Progress in the separation and extraction of rice protein

 Abstract: The methods, conditions and factors affecting the separation and extraction of rice protein are described. The methods of rice protein separation and extraction mainly include alkaline method, single enzyme method, multi-enzyme method and stepwise hydrolysis method. The selection of protease, solid-liquid ratio, amount of enzyme, pH value, temperature, time and other conditions of enzymatic hydrolysis are the main factors affecting the hydrolysis of rice protease. Suitable physical effects and the presence of non-proteolytic enzymes can improve the hydrolysis effect.

 

The production of caramel, glucose, starch and dextrin from rice will produce a large amount of rice residue, in which the protein content is as high as 35% ~ 40%, which is 3 ~ 4 times of the raw material rice, and it is an excellent resource for the extraction of rice protein. Rice protein is mainly composed of four kinds of proteins: albumin, globulin, alcohol-soluble proteins and glutenin, etc. The main endosperm proteins in rice residue are albumen proteins, which are composed of albumin (4% ~ 9%), salt-soluble globulin (10% ~ 11%), alcohol-soluble glutenin (3%) and alkali-soluble glutenin (66% -78%) [1]. Among the cereal proteins, rice proteins have a raw material price (B . V . ) and protein price (P . V . (B.V.) and protein price (P.V.) are higher than those of other proteins, with a high amino acid content and a balanced composition. Rice protein is hypoallergenic compared to soybean and milk proteins [2], and is the least allergenic of the known cereal proteins [3].

 

Rice protein is a high-quality cereal protein, although it has not yet been well developed and utilized in our country, but its many excellent characteristics determine its broad application prospects. In addition to direct feed, rice protein can be processed into soy sauce, high protein powder, protein drinks, peptones and protein foaming powder, etc. If it is degraded into short peptides or amino acids, it can be made into amino acid nutrient solution with high nutritional value, which can be used in health drinks, condiments, cosmetics and detergents, etc. [4]. Due to its hypoallergenic nature, rice protein is well suited for the development of foods for infants and young children. In addition, the South Louisiana Research Center in the United States has developed edible films with a certain tensile strength and water vapor resistance from a combination of rice protein concentrate and pullulan polysaccharide, which are used as carriers for flavor substances and nutritional additives, or as separators, protectors, and preservatives [1].

 

Compared with other cereals, it is more difficult to separate rice proteins due to the small starch granules and almost all of them exist in the state of compound grain, the protein and starch granules are tightly bonded with the envelope, and the internal structure of rice endosperm is tightly packed. Rice gluten, which accounts for more than 80% of rice protein, is a very large molecular weight, and can only be dissolved in solutions with a PH value of less than 3 or more than 10. At present, the separation and extraction of rice proteins are mainly by alkaline and enzymatic methods.

 

1 Alkaline extraction of rice protein

The research on rice protein extraction by alkali method started earlier. More than 80% of rice proteins are alkali-soluble rice gluten, and dilute alkali can make the compact amylopectin structure of rice become loose, and alkali has a degrading effect on the large molecules of rice gluten, so that the proteins in the rice starch particles are dissolved and separated [5]. The effect of dilute alkali on the mixed system of proteins and starch is complex, and the effects of pH, temperature and time on the properties of proteins and starch can cause changes in the properties of the extraction system and the extract, which in turn cause changes in the extraction rate of proteins [5]. Experiments showed that the extraction rate of proteins with 0.1 mol/L NaoH solution at room temperature was decreased by 0.1 mol/L NaoH solution. It was shown that the protein was extracted with 0.1 mol/L NaoH at room temperature for 1.5 h at a liquid-solid ratio of 10:1. The extraction rate of protein could reach 98% after 1.5 h of extraction with 0.1 mol/L NaoH at 10:1 liquid-solid ratio at room temperature [6]. Currently, alkaline extraction is the most common method for plant protein extraction.

 

Alkaline extraction is a simple process, but it has a destructive effect on amino acids, and has the disadvantages of high starch content in the extract, large extraction liquid-solid ratio, large amount of acid consumption for isoelectric point precipitation, difficulty in desalination and purification, and low concentration of proteins in the extract, etc., and it requires a large amount of alkali and water, which makes it difficult to be applied in industrial production [7]. Although the extraction rate of protein extracted by alkali method can reach more than 90%, the high alkali conditions will lead to protein denaturation and hydrolysis; intensification of the Maillard reaction, the production of black-brown substances; increase in the content of non-protein substances in the extract, and reduce the separation effect of a number of adverse effects. In addition, alkali extraction can cause some changes in the properties of proteins, destroy the structure of amino acids, reduce the nutritional value of proteins, and even form toxic substances such as LysinoalninE, which can damage the function of the kidney [8].

 

2 Enzymatic extraction of rice protein

Enzymatic extraction utilizes proteolytic enzymes to degrade and modify rice proteins, turning them into soluble peptides and extracting them. Enzymatic extraction reaction conditions are mild, protein polypeptide chains can be hydrolyzed to short peptide chains, improving the solubility of proteins, while the reaction of the liquid-solid ratio is small, increasing the solids content of the extracted liquid, reducing the energy consumption used to remove the water of the extracted liquid ［7］, creating favorable conditions for industrial production.

 

During enzymatic extraction, partial enzymatic digestion of rice protein not only improves the nutritional value and facilitates digestion and absorption, but also the degradation of specific peptide chains of rice protein may produce biologically active peptides with a variety of activities, for example, the GYPMYPLR peptide sequence of rice albumin has immunological activity, and the YPMYPLPR peptide sequence of tryptic digest of rice protein has opioid-like activity, etc. [9]. YPMYPLPR peptide sequence in the tryptic digest of rice protein has opioid-like activity [9].

 

Studies on the functional properties of proteins revealed that enzymatic rice protein isolates had lower surface hydrophobicity values, emulsifying activity and stability than alkaline rice protein isolates, but better solubility [10]. The solubility of the former was 40%, 6%, 13%, 17% and 84% at PH 3, PH 5, PH 7, PH 9 and PH 11, respectively, while the solubility of the latter was 28%, 6%, 10%, 13% and 56%, respectively. Enzymatic hydrolysis of proteins increases their hydrophobicity, which can effectively improve their solubility, emulsification and foaming properties [7].

 

2 . 1 Selection of proteases

According to their mode of action, proteases are classified into endoproteases and exoproteases. Exonucleases cut a single amino acid residue from either end of the peptide chain; commercial proteases are mainly endonucleases, which break the peptide bond inside the polypeptide chain and produce a series of peptides with different molecular weights depending on the degree of hydrolysis [11]. Differences in the mode of action of endoproteases and exoproteases on the substrate affect the extraction of proteins. Proteases can be categorized as acidic, neutral, or alkaline proteases according to their conditions of action. In order to improve the extraction efficiency and nitrogen yield of enzymatic extraction, Wang Wenggao studied three kinds of proteases, namely, complex flavor enzyme, neutral protease and alkaline protease, and the results showed that the type of enzyme had the greatest influence on the protein extraction rate [7].

 

Currently, there are many studies on the hydrolysis of proteins by individual different proteases as well as the hydrolysis of proteins by multiple enzyme combinations, and some of the results are shown in Table 1. The selection of protease, solid-liquid ratio, amount of enzyme added, pH value, temperature and other conditions of enzymatic hydrolysis are the main factors affecting the enzymatic hydrolysis of rice proteins. As shown in Table 1, acid protease, complex protease, papain, neutral protease and alkaline protease can be used to extract rice protein, among which the nitrogen yield of multi-enzyme complex enzyme hydrolysis is the highest, which is far beyond the reach of other single enzyme hydrolysis except alkaline protease.

 

2 . 2 Stepwise hydrolysis

Using alkaline enzyme two-step method to extract rice protein step by step can get better results. In this method, part of the protein is first extracted by alkaline solubilization, and then the residue is slightly hydrolyzed by alkaline protease to improve the solubility of the protein and carry out the second extraction of the protein, so that the extraction rate of protein reaches 78.8%. The protein extraction rate reached 78.8%. The conditions of alkaline extraction were as follows: PH value 12, time 2 h, temperature 40 ℃, material-liquid ratio 1 : 12, and the extraction rate of protein could reach 49.9%. The residue after alkaline extraction still contained 25% ~ 35% of protein, and then hydrolyzed by alkaline protease for secondary extraction, the secondary extraction rate of up to 28.9%. 9%. Enzymatic conditions were 40 ℃, PH value 10.5, enzyme dosage 140 U/kg. 5, enzyme dosage 140 U/ml, time 2 h [18].

 

In order to overcome the shortcomings of single enzyme hydrolysis and the limitations of multi-enzyme hydrolysis, Xiong Shanbai et al. firstly proposed a step-by-step enzyme hydrolysis process in the study of enzymatic hydrolysis of otter chickens [19], and then the step-by-step enzyme hydrolysis process was successfully applied to the extraction of rapeseed cake meal proteins. In the study of protein enzymatic hydrolysis of defatted rapeseed cake meal, it was found that under the optimal operating temperature and starting PH value of alkaline protease and papain, the hydrolysis degree and nitrogen yield of hydrolyzed products obtained by the stepwise hydrolysis method of hydrolysis with alkaline protease and then papain were very high, and the hydrolyzed products had a better solubility within the range of PH 3 ~ 9, with the nitrogen solubility index of 76.92% or more. The solubility of the hydrolyzed product was good in the PH range of 3 ~ 9, and the nitrogen solubility index was above 76.92%. 26 g/100 ml) [20]. The extraction of rice protein by stepwise enzymatic hydrolysis needs to be further investigated.

Table 1 Selection of proteases in enzymatic extraction of rice proteins, etc. [14 ~ 18]

 

Protease or protease combination	substrate			Optimum conditions for enzymatic action					Enzymatic effect * * * %
		Temperature ℃	PH value	Amount of enzyme substrate added g-(100 g)- 1			Time h	liquid-solid ratio
537 Acid protease	rice dregs	50	4	10			12	16 . 5 : 1	Extraction rate 71
complex protease	rice dregs	50	6	0 . 6			2	10 : 1	0 . 39
papain	rice dregs	70	6	0 . 3			3	10 : 1	0 . 41
papain	rice dregs	50	7	0 . 15			3	—	0 . 70
Bacillus subtilis protease	Soybean isolate	45	7	2 000 U/g			6	Concentration of substrate 3%	Degree of hydrolysis 60 or more
neutral protease	dregs of beer	50	7	0 . 3 ml/100 g			4	10 : 1	Degree of hydrolysis 68 . 6
neutral protease	rice dregs	45	7 . 6	2 . 0 ml/100 g			4	10 : 1	0 . 56
alkaline protease	rice dregs	45	8 . 2	0 . 6			3	10 : 1	1 . 53
Papain * Alkaline protease	rice dregs	50	7 . 6	0 . 6 0 . 6			3	10 : 1	1 . 43
Neutral protease * Alkaline protease	rice dregs	45	7 . 6	2 . 0 ml/100 g 0 . 6			3	10 : 1	1 . 87
Alcalase * * Papain	Gallus gallus domesticus Brisson	50 55	7 . 5 7	1 000 U/ 4 000 U/	g g	Protein. Protein.	1 4	Protein content 8%	Degree of hydrolysis 31 . 99 Nitrogen yield 77 . 15
Alkaline protease * * Papain	Rapeseed Biscuit meal	40 50	10 5~6	3 000 U/g protein 3 000 U//g protein			4 4	Substrate concentration 8%	Degree of hydrolysis 30 . 95 Nitrogen yield 90 . 12

 

Note: * Multi-enzymatic complex digestion,* * Multi-enzymatic stepwise digestion,* * * Not indicated are dry basis ammoniacal nitrogen amounts.

 

2 . 3 Effect of physical treatments on enzymatic extraction of rice proteins

S. Tang et al. from the Department of Food Science, University of Arkansas, USA. Tang et al. [21] showed that physical treatments can provide a suitable microenvironment for solubilization and extraction of proteins and increase the rate of protein extraction. This study evaluated the effect of sonic treatment, freeze-thaw, high pressure and high rate homogenization with and without enzymes on the extraction of proteins from rice bran. Sonic treatment, freeze-thaw, high pressure and high speed homogenization extracted 15%, 14%, 11% and 16% of the protein from rice bran, respectively. At 0%, 20%, 40%, 80% and 100% output power (750 W), 25.6% to 33.6% of the protein was extracted with the addition of saccharase only. 6% to 33.9% of protein was extracted with the addition of saccharase only. 9% of protein was extracted with the addition of amylase and protease, while 54.0% to 57.0% of protein was extracted with the addition of amylase and protease. 0% ~ 57 . 8%.

 

High pressure treatments at 0, 200, 500 and 800 Mpa extracted 10 . 5 %, 11 . 1% of protein, and 61 .8%-66 .5% with the addition of amylase and protease. 8%-66 . 6% of protein could be extracted with the addition of amylase and protease. Under the high speed homogenization treatment, 5% more protein was extracted than the control without this treatment (9 . 9%). 9%). These results indicate that the combination of physical and enzymatic treatments is favorable for the extraction of rice proteins.

 

2 . 4 Role of non-proteolytic enzymes in rice protein extraction

Tang S, Hettiarachchy N S et al. studied the role of amylase, cellulase and pentosan complex enzymes in protein extraction and found that heat treatment leads to protein denaturation and protein-starch interactions, that amylase hydrolyzes starch and releases bound proteins, and that α-amylase is more potent than cellulase, releasing most of the water-soluble proteins [22]. α-amylase is more efficient than cellulase, and α-amylase releases most of the soluble proteins [22]. In addition, combined enzymatic treatment with saccharase and protease can increase protein extraction up to 80% [23].

 

In summary, alkaline, single-enzyme, multi-enzyme and stepwise hydrolysis methods can be used for the separation and extraction of rice protein. The amount of enzyme added, pH value, temperature and time are the main factors affecting protein extraction. Suitable physical treatments and the presence of non-proteolytic enzymes can improve the extraction of proteins.

 

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