How to Improve the Solubility of Rice Protein by Dual Enzymatic Hydrolysis?

 Abstract : Objective:To investigate the conditions for enzymatic hydrolysis of rice protein and to improve the solubilization, emulsification and foaming properties of rice protein. [Methods] The enzyme incorporation was determined by the enzyme-catalyzed reaction process, and the reaction conditions of the enzymes were determined by a homogeneous design of experiments and Mathematica mathematical software. O [Results] The results showed that alkaline protease and complex protease hydrolyzed rice protein more effectively than a single enzyme preparation; during the enzyme-catalyzed reaction process, the indexes of solubility, emulsification, and foaming properties of rice protein varied in different tendencies, and there was no correlation between the degree of hydrolysis and the indexes mentioned above. There was no corresponding relationship between the hydrolysis degree and the above indexes. [The conclusion is that the two-enzyme hydrolysis is more suitable for improving the solubility of rice protein; there is a certain synergy between the two enzymes, and appropriate control of the reaction conditions can lead to rice protein hydrolysates with significant solubility, emulsification or foaming properties.

 

Rice dregs are the by-products produced when producing starch sugar or organic acid from early indica rice or broken rice, its protein content is as high as 50% or more, because of its poor solubility, it can not be used in the food industry, and it is mainly used in animal feed, and the high-quality protein resources have not been reasonably utilized[1] . Enzymatic hydrolysis is a common method to improve protein solubility and emulsification, foaming and other properties[2,3] , alkaline protease has a better hydrolysis effect than other proteases, but a single alkaline protease preparation can only make a small amount of rice protein solubility, and the insoluble protein component still maintains a high relative molecular mass[4] . According to the characteristics of different proteases with different hydrolysis sites, the use of more than two proteases for hydrolysis may have better results. The effects of hydrolysis with alkaline protease and complex protease on the solubility, emulsification and foaming of rice proteins are reported here.

 

1 Experimental materials and methods

1.1 Experimental materials and main instruments

Rice protein powder prepared by the author's laboratory to amylose production of rice residue as raw materials through the purification method, the protein content of 82%; alkaline protease (A1calase) and complex protease (protamex) is a product of Novozymes.

 

1.2 Experimental Procedures and Analytical Methods

1.2.1 Catalytic process of different proteases

The experiment was carried out in March-June, 2006 at Zhengzhou Institute of Light Industry. The rice protein powder (crushed through 100 mesh sieve) was placed in a beaker, and the suspension was prepared according to the designed concentration and the high concentration.

After rapid dispersion (10000r/min,2min), transfer to the enzyme reactor, adjust to the optimum PH and temperature of each protease with NaoH solution, time immediately after adding enzyme preparation, take samples at specified intervals, 3.0 ml each time, put the samples into test tubes and immediately inactivate the enzyme in boiling water bath for 3min; centrifugate the samples at 1500r/min,5min. The protein content of the supernatant was determined by Folinol reagent method and the enzyme reaction curve was plotted.

 

1.2.2 Study of enzyme reaction conditions

A homogeneous design of experiments was used to set the concentration of rice protein in the reaction solution at 10%, the pH 8.2, and the reaction temperature at 55 ℃. The solubilization, emulsification and foaming properties of rice protein were investigated under the conditions of different addition amounts and different reaction times of the two enzymes. At the end of the reaction, the enzymes were inactivated by boiling water bath heating, centrifuged at 2000r/min for 10min, and the supernatant was analyzed.

 

1.2.3 Determination of emulsifying and foaming properties

The emulsification was determined by the turbidity method[5] , 30ml of 0.25% protein suspension was prepared with phosphate buffer, adjusted to PH8.0, and then 10ml of soybean oil was added, dispersed at 10000rPm for 30s, and then 40μl of emulsion was sucked out from the bottom of the solution with a microsyringe, and then added to 5ml of 0.1% SDS, and then the light absorbance value A0 was shaken well, and then measured at 500nm. The light absorption value A0 was measured at 500 nm, which is the emulsification activity index (EAI); the foaming ability (FA) was expressed as the foaming volume (V0) of the 4% protein test solution stirred at 10000 rPm [3,5].

 

1.2.4 Determination of degree of hydrolysis (DH)

The classical PH-stat method was used [6,7].

 

2 Test results and discussion

2.1 Influence of enzyme addition method on the reaction process

Different proteases have different cleavage sites and catalytic properties. The use of two or more proteases may hydrolyze proteins into smaller molecules, thus improving the solubility of proteins. The author firstly examined the effect of different methods of adding alkaline protease (A) and complex protease (P) on the reaction process and the maximum solubility of protein. The experimental conditions were 8% protein concentration, 0.2% Alcalase and 0.2% Protamex (E/S), and the results are shown in Fig. 1. The results are shown in Fig. 1. It can be seen that the maximum solubility achieved when the two enzymes were added at the same time and sequentially was close to that achieved when the two enzymes were added in order, and was higher than that achieved when the two enzymes were hydrolyzed alone, indicating that the two enzymes have certain synergistic effect in co-catalyzing the hydrolysis.

 

2.2 Optimization of two enzyme hydrolysis conditions

The homogeneous design method was used to investigate the effects of enzyme addition and reaction time on the solubilization properties of rice proteins, and to analyze the emulsification and foaming properties of the enzyme digests as well as the hydrolysis degree, and the results are shown in Table 1. The advantage of the homogeneous design method is to use as few tests as possible to analyze the effect of enzyme addition and reaction time on rice proteolysis.

The effect of reaction conditions was studied more frequently. From the experimental results, it can be seen that with

The changes of solubility, emulsification and foaming of proteins under different reaction conditions show that the enzyme digestion conditions have different effects on the above indexes.

In order to quantitatively characterize the effects of the two enzyme additions (X1 and X2, respectively) and the reaction time (X3) on the protein solubility, the experimental data were processed by Mathematica software, and the resulting regression equations were as follows.

y = 15.2967 +3.8378x1 +5.2568x2 +6.9716x3- 0.6155x1x2-0.9065x2x3

 

From the F-test, the equation was found to have a goodness of fit of 99.99% at 97.67% confidence level with a correlation coefficient of R2 = 0.999811. From the regression analysis, it was found that the interaction between alkaline protease and reaction time was higher than that between complex protease and alkaline protease. The equation was solved within the constraints and levels of this test and the results were as follows.

X1=6.55, X2=1.76, X3=6.15, at this time, ymax = 75.63, i.e., when the dosage of complex protease and alkaline protease is 0.03% and 0.048% respectively, and the reaction time is 3.1h, the concentration of protein that can be solubilized reaches 75.63% at the highest. According to this condition, two repeated experiments were arranged, and the results coincided with this value, indicating that the equation is reliable.

Similarly, the Mathematica software was used to obtain the relationship between the enzymatic conditions and the emulsification properties of the digests as follows.

y = -0.0192 +0.0536X1 +0.0593X2 +0.0630X3-0.0074X1X1-0.0078X2X3

The optimized set of solutions was obtained by solving the equation as follows: X1 = 3.6, X2 = 7, X3 = 7, ymax = 0.552. That is to say, when the amount of complex protease and alkaline protease is 0.018% and 0.1% respectively, and the reaction time is 3.5h, the protein hydrolysate with emulsification capacity of 0.552 can be obtained.

The above results indicate that different enzymatic conditions are required to obtain rice protein hydrolysates with the best solubility, emulsification or foaming properties.

 

2.3 Relationship between the degree of hydrolysis and the nature of enzymatic hydrolysates

Proteins are hydrolyzed by enzymes to form smaller molecules, and the degree of hydrolysis (DH) value is an important indicator of the degree of protein hydrolysis. The relationship between the DH value of rice protein and its solubilization, emulsification and foaming properties is shown in Figure 2.

It can be seen that, with the increase of DH, the solubility, emulsification and foaming of proteins do not have the same trend, especially the solubility of proteins and the degree of hydrolysis is not parallel, which is different from the hydrolysis of soluble proteins (e.g., soybean proteins) [8]. According to the method of hydrolysis, there are two sources of DH in the enzymatic reaction system of insoluble rice proteins: the dissolution of insoluble rice proteins into solution after enzymatic hydrolysis, and the further degradation of solubilized proteins. When the amount of enzyme and the reaction time of the two enzymes are different, even if the DH is the same, the molecular composition of the protein in the reaction system may be different, thus showing different solubilization, emulsification and foaming properties. Generally speaking, the emulsification and foaming properties of proteins require appropriate molecular size and molecular polarity, and higher solubility is often an important reflection of smaller protein molecules.

 

3 Summary

In improving the solubility of rice proteins, the combined hydrolysis of alkaline protease and complex protease was more effective than the use of the two enzymes alone. During the enzyme-catalyzed reaction, the solubility, emulsification and foaming of rice proteins showed different trends, suggesting that there was some synergy between the two enzymes. Due to the different molecular bases required for protein solubilization, emulsification and foaming, it is necessary to use different enzymatic conditions to obtain rice protein hydrolysates with outstanding properties.

 

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