What Are the Rice Protein Powder Extraction Methods?

Rice is one of the main food crops in China. In 2018, about 481 million tons of rice were produced worldwide [1], and it is expected that the demand for rice will increase by 40% by 2030 [2]. The main components of rice are starch and protein [3], which account for about 80% and 8% of the weight of rice, respectively. Among them, rice starch has the advantages of fine particles, uniform particle size, and good freeze-thaw stability, and has unique properties and uses [4]; rice protein contains 18 amino acids, such as methionine, proline, lysine, isoleucine, phenylalanine, leucine and tryptophan and threonine, etc. Eight essential amino acids, a reasonable amino acid composition, high nutritional value, and close to the WHO / FAO recommended nutritional model [5], rice protein has a biological potency of up to 77, similar to beef and fish, and is a high-quality vegetable protein [6].

Rice protein also has a unique hypoallergenicity, and the hydrolyzed polypeptides have health benefits such as lowering blood pressure and cholesterol [7]. Therefore, the comprehensive utilization of rice mainly focuses on protein modification [8] and the development of nutritional value [9]. China has long been the world's largest rice producer [10], but China's efforts to develop rice have been modest and research has not been in-depth, which has resulted in a serious waste of China's rice resources. With the continuous development of food technology, research on rice resources should be increased, so that low-value rice resources can be reasonably transformed into high-value products, thereby achieving greater economic and social benefits. This paper provides an overview of the extraction technology, structural properties, functional properties and comprehensive utilization of rice protein.

1 Extraction methods for rice protein powder

1.1 Alkali extraction and acid precipitation

The alkali method is currently the most common and traditional method for extracting rice protein. It is based on the principle that rice protein contains more than 80% alkali-soluble protein. The protein obtained by alkali extraction is relatively pure, but the use of a high concentration alkali solution in the extraction process has a certain impact on the extraction rate and physicochemical properties of rice protein. Wang [11] investigated the effect of different concentrations of NaOH solution on the extraction rate and physicochemical properties of rice protein. The study showed that under the conditions of high alkali concentration extraction, the protein was severely denatured, the Maillard reaction was severe, and lysine and alanine underwent a condensation reaction to form toxic substances. At the same time, brown substances were produced, which affected the color of the product. This shows that the concentration of the alkali solution has a significant effect on rice protein. At the same time, factors such as temperature, time, and material-to-liquid ratio also have a significant effect on the extraction rate of rice protein [12]. Wang Yalin et al. [13] and Wang Liying et al. [14] used the alkali extraction and acid precipitation method to extract rice protein, and more protein components can be obtained under optimal conditions. The alkali method is simple to extract, but it consumes a lot of water and alkali, so it is difficult to apply the alkali method to industrial production.

1.2 Enzyme method

Enzyme extraction can be divided into protease extraction and non-protease extraction. Protease extraction uses the degradation and modification of rice proteins by relevant enzymes to make rice proteins soluble peptides and then extracted. Common proteases include neutral proteases, alkaline proteases, and complex proteases. Among them, alkaline proteases have the best extraction effect [15]. Zhang Juanjuan et al. [16] used papain, alkaline protease, neutral protease, and bromelain to extract proteins from rice residue, and found that the extraction effect of alkaline protease and neutral protease was better than that of papain and bromelain.

According to the different types of protease hydrolysis, proteases are divided into endopeptidases and exopeptidases. Endopeptidases react with the peptide bonds inside proteins to hydrolyze proteins into polypeptides, such as alkaline proteases and neutral proteases. Exopeptidases break down proteins into individual amino acids from either end of the peptide chain, such as flavor proteases and papain. Hamada et al. [17] used alkaline protease to extract rice bran protein, and at a hydrolysis degree of 10%, the rice bran protein extraction rate reached 92%, which is relatively high. The non-protease extraction method refers to the use of amylase, cellulase, etc., to remove the non-protein components from the rice and retain the protein components, so as to extract the rice protein. Shih et al. [18] used α-amylase to enzymolyze rice bran, and the protein recovery rate and protein purity obtained were higher than those of rice bran protein prepared by the protease method. This is similar to the results of Tang et al. [19], who used amylase to hydrolyze starch and improve the extraction rate of rice protein.

1.3 Osborne classification

Osborne classifies proteins according to their solubility into four types: albumins, globulins, alcohol-soluble proteins and glutenins. Albumins are proteins that can be dissolved in water, and globulins are proteins that can be dissolved in dilute salt solutions in addition to water-soluble proteins. Albumins and globulins are the active proteins in rice, but their content is relatively low. In addition to the above two proteins, the protein that can be dissolved in 50% to 90% ethanol is alcohol-soluble protein, and the protein that can only be dissolved in acid or alkali is gluten. Gluten and alcohol-soluble protein are storage proteins and the main components of rice protein, of which gluten accounts for about 80% and alcohol-soluble protein accounts for about 10% [20]. Osborne first used the difference in the solubility of wheat proteins at the beginning of the century to extract wheat proteins continuously, and it was later widely used to extract rice proteins in a graded manner. Wang Yanling et al. [21] used Osborne's graded extraction method to extract four types of proteins from rice bran, obtaining a total protein extraction rate of 96%. The advantage of this method is that the four proteins can be extracted separately to study their properties. The disadvantage is that it requires more equipment and is more complicated to operate.

1.4 Physical assisted method

Physical assisted extraction generally uses physical methods such as ultrasound, freeze-thaw, high pressure and high-speed homogenization to assist the alkaline or enzymatic method to extract proteins from rice. The advantage of the physical assisted extraction method is that it can improve the extraction rate of rice proteins. Ultrasonic-assisted extraction uses the cavitation, mechanical and thermal effects of sound waves to increase the speed and frequency of molecular motion in the extraction agent, so that solvent molecules can more quickly and easily penetrate the extract cells and the target substances can be dissolved more quickly. Cai Sha et al. [22] and Liu Haifei et al. [23] studied the ultrasonic-assisted alkaline method for extracting rice protein. Both found that ultrasonic waves can increase the protein extraction rate. The principle may be that  Ultrasound propagates in liquid, causing the liquid medium to be constantly stretched and compressed, creating a cavitation effect. This cavitation effect can destroy the cell and cell membrane structure of the rice, thereby increasing the ability of the rice protein to penetrate through the cell membrane.

The freeze-thaw principle is that when cells are repeatedly frozen and thawed, ice crystals form in the water, and the salt concentration in the remaining liquid increases, which causes the cells to swell and the cell walls to break, making it easier for the rice protein to dissolve. Cui Suping et al. [24] used the freeze-thaw method to treat rice slurry, and then extracted the protein from the rice using an alkali method. The results showed that the protein extraction rate of rice slurry after freeze-thaw treatment was 2.6% higher than that of the alkali method. Choi et al. [25] used a freeze-thaw assisted protease method to remove protein to obtain rice starch. Compared with the alkali method and the protease method, the removal rates of rice protein were increased by 2.5% and 7.57%, respectively. It is speculated that the freeze-thaw effect can change the internal equilibrium of the rice, partially changing the structure, thereby increasing the protein extraction rate.

In high-pressure assisted extraction, when the pressure reaches a certain level, the complex chemical bonds between the starch and proteins in the rice break[26], and the four-dimensional structure of the protein changes, thereby increasing the extraction rate of rice protein. Xi Haiyan et al.[27] used ultra-high pressure assisted alkaline enzyme method to extract rice protein, and found that when the pressure was 400 MPa, the extraction rate of rice protein was 7.82% higher than that of alkaline enzyme method. Zhao Congcong et al. [28] found that after treating rice particles with high pressure, the protein extraction rate was increased by 24% compared to the alkaline extraction method. High pressure can cause a certain degree of change in the properties of the material, thereby increasing the protein extraction rate.

High-speed homogenization uses mechanical forces such as high-speed shearing, high-frequency oscillation, cavitation, and convective impingement, as well as corresponding thermal effects, which are caused when the material quickly passes through the homogenization chamber. The mechanical and chemical effects thus induced can induce changes in the physical, chemical, and structural properties of the material's macromolecules, ultimately achieving the effect of increasing the protein extraction rate. molecular physical, chemical and structural properties change, and ultimately achieve the effect of improving the protein extraction rate. Shi Xuanming et al. [29] investigated the effect of different extraction methods on the protein extraction rate of rice and found that high-pressure homogenization treatment can improve the protein extraction rate of rice.

2 Structural characteristics of rice protein powder

The endosperm of rice has a compact internal structure, with protein bodies encapsulated and bound to small starch granules. Disulfide bonds and hydrophobic groups are cross-linked and aggregated between molecules [30]. According to the state of protein body existence, it can be divided into PB-I type and PB-Ⅱ type. By observing with an electron microscope, it can be seen that the PB-I type has a compact structure with granular layers, with a diameter of 0.5 to 2 μm; PB-Ⅱ type has a uniform texture without layering and is ellipsoidal in shape, with a diameter of about 4 μm [31]. Alcohol-soluble egg white is mainly PB-I type, while globular egg white and glutine egg white are mainly PB-Ⅱ type.

The albumin is composed of a single peptide chain with a wide range of molecular weights, and the subunits are mainly distributed between 18 and 20 kDa [32]. Due to its high water solubility and high lysine content, it has a higher nutritional value than other proteins, and is therefore particularly valued as a nutrient-rich component of rice protein [33]. globulin polypeptides include four polypeptides: a, b, c and d, with molecular weights of 25.5, 15, 200 and over 200 kDa, respectively [34]. Among them, a-globulin polypeptide is the main polypeptide of rice globulin and is stored in the endosperm of mature rice seeds [35]. The albumins and globulins have a significant effect on the physiological activity of rice and play a key role during the growth period of rice [36].

Alcohol-soluble proteins and glutenins are storage proteins in rice. Alcohol-soluble proteins have a relatively low molecular weight, but there are many different types. They can be divided into 10 kDa alcohol-soluble proteins (RP10), 13 kDa alcohol-soluble proteins (RM1, RM2, RM4 and RM9) and  16 kDa alcohol-soluble protein (RP16) [37]. The 10 kDa alcohol-soluble protein consists of 110 amino acids. The N-terminal of its precursor protein contains a signal peptide sequence composed of 24 amino acids. There are many types of 13 kDa alcohol-soluble protein, and their amino is not the same, and the signal peptide of the precursor protein consists of 18 to 19 amino acids; 16 kDa alcohol-soluble proteins generally consist of 130 to 140 amino acids, and the signal peptide of the precursor protein consists of 18 to 19 amino acids [38]. According to the molecular weight, glutenin is divided into 57 kDa glutenin, 37–39 kDa glutenin and 20–22 kDa glutenin. According to the isoelectric point, 37–39 kDa glutenin is called acidic subunit , and the 20–22 kDa glutenin is called the basic subunit. Glutenin can also be divided into four subunits based on the similarity of the amino acid sequence: GluA, GluB, GluC, and GluD [37]. The amino terminus (N-terminus) of the gluten precursor contains a signal peptide composed of 24 amino acid residues, and the sequence of this signal peptide plays an important role in the synthesis of the gluten precursor [30].

3 Functional properties of rice protein powder

3.1 Water retention

The water retention of a protein is the mass of water remaining in the protein after centrifugation of a protein solution of a certain concentration. Protein conformation, amino acid composition, surface polarity and surface hydrophobicity all affect the water retention of rice protein [39]. The backbone of the protein's peptide chain can retain more water, and the amount of water retained is related to the looseness of the peptide chain backbone. The looser the peptide chain, the more water it retains. In addition, some polar groups in the rice protein can interact with ions in the raw material, making the structure around the peptide chain looser and the water retention effect increase [40]. Zhu et al. [41] used high pressure to treat rice bran protein and found that high pressure treatment significantly enhanced the water retention of rice bran. This is because as the pressure increases, the structure of rice bran protein tends to unfold, exposing more hydrophilic groups and providing more water binding sites. The higher the pressure, the more amino acid groups are exposed, and the better the water retention.

3.2 Foaming properties and foam stability

Foaming properties refer to the interfacial area formed by the protein during foaming, while foam stability refers to the ability to keep the bubbles from bursting. Rice protein has good foaming properties and foaming stability because it contains both hydrophilic and hydrophobic groups. After high-speed homogenization, the spatial structure of rice protein unfolds and the hydrophobicity of the surface increases, which improves the formation of foam [42]. This leads to rapid adsorption of the protein at the air-water interface, forming a layer of cohesive protein [43]. Wan Hongxia et al. [44] found that the foaming properties of rice protein increased significantly after dynamic ultra-high pressure microjet homogenization, which was attributed to the exposure of hydrophobic groups and increased molecular cross-linking, which improved the foaming properties and stability to varying degrees.

3.3 Emulsifying properties and emulsion stability

Emulsifying properties refer to the ability of proteins to rapidly absorb polar and non-polar ingredients at the water/oil interface by preventing binding, while emulsion stability reflects the ability of emulsions to remain emulsified for a measurable period of time. The emulsifying and stabilizing properties of proteins depend on the balance between hydrophilic and lipophilic properties [45].

Wang et al. [46] reported the effect of surface hydrophobicity on the emulsifying ability of rice bran protein and found that the unfolding of the spatial structure of rice bran protein and the exposure of hydrophobic groups can increase the absorption of rice bran protein at the absorption at the oil/water interface increased, emulsifying properties improved, and the unfolding of the rice bran egg structure exposed more hydrophilic and lipophilic groups, which was beneficial for the interaction between the protein and the solvent and prevented the coalescence of oil droplets, and emulsifying stability was also improved.

In fact, there are many factors that restrict the emulsifying stability of proteins, which leads to different trends in the emulsifying capacity and emulsifying stability of proteins. Liu Fang et al. [47] found that using rice protein as a raw material, the emulsifying properties and emulsion stability increased with increasing heat treatment temperature. Zhang Jing et al. [48] found that the emulsifying properties of rice protein increased with increasing pressure, and then decreased, while the emulsion stability decreased continuously.

4 Applications of rice protein powder

4.1 High-protein nutritional supplements

Due to its reasonable amino acid composition, unique hypoallergenicity and high biological potency, rice protein is the first choice of high-quality plant protein for special groups of people when supplementing nutrition. Some plant proteins or animal proteins have anti-nutritional factors, such as trypsin inhibitors, raffinose and hemagglutinin in soybeans, mucin in eggs, and β-lactoglobulin in cow's milk. Compared with the above proteins, rice protein has extremely low allergenicity and can be added to infant foods as a substitute for other plant proteins such as soy protein and animal proteins. It is an important protein source for infant foods [49]. On the other hand, rice protein powder has also been widely studied in animal feed and has been widely used in the aquaculture and animal husbandry industries.

4.2 Active peptides

Rice protein hydrolysate contains a variety of small, physiologically active peptide fragments that have the effect of lowering blood pressure, anti-aging, lowering cholesterol levels, and enhancing the body's immune system [50]. Yang et al. [7] studied the effects of alcohol-soluble proteins on the in vitro antitumor immune response and the growth of leukemia in mice in vivo, and found that alcohol-soluble proteins can effectively promote antitumor immunity and inhibit the growth of leukemia without significant toxicity. Wang et al. [46] studied whether the different components of rice glutenin and alcohol-soluble proteins could exert different in vitro antioxidant activities. They found that after pepsin-trypsin digestion, glutenin showed stronger antioxidant reactions in terms of free radical scavenging activity, metal chelating activity and reducing power, while alcohol-soluble proteins produced weaker antioxidant capacity.

4.3 Edible film

Edible films are a new type of food packaging material that has rapidly developed in response to consumer demand for convenient and environmentally friendly food packaging. They have good gas barrier properties, oil barrier properties and aroma retention properties [51]. The hydrophilicity of rice protein itself means that the water resistance of edible films based on rice protein is relatively poor. Therefore, the high water permeability rate means that rice protein edible films can only be used for foods with relatively low moisture content, such as candy, preserved fruits and nut foods [52]. The research on rice edible films not only conforms to the needs of the international trend of environmental protection, but also a forward-looking exploration of the comprehensive development and utilization of rice protein. The use of rice protein to make green plastics and edible packaging film materials is one of the important aspects of the comprehensive utilization technology of rice.

5 Conclusion

Protein is an important component of all cells and tissues in the human body and is one of the seven essential nutrients. Currently, animal protein is the main source of protein in the diet. With the improvement of living standards and people's constant pursuit of health, protein of plant origin is becoming increasingly important in dietary supplements and food processing.

Rice protein powder, as a high-nutrition, low-allergenic, high-quality plant protein, is very suitable for children, the elderly and the sick. However, due to the low protein content in rice, the development of the functional nutritional properties of rice protein has not been fully appreciated for a long time. At present, most research on rice protein focuses on the extraction process. Although the alkaline method for extracting rice protein is relatively mature, it has not been truly applied in industrial production due to various shortcomings. Although the enzymatic method for extracting rice protein has better performance in all aspects, its solubility is not optimistic. Other auxiliary or high-tech methods are only suitable for laboratory research.

The proteins in rice can be divided into albumins, globulins, alcohol-soluble proteins and glutenins according to their solubility. Different types of protein components have different practical application fields. As the most abundant protein component in rice, glutenin is relatively widely used in the processing of functional foods. However, glutenin has poor solubility, so some research is currently focused on modifying rice protein to improve its solubility, foaming properties, emulsifying properties, oil absorption, etc., and enhance its functional properties. This will enable the production of rice protein powders, rice protein films and other rice protein products with high functionality and good processing indicators.

With the deepening of research and the involvement of high-tech, the extraction rate and functional properties of rice protein will surely be improved. The development and utilization of rice protein will surely achieve the reasonable transformation of low-value resources into high-value resources, thereby obtaining greater economic and social benefits.

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