Production of Rice Protein by Alkaline Extraction Improves Its Digestibility

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1 J Nutr Sci Vitaminol, 52, , 2006 Production of Rice Protein by Alkaline Extraction Improves Its Digestibility Takehisa KUMAGAI 1, Hiroyuki KAWAMURA 1, Takao FUSE 1, Toshiyuki WATANABE 1, Yuhi SAITO 2, Takehiro MASUMURA 2, Reiko WATANABE 3 and Motoni KADOWAKI 4 1 Kameda Seika Co., Ltd., Niigata , Japan 2 Laboratory of Genetic Engineering, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto , Japan 3 Course of Food and Nutrition, Department of Human Life Environments, Niigata Women s College, Niigata , Japan 4 Laboratory of Nutritional Regulation, Faculty of Agriculture, Niigata University, Niigata , Japan (Received May 2, 2006) Summary Rice seed endosperm has two types of protein bodies (PB). Type I protein body (PB-I) accumulates prolamin and is hard to digest, while type II protein body (PB-II) mainly consists of glutelin, an easily digestible protein. A simple method to process rice protein and improve its digestibility was tested from the viewpoint of its application to food manufacturing. Rice protein prepared by alkaline extraction followed by neutralization sedimentation (AE-RP) was compared with that prepared by starch degradation by -amylase (SD-RP). The crude protein content of AE-RP and SD-RP was 84.7% and 78.2%, respectively. There were no major differences in protein composition among AE-RP, SD-RP and rice flour by SDS-PAGE, except 16 kda polypeptide. With respect to amino acids, all the groups showed quite similar compositions, although cysteine and methionine were lower in AE-RP. In an in vitro digestion study with pepsin and pancreatin, both the SDS-PAGE analysis of protein pattern and the crude protein content of undigested residue clearly demonstrated that AE-RP has a higher digestibility than SD-RP. To find the cause of the difference in digestibility, the structural property of protein bodies by two production methods was compared using electron microscopy. PB-II of AE-RP was transformed into small, amorphous granules, while that of SD-RP was still kept partial protein body structures. PB-I of AE-RP kept its protein body structure, but produced double layers. From the finding that glutelin-gold was detected by immunochemistry not only in small, amorphous granules but also in PB-I, mainly the cortex layer, in AE-RP, it became clear that PB-I was swollen and fragile as a result of alkali treatment. These results strongly indicate that the improvement in digestibility of AE-RP is a result of the structural change of PB-I and -II caused by alkaline extraction. Key Words rice protein, alkaline extraction, starch degradation, in vitro digestion, electron microscopy Rice seed endosperm has two types of protein bodies and these protein bodies accumulate as storage protein to provide a nitrogen source for germination. Prolamin is stored in the type I protein body (PB-I), and glutelin and globulin are stored in the type II protein body (PB- II) (1). Prolamin is soluble in alcohol, alkaline and/or acidic solution (2, 3), and separates into three polypeptide bands (molecular sizes, 10, 13 and 16 kda) with SDS-PAGE (2, 4 6). Glutelin can be dissolved in alkaline and/or acidic solution, and consists of acidic subunits (37 39 kda) and basic subunits (22 23 kda) that occur in the processing of proglutelin (57 kda) in protein storage vacuoles (7). Protein particles were found in the feces of humans who consumed cooked rice (8, 9). The amount of fecal protein particles decreased when they changed to t_kumagai@kameda.co.jp steamed sweet potato, then it increased again when they returned to the rice diet. The fecal protein particles were thought to be the same as PB-I because of the following evidence: the electron micrograph of the fecal protein particles resembled that of PB-I, and the fecal protein particles and PB-I showed an equal digestibility for pepsin in an in vitro study (10). These studies suggested that PB-I including prolamin shows poor digestibility in the human digestive tract. The true protein digestibility of cooked rice was 85 90% in humans (11), suggesting that the true digestibility of rice protein may be due to the low digestibility of PB-I. For food manufacturing purposes, rice protein had been isolated by heat-resistant -amylase (12), but it exhibited a lower digestibility than casein. On the other hand, in the general manufacturing method for rice starch, polished rice is dissolved in alkaline solution and the fraction of starch is prepared by centrifuging. The supernatant contains a large quantity of protein and 467

2 468 KUMAGAI T et al. therefore the rice protein, except for soluble protein, can be easily collected by neutralization. As a result of alkaline extraction, PB may suffer from structural change and show different properties from native rice protein. We examined the improved digestibility of rice protein from the viewpoint of its application to food manufacturing. Rice protein was prepared by the alkaline extraction/neutralization method and by the heat-resistant -amylase method through amylolysis. The two types of proteins obtained were compared using the following analyses: amino acid analysis, SDS-PAGE, in vitro digestion study, and electron microscopic observation. MATERIALS AND METHODS Preparation of rice protein. Rice flour (10% polished fraction) of Oryza sativa L. cv. Koshihikari was purchased from Niigata Seihun (Niigata, Japan). Rice protein was prepared according to the following methods. Preparation of rice protein by alkaline extraction (AE- RP): rice flour (1 kg) was mixed with 0.2% NaOH solution (5 L, approximately ph 13) and the mixture was stirred at room temperature for 1 h and then left overnight. The mixture was then centrifuged at 3,000 g for 10 min and the supernatant was collected. The precipitate was extracted again and the second supernatant was combined with the first one. The ph of the combined extracts was adjusted to 6.0 with 1 N HCl and the rice crude protein was precipitated. The precipitate was collected by centrifugation at 3,000 g for 10 min and was washed 3 times with distilled water. Finally, the precipitate was freeze-dried. Preparation of rice protein by starch degradation (SD-RP): rice flour (1 kg) was mixed with a heat-resistant -amylase solution (3 g in 5 L, amylase AD, Amano, Japan), and the mixture was stirred at 80 C for 30 min and heated to 90 C and kept for 10 min. After lowering the temperature below 50 C, the mixture was centrifuged at 3,000 g for 10 min to collect the precipitate. The precipitate was treated with the heat-resistant -amylase again. The precipitate of rice crude protein was washed and dried in the same manner as AE- RP. Chemical analyses. The crude protein content of both preparations was determined according to the Kjeldahl method using an N-to-protein factor of 5.95 (13). Amino acid composition was determined using an amino acid analyzer (Hitachi L-8800, Japan) according to the method of Chang et al. (14). SDS-PAGE was carried out according to Laemmli (15) on a 15% acrylamide gel. SDS-urea solution for protein extraction contained 8 M urea, 2% SDS, 5% 2-mercaptoethanol, and 10% sucrose in 50 mm Tris-HCl buffer (ph 6.8) (16). The samples (40 mg as protein) of AE-RP, SD-RP and rice flour were immersed in 1 ml of SDS-urea solution and heated at 100 C for 10 min. After centrifugation at 3,000 g for 10 min, 2 L of supernatant was subjected to SDS-PAGE. The gel was stained with Coomasie Brilliant Blue R 250 (Merck, USA). The densities of proteins were measured with the NIH image software. In vitro digestion study. The in vitro digestion study was based on the Akeson and Stahmann method (17). The samples (1 g) of AE-RP, SD-RP and casein (Merck) were placed in a flask with a cap. Artificial gastric juice (50 ml) was added to the samples before incubation in a water bath at 37 C for 2 h with shaking. The artificial gastric juice consisted of pepsin (10 mg, from stomach mucosa, Sigma, USA) in distilled water (50 ml) at ph 2 lowered by HCl. Then, the digested samples were neutralized with 6 N NaOH, supplemented with artificial intestinal juice (50 ml), and incubated at 37 C for 24 h. The artificial intestinal juice consisted of pancreatin (100 mg, from porcine pancreas, Sigma) in 50 mm Tris-HCl buffer (50 ml, ph 8.8). The digested samples of AE-RP and SD-RP were heated at 100 C for 10 min and centrifuged at 3,000 g for 10 min to obtain the precipitates. After lyophilization, the precipitates, i.e., undigested residues (6.2 mg as N) were immersed in 1 ml of SDS-urea solution and analyzed using SDS- PAGE in a similar manner as above. The dry weight and protein content of the precipitates were measured. The digested samples of casein were analyzed using SDS- PAGE after heating and lyophilization. Because casein was more densely stained than rice protein, the casein samples were 5-fold diluted with SDS-urea solution and subjected to analysis. Electron microscopy (18). Rice flour, AE-RP and SD- RP were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (ph 7.2) and placed in the fixative for 1 h at room temperature. After washing with the same buffer, the samples were supplemented with 1% agar solution. The agar blocks were dehydrated in graded ethanol series and embedded in LR White resin (London Resin Co. Ltd., Hampshire, UK). The blocks were then polymerized at 55 C for 48 h. Ultra thin sections were cut with a diamond knife using a Leica Ultracut UCT (Leica, Heidelberg, Germany) and mounted on nickel grids. The sections were stained with 2% uranyl acetate. After staining, the sections were examined under a transmission electron microscope (JCM-1220, JEM, Tokyo, Japan) at 100 kv. Immunoelectron microscopy: the sections were soaked in blocking solution consisting of 1% bovine serum albumin in 0.1 M phosphate buffer (ph 7.2) for 30 min at room temperature, and were incubated with antibodies raised against 10 kda prolamin (diluted with blocking solution 1 : 1,000), 13 kda prolamin (diluted 1 : 10,000) and glutelin (diluted 1 : 10,000) overnight at 4 C (antibody raised against 10 kda prolamin was provided by Dr. M. Kuroda, Hokuriku Research Center). The sections were washed with 0.1 M phosphate buffer (ph 7.2), and incubated with 10 nm gold-labeling goat anti-rabbit antibody (diluted 1 : 50, Amersham Biosciences, Buckinghamshire, UK) in blocking solution for 1 h at room temperature. Staining and electron microscope observation were performed as described above. Statistical analyses. Statistical analyses were performed with Statcel for Windows XP. The data were expressed as means SE and evaluated using one-way

3 Rice Protein Produced by Alkaline Extraction 469 Table 1. Percentage of major polypeptides of rice flour, AE-RP and SD-RP. Polypeptides Rice flour AE-RP SD-RP % 57 kda proglutelin kda glutelin kda globulin kda glutelin kda prolamin a b b 13 kda prolamin kda prolamin Total Fig. 1. SDS-PAGE pattern of rice flour, AE-RP and SD- RP. Lane 1, rice flour; lane 2, AE-RP, rice protein by alkaline extraction; lane 3, SD-RP, rice protein by starch degradation; M, marker (kda). analysis of variance followed by Fisher s PLSD post-hoc analysis, and using Student s t-test. A level of p 0.05 was used as a criterion for statistical significance. RESULTS AND DISCUSSION Characterization of rice protein The yield of AE-RP and SD-RP from rice flour (1 kg) was 38.0 g and 59.8 g, and the crude protein (CP) content was 84.7% and 78.2%, respectively. Since the protein content of rice flour was 52.0 g/kg, the recovery of protein was calculated to be 61.9% and 89.9%, respectively. The recovery of SD-RP was higher than that of AE-RP. The SDS-PAGE pattern of rice flour, AE-RP and SD-RP is shown in Fig. 1. From all samples, major polypeptides of 13 kda prolamin, 26 kda globulin, kda and kda glutelin, and small quantities of 10 kda and 16 kda prolamin and 57 kda proglutelin were observed. Six polypeptides other than 57 kda proglutelin were identified with specific antibodies by Western blotting method (data not shown). Densitometric analysis of rice flour, AE-RP and SD-RP for Fig. 1 is shown in Table 1. No quantitative difference in polypeptide bands was seen among the groups except 16 kda prolamin. The percentage of 16 kda prolamin in rice flour was significantly higher than that of AE-RP and SD-RP. Since it is reported that a 16 kda polypeptide extracted by dilute Tris-HCl buffer (ph 7.5) from rice grain is albumin, not prolamin (6), albumin may have been removed by washing during preparation of AE-RP and SD-RP. Therefore, a 16 kda polypeptide in rice flour may include albumin together with prolamin. Morita et al. (12, 19) reported preparations of rice protein according to similar methods, i.e., alkaline extraction and starch degradation. In their study, rice protein was washed with ethanol in order to dry. This is a different step from our method. The prolamin content of alkaline-extracted rice protein in their study was considerably lower than that of the rice flour. Prolamin is dissolved in alcohol (2, 3). We were able to prepare AE-RP and SD-RP, which consist of almost the same protein The bands of Fig. 1 were quantitated densitometrically and shown as percentage of sum of 7 major polypeptides. Values represent means SE (n 6). The data were evaluated using one-way analysis of variance followed by Fisher s PLSD post-hoc analysis. a, b Significantly different in each polypeptide (p 0.05). Table 2. Amino acid composition of rice flour, AE-RP and SD-RP. Rice flour AE-RP SD-RP g/mg protein Asp Thr Ser Glu Gly Ala Val Cys a b a Met a b a Ile Leu b a b Tyr Phe b a b Lys His Arg Pro Trp Total Values represent means SE (n 3). The data were evaluated using one-way analysis of variance followed by Fisher s PLSD post-hoc analysis. a, b Significantly different in each amino acid (p 0.05). composition as rice flour, by taking account of the solubility of prolamin and glutelin. The amino acid composition of rice flour, AE-RP and SD-RP is shown in Table 2. The composition was nearly the same among the samples except for sulfur-containing amino acids, leucine and phenylalanine. The relative contents of cysteine and methionine in AE-RP were significantly lower than that of SD-RP and rice flour,

4 470 KUMAGAI T et al. Fig. 2. SDS-PAGE of in vitro digestion study. Lane 1 5, AE-RP; lane 6 10, SD-RP; lane 11-13, casein; lane 1, 6 and 11, non-digestion; lane 2, 7 and 12, after pepsin for 2 h; lane3, 8 and 13, after pepsin for 2 h and pancreatin for 2 h; lane 4 and 9, after pepsin for 2 h and pancreatin for 2 h; lane 5 and 10, after pepsin for 2 h and pancreatin for 4 h; M, marker (kda). Fig. 3. Change in total weight and protein content of undigested residue by in vitro digestion study. 1, nondigestion; 2, after pepsin for 2 h; 3, after pepsin for 2 h and pancreatin for 1 h; 4, after pepsin for 2 h and pancreatin for 2 h; 5, after pepsin for 2 h and pancreatin for 4 h; 6, after pepsin for 2 h and pancreatin for 24 h. Values represent means SE (n 3). The data were evaluated using Student s t-test. Significantly different from # AE-RP (* Total weight, p 0.05; Protein content, p 0.05). while leucine and phenylalanine were higher in AE-RP. During preparation of rice protein, a sulfur smell was detected at the steps from neutralization to drying only from AE-RP. It was reported that mild alkali treatment of a sulfur-rich protein solution resulted in degradation of cystine, and H 2S occurred when the solution was acidified (20, 21). Thus, the sulfur compounds appeared to have been eliminated from rice protein in the alkaline solution. In vitro digestion study Since the qualities of rice protein are not so different between AE-RP and SD-RP from the above criteria, the digestibility was compared by an in vitro digestion study. The SDS-PAGE of the in vitro digestion study is shown in Fig. 2. Digestion was started by pepsin treatment for 2 h, but most of the protein bands still remained when compared with non-digested sample. In AE-RP, the protein bands disappeared after further digestion with pancreatin for 1 h except for prolamin. The digestion of SD-RP was clearly slower than that of AE-RP. The digestion of glutelin was much faster than that of prolamin. In casein digestion, which was observed as a control, protein bands completely disappeared with pepsin after 2 h and pancreatin after 1 h. The change in the weight and protein content of the undigested residue is shown in Fig. 3. Both the weight and protein content of the undigested residue in AE-RP and SD-RP decreased over time, and more protein remained in the undigested residue of SD-RP than that of AE-RP. Other components of AE-RP decreased over time, but those of SD-RP did not change in digestion longer than 1 h with pancreatin. As for casein, trace amounts were detected by pepsin for 2 h and pancreatin for 1 h, and no bands were detected at all after further digestion (data not shown). Electron microscopy The rice protein obtained by the alkaline extraction method was improved its digestibility compared with that obtained by the starch degradation method. Since there were no significant differences in their polypeptide pattern or amino acid composition, the reason may be due to the alteration in their higher structures. The protein bodies of both preparations were morphologically investigated. The electron micrographs of rice flour, AE- RP and SD-RP are shown in Fig. 4. Rice flour showed two typical types of spherical protein bodies, PB-I and PB-II, among the starch granules (Fig. 4A). Electron lucent particles are PB-I and electron dense particles are PB-II (1). In AE-RP, spherical particles with the same electron density as PB-I were observed, and the larger particles showed a double layer structure (PB-I like structure) (Fig. 4B). However, typical PB-II like particles disappeared completely; instead, many small and amorphous granular structures of high electron density

5 Rice Protein Produced by Alkaline Extraction 471 Fig. 4. Electron micrographs of rice flour, AE-RP and SD-RP. A, rice flour; B, AE-RP; C, SD-RP; PB-I, Type I protein body; PB-II, Type II protein body; SG, starch grain; CW, cell wall. White arrows indicate PB-I like structure. Fig. 5. Immunoelectron micrographs of AE-RP and SD-RP. A, AE-RP reacted with 10 kda prolamin antibody; B, AE-RP reacted with 13 kda prolamin antibody; C, SD-RP reacted with glutelin antibody; D, AE-RP reacted with glutelin antibody. Small black particles indicate immunogold particles reacted with specific antibodies. were distributed all over the field. This disaggregation of PB-II into small amorphous granules may be a major cause of improvement in the digestibility of AE-RP. On the other hand, in SD-RP, although all of starch granules disappeared, typical PB-I, distorted PB-II and filmy materials were observed (Fig. 4C). Compared to AE-RP, both PB-I and PB-II were less subjected to denaturation. The characteristics of both preparations of rice protein were further examined using immunoelectron microscopic analysis (Fig. 5). The PB-I like structure was demonstrated to be prolamin particles using 10 kda prolamin antibody (Fig. 5A). Immuno-gold particles were distributed only in the PB-I structure. The same distribution was also confirmed with 13 kda prolamin antibodies (Fig. 5B). On the other hand, in SD-RP, immuno-gold particles with glutelin antibody were distributed only in high electron dense particles, which are PB-II (Fig. 5C). Interestingly, when glutelin antibody was applied to AE-RP, gold particles with glutelin antibody were distributed not only in small and amorphous granular structures which were regarded to be derived from PB-II, but also in PB-I like structures, especially their outer layers (Fig. 5D). In rice flour (data not shown) and SD-RP, gold particles of glutelin were only seen in PB-II but never detected in PB-I, prolamin, particles. Therefore, it seemed that the intermolecular force of prolamin aggregates in PB-I structure was weakened and swollen by alkaline treatment, and then glutelin was inserted into the surface of PB-I. The alteration in the structure described above must have caused the improvement of the digestibility of AE-RP. As suggested by Morita et al. that the rice protein from alkaline extraction had its digestibility improved over that from amylase digestion (19), our results confirmed that the in vitro digestibility of AE-RP is superior to that of SD-RP. The improved digestibility seems to be the result of an alteration in the PB structure, mainly disaggregation of PB-II and possibly loosening of PB-I, by alkaline extraction. Our preliminary results for a rat growth study support our in vitro digestion study (Kumagai et al., manuscript in preparation). Therefore, it is concluded that the rice protein produced by alkaline extraction is superior in its digestibility to rice flour and rice protein from starch degradation. It would be favorable to raise the nutritive value by adding the rice protein to high starch foods, especially for the elderly.

6 472 KUMAGAI T et al. Several foodstuffs containing AE-RP, such as bread, rice crackers, and cookies, have already been made as trial products. Rice protein is easily extracted by alkali treatment industrially and is made from a by-product of starch production. Such rice protein can be available as a high quality plant protein source for food manufacturing. Acknowledgments The authors thank Dr. M. Kuroda of Hokuriku Research Center for providing the antibody raised against 10 kda prolamin. This work was supported in part by the Technical Development Program for building agribusiness in the form of utilizing concentrated know-how from the private sector. REFERENCES 1) Tanaka K, Sugimoto T, Ogawa M, Kasai Z Isolation and characterization of two types of protein bodies in the rice endosperm. Agric Biol Chem 44: ) Mitsukawa N, Konishi R, Kidzu K, Ohtsuki K, Masumura T, Tanaka K Amino acid sequencing and cdna cloning of rice seed storage proteins, the 13 kda prolamins, extracted from type protein bodies. Plant Biotechnol 16: ) Sugimoto T, Tanaka K, Kasai Z Improved extraction of rice prolamin. Agric Biol Chem 50: ) Masumura T, Shibata D, Hibino T, Kato T, Kawabe K, Takebe G, Tanaka K, Fujii S cdna cloning of an mrna encoding a sulfur-rich 10 kda prolamin polypeptide in rice seeds. Plant Mol Biol 12: ) Mitsukawa N, Konishi R, Uchiki M, Masumura T, Tanaka K Molecular cloning and characterization of cysteine-rich 16.6 kda prolamin in rice seeds. Biosci Biotechnol Biochem 63: ) Yamagata H, Sugimoto T, Tanaka K, Kasai Z Biosynthesis of storage proteins in developing rice seeds. Plant Physiol 70: ) Yamagata H, Tanaka K The site of synthesis and accumulation of rice storage proteins. Plant Cell Physiol 27: ) Tanaka Y, Hayashida S, Hongo M The relationship of the feces protein particles to protein body. Agric Biol Chem 39: ) Tanaka Y, Resurreccion AP, Juliano BO, Bechtel DB Properties of whole and undigested fraction of protein bodies. Agric Biol Chem 42: ) Ogawa M, Kumamaru T, Satoh H, Iwata N, Omura T, Kasai Z, Tanaka K Purification of protein body-i of rice seed and its polypeptide composition. Plant Cell Physiol 28: ) WHO Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert consultation. Tech. Rep. Ser WHO, Geneva. 12) Morita T, Kiriyama S Mass production method for rice protein isolate and nutritional evaluation. J Food Sci 58: ) Miller L, Houghton JA The micro-kjeldahl determination of the nitrogen content of amino acid and proteins. J Biol Chem 159: ) Chang KC, Lee CC, Brown G Production and nutritional evaluation of high-protein rice flour. J Food Sci 51: ) Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: ) ida S, Amano E, Nishino T A rice (Oryza sativa L.) mutant having a low content of glutelin and high content of prolamin. Theor Appl Genet 87: ) Akeson WR, Stahmann K A pepsin pancreatin digest index of protein quality evaluation. J Nutr 83: ) Takahashi H, Saito H, Kitagawa T, Morita S, Masumura T, Tanaka K A novel vesicle derived directly from endoplasmic reticulum is involved in the transport of vacuolar storage protein in rice endosperm. Plant Cell Physiol 46: ) Morita T, Oh-hashi A, Kasaoka S, Ikai M, Kiriyama S Rice protein isolates produced by two different methods lower serum cholesterol concentration in rats compared with casein. J Sci Food Agric 71: ) Nashef AS, Osuga DT, Lee HS, Ahmed AI, Whitaker JR, Feeney RE Effect of alkali on proteins. Disulfides and their products. J Agric Food Chem 25: ) Florence TM Degradation of protein disulphide bonds in dilute alkali. Biochem J 189: