CN110592701A

CN110592701A - Method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate

Info

Publication number: CN110592701A
Application number: CN201910831968.1A
Authority: CN
Inventors: 李良; 安迪; 赵添; 胡安娜; 洪瑞
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-09-04
Filing date: 2019-09-04
Publication date: 2019-12-20

Abstract

The invention provides a method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysis products, and particularly relates to nanofibers formed by self-assembly of enzymatic hydrolysis products after trypsin enzymolysis. The method comprises the following steps: selecting enzyme and measuring enzyme activity; enzymolysis of soybean protein: adding 0.02g trypsin (1:100) into protein solution, and performing enzymolysis at 37 deg.C and pH7 for 18min and 40 min; centrifuging the enzymolysis liquid; preparing the nano-fibers: the concentrations of the 1% and 3% hydrolysis supernatants were diluted to 1%, 3% (W/V) using NaCl solution, the final concentration of NaCl being 20 mM. The pH value of the solution is 2, and the solution is heated for 8 hours at 85 ℃ to prepare the nano fiber; preserving at 4 ℃. The method uses trypsin enzymolysis soy protein as a construction fragment for preparing the nano-fiber, the obtained nano-fiber has high reaction activity, and partial function loss caused by reverse reaction aggregation of the nano-fiber can be obviously inhibited.

Description

Method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate

Technical Field

The invention belongs to the food protein processing technology, and particularly relates to a method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate.

Background

Self-assembly is a structurally well-defined aggregate in which molecules spontaneously join together through non-covalent bonds under equilibrium conditions. Self-assembly is a building tool for nanomaterials, which can spontaneously assemble molecular, nano, micro or larger substances into ordered structures. Self-assembly not only confers structural functions to the protein, such as good stability, but also plays a key role in regulating biological functions. The nanofibers formed by self-assembly generally have diameters on the nanometer scale and lengths on the micrometer scale, and morphologically appear as distinct linear fibers. It has been demonstrated that nanofiber production depends primarily on noncovalent interactions, which are the result of synergistic interactions such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, and the like.

Peptides generated by the self-assembly process are widely accepted as building blocks for thermally induced nanofibers. Thus, pre-hydrolyzed protein is used to make nanofibers rather than intact protein. More importantly, the hydrolysis kinetics of the produced peptides influence the growth kinetics of the nanofibers, and thus the prior enzymatic hydrolysis has an important influence on the formation of soy protein nanofibers.

Soy protein is an important food ingredient, and good functionality has been widely used in food research due to its extremely high nutritional value. Long-term studies have shown that nanofibers can be formed from soy protein heated under acidic conditions at pH 2. And soybean protein can be made into a fibrous component having a nano-scale as an excellent building unit for forming nanofibers. Most of the characteristics of the soybean protein nanofiber are equivalent to those of whey protein which is a hot point in the current research, and even the soybean protein nanofiber is superior to the whey protein in some aspects, so that the soybean protein self-assembly nanofiber has a wide research prospect.

The method uses trypsin enzymolysis soy protein as a construction fragment for preparing the nano-fiber, not only can increase the number of the construction fragments, but also can generate fragments with excellent self-assembly performance. Compared with the traditional method of directly heating the soybean protein under the acidic condition, the new method obviously improves the functionality of the nano-fiber, increases the yield of the nano-fiber, has high reaction activity of the obtained nano-fiber, and can obviously inhibit partial function loss caused by reverse reaction aggregation of the nano-fiber.

Disclosure of Invention

The invention provides a method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate, which achieves the purposes of improving the physicochemical properties of the nanofibers, standardizing the process and improving the application value of the nanofibers.

The technical problem to be solved by the invention is realized by the following technical scheme: firstly, screening enzyme; secondly, enzymolysis of soybean protein; and thirdly, preparing the nano-fibers.

The method for preparing the nano-fiber by utilizing the soybean protein enzymolysis product comprises the following steps:

firstly, enzyme activity determination is carried out on alkaline protease, neutral protease, trypsin and papain.

And secondly, screening four enzymes of alkaline protease, neutral protease, trypsin and papain to determine the trypsin as the enzyme for the subsequent experiment.

Thirdly, enzymolysis of the soybean protein: 2g of soy protein was dissolved in 50mL of deionized water, magnetically stirred for 2h, and then put into a refrigerator at 4 ℃ to be hydrated overnight. Adding 0.02g trypsin (1:100) into the soybean protein solution, adjusting pH to 7, performing enzymolysis reaction in a water bath at 37 deg.C for 18min and 40min, and controlling the hydrolysis degree of the solution at 1% and 3%. After the enzymolysis reaction is finished, putting the enzymolysis liquid in a boiling water bath for 10min to stop the enzymolysis reaction.

Fourthly, cooling the enzymolysis liquid to room temperature, centrifuging for 15min at 7500rpm, and taking the supernatant for standby.

Fifthly, preparing the nano fibers: and (3) diluting the concentration of the supernatant to 1% by using a NaCl solution, measuring the protein content of the supernatant by a BCA method, wherein the final concentration of NaCl in the solution is 20mM, accurately adjusting the pH value of the solution to 2 by using 2mol/L and 0.1mol/L HCl, then placing the solution in a water bath at 85 ℃ for heating for 8h, and placing the solution in an ice bath to stop the reaction after the reaction is finished to obtain the self-assembled soybean protein nanofiber.

The specific implementation mode is as follows:

EXAMPLE 1 preparation of nanofibers

Thirdly, enzymolysis of the soybean protein: 2g of soy protein was dissolved in 50mL of deionized water, magnetically stirred for 2h, and then put into a refrigerator at 4 ℃ to be hydrated overnight. Adding 0.02g trypsin (1:100) into the soybean protein solution, adjusting the pH value of the solution to 7, and carrying out enzymolysis reaction in a water bath at 37 ℃ for 18 min. The degree of hydrolysis of the solution was controlled to 1%. After the enzymolysis reaction is finished, putting the enzymolysis liquid in a boiling water bath for 10min to stop the enzymolysis reaction.

Fifthly, preparing the nano fibers: and (3) diluting the concentration of the supernatant to 1% (W/V) by using a NaCl solution, determining the protein content of the supernatant by a BCA method, wherein the final concentration of NaCl in the solution is 20mM, accurately adjusting the pH value of the solution to 2 by using 2mol/L and 0.1mol/L HCl, then placing the solution in a water bath at 85 ℃ for heating for 8h, and placing the solution in an ice bath to stop the reaction after the reaction is finished to obtain the self-assembled soybean protein nanofiber.

EXAMPLE 2 preparation of nanoparticles

Thirdly, enzymolysis of the soybean protein: 2g of soy protein was dissolved in 50mL of deionized water, magnetically stirred for 2h, and then put into a refrigerator at 4 ℃ to be hydrated overnight. Adding 0.02g trypsin (1:100) into the soybean protein solution, adjusting the pH value of the solution to 7, and carrying out enzymolysis reaction in a water bath at 37 ℃ for 40 min. The degree of hydrolysis of the solution was controlled to 3%. After the enzymolysis reaction is finished, putting the enzymolysis liquid in a boiling water bath for 10min to stop the enzymolysis reaction.

Fifthly, preparing the nano fibers: and (3) diluting the concentration of the supernatant to 3% (W/V) by using a NaCl solution, measuring the protein content of the supernatant by a BCA method, wherein the final concentration of NaCl in the solution is 20mM, accurately adjusting the pH value of the solution to 2 by using 2mol/L and 0.1mol/L HCL, then placing the solution in a water bath at 85 ℃ for heating for 8h, and placing the solution in an ice bath to stop the reaction after the reaction is finished to obtain the self-assembled soybean protein nanofiber.

Claims

1. A method for preparing nanofibers by self-assembly of soybean protein enzymatic hydrolysate is characterized in that the preparation of the nanofibers with excellent performance by enzymatic hydrolysis of soybean protein by trypsin is realized by the following steps: firstly, the study of enzymolysis products with different degrees of hydrolysis obtained by enzymolysis of soybean protein by trypsin: obtaining the optimal reaction condition of the enzymolysis process by controlling the hydrolysis degree, and obtaining the relationship between the two by researching the hydrolysis degrees under different enzymolysis times; secondly, research on preparation of nano-fibers by enzymatic hydrolysate: diluting the enzymolysis liquid to a specific concentration by using a NaCl solution, wherein the diluted enzymolysis liquid is used for preparing the nano-fibers, and the final concentration of NaCl is 20 mM; adjusting the pH value of the solution to 2, and heating the solution at 85 ℃ for 8h to prepare nano fibers; to terminate the reaction, the reacted sample was cooled in an ice bath and stored in a refrigerator at 4 ℃.

(1) The method for preparing the nanofibers by self-assembly of the soybean protein enzymatic hydrolysate according to claim 1, wherein in the step one, in the step of performing enzymolysis on the soybean protein by using the trypsin, the soybean protein solution is placed in a water bath kettle at 37 ℃, the pH value is adjusted to 7, 0.02g of the trypsin (1:100) is added for reaction for 18min and 40min, and the hydrolysis degree is measured to be 1% and 3%.

(2) The method for preparing the nanofiber by self-assembly of the soybean protein enzymolysis product according to claim 1, wherein in the second step, the concentration of the enzymolysis solution with the hydrolysis degree of 1% and the concentration of the enzymolysis solution with the hydrolysis degree of 3% are respectively diluted to 1% and 3% (W/V) by using a NaCl solution, and the final concentration of NaCl is 20 mM; heating at pH2 and 85 deg.C for 8h to prepare nanofiber.