3 Rice Protein Extraction Methods

Rice is one of the most important food crops on the planet. China is the “kingdom of rice” among the more than 100 rice-producing countries in the world, with an annual rice production accounting for about 34% of the world's total annual rice production, ranking first in the world. However, in China, rice is used mainly as a food crop, and the utilisation rate of deep processing is very low. Every year, China produces more than 30 million tons of reusable rice by-products, which concentrate 64% of the nutrients in rice. However, these substances are not effectively utilised. In the United States, Japan and other countries with well-developed rice processing industries, the added value of rice resources in the processing industry has reached about 1:4 to 1:5; among them, rice protein has become an important direction for the rice processing industry. The development and rational use of rice protein is based on the deep processing and comprehensive utilization of rice processing products and by-products [1].

1. Nutritional value of rice protein

The quality of rice protein is recognized as the best among cereal proteins. It is rich in essential amino acids, with lysine, the first limiting amino acid, having a higher content than other cereals. The amino acid composition pattern is close to the recommended pattern of the WTO/FAO and is easily digested and absorbed by the human body. Compared with other cereal proteins, rice protein has a higher biological value (BV) and protein utilization rate (PER). The biological value can be as high as 77, and the protein utilization rate is 1.36% to 2.56%, ranking first among all kinds of grains. The most important characteristic of rice protein is its hypoallergenicity, which makes it very suitable as a protein supplement for infants or special populations. It is therefore a favorite in the world protein market, fetching up to 30,000 yuan per ton [2].

2 Extraction of rice protein

The main purpose of extracting rice protein is to obtain high-purity rice protein. At present, the main methods for extracting rice protein at home and abroad are: alkaline extraction, enzymatic extraction, and composite extraction.

2.1 Alkaline extraction

Rice protein contains 80% alkali-soluble protein. The alkali method is based on the principle that alkali-soluble protein can dissolve in an alkali solution. The alkali solution loosens the tight structure of the rice starch in the rice that is bound to the rice protein. At the same time, the alkali solution has a destructive effect on the secondary bonds of the protein molecules, especially the hydrogen bonds, and can cause the dissociation of some polar groups groups dissociate, giving the protein molecule surface the same charge, thus solubilizing the protein molecule and promoting the separation of starch and protein, and impurities are removed by centrifugation; the pH of the supernatant is then adjusted to the isoelectric point of the rice protein, allowing most of the protein to precipitate.

HABA Pepe Francois et al. used indica rice as the raw material to separate rice protein and starch by the alkali method, and obtained the optimal process conditions for separating rice protein and starch through orthogonal experiments: the concentration of sodium hydroxide solution was 0.075 mol/L; the extraction 6h; liquid-to-material ratio 1:7g/ml; temperature 40℃; the resulting protein purity was higher than 80%, the protein extraction rate was 86.23%, and the starch recovery rate was 92.12% [4]. Wang Wei et al. used response surface methodology to optimize the alkaline extraction process of rice protein. The experimental results showed that the mass fraction of NaOH was 0.35%, the extraction time was 2–3 h, and the water-to-material ratio was 7–8. The extraction rate of rice protein was ideal, and the protein purity was higher than 85% [4]. The process flow of the alkali method for extracting rice protein is as follows: rice flour → alkali leaching → centrifugation → pH adjustment of the supernatant → centrifugation → washing and precipitation → freeze-drying → rice protein.

The main factor affecting the alkali method is the concentration of the alkali solution. When the concentration is low, the extraction rate of rice protein is low, but when the concentration of the alkali solution is too high, the starch is easily gelatinized, and the color of the rice protein will be affected and turn yellow. Sun Qingjie et al. selected different concentrations of NaOH to act on rice. The experimental results showed that when the concentration of NaOH was 0.02 mol/L, the extraction rate of rice protein was only 22.37%; when the concentration reached 0.06 mol/L, the extraction rate could reach 78.15%; and when the concentration was 0.09 mol/L, the extraction rate of rice protein could be as high as 90.10% [5]. Evelynmae et al. used 0.1 mol/L NaOH at room temperature with a 1:10 liquid-to-liquid ratio to extract for 1.5 hours, and the protein extraction rate could reach 98% [6].

The alkaline method of extracting rice protein is simple, has a high extraction rate, and the resulting product has a better color and texture. It is also widely used in industrial production due to its low cost. However, the alkali method also has many disadvantages: the reaction liquid-solid ratio is large, requiring the consumption of large amounts of alkali and water; a large amount of acid is consumed during isoelectric precipitation; desalination and purification are difficult; under the action of high concentrations of alkali, the protein is easily denatured, which also changes the properties of the starch; and some amino acids undergo condensation reactions to produce toxic substances, posing a hidden danger to food safety.

2.2 Enzyme extraction

Enzymatic extraction of rice protein mainly involves treating rice with proteases or non-proteases to separate the rice protein from other components and increase its solubility, so that the rice protein can be more easily extracted. Enzymes can be classified as proteases or non-proteases according to their function.

The protease method involves the use of proteases to degrade and modify the rice protein, so that it can be extracted as soluble peptides. Currently, the proteases commonly used to extract rice protein include acid protease, neutral protease, alkaline protease, and composite protease. Many publications report that alkaline protease is the optimal enzyme for extracting rice protein. Song Na et al. used four different proteases to extract and hydrolyze rice, and studied the change of the degree of hydrolysis of rice protein with hydrolysis time. The results showed that the extraction rate of alkaline protease hydrolyzed rice protein was higher than that of the other three proteases. At pH 9 .5, temperature 60°C, enzyme addition amount (E/S) 1.5%, material to liquid ratio 1:6, hydrolysis time 4h, the extraction rate of rice protein can reach 76.42% [7].

However, due to the fact that the enzymes used in different studies come from different manufacturers, enzyme activity varies, and the raw materials and extraction processes used are not the same, there are certain differences in the conclusions of the studies. Deng Jing and others found in their research on the enzymatic extraction of crude rice bran protein that the extraction efficiency of the complex protease was significantly better than that of the alkaline protease and the acid protease [8]. Others have also obtained good results using neutral protease to process rice materials to obtain rice protein, and believe that the neutral protease extract is superior to the alkaline protease extract in terms of color, flavor and protein purity.

Another idea for enzymatic extraction is to remove impurities and retain the main ingredient. Appropriate enzymes are used to remove non-protein components from rice, and the protein is separated from other components by separation and purification. Amylase is more commonly used to recover protein during starch extraction. The rice protein content in the precipitate obtained by this method is about 40%. Because this method is mainly used in industry to produce starch sugar, liquefaction is not very thorough in order to avoid producing too much reducing sugar equivalent in the supernatant and reducing the yield of maltose. This results in a high-protein rice flour with some unhydrolyzed starch and sugars, which makes its protein content much lower than that of rice protein concentrate, thus limiting the application of this method in the industrial production of rice protein.

In addition, because rice residue and bran are mixed with cellulose, hemicellulose, xylan and other non-starch polysaccharides, non-starch enzymes are also commonly used to extract protein. Morita used large rice flour as the raw material and reacted it with high-temperature liquefied amylase at 97 °C for 2 h, and then filtered to remove the sugars to obtain rice isolated protein with a protein content of more than 90% [9]. Ji Fengdi and others used high-temperature and medium-temperature amylases to purify rice dregs protein, obtaining rice protein contents of 78% and 79%, respectively [10].

Enzymatic extraction has mild reaction conditions, basically does not damage nutrients, and hydrolyzes protein polypeptide chains into short peptide chains, which improves the digestibility of the protein. At the same time, the reaction has a small liquid-to-solid ratio, which not only saves the consumption of alkali and water, but also increases the content of solids in the extract, thereby reducing the energy consumption for removing the moisture from the extract, creating certain conditions for industrial production. However, the high price of enzyme preparations makes production costs too high, hindering industrial production.

Alkaline extraction is fundamentally different from enzymatic extraction. Alkaline extraction mainly extracts the alkaline-soluble protein, which accounts for more than 80% of the total protein in rice and is a large protein molecule; enzymatic extraction, on the other hand, extracts more water-soluble protein, alcohol-soluble protein, poorly soluble protein, as well as water-soluble small molecule active peptides and free amino acids. Therefore, the proteins extracted by the two methods are very different in nature.

Guo Rongrong et al. compared the functional properties of rice proteins extracted by the alkali method and the enzymatic method. The rice protein extracted by the alkali method has better water retention, oil absorption and foaming properties than the rice protein extracted by the enzymatic method, but its solubility, emulsion stability and foam stability are far inferior to the rice protein extracted by the enzymatic method; and the emulsifying ability of the two protein products is quite similar [11]. Secondly, the color and flavor of the protein extracted by the alkali method are inferior to those of the enzymatic method. In summary, the properties of the protein obtained by enzymatic hydrolysis are superior to those of the protein obtained by alkaline hydrolysis.

2.3 Composite extraction method

Since both the enzymatic and alkaline methods have their own problems, research is now shifting to the combined extraction method, in the hope of improving product quality and yield while reducing production costs. Wang Yalin et al. studied the two-step alkaline-enzyme method for extracting rice protein from rice bran. First, alkaline solubilization was used to partially extract the protein, and then alkaline protease was used to slightly hydrolyze the residue to improve protein solubility and carry out secondary protein extraction, resulting in a protein extraction rate of 78.8% [12]. Some people also supplement the traditional method with a physical method to extract the protein. Xi Haiyan and others used an ultra-high pressure assisted enzyme method to extract rice protein. The extraction rate was 70% when the alkaline protease was used alone, but after being supplemented with 400 MPa pressure, the protein extraction rate increased to 78.72% [13].

3 Development status of rice protein products at home and abroad

For a long time, people have not paid much attention to rice protein. However, with the recognition of the nutritional value and hypoallergenicity of rice protein and the attention paid to it by the international community, rice protein has stood out among cereal proteins and become a hot spot in the industry. At present, the main forms of rice protein products are: food additives, high-protein nutritional powders, edible coatings, bioactive peptides with special functions, etc. Rice protein has functional properties such as solubility, emulsification, foaming, water retention and oil retention. Although it has poor solubility, hydrolysis can release certain amino and carboxyl groups, increase the polarity of the protein molecule, and not only increase its solubility, but also bring out its foaming and emulsifying properties, making it easier to develop and use. Rice protein added to food not only improves food processing performance, but also greatly increases the nutritional value of the food [14].

In recent years, research on rice protein products in the United States has focused on the development of various nutritional foods and fast foods containing rice protein, such as the development by the American ABBOTT laboratory in June 2003 of a nutritional tablet for infant milk feeding containing 10-20 wt% of a blend of proteins including rice protein, and soybean protein; NATURE'S ONE INC researched a ready-to-drink nutritional drink in April 2002, which contains 10-30μg/L of rice protein, and can provide supplementary nutrition for patients, infants, and young children. In Japan, research has begun to focus on extracting therapeutic foods with adjuvant therapeutic effects from rice protein. DOK UR ITSU GYO SEI HOJIN SANGYO GIJUJTSU SO and OKINAWA SHOKURYO KK, in collaboration with other units, researched a novel peptide-containing angiotensin-converting enzyme inhibitor and its salts, which are hydrolyzed from rice protein, in November 2001, and can be used to assist in the treatment of hypertension.

In recent years, research has also begun in China on the production processes for rice protein powder, modified rice proteins, high value-added peptides, resistant proteins, and edible coatings. Shan Chengjun and others studied the process conditions for edible rice protein membranes and determined the tensile strength, elongation, and water permeability of the membranes. The results showed that when the mass fraction of rice protein was 5%, the mass fraction of glycerol added was 3%, the addition amount of glutamine transaminase is 0.2% by mass, the enzyme reaction time is 90 min, the pH of the membrane solution is adjusted to 11.5, and the treatment is carried out at 80 °C for 40 min to obtain a membrane with better properties [15].

4 Outlook

China is a major rice-producing country with abundant rice resources. Rice bran and rice dregs from the production of starch sugar from early indica rice and broken rice can also be used as raw materials to obtain rice protein products with good performance through extraction. After comparing several extraction methods, the alkaline enzyme method is still the most practical method in industry at present, which is simple to operate and relatively economical. With further research, the composite method is also gradually being widely adopted. Because the solubility of rice protein is relatively poor compared to other proteins, this also affects other properties, limiting the wide application of rice protein. In addition, rice protein also has a certain peculiar smell, so finding a scientific and reasonable modification method to modify rice protein will be one of the focuses of future work.

The development and utilization of rice protein resources can compensate for the shortcomings of animal protein and prevent the occurrence of a series of rich diseases caused by excessive intake of animal fat and cholesterol. More importantly, further research and utilization of high-quality rice protein resources is conducive to the comprehensive utilization of deep processing of rice, increase added value, and promote the development of China's food industry with the support of science and technology.

References

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