Preparation and characterization of extruded rice protein-glucose graft complexes

 Abstract: The preparation of grafted complexes of extruded rice protein and glucose and the determination of their properties were investigated. The effects of different extrusion conditions on the grafting degree of sugar grafting reaction were analyzed by taking the extrusion temperature, moisture content of rice protein and screw rotational speed as the investigating factors. The rice protein extrusion process was optimized by using the Design-Expert 8.0 software, and the grafted complexes of rice protein and glucose obtained by the optimal extrusion process were prepared, and the solubility, emulsification activity, and emulsion stability of the grafted complexes were determined, which were analyzed and observed by infrared spectrometry and scanning electron microscopy. The solubility, emulsification activity and emulsification stability of the grafted complexes were determined, and analyzed and observed by infrared spectroscopy and scanning electron microscopy. The results showed that the grafted complexes of extruded rice protein and glucose were prepared at an extrusion temperature of 90 ℃, moisture content of rice protein of 36%, and screw speed of 182 r/min, and the grafting degree reached the maximum value of 31.0%±0.3%. The solubility, emulsification and emulsion stability of the grafted complexes of extruded rice protein and glucose were increased by 76.6% and 67.0%, respectively, compared with those of natural rice protein. Fourier infrared spectroscopy and scanning electron microscopy showed that the extruded glucose grafting reaction significantly changed the protein structure and could improve the application value of rice protein.

 

Rice protein is a high-quality plant protein with hypoallergenic and high bioavailability properties, but its application is limited by the large amount of hydrophobic glutenin [1-2]. Therefore, how to change the use value of rice protein has become a hot research topic.

 

Extrusion technology is widely used in the food industry, and moderate extrusion can improve the physical properties of protein such as water absorption and emulsification, but the aggregation and solubility of protein molecules are seriously reduced after extrusion [3-5], which affects the application of extruded rice protein in other areas. The grafting reaction between protein and sugar can significantly change the functional properties of protein, but due to the long time of sugar grafting reaction, microwave and ultrasonic methods have been applied to improve the process of sugar grafting reaction [6-8]. Few studies have been reported on the improvement of the functional properties of extrusion-modified rice protein with sugar grafting reaction. Therefore, the aim of this paper is to study the extrusion preparation process of extrusion-modified rice protein-glucose grafted complexes, and to determine the solubility, emulsification, and emulsion stability changes, and to investigate the structural changes of the extruded rice protein-glucose grafted complexes by Fourier infrared spectroscopy and scanning electron microscopy, in order to provide a theoretical basis for expanding the application of rice protein.

 

1 Materials and Methods

1.1 Materials and reagents

Rice protein powder (protein content 82.5%): Jiangxi Jinnong Bio-technology Co., Ltd; OPA: Sigma Company; other reagents were analytically pure.

 

1.2 Instrumentation

DS56-Ⅲ Twin Screw Extruder and Expander (screw diameter of 25 mm, L/D ratio of 16:1, circular grinding head inner diameter of 4 mm): Jinan Saixin Expansion Machinery Co.

 

1.3 Test methods

1.3.1 Process flow

Adjust the moisture content of rice protein and mix it thoroughly, adjust the screw speed and extrusion temperature of the twin-screw extruder and extrude the rice protein, dry the extruded product at room temperature, pulverize it and pass it through a 100-mesh sieve. The conditions of sugar grafting reaction were referred to the previous experiments and references [8-9], the protein concentration of extruded rice protein was 10 g/L, the pH was adjusted to 11.0 with 0.2 mol/L NaOH, glucose was added in the ratio of 3:1 with protein, and it was mixed thoroughly, and the reaction was carried out at 90 ℃ for 30 min in a water bath, and the modified mixture was dialyzed for 24 h, and then made into powder by freeze-drying. The modified mixture was dialyzed for 24 h, freeze-dried and then powdered to obtain the extruded rice protein-glucose grafting complex sample.

 

1.3.2 One-way tests

Extrusion temperature, protein moisture content and screw speed were taken as factors, and the grafting degree of extruded rice protein-glucose graft complex was taken as an index, and the values of each extrusion factor were fixed as extrusion temperature 90 ℃, protein moisture content 35% and screw speed 180 r/min, respectively, to examine the effects of each factor on the index.

 

1.3.3 Response surface tests

On the basis of the one-way test, a response surface center combination design was adopted. The parameters of extruded rice protein extrusion process were optimized by taking three factors, namely extrusion temperature, protein moisture content and screw speed as independent variables (X), and the grafting degree of extruded rice protein and glucose grafting reaction complex (Y) as response values. The coding table of factor levels is shown in Table 1.

 

1.3.4 Determination of test indicators and properties

The grafting degree was calculated by the method of Church et al [10]. The solubility of the protein was determined by the method of [11], and the solubility was calculated according to the protein content of the sample and the protein content of the solution, which was expressed as the nitrogen solubility index (NSI%). The emulsification and emulsion stability of the protein were determined by the turbidity method [12]. Fourier infrared spectroscopy and scanning electron microscopy were performed according to the method and parameters of [13].

 

1.4 Data processing analysis

Data were processed using Design-Expert 8.0 and SPSS 20.0 software and plotted using Origin 8.5.

 

2 Analysis of results

2.1 Univariate tests

2.1.1 Effect of extrusion temperature on grafting degree of grafted complexes

As shown in Figure 1, the grafting degree of the grafting complex increases and then decreases with the increase of extrusion temperature, and the grafting degree of the grafting complex of rice protein and glucose extruded at the extrusion temperature of 90 ℃ is 30.5%±0.3%, which may be attributed to the fact that rice protein is subjected to the effects of extrusion, shear, and high pressure, protein denaturation, and the protein molecules partially aggregated and unfolded, which partially exposes free amino groups [14], and when the grafting reaction with glucose is carried out with the use of sugar grafting reaction. This may be due to the fact that when rice protein is subjected to extrusion, shear and high pressure, the protein is denatured, the protein molecules are partially aggregated and unfolded, and the free amino group is partially exposed [14], which can be utilized in the grafting reaction with glucose. Based on the experimental results, 80~100 ℃ was selected as the range of response surface.

 

2.1.2 Effect of moisture content of extruded rice protein on the grafting degree of the grafting complexes

It can be seen from Figure 2, with the increase of rice protein moisture content, the grafting degree of the grafting complex first increased and then decreased, in the protein moisture content of 35%, the grafting degree of the extruded rice protein and glucose grafting complex was 29.8% ± 0.3%, probably because when the moisture content is low, the viscosity of the rice protein is larger, and it stays in the extruder for a long time, and it is denatured by the high temperature and high pressure effect, and the moisture content is too large. Too large, the water molecules lubrication effect of the material viscosity decreases, the resistance to flow is reduced, so that the material in the extruder inside the residence time is less, the degree of denaturation is small [6], so the moisture content of extruded protein has a great impact on the degree of protein denaturation. According to the experimental results, 25%~45% was selected as the range of response surface research.

 

2.1.3 Effect of extrusion screw speed on the grafting degree of grafted complexes

It can be seen from Figure 3, with the increase of extrusion screw speed, the grafting degree of the grafting compound increases and then decreases, the grafting degree of rice protein and glucose grafting compound extruded under the condition of extrusion screw speed of 180 r/min is 30.0%±0.3%, probably because when the rotational speed is low, the protein is subjected to extrusion, the energy is aggregated, and the degree of protein molecules partially aggregated and degraded is small, and increasing the rotational speed, the material residence time is short, high screw speed, the protein extrusion is subjected to stronger pressure, so the protein denaturation is serious and the degree of protein molecules aggregated is too large [3], which is not conducive to the subsequent grafting reaction with glucose. Material residence time is short, high screw speed, protein extrusion is subjected to stronger pressure, so the protein denaturation is serious, protein molecules aggregation is too large [3], is not conducive to the later grafting reaction with glucose, according to the results of the test, 160~200 r/min was selected as the range of the response surface study.

 

2.2 Response surface optimization test

The response surface results are shown in Table 2, and the numerical model equations are analyzed and organized by the software Design-Expert 8.0:

y=31.22-0.74x1-0.83x2+1.03x3-0.95x1x2+ 1.11x1x3+0.65x2x3-5.08x12-4.14x22-1.15x32

The results of the ANOVA of the quadratic regression equation are shown in Table 3, from which it can be seen that the P value of the model is less than 0.01, the model equation is highly significant, and the out-of-phase P value is more than 0.05, which is not significant, and the model R2=0.988 4 ＞ 0.800 0, which indicates that the equation is well fitted to the experiment, the linear relationship between the independent variable and the response value is significant, the experimental error is small, and the model can reflect the data pattern better. At the same time, the model can fully show the relationship among the factors, X1, X2, X3, X1X2, X1X3 have significant effects on the grafting degree of the grafted complex, and the other factors have no significant effects. From the F-value test, it can be obtained that the contribution rate of the factors is X3＞X2＞X1, i.e., screw speed＞rice protein moisture content＞extrusion temperature.

 Figs. 4-5 intuitively reflect the effects of the modeled extrusion temperature and moisture content, extrusion temperature and screw speed on the grafting degree of the grafted compound. The interaction of extrusion temperature and moisture content is significant, as shown in Fig. 4, which indicates that the grafting degree of the grafted compound shows a tendency to increase and then decrease as the extrusion temperature increases, and the grafting degree shows a tendency to increase and then decrease as the moisture content increases, and the contours of the two factors tend to be elliptical and close to each other. And the contour lines of the two factors tended to be elliptical and close to each other; the interaction effect of extrusion temperature and screw speed was significant, as shown in Fig. 5, which showed that the grafting degree of the grafted compound tended to increase and then decrease with the increase of extrusion temperature, and tended to increase and then decrease with the increase of screw speed, but the interaction effect was not as large as that of the extrusion temperature and screw speed on the grafting degree.

 

The model was further analyzed by using Design-Expert 8.0 software, and the optimal extrusion conditions for the grafted complex were extrusion temperature 89.40 ℃, moisture content 35.75%, and screw speed 182.88 r/min. In order to verify the reliability of the response surface methodology, validation experiments were carried out, and the corrected extrusion parameters were extrusion temperature 90 ℃, protein moisture content 36%, screw speed 182 r/min, and three tests were carried out in parallel, and the predicted value was 31.0%±0.3%. In order to check the reliability of the response surface method, a validation test was carried out with the modified extrusion parameters of extrusion temperature 90 ℃, protein moisture content 36%, and screw speed 182 r/min.

 

2.3 Properties of Extruded Rice Protein and Glucose Grafted Complexes

In order to investigate the effects of extrusion and sugar grafting reactions on the properties of extruded rice protein-glucose graft complexes, three proteins, namely, natural rice protein (NRP), extruded rice protein (ERP), and extruded rice protein-glucose graft complex (ERPG), were analyzed in terms of their structures and properties.

 

2.3.1 Research on functional properties

The solubility, emulsification and emulsion stability of NRP, ERP and ERPG are shown in Table 4. It can be seen from Table 4 that the solubility of ERP was decreased by 9.1%, the emulsification activity and emulsion stability of ERP were increased by 14.3% and 15.0%, respectively, while the solubility of ERPG was increased by 76.6%, and the emulsification activity and emulsion stability of ERPG were increased by 67.0% and 52.6%, respectively, in comparison with NRP. 52.6%, respectively. This may be due to the fact that high pressure and high shear changed the structure of rice protein molecules, the protein molecules were partially unfolded or aggregated, and the rice protein molecules formed macromolecular aggregates through non-covalent interactions, which showed a tendency of decreasing the solubility of the protein [13], and the hydrophobic groups embedded inside the molecules were exposed [15], which enhanced the lipophilicity, and then increased the emulsification.

 

After extrusion of rice protein, due to the change of protein structure, the amino group for the grafting reaction with glucose was exposed, so that the grafting reaction was carried out smoothly. The introduction of glucose caused the secondary bond to be opened, the protein molecules were further stretched, and a hydrated layer was formed on the surface of the molecules, which increased the number of hydrophilic hydroxyl groups in the protein structure and changed the solubility of the protein [7], and part of the protein could be effectively absorbed in the oil-water interface, which increased the emulsification activity of the grafted complex. Some proteins can be effectively adsorbed at the oil-water interface, thus increasing the emulsifying activity of the graft complex, while the generation of glucose-protein graft complexes reduces the interfacial tension and stabilizes the emulsion [9]. Therefore, the grafting reaction of modified rice protein with dextran can change the solubility, emulsification and emulsion stability of the original rice protein to a certain extent by extruding it appropriately.

 

2.3.3 Scanning Electron Microscopy Analysis

The SEM results of NRP, ERP and ERPG are shown in Fig. 7, which indicate that the surface of NRP is in the form of lumps, the ERP is in the form of whole thin layers with interspersed pores, and the ERPG is in the form of disorganized and irregular fragments. This also indicates that rice protein undergoes different degrees of degradation and polymerization during the extrusion process, and its natural ordered structure is broken, and the protein molecules degrade and polymerize with each other to form a lamellar structure [15]. Sugar grafting alters the surface structure of rice protein, which results in the extension of the peptide chain of rice protein and the diffusion of the molecules [9]. Therefore, the highly hydrophobic rice protein is more favorable for grafting reaction with glucose after extrusion, and the functionality of the grafted complexes with sugar grafting reaction after extrusion is improved, which also indicates that extrusion has a certain effect on the modification of rice protein.

 

3 Conclusion

In this study, the influence of extrusion process parameters on the grafting degree of sugar grafting reaction was determined by screw speed, moisture content and extrusion temperature, and the optimal conditions for the preparation of extruded rice protein-glucose graft complexes were extrusion temperature of 90 ℃, moisture content of rice protein of 36%, and screw speed of 182 r/min, and the maximum grafting degree of 31.0%±0.3% was achieved under these conditions. Compared with natural rice protein, the solubility of the grafted complex was significantly increased by 76.6%, and the emulsification and emulsion stability were increased by 67.0% and 52.6%, respectively. Therefore, the change in functionality of the graft-modified extruded rice protein sugars is of significance for the continued processing and utilization of rice protein.

 

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