What Are the Extraction Methods of Brown Rice Protein Powder?

Rice is an important food crop. About half of the world's population relies on rice as their staple food. The main producing and consuming regions of rice in the world are in Asia, which accounts for 91% of the world's total consumption. China's rice production accounts for 37% of the total, ranking first in the world[1]. The deep processing and comprehensive utilization of rice is currently a research hotspot. The by-products of processing – broken rice, rice residue, rice bran, etc. – contain many ingredients of great development value. Rice residue is a by-product of the fermentation industry's production of monosodium glutamate, lactic acid, citric acid, etc., and the production of starch sugar. It contains essential amino acids required by organisms and has great recycling value[2]. Due to the hypoallergenic properties and high nutritional value of rice protein, the food market has a relatively bright future.

At present, there is a lot of research on brown rice protein, and its applications are becoming more and more widespread. For example, it is used to produce brown rice protein powder, hydrolyzed brown rice protein, modified brown rice protein, high value-added peptides, bioactive peptides and resistant proteins[3]. It can also be used as an additive in blended drinks, puddings, ice cream, baby food, etc. Brown rice protein not only has unique nutritional functions, but also has health-promoting functions such as reducing serum cholesterol levels[4]. Although the amount and demand for brown rice protein is increasing, its application in the food industry is very limited, mainly due to its imperfect extraction method, poor water solubility and functional properties.

Brown rice protein is extracted to obtain a high-purity brown rice protein product, which is generally divided into brown rice protein concentrate (RPC, protein content 50%–89%) and brown rice protein isolate (RPI, protein content 90% or more) [5]. Rice flour, rice residue, rice bran and other raw materials can be used to prepare brown rice protein. Researchers have proposed various preparation methods for the development and utilization of rice protein.

1. Solvent extraction method

Proteins exist in the form of protein bodies and are tightly surrounded by starch granules. More than 80% of the proteins in the endosperm are poorly soluble gluten, so it is relatively difficult to extract brown rice protein. At present, the solvents commonly used to extract brown rice protein are: (1) surfactants: SDS, cetyltrimethylammonium bromide; (2) fatty acid salts; (3) weak acids: acetic acid, lactic acid; (4) hydrogen bond disruptors: urea, guanidine hydrochloride; (5) alkali: 0.1 N NaOH, 0.1 N KOH; (6) reducing agents: mercaptoethanol, etc. When rice protein is extracted for food purposes, 0.1 N NaOH is the most widely used.

Wang Zhangcun [6] and others compared the methods of extracting rice protein from rice bran using non-alkaline solvents and alkali methods, respectively. It was found that the protein content of the product extracted with a non-alkaline solvent was equivalent to that extracted with an alkali method, but the protein recovery rate was significantly higher, reaching 95.2%, while the highest recovery rate of the alkali method was only 40%. Clearly, there are certain advantages to extracting rice protein using this non-alkaline solvent, but the extraction solvent is not easy to remove, and there are safety issues with product application, especially in food applications. Moreover, production costs will increase considerably due to the use of the extraction solvent. The main drawback of the alkaline method is that when extracted at high alkali concentrations, it can cause severe denaturation of the protein, as well as an increase in the Maillard reaction, which produces brown substances. This, coupled with the solubilisation of non-protein substances at high alkali concentrations, can affect the quality of the final protein product. At the same time, an alkaline environment can cause condensation reactions between certain amino acids such as lysine and alanine or cystine, producing toxic substances. The nutritional value of lysine is greatly reduced, and the finished product has a dark color, a bitter taste, and is of poor palatability.

2.Protease extraction method

The proteases commonly used to extract brown rice protein include acid proteases, alkaline proteases, neutral proteases, complex enzymes and pepsin. At present, there has been much research on the enzymatic hydrolysis of proteins. The main process is as follows: raw material → soaking → pulping → enzymolysis → enzyme inactivation → centrifugation → hydrolysate drying → protein powder. Ge Na [7] and others extracted rice protein using acid protease and neutral protease, respectively, with extraction rates of 84% and 43%, respectively; Wang Shenglin [8] extracted rice protein using acid protease, with an extraction rate of 75% to 80%; Wang Wengao [9] used alkaline protease and flavor compound enzyme to extract brown rice protein, with an extraction rate of 81.6% and a product purity of 85%; Huang Taihua [10] used pepsin to extract brown rice protein from rice residue, with an extraction rate of 72.4%. In fact, the process of extracting brown rice protein using protease is not mature. Although the use of protease can improve the protein properties, the low yield and high price of protease have limited the development of the process of extracting brown rice protein using protease.

Which protease is more suitable for preparing rice protein? Since different researchers use enzymes from different manufacturers, enzyme activity, composition, raw materials used, extraction processes are not the same, and many other factors, the conclusions differ.

For example, Wang Wengao [11] and others studied the types of enzymes (Novozyme's Flavourzy 500MG composite flavour proteinase from Denmark, with 500 LAPU/g activity units; Neutrase 0.5 L neutral proteinase, with 0.5 AU/g activity units; and Alcalse 2.4 L, 2,4 AU/g), liquid-to-solid ratio, disulfide bond breaker, and degree of hydrolysis. A four-factor, three-level orthogonal experiment was conducted using rice as the raw material to prepare rice protein. The results showed that the type of enzyme had the greatest effect on the protein extraction rate, with the alkaline protease having the highest extraction rate. Ge Na [7] and others compared the acidic protease produced by Wuxi Enzyme Factory (50,000 U/g activity unit), the neutral protease (0.5 AU/g activity unit), alkaline protease (activity unit 2.4 AU/g), and complex protease (activity unit 1.5 AU/g), and found that the extraction rate of brown rice protein by acidic protease was the highest. The extraction rate can reach 91.25%, followed by alkaline protease, and the extraction effect of flavor protease and neutral protease is the worst.

3. Physical separation method

Kang Yanling et al. [12] introduced a method for extracting brown rice protein by crushing the structure and dissolving the protein through colloid milling and homogenization. After brown rice is micronized and homogenized, the protein dissolution concentration is 75% higher than that extracted from a simple aqueous solution. Treating the raw material with physical methods can increase the protein dissolution concentration by 18.7%. Pulping and homogenization can also cause the molecular weights of the dissolved components to vary greatly. Therefore, it is feasible to use physical methods to increase the protein extraction rate.

In 2005, World Agriculture reported that after four years of research, American scientists had invented a new method with a higher price-performance ratio. That is, the high pressure generated by a special homogenizer is used to physically break up the starch and protein aggregates in brown rice into chunks, and the brown rice only needs to pass through this device once to produce watery particles of uniform starch and protein micromolecules, which are then separated from the starch and protein by a conventional density-based separation process. This new process retains the original quality of the extracted rice protein and starch, and the resulting protein and starch have better integrity and functionality than traditional processing methods. American scientists believe that this new method has the potential to revolutionize the rice starch and protein production industry, and they are also researching the use of the same process to extract protein, oil and starch from rice bran.

4 Amylase method

The extraction of brown rice protein using amylase actually involves removing as much of the non-protein components of the rice as possible, and the remaining substance is rice protein. In the extraction of brown rice protein, the brown rice is ground and mixed with water to form a slurry, amylase is added and the mixture is kept warm for a period of time. The supernatant is then separated from the solid matter to obtain a solution that is added to saccharifying enzyme to produce maltose.

The precipitate is high-protein powder, with a protein content of about 50% to 60%. The protein powder obtained using this process contains some starch and sugars that have not been enzymatically hydrolyzed, which means that it does not meet the requirements for concentrated protein and limits its scope of application. In order to increase the protein content, some researchers have added other separation steps while ensuring that the starch in the brown rice is liquefied to the greatest extent. Liu Bin [13] and others used rice residue as a raw material, washed it at high temperature, and then added amylase again to prepare concentrated protein, with a protein purity of over 85%. Frederick·F·S [14] and others carried out alcohol washing on the precipitate separated from the solid-liquid mixture after liquefaction, increasing the protein content to 70%–80%.

5. Compound extraction method

There are many problems with the simple use of lye or protease extraction, such as low extraction rate, insufficient purity, and potential food safety risks. Researchers have begun to consider compound extraction methods to optimize the extraction process. Wang Yalin [15] and others used a two-step alkaline enzyme method to extract rice protein from rice bran in a stepwise manner, with good results. This method first uses an alkali solution to extract some of the protein, and then uses alkaline protease to slightly hydrolyze the residue to improve the solubility of the protein and carry out a secondary protein extraction, achieving a protein extraction rate of 78.8%.

Liu Ji [16] and others also used rice residue as a raw material and similarly used a two-step method of alkali and protease to extract brown rice protein, with a purity and yield of 70.2% and 57.1%, respectively. Chen Ji-wang [17] and others used a two-step method of alkali and protease to extract brown rice protein from broken rice. The process is as follows: broken rice → crushing → dilute alkali extraction → centrifugation → protein solution → amylase hydrolysis → enzyme inactivation → centrifugation → precipitation → freeze-drying → brown rice protein. The brown rice protein extraction rate was 73.22%, and the purity was 88.75%; Chen Ji-wang [18] and others used brown rice protein concentrate using two-stage cyclone separation to remove impurities, which can reduce ash content to 1.52%. Combined hydrolysis with cellulase and lipase can reduce the fat content in the protein to 2.72%. After purification, the protein content of brown rice protein concentrate reaches 70%, with a yield of 73.96%.

6 Conclusion

As people become more aware of the nutritional value and hypoallergenic properties of brown rice protein, and as the international market pays more attention to rice protein, it is certain to attract many researchers to conduct research and development. In recent years, researchers have concluded that the non-alkali extraction method in the solvent method has problems such as high production costs and food safety risks; the alkali method consumes a large amount of lye and has environmental protection problems; the protease method also has problems of low extraction rate and high cost; and physical separation methods do not have mature technical capabilities in China at present. Therefore, in future research work, the extraction method should be determined in a targeted manner according to the raw material, and an optimized combination of various extraction methods should be used to complement each other's advantages.

References:

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