How to Prepare Rice Protein by Alkaline Separation Method?

Abstract: Rice is one of the most important food crops on earth, and the effective processing of its protein is of great significance. Using commercially available broken rice as raw material, the rice protein was separated and prepared by the alkaline method. The isoelectric point of the rice protein prepared by the alkaline method was determined, and the effects of factors such as the liquid-to-material ratio, reaction pH, reaction temperature, and reaction time on the extraction rate of rice protein were investigated, and the process conditions were optimized. The results showed that the optimal process conditions for preparing rice protein by the alkali method were a liquid-to-solid ratio of 1:8, a reaction pH of 11.5, a reaction temperature of 45 °C, and a reaction time of 1.0 h. Under these conditions, the extraction yield of rice protein was 67%.

 

Rice is an important food crop. According to statistics, about 50% of the world's population rely on rice as their staple food, and 21% of the world's population's food calories come from rice[1]. Rice is the number one food crop in China, and China is the “kingdom of rice” among the more than 100 rice-producing countries in the world. China's annual rice production accounts for about 34% of the world's total annual rice production, ranking first in the world[2]. China produces about 1.85×108 t of rice annually, accounting for 42% of total grain production. The development and utilization of rice protein is of far-reaching significance for expanding the application of cereal protein in food, increasing the added value of food products, and promoting the development of the food industry [3].

 

Rice protein is a protein with extremely high nutritional value. Rice protein is recognized as high-quality edible protein, mainly because its amino acid composition is balanced and reasonable, in line with the ideal model recommended by WHO/FAO. Among them, the content of methionine is relatively high, which is unmatched by other plant proteins [4].

 

This study used commercially available broken rice as the raw material, and the alkaline solubilization and acid precipitation method was used to study the isolation and preparation of rice protein.

 

1 Materials and methods

1.1 Materials

Commercially available broken rice.

1.2 Experimental plan

The process flow of alkaline solubilization and acid precipitation for the preparation of rice protein:

broken rice → crushing → alkaline solubilization → centrifugation → supernatant → centrifugation → precipitation → centrifugation → drying → rice protein.

1.3 Preparation process

(1) Pretreatment: The commercially available broken rice is crushed into small particles in advance using a grinder. 4 g of the crushed broken rice is taken and ground further in a mortar until it is powdered. The liquid-to-material ratio is 1:8. The mixture is soaked overnight in water to make a slurry.

(2) Alkali extraction: Adjust the pH of the soaked slurry to 11, and extract it at a constant temperature of 50 °C in a water bath for 2 h to fully separate the protein from the starch and cellulose, and dissolve the protein in the reaction solution.

(3) Centrifugation: The reaction solution was poured into a centrifuge tube and centrifuged at 6,000 r/min for 10 min to obtain the supernatant containing rice protein. The precipitate was discarded.

(4) Isoelectric point sedimentation of protein: The pH of the supernatant after centrifugation was adjusted to the isoelectric point (pH 5.5), and the protein was allowed to settle for several hours to complete the sedimentation.

(5) Separation and drying: Pour the above mixture into a centrifuge tube and centrifuge at 6,000 r/min for 10 min to obtain a protein precipitate. Discard the supernatant. Dry the precipitate at 60 °C until it reaches a constant mass. The rice protein powder is obtained once the drying is complete.

1.4 Determination of isoelectric point by acid precipitation

The supernatant containing rice protein was obtained by centrifuging the alkaline extract obtained under certain conditions, and the precipitate was discarded. The pH of the supernatant was precisely adjusted to 6.5, 6.0, 5.5, 5.0, and 4.5, respectively. After standing for a period of time, it was centrifuged and dried to obtain rice protein powder.

1.5 Single factor test

Other conditions remain unchanged. Single factor tests are carried out on the liquid-to-material ratio, pH value, reaction temperature and reaction time. The liquid-to-material ratios are 1:4, 1:6, 1:8, 1:10 and 1:12, respectively; the pH values are 9.0 , 10.0 , 11.0 , 12.0; reaction temperature was selected as 40, 50 , 60 ,70 ℃; reaction time was selected as 0.5 , 1.0 , 2.0, 3.0 ,4.0 h.

1.6 Orthogonal test

investigate the combined effect of the four factors of the alkali method for preparing rice protein, namely the liquid-to-solid ratio, reaction pH, reaction temperature and reaction time, on the extraction rate of rice protein. Three levels were selected for each factor, and an L9 (34) orthogonal array was used to carry out further experimental research.

The orthogonal test factors and level design are shown in Table 1.

2 Results and analysis

2.1 Determination of the isoelectric point of acid precipitation

Under certain conditions, rice protein was isolated and prepared, and the pH of the supernatant was accurately adjusted to 6.5, 6.0, 5.5, 5.0, and 4.5, respectively. The study determined the optimal pH for the alkaline method of isolating and preparing rice protein supernatant for acid precipitation, that is, the isoelectric point of the alkaline method of preparing rice protein.

The effect of different acid precipitation pH values on the extraction rate of rice protein is shown in Figure 1.

As can be seen from Figure 1, when the pH value is greater than 5.5, the extraction rate of rice protein continues to increase as the pH value decreases; when the pH value drops to 5.5, the extraction rate of rice protein reaches a maximum; after that, when the pH value continues to decrease, the extraction rate of rice protein decreases rapidly again, so it can be determined that the isoelectric point for the alkaline method of separating and preparing rice protein is pH 5.5.

During the experiment, precipitation occurred continuously as the pH value was adjusted from alkaline to acidic, and the supernatant became turbid. When the pH value reached a certain value, a milky white flocculent precipitate began to form, and it vibrated slightly. The flocculent precipitate rolled violently, and when the pH value continued to decrease, the milky white flocculent precipitate rapidly decreased until it disappeared. Experimental research shows that when the production of milky white flocculent sediment reaches its maximum, the extraction rate of rice protein also reaches its maximum.

 

2.2 Effect of liquid-to-material ratio on extraction rate

The effect of liquid-to-material ratio on the extraction rate of rice protein is shown in Figure 2.

As can be seen from Figure 2, when the liquid-to-material ratio is 1:8, the extraction rate is already high. Although the extraction rate of rice protein increases with the increase of the liquid-to-material ratio, the increase in extraction rate is not significant. Therefore, the liquid-to-material ratio is tentatively selected as 1:8.

 

2.3 Effect of pH of dissolution reaction on protein extraction rate

The effect of pH on the extraction rate of rice protein is shown in Figure 3.

As can be seen in Figure 3, when the reaction pH is low, the extraction rate of rice protein increases with increasing pH. When the pH is 11.0, the extraction rate reaches a maximum, which is related to the fact that more than 80% of rice protein is alkali-soluble protein. Normally, rice protein is tightly bound to starch in the endosperm and is difficult to dissolve. However, an alkali solution can loosen this tight structure and promote the separation of protein and starch. However, as the pH value increases, the extraction rate decreases. This may be because the high concentration of alkali solution makes the starch easily gelatinize, which is not conducive to the extraction of rice protein. Therefore, the reaction pH value is temporarily set at 11.0 as the optimal reaction condition.

 

2.4 Effect of reaction temperature on extraction rate

The effect of reaction temperature on the extraction rate of rice protein is shown in Figure 4.

As can be seen from Figure 4, at lower reaction temperatures, the extraction rate of rice protein increases with increasing reaction temperature. When the reaction temperature is 50 °C, the extraction rate reaches a maximum; after that, when the reaction temperature increases, the extraction rate decreases instead. When the temperature reaches 70 °C, rice protein cannot be extracted using this method, mainly because when the temperature is greater than 60 °C, the starch begins to gelatinize, hindering the extraction of rice protein, so the extraction rate begins to decrease to the point where it cannot be extracted. Overall, 50 °C is tentatively taken as the optimal reaction temperature.

 

2.5 Effect of reaction time on protein extraction rate

The effect of reaction time on the extraction rate of rice protein is shown in Figure 5.

It can be seen from Figure 5 that the extraction rate of protein increases with the increase of reaction time. When the reaction time is 1.0 h, the extraction rate of rice protein prepared by alkali method is already high; then, although the extraction rate of rice protein increases when the reaction time continues to increase, the increase is not obvious, and even if the time is too long, the extraction rate will decrease instead. Therefore, 1.0 h was tentatively selected.

2.6 Results and analysis of orthogonal experiments

The results and analysis of the orthogonal experiments are shown in Table 2, and the analysis of variance is shown in Table 3.

Tables 2 and 3 show that the order of importance of the four factors A (liquid-to-material ratio), B (reaction pH), C (reaction time) and D (reaction temperature) on the extraction yield of rice protein by the alkali method is A>B>D>C. The liquid-to-material ratio and pH have a highly significant effect on the extraction yield of rice protein, while the reaction temperature is generally significant. The analysis of variance showed that the optimal conditions were A2B3C3D1. However, considering that the effect of reaction time on the extraction rate of rice protein was not significant, the conditions of A2B3C2D1 were selected for the verification test, which yielded a protein extraction rate of 67.0%, which was slightly better than the optimal conditions of the test group (the extraction rate of the 5th group was 63.2%). group, which had an extraction rate of 63.2%), and therefore the final optimal conditions were selected as A2B3C2D1, i.e. a liquid-to-solid ratio of 1:8, a reaction pH of 11.5, a reaction temperature of 45 °C, and a reaction time of 1.0 h.

 

3 Conclusion

The acid precipitation and alkali dissolution method is simple to use and produces good results. The optimal conditions for the alkali method of extracting rice protein were found to be a liquid-to-material ratio of 1:8, a reaction pH of 11.5, a reaction temperature of 45°C, and a reaction time of 1.0 h.

 

References:

[1] Yang Zhongqiu, Lin Qinlu, Liu Xing, et al. Extraction of rice protein and product development [J]. China Food and Nutrition, 2009 (3): 27-29.

[2] Wang Zhangcun, Shen Ruiling. Research progress of rice protein [J]. China Cereals and Oils Journal, 2004 (4): 11-13.

[3] Han Xiuli, Zhang Ruyi, Ma Xiaojian, et al. Research progress on the extraction process of rice protein [J]. Food Research and Development, 2007, 28(2): 161-163.

[4] Wei Mingying, Wu Yinglong. Research progress of rice protein [J]. Grain and Feed Industry, 2003(3): 44-45.