysis bag, place it in 30 times the volume of pure water for 24h, remove the uncoated VE, get the composition, and freeze it at -45°C for 20h using a vacuum freeze dryer. A dry β-lactoglobulin-vitamin conjugate product containing sugar alcohol is obtained.
  8. 如权利要求7所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，包括如下步骤：The method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 7, comprising the following steps:
    （11）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；(11), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH 7.4;
    （12）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs；(12) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs;
    （13）、将步骤（12）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用；(13). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (12) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water for reconstitution, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1mg/mL, place the obtained secondary high-pressure pulse glycosylation-treated β-lactoglobulin at -20℃ for use;
    （14）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为30mg:3mg的β-乳球蛋白和VE，加入30ml纯水，5g山梨糖醇，10g麦芽糖醇，涡旋混合60s，将得到的混合液在50℃下磁力作用90min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(14) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment by combining the following methods: Weigh the β-lactoglobulin with a mass ratio of 30mg: 3mg and VE, add 30ml of pure water, 5g sorbitol, 10g maltitol, vortex to mix for 60s, put the resulting mixture under magnetic force at 50 ℃ for 90min, put the solution into the dialysis bag and put it in 30 times volume After dialysis in pure water for 24h to remove uncoated VE, the composition was obtained and frozen at -45°C for 20h using a vacuum freeze dryer to obtain a dried β-lactoglobulin-vitamin conjugate product containing sugar alcohol.
  9. 如权利要求7所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，包括如下步骤：The method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 7, comprising the following steps:
    （21）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；(21), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH 7.4;
    （22）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs；(22) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs;
    （23）、将步骤（22）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用；(23). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (22) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water to reconstitute it. Then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1mg/mL, place the obtained secondary high-pressure pulse glycosylation-treated β-lactoglobulin at -20℃ for use;
    （24）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取质量比为40mg:4mg的β-乳球蛋白和VE，加入40ml纯水，10g山梨糖醇，20g麦芽糖醇，涡旋混合40s，将得到的混合液在50℃下磁力作用100min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(24) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second high-pressure pulse glycosylation treatment by combining the following methods: Weigh the β-lactoglobulin with a mass ratio of 40mg: 4mg and VE, add 40ml pure water, 10g sorbitol, 20g maltitol, vortex to mix for 40s, put the resulting mixture under magnetic force at 50 ℃ for 100min, put the solution into the dialysis bag and place it in 30 times volume After dialysis in pure water for 24h to remove uncoated VE, the composition was obtained and frozen at -45°C for 20h using a vacuum freeze dryer to obtain a dried β-lactoglobulin-vitamin conjugate product containing sugar alcohol.
  10. 如权利要求7所述的含有糖醇的β-乳球蛋白-维生素结合物的制备方法，其特征在于，包括如下步骤：The method for preparing a β-lactoglobulin-vitamin conjugate containing sugar alcohol according to claim 7, comprising the following steps:
    （31）、将β－乳球蛋白用0.01mol/L、pH7.4的磷酸盐缓冲溶液配成1mg/mL的β－乳球蛋白溶液；(31), β-lactoglobulin is made into 1mg/mL β-lactoglobulin solution with 0.01mol/L phosphate buffer solution at pH 7.4;
    （32）、将β－乳球蛋白溶液放置在电场强度为30kV/cm，电场波形为单极性方波的高压电场中高压脉冲处理80μs；(32) Place the β-lactoglobulin solution in a high-voltage electric field with an electric field strength of 30 kV/cm and an electric field waveform of a unipolar square wave in high-voltage pulse processing for 80 μs;
    （33）、将步骤（32）得到的一级高压脉冲糖基化处理过的β-乳球蛋白与VE、山梨糖醇、麦芽糖醇按质量比1:0.01:2混匀后冻干，再将冻干后的粉末装于离心管后置于培养箱中，在温度65℃，相对湿度65%的质量比59%饱和碘化钾溶液中反应3h，反应结束后将离心管立即置于冰浴中终止反应，待反应结束后，再加入预先冷却的超纯水复溶，然后采用截留分子量为10ku的超滤离心管除去溶液中的盐和多余的糖，最终将溶液中的蛋白质的浓度调为 1mg/mL，将得到的二级高压脉冲糖基化处理后的β-乳球蛋白置于-20℃待用；(33). The first-level high-pressure pulse glycosylation-treated β-lactoglobulin obtained in step (32) is mixed with VE, sorbitol, and maltitol at a mass ratio of 1:0.01:2, lyophilized, and then Place the lyophilized powder in a centrifuge tube and place it in an incubator, react at a temperature of 65°C, a relative humidity of 65% and a mass ratio of 59% saturated potassium iodide for 3 hours. After the reaction, place the centrifuge tube in an ice bath immediately Terminate the reaction. After the reaction is completed, add pre-cooled ultrapure water to reconstitute it. Then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 10ku to remove the salt and excess sugar in the solution. Finally, adjust the protein concentration in the solution to 1mg/mL, place the obtained secondary high-pressure pulse glycosylation-treated β-lactoglobulin at -20℃ for use;
    （34）将二级高压脉冲糖基化处理后的β-乳球蛋白、VE、山梨糖醇、麦芽糖醇通过以下方式组合在一起：称取一定质量比为25mg:2.5mg的β-乳球蛋白和VE，加入25ml纯水，15g山梨糖醇，15g麦芽糖醇，涡旋混合50s，将得到的混合液在55℃下磁力作用70min，将作用后的溶液放入透析袋中，置于30倍体积的纯水中透析24h，去除未被包被的VE，得到组合物，利用真空冷冻干燥机在-45℃下冷冻20h，得到干燥的含有糖醇的β-乳球蛋白-维生素结合物产品。(34) Combine the β-lactoglobulin, VE, sorbitol, and maltitol after the second-stage high-pressure pulse glycosylation treatment in the following way: Weigh a certain mass ratio of 25mg: 2.5mg β-lactoglobulin Protein and VE, add 25ml of pure water, 15g sorbitol, 15g maltitol, vortex to mix for 50s, put the resulting mixture under magnetic force at 55℃ for 70min, put the solution into the dialysis bag and place in 30 Dialysis of double volume of pure water for 24h to remove uncoated VE to obtain the composition, and freeze it at -45°C for 20h using a vacuum freeze dryer to obtain a dried sugar alcohol-containing β-lactoglobulin-vitamin conjugate product.
PCT/CN2019/120410 2018-12-24 2019-11-22 β-LACTOGLOBULIN-VITAMIN CONJUGATE COMPRISING SUGAR ALCOHOLS AND PREPARATION METHOD THEREOF WO2020134772A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811585476.0A CN109535242A (en) 2018-12-24 2018-12-24 A kind of beta lactoglobulin containing sugar alcohol-vitamin conjugate and preparation method thereof
CN201811585476.0 2018-12-24

Publications (1)

Publication Number Publication Date
WO2020134772A1 true WO2020134772A1 (en) 2020-07-02

Family

ID=65856964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/120410 WO2020134772A1 (en) 2018-12-24 2019-11-22 β-LACTOGLOBULIN-VITAMIN CONJUGATE COMPRISING SUGAR ALCOHOLS AND PREPARATION METHOD THEREOF

Country Status (2)

Country Link
CN (1) CN109535242A (en)
WO (1) WO2020134772A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109535242A (en) * 2018-12-24 2019-03-29 浙江华康药业股份有限公司 A kind of beta lactoglobulin containing sugar alcohol-vitamin conjugate and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103783169A (en) * 2014-02-25 2014-05-14 安徽农业大学 Preparation method of fortified vitamin E-containing fresh cheese
CN109535242A (en) * 2018-12-24 2019-03-29 浙江华康药业股份有限公司 A kind of beta lactoglobulin containing sugar alcohol-vitamin conjugate and preparation method thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103918871B (en) * 2014-04-15 2016-01-20 南昌大学 A kind of impulse electric field processing method for changing milk allergen protein immunization characteristic
CN103931874B (en) * 2014-05-07 2016-05-25 河南工业大学 A kind of glycosylation and the enzymolysis modified compound method of preparing subsensitivety soyabean protein powder
CN108497027A (en) * 2018-07-04 2018-09-07 安徽盼盼食品有限公司 A kind of preparation method of polysaccharides health care bread

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103783169A (en) * 2014-02-25 2014-05-14 安徽农业大学 Preparation method of fortified vitamin E-containing fresh cheese
CN109535242A (en) * 2018-12-24 2019-03-29 浙江华康药业股份有限公司 A kind of beta lactoglobulin containing sugar alcohol-vitamin conjugate and preparation method thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KADOYA, S. ET AL.: "Freeze-Drying of Proteins with Glass-Forming Oligosaccharide-Derived Sugar Alcohols", INTERNATIONAL JOURNAL OF PARMACEUTICS, vol. 389, 25 January 2010 (2010-01-25), pages 107 - 113, XP028308326, DOI: 20200212130053A *
NATIONAL HEALTH AND FAMILY PLANNING COMMISSION OF PRC: "GB2760-2014 National Food Safety Standard for Uses of Food Additives", NATIONAL STANDARDS OF THE PEOPLE’S REPUBLIC OF CHINA, 24 December 2014 (2014-12-24), DOI: 20200212130315Y *
TIAN, MING ET AL.: "Effect of Glycation Combined with Pulsed Electric Field Pretreatment on Allergenicity and Functional Properties of β-Lactoglobulin", FOOD SCIENCE, vol. 38, no. 13, 31 December 2017 (2017-12-31), DOI: 20200212124452Y *
TIAN, MING: "Effect of Pulsed Electric Fields Treatment on Structure, Immunogenicity and Glycation Properties of β-Lactoglobulin", SCIENCE-ENGINEERING (A), CHINA MASTER’S THESES FULL-TEXT DATABASE, no. 02, 15 February 2018 (2018-02-15), DOI: 20200212124913Y *
TU, ZONGCAI ET AL.: "Effect of the Pretreatment Combining Glycation and Pulsed Electric Fields on the Antigenicity and Structure of β-Lactoglobulin", MODERN FOOD SCIENCE AND TECHNOLOGY, vol. 32, no. 11, 31 December 2016 (2016-12-31), DOI: 20200212125158Y *
YOU, XIN: "The Function and the Developing Prospect in Industry Use of Sugar Alcohols", CHINA FOOD ADDITIVES, 31 December 2010 (2010-12-31), pages 45 - 52, DOI: 20200212125828Y *

Also Published As

Publication number Publication date
CN109535242A (en) 2019-03-29

Similar Documents

Publication Publication Date Title
Wu et al. Effect of a polysaccharide from Poria cocos on humoral response in mice immunized by H1N1 influenza and HBsAg vaccines
CN105017412B (en) A method of the separating high-purity bovine serum albumin(BSA) from cow's serum
JP6007209B2 (en) Microparticle-soluble glucan preparation
Wu et al. Ball-milling is an effective pretreatment of glycosylation modified the foaming and gel properties of egg white protein
CN106434804B (en) A kind of protein peptides with antioxidation activity and preparation method thereof
Aviles et al. Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection
Lv et al. Changes in structure and allergenicity of shrimp tropomyosin by dietary polyphenols treatment
CN109965167B (en) Octacosanol sports drink and preparation method thereof
WO2020134772A1 (en) β-LACTOGLOBULIN-VITAMIN CONJUGATE COMPRISING SUGAR ALCOHOLS AND PREPARATION METHOD THEREOF
Tao et al. Improving the antioxidant activity, in vitro digestibility and reducing the allergenicity of whey protein isolate by glycosylation with short-chain inulin and interaction with cyanidin-3-glucoside
US20150173406A1 (en) Hypoallergenic food-grade protein matrices and uses thereof
Zhao et al. Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review
CN106701875A (en) Preparation method of freshwater fish antioxidant bioactive peptide
CN109680027A (en) A kind of grifola frondosus small peptide iron chelate and its preparation method and application
CN103966292A (en) Industrial manufacturing method and use of allergen-eliminated partly appropriately-hydrolyzed casein peptide
US20100159089A1 (en) Oxidation Stability Using Natural Antioxidants
Dai et al. Extraction optimization, preliminary characterization and antioxidant activity of glycoproteins from the muscle of Sepia pharaonis
BR112021010435A2 (en) Methods for making mixed allergen compositions
Hirano et al. Biologically active glycopeptides in human colostrum
NL2029200B1 (en) Gluten-free hypoallergenic soy protein flour and preparation method thereof
CN108753897A (en) A kind of preparation method of sea cucumber internal organ polypeptide
CN108813090A (en) The preparation method of poultry plasma protein
CN110393256A (en) It is a kind of using brewing yeast cell wall as the mould inorganic agent of carrier
CN110326795B (en) Eggplantin complex for improving immunity of organism and preparation method thereof
AU2019100971A4 (en) Lactoferrin and sialic acid-modulated milk powder and preparation method thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19901894

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19901894

Country of ref document: EP

Kind code of ref document: A1