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Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process
Journal of Bioscience and Bioengineering VOL. 119 No. 2, 206e211, 2015 www.elsevier.com/locate/jbiosc
Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process Masanori Watanabe,1, * Isamu Maeda,2 Masahiro Koyama,3 Kozo Nakamura,3 and Kazuo Sasano4 Department of Food, Life and Environmental Science, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan,1 Graduate School of Agricultural Science, Utsunomiya University, 350 Minemachi, Utsunomiya 321-8505, Japan,2 Department of Bioscience and Biotechnology, Graduate School of Agriculture, Shinshu University, Minamiminowamura, Nagano 399-4598, Japan,3 and Shokkyo Co., Ltd., 1-10-28 Fukawa, Asakita-Ku, Hiroshima 739-1751, Japan4 Received 19 April 2014; accepted 23 July 2014 Available online 27 August 2014
We studied a process that enables simultaneous recovery of protein and phosphorus compounds from rice bran. Phosphorus substances in full-fat and defatted rice bran such as phytic acid and inorganic ions were solubilized under acidic conditions in the first step. After that, inorganic and/or organic phosphate salts were recovered in insoluble form under weak alkaline conditions. Furthermore, protein fractions obtained after phosphorus compounds had been removed were solubilized under alkaline conditions. After solubilization, protein fractions with high content were recovered by isoelectric precipitation (IP) followed by electrolyzed water treatment (EWT). The highest protein content (52.3 w/w%) was attained when machine defatted rice bran was treated through the process. Energy-dispersive X-ray spectroscopy (EDX) and inductively coupled plasma atomic emission spectrometry (ICP-AES) analyses demonstrated efficient desalting from the protein fractions by EWT and higher phosphorus contents (15.1e16.4 w/w% P) in the phosphorus fractions compared with commercial phosphate rock. In addition, no heavy metal ions in either protein or phosphorus fractions were detected. These results suggest that the newly developed process is suitable for practical recovery of highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Full-fat rice bran; Machine defatted rice bran; Isoelectric precipitation; Electrolyzed water; Rice protein; Phosphorus compounds]
World rice production in 2008 was approximately 661 million metric tons (1), and the weight ratio of bran to whole rice particles was about 10% (2). Therefore, rice bran has been focused on as a potential resource of nutrients and elements all over the world. Rice bran is known to be a major by-product of rice polishing that contains germ and several histologically identifiable soft layers such as pericarp, seed coat, nucellus, and aleurone layers (3). Rice protein has a great deal of potential to be a functional food when it is used in ingredients and nutritional supplements because its protein efficiency ratio (1.6e1.9) is comparable to that of casein (2.5) (1). Thus, protein production from rice bran has been extensively studied. Physicochemical treatments that include solvent extraction (4,5), compressed hot water extraction (6), and enzymatic treatments using endoprotease, exoprotease, xylanase, phytase, amylase, and carbohydratase (1), are currently being used for effectively extracting protein from rice bran. However, these known methods involve high levels of energy consumption, harmful chemicals, and high labor costs. Therefore, the rice processing industry has to consider commercialization in extracting proteins from this material while keeping low energy consumption and production costs low.
* Corresponding author. Tel./fax: þ81 235 282848. E-mail address: [email protected] (M. Watanabe).
Rice bran also includes a relatively large amount of phosphorus compounds (2). Phytic acid is particularly known to be a major constituent of the organic phosphates in rice bran. It serves as a phosphorus storage compound in plant seeds as well as a natural antioxidant because of its chelating property in iron and zinc as well as its ability to decrease the catalytic activities of many divalent transition metals (7,8). In addition, the expected global peak of phosphorus production is predicted to occur around 2030 (9). It is widely acknowledged within the fertilizer industry that the quality of remaining phosphate rock is decreasing and production costs are increasing. Technologies for recycling phosphorus from waste and extracting it from unused resources have attracted a great deal of attention because of this difficulty with producing phosphorus predicted in the future. Considering the potential of rice bran as protein and phosphorus sources, the development of safe and lowcost methods to recover these nutrients may partly contribute to establishment of sustainable use of renewable resources. However, simultaneous recovery of proteins and phosphorus compounds from rice bran by using solvent-free process has not been demonstrated. We developed a novel process in this study that could produce highly concentrated proteins and phosphorus compounds simultaneously without organic solvents that can be applied to commercial use. We analyzed their concentrations, recovery ratios, and chemical compositions. In addition, a technique of isoelectric precipitation combined with electrolyzed-water treatment (IP-EWT)
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.07.009
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was intensively focused on in terms of protein content and efficiency of desalination and protein purification. MATERIALS AND METHODS Materials Raw (full-fat) rice bran (fat content: 20 w/w%) was prepared from brown rice (Oryza sativa, cv. Koshihikari, harvested in Niigata prefecture, Japan) by using a rice polishing machine (NCP100B; Satake Co., Ltd., Higashi-Hiroshima, Japan) (10). The raw rice bran (supply rate of 120 kg/h) was machine defatted to prepare defatted rice bran (approximately 50% defatted) by using an oil press (V-05W;
207
Suehiro EPM Co. Ltd., Yokkaichi, Japan) after pre-heating the raw rice bran at 110e125 C. Experimental procedures Rice protein and phosphorus concentrates were prepared using acid and alkaline extraction, followed by the isoelectric precipitation (IP) and a washing process with electrolyzed/distilled water, as shown in Fig. 1. Thirty grams of raw rice bran or a defatted rice bran sample and 300 ml of deionized water (less than 2 mS cm1) were placed into a vessel, and pH was adjusted to 3.5 by using a 6 N HCl solution to elute the phosphorus compounds from the rice bran. After that, the vessel was centrifuged at 5000 g for 10 min at ambient temperature to separate eluted phosphorus compounds (supernatant) from rice bran residue (precipitate). The supernatant was adjusted to pH 7.5 with 6 N NaOH to
FIG. 1. Flow diagram for recoveries of phosphorus compound and protein concentrates from rice bran.
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precipitate the phosphorus compounds and centrifuged at 5000 g for 10 min at ambient temperature. The precipitated fraction containing the phosphorus compounds was heat dried at 80 C and stored in a desiccator for later analysis. The supernatant obtained after the phosphorus compounds had been precipitated was mixed with the rice bran residue at a setting of 500 rpm on a high torque stirrer (SWRS077, Nisshin Rika Co., Ltd., Tokyo, Japan) for an arbitrary time. NaOH was added to the mixture to dissolve and restore rice bran protein into the supernatant. The mixture was then centrifuged at 5000 g for 10 min at ambient temperature and the supernatant was collected as protein extracts and used to quantitative analyze the extracted protein. Isoelectric precipitation of protein from the protein extracts was done by adjusting the pH with acetic acid within a pH range of 3e6. Rice crude protein was precipitated by centrifugation at 5000 g for 10 min at ambient temperature, and washed with pH-adjusted electrolyzed water (EW) or distilled deionized water (DW) to remove water-soluble impurities. EW was prepared by using an electrolyzed water ionizer (TK8051, Panasonic Electric Works Co., Ltd., Shiga, Japan). This apparatus generated different pHs for EW by electrolysis with a tap water system that had a volume flow rate 2.5 l min1. Four kinds of EW were used in the experiment: EW with pH 3, oxidative-reduction potential (ORP) þ748 mV; EW with pH 4, ORP þ909 mV; EW with pH 5, ORP þ964 mV; and EW with pH 6, ORP þ1025 mV. Element analysis of protein and phosphorus concentrates The elements in the rice protein and phosphorus concentrates were detected and semi-quantitatively analyzed with a scanning electron microscope (T-3000, Hitachi Co., Ltd, Japan) equipped with an energy dispersive X-ray spectroscopy (EDX) detector (Quantax 70, Bruker AXS Microanalysis GmbH, Germany). A working distance of 25 mm, an accelerating voltage of 20 kV, and a beam current of approximately 1 mA (about 1300 cps) were used. A time of 400 s for each measurement was used for identification. Samples were dried by using the tert-butyl alcohol freeze-drying before the measurements (11). Heavy metal analyses of both concentrates were performed with an inductively coupled plasma atomic emission spectrometer (ICPS-7510, Shimadzu Co., Ltd, Japan). Samples were prepared by using open digestion with nitric acid and hydrochloric acid (12). Quantification of protein and phytic acid The protein content in rice bran and extracted rice protein concentrates was determined by using a total nitrogen analyzer (variMAX CN; Elementar Analysensysteme GmbH, Denmark) in compliance with the improved Dumas method. The percentages of total protein content were read from the output of the instrument by using a conversion factor of 5.95 (13). The protein recovery ratio (%) from both types of rice bran was calculated as: Protein recovery ratio (%) ¼ 100 (Cf A)/(Cb B)
(1)
where A is the value of the dry weight of the recovered protein fraction (see in Fig. 1) derived from 30 g of both type of rice bran, B is the value of the dry weight of total protein of 30 g of both type of rice bran, Cf is the percentages of total protein content (w/w%) of recovered protein as a crude or purified protein fraction and Cb is the percentages of total protein content (w/w%) of rice bran. The phytic acid content in the rice phosphorus concentrates was determined by colorimetry according the method by Haug et al. (14).
RESULTS AND DISCUSSION Effect of NaOH concentration and extraction time on solubilization of protein First, we examined what effects sodium hydroxide concentration had on solubilization of the rice bran protein after the phosphorus compounds had been removed when the extraction time was fixed to 2 h. As can be seen in Fig. 2A, the concentrations of protein extracted from full-fat and defatted rice bran attained nearly a saturated level under sodium hydroxide concentrations of 0.5 w/v% for the former and 1.0 w/v% for the latter. In addition, pH values of both extracts were 12.1 for the former and 12.8 for the latter. Fig. 2B shows the concentrations of extracted protein from full-fat and defatted rice bran with the changes in extraction time under sodium hydroxide concentration of 0.5 and 1.0 w/v%, respectively. A minimum extraction time of 1 h was required for full-fat rice bran and 2 h was required for defatted rice bran. The protein extracted from defatted rice bran in this time of 2 h was about 90% of that extracted in 4 h. Hence, 2 h was selected as a standard time for protein extraction throughout the experiment from the viewpoint of cost benefits such as reducing process energy. Effect of removing phosphorus compounds from rice bran on extracted protein content Rice bran contains inorganic and organic forms of phosphorus compounds (2). Separation of
FIG. 2. Effect of NaOH concentration (A) and mixing time (B) on solubilization of rice bran protein. Mixing time was fixed to 2 h (A). Bars indicate standard deviations (n ¼ 3). Incubation was carried out at 20 C in 500-ml conical flask containing 30 g of rice bran and 300-ml NaOH solution. Symbols: closed circles, machine defatted rice bran; open circles, full-fat rice bran.
such phosphorus compounds from rice bran was considered as they are impurities in recovering high quality protein from it and they are also new phosphorus resources that have remained unused in the past. We examined what effect removing phosphorus compounds from full-fat and machine defatted rice bran would have on the protein content and recovery ratio. Higher protein content was observed in the phosphorus-removed protein fractions (PhosR þ IP) than in the protein fractions without phosphorus removal (IP) within a pH range of 3e5, as is summarized in Table 1. However, phosphorus removal had no effect on increasing the protein content at pH 6. An increasing protein recovery ratio was also observed within a pH range of 3e5 in the PhosR þ IP of defatted rice bran. This suggests that the phosphorus removal process before recovering protein was recovered was effective to decrease the insoluble form of phosphorus compounds in purified protein fractions, as the result, extraction of high quality protein might be attained. Effect of electrolyzed water treatment on extracted protein content and protein recovery ratio EW has been regarded as a new sanitizer in recent years. It possesses antimicrobial properties against a variety of microorganisms (15). EW is generated by the electrolysis of deionized or tap water containing a low concentration of sodium chloride or potassium chloride in an electrolysis chamber where anode and cathode electrodes are separated by an ion-permeable exchange diaphragm. Acidic EW with low pH is produced from the anode and alkali EW with high pH is produced from the cathode (16). The pH of EW can be simply adjusted by using the quantitative ratios of acidic to alkali EW. Therefore, electrolytes in the protein concentrate except for protein can be removed by using EW adjusted to pH values used in the IP process without having to add acids or alkalis while keeping the precipitated protein resuspended. We clarified the ability of isoelectric precipitation and electrolyzed water treatment (IP-EWT) when used for protein recovery and purification after the IP process. The highest protein content (52.3 w/w%) of the protein fraction derived from machine defatted rice bran was attained by using a simultaneous recovery process of protein and phosphorus compounds that included IPEWT at pH 5 (Table 2). In addition, the protein recovery ratios obtained with IP-EWT were efficiently improved within the pH range of 3e5, compared to those with IP-distilled deionized water treatment (IP-DWT) (Table 2). The protein content and recovery ratios exceed those archived with reported physical
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TABLE 1. Effect of removing phosphorus compounds from full-fat and machine defatted rice bran on extracted protein content (w/w%) and protein recovery ratio (%). Material
Procedure
IP pH value 3
FFRB FFRB MDRB MDRB
IP PhosR þ IP IP PhosR þ IP
32.4 40.8 40.9 44.0
1.8 2.3 1.9 2.1
(42.3 (45.9 (38.8 (50.4
4
3.6) 2.9) 2.2) 1.4)
35.6 39.8 36.2 40.7
1.5 1.3 3.5 3.2
(41.4 (43.5 (34.4 (51.7
5
2.8) 3.4) 1.7) 2.1)
38.2 40.0 34.6 35.9
0.8 1.5 2.6 1.8
(40.1 (41.2 (32.8 (45.3
6
2.2) 1.3) 3.1) 0.9)
37.4 37.7 31.7 31.1
3.0 1.7 1.0 0.4
(41.7 (40.9 (25.1 (19.5
4.3) 3.8) 0.8) 0.3)
Content (w/w%) together with recovery ratio (%) in parenthesis are shown. FFRB, full-fat rice bran; MDRB, machine defatted rice bran; IP, isoelectric precipitation; PhosR, phosphorus removal.
Stoichiometry of the phosphorus fractions in Table 5 indicated large differences from the molar ratio of Mg, N, and P in magnesium ammonium phosphate (MAP: MgNH4PO4$6H2O) (17). The formation of MAP also requires higher pH (pH8e9) than the pH values of our phosphorus compound recovery process (pH7.5, see Fig. 1) and hydroxyapatite (HAP: Ca5OH(PO4)3) formation under aquatic conditions (18,19). In addition, phytic acid content in the phosphorus fractions derived from full-fat and defatted rice bran was equal at 46 w/w%. Moreover, the phosphorus fractions indicated relatively high contents of C and O (Table 5), which are assumed to be parts of the carbon cyclic skeleton and phosphate ions of phytic acid. Thus, phosphorus fractions derived from rice bran might be composed of both organic and inorganic forms of phosphorus such as phytic acid and insoluble phosphate salt except for MAP. In relation to that, phytase treatment increases the extracting yield of rice bran protein to about 57% (10). When phytase is used in combination with xylanase, the yield reaches 74.6%. These positive effects of phytase on improving the extracting yield are caused by its catalytic activity to digest phytic acid in an insoluble proteinphytate complex (20). The phosphorus removal process reported here may have almost the same effect on the cleavage of the bonds considered in phytase treatment. Additionally, at pH values above the isoelectric point (approximately 5) of rice bran protein, both proteins and phytic acid have net negative charge, according to de Rham et al. (21). However, phytic acid at this pH can still bind with proteins in the presence of multivalent cations such as calcium through forming a ternary complex of protein-cation-phytic acid. The formation of complex may hinder the effects of DWT and EWT on decreasing the phosphorus weight ratios in protein fraction (Tables 3 and 4). We studied the simultaneous recovery process of protein and phosphorus compounds based on combinations of the recovery (removal) of phosphorus compounds and the IP-EWT method for protein recovery and purification. The highest protein content (52.3 w/w%) was attained in the protein fraction derived from machine defatted rice bran by using this process. In addition, higher values for phosphorus content (15.1 and 16.4 w/w% P) were also obtained in the phosphorus fractions compared with that of commercial phosphate rock. These results suggested that this process is suitable in practice for recovering highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants.
treatment processes, which have included colloid milling (recovery yield: 15% and purity: 7.5%), colloid milling combined with homogenization (recovery yield: 16% and purity: 3.2%), freezethawing (recovery yield: 12%), high pressure (recovery yield: 11%), and sonication (recovery yield: 11%), and seem to be similar to those of the enzyme treatment process using amylase and protease (1). Contrary to the increases in the protein content and recovery ratio by IP-EWT obtained with defatted rice bran, its effects were limited when full-fat rice bran was used (Table 2). Machine defatted rice bran contains approximately 50 w/w% of rice bran fat (11 w/w% in rice bran (data not shown)). Consequently, due to the reduction in the remaining amount of rice bran fat, the protein content in the protein fractions derived from the defatted rice bran had higher values than those obtained from the full-fat rice bran. These results suggest that IP-EWT can effectively recover protein concentrates with higher content and recover protein with higher efficiency from machine defatted rice bran. Element analysis of protein and phosphorus fractions The compositions and weight ratios of elements derived from full-fat and defatted rice bran were analyzed with respect to the phosphorus removed protein fraction (Tables 3 and 4) and the phosphorus fractions (Table 5). Decreases in the weight ratios of Na, K, and Cl in both protein fractions obtained with IP-DWT and IP-EWT were observed under all pH conditions compared with those obtained with IP, as listed in Tables 3 and 4. DWT and EWT did not reduce the phosphorus ratios. These results suggest that the IP-EWT process not only increased the protein content and recovery ratio but also contributed to desalination. The composition and weight ratio of elements in the phosphorus fractions (phosphorus fraction derived from full-fat rice bran (PFFRB) and phosphorus containing fraction derived from machine defatted rice bran (P-MDRB)) are summarized in Table 5. We found 15.1 w/w% of phosphorus in P-FFRB and 16.4 w/w% in PMDRB. These values are higher than the average phosphorus content of commercial phosphate ore (less than 13 w/w% P) (9). In addition no heavy metal ions were not detected (less than 0.005 w/w%) from the results of ICP-AES analysis from either fractions (P-FFRB and P-MDRB) throughout our study (data not shown). Thus, P-FFRB and P-MDRB might be used as an alternative ingredient or nutritional supplements to promote phosphorus intake.
TABLE 2. Effect of the electrolyzed water treatments (EWT) for the phosphorus compound removed protein recovery ratio (%) of protein fraction derived from full-fat and machine defatted rice bran. Material
Procedure
IP pH value 3
FFRB FFRB MDRB MDRB
PhosR-DWT PhosR-EWT PhosR-DWT PhosR-EWT
40.3 41.1 44.1 44.6
1.0 2.8 2.6 0.9
(37.3 (43.0 (40.2 (51.1
4
2.1) 1.7) 1.6) 3.3)
43.5 44.7 45.0 50.1
1.4 3.7 1.8 2.2
(39.4 (43.3 (37.6 (50.3
5
2.3) 2.5) 0.4) 2.4)
45.2 45.8 46.2 52.3
2.5 1.9 3.3 1.2
(38.1 (41.4 (35.7 (44.2
6
3.6) 2.2) 2.3) 2.5)
44.3 44.7 46.0 46.3
2.3 0.7 1.9 1.3
(31.6 (35.1 (17.1 (17.0
5.3) 3.9) 4.8) 3.1)
Content (w/w%) together with recovery ratio (%) in parenthesis are shown. FFRB, full-fat rice bran; MDRB, machine defatted rice bran; IP, isoelectric precipitation; PhosR, phosphorus removal; DWT, distilled deionized water treatment.
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TABLE 3. Composition and weight ratios of elements in the protein fractions recovered after phosphorus and protein extraction from full-fat rice bran. Element (w/w%)
Na Mg Al Si P S Cl K Ca Fe
pH 3
pH 4
pH 5
pH 6
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
1.1 0.1 N.D. N.D. 2.0 0.4 0.5 0.4 N.D. N.D.
0.9 0.1 N.D. N.D. 2.3 0.6 0.3 0.2 N.D. N.D.
0.7 0.2 N.D. N.D. 2.5 0.6 0.2 0.2 N.D. N.D.
2.2 0.2. N.D. N.D. 1.5 0.4 1.1 0.6 N.D. N.D.
1.1 0.2 N.D. N.D. 1.7 0.5 0.5 0.3 N.D. N.D.
0.9 0.2 N.D. 0.1 1.9 0.6 0.6 0.2 N.D. 0.1
2.2 0.2 N.D. 0.1 1.0 0.5 1.4 0.2 N.D. N.D.
1.2 0.2 N.D. 0.1 1.0 0.5 0.5 0.2 N.D. N.D.
0.9 0.2 N.D. N.D. 1.0 0.5 0.4 0.2 N.D. N.D.
2.7 0.3 N.D. 0.1 1.0 0.4 1.4 0.8 N.D. N.D.
1.2 0.2 N.D. 0.1 0.7 0.5 0.6 0.2 N.D. 0.1
0.9 0.1 N.D. N.D. 0.5 0.5 0.4 0.3 N.D. N.D.
N.D., not detectable; IP, isoelectric precipitation.
TABLE 4. Composition and weight ratios of elements in the protein fractions recovered after phosphorus and protein extraction from machine defatted rice bran. Element (w/w%)
Na Mg Al Si P S Cl K Ca Fe
pH 3
pH 4
pH 5
pH 6
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
2.2 0.1 N.D. 0.1. 2.6 0.7 1.0 0.5 0.1 0.1
0.9 N.D. N.D. N.D. 3.4 0.8 0.5 0.3 N.D. N.D.
0.7 N.D. N.D. 0.2. 3.3 0.7 0.4 0.2 0.2 0.1
5.2 0.3. N.D. 0.1 1.6 0.5 3.7 0.7 N.D. N.D.
1.4 0.2 N.D. 0.1 1.6 0.6 0.4 0.3 N.D. 0.1
1.4 0.2 N.D. 0.1 1.6 0.5 0.4 0.2 N.D. 0.2
6.4 0.3 N.D. 0.1 0.8 0.4 3.2 0.5 N.D. N.D.
1.9 0.1 N.D. 0.1 1.1 0.7 0.6 0.3 N.D. N.D.
2.2 0.2 N.D. 0.1 1.1 0.6 0.7 0.4 N.D. 0.2
6.8 0.3 N.D. 0.1 1.2 0.6 3.0 1.0 N.D. N.D.
1.9 0.2 N.D. 0.2 0.3 0.5 0.7 0.3 N.D. N.D.
1.9 0.1 N.D. 0.1 0.4 0.6 0.6 0.4 N.D. 0.1
N.D., not detectable; IP, isoelectric precipitation.
TABLE 5. Composition and weight ratios of elements in the phosphorus fractions extracted from full-fat rice bran (P-FFRB) and machine defatted rice bran (P-MDRB). Element (w/w%) C N O Na Mg Al Si P S Cl K Ca Fe
P-FFRB
P-MDRB
16.6 0.1 49.0 2.7 9.5 N.D. 0.1 15.1 0.1 1.7 4.8 0.5 N.D.
13.6 0.2 49.4 2.8 9.8 N.D. 0.1 16.4 N.D. 1.7 5.4 0.4 0.1
N.D., not detectable.
From the viewpoints of nutritional value and food safety, detailed information such as protein composition and rice allergens would be necessary for practical application of protein recovered with this process. These approaches are currently being investigated.
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20. Wang, M., Hettiarachchy, Qi. M., Burks, M., and Siebenmorgen, T.: Preparation and functional properties of rice bran protein isolate, J. Agric. Food Chem., 47, 411e416 (1999). 21. de Rham, O. and Jost, T.: Phytate-protein interactions in soybean extracts and low phytate soy protein products, J. Food Sci., 44, 596e600 (1979).
Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process Masanori Watanabe,1, * Isamu Maeda,2 Masahiro Koyama,3 Kozo Nakamura,3 and Kazuo Sasano4 Department of Food, Life and Environmental Science, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan,1 Graduate School of Agricultural Science, Utsunomiya University, 350 Minemachi, Utsunomiya 321-8505, Japan,2 Department of Bioscience and Biotechnology, Graduate School of Agriculture, Shinshu University, Minamiminowamura, Nagano 399-4598, Japan,3 and Shokkyo Co., Ltd., 1-10-28 Fukawa, Asakita-Ku, Hiroshima 739-1751, Japan4 Received 19 April 2014; accepted 23 July 2014 Available online 27 August 2014
We studied a process that enables simultaneous recovery of protein and phosphorus compounds from rice bran. Phosphorus substances in full-fat and defatted rice bran such as phytic acid and inorganic ions were solubilized under acidic conditions in the first step. After that, inorganic and/or organic phosphate salts were recovered in insoluble form under weak alkaline conditions. Furthermore, protein fractions obtained after phosphorus compounds had been removed were solubilized under alkaline conditions. After solubilization, protein fractions with high content were recovered by isoelectric precipitation (IP) followed by electrolyzed water treatment (EWT). The highest protein content (52.3 w/w%) was attained when machine defatted rice bran was treated through the process. Energy-dispersive X-ray spectroscopy (EDX) and inductively coupled plasma atomic emission spectrometry (ICP-AES) analyses demonstrated efficient desalting from the protein fractions by EWT and higher phosphorus contents (15.1e16.4 w/w% P) in the phosphorus fractions compared with commercial phosphate rock. In addition, no heavy metal ions in either protein or phosphorus fractions were detected. These results suggest that the newly developed process is suitable for practical recovery of highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Full-fat rice bran; Machine defatted rice bran; Isoelectric precipitation; Electrolyzed water; Rice protein; Phosphorus compounds]
World rice production in 2008 was approximately 661 million metric tons (1), and the weight ratio of bran to whole rice particles was about 10% (2). Therefore, rice bran has been focused on as a potential resource of nutrients and elements all over the world. Rice bran is known to be a major by-product of rice polishing that contains germ and several histologically identifiable soft layers such as pericarp, seed coat, nucellus, and aleurone layers (3). Rice protein has a great deal of potential to be a functional food when it is used in ingredients and nutritional supplements because its protein efficiency ratio (1.6e1.9) is comparable to that of casein (2.5) (1). Thus, protein production from rice bran has been extensively studied. Physicochemical treatments that include solvent extraction (4,5), compressed hot water extraction (6), and enzymatic treatments using endoprotease, exoprotease, xylanase, phytase, amylase, and carbohydratase (1), are currently being used for effectively extracting protein from rice bran. However, these known methods involve high levels of energy consumption, harmful chemicals, and high labor costs. Therefore, the rice processing industry has to consider commercialization in extracting proteins from this material while keeping low energy consumption and production costs low.
* Corresponding author. Tel./fax: þ81 235 282848. E-mail address: [email protected] (M. Watanabe).
Rice bran also includes a relatively large amount of phosphorus compounds (2). Phytic acid is particularly known to be a major constituent of the organic phosphates in rice bran. It serves as a phosphorus storage compound in plant seeds as well as a natural antioxidant because of its chelating property in iron and zinc as well as its ability to decrease the catalytic activities of many divalent transition metals (7,8). In addition, the expected global peak of phosphorus production is predicted to occur around 2030 (9). It is widely acknowledged within the fertilizer industry that the quality of remaining phosphate rock is decreasing and production costs are increasing. Technologies for recycling phosphorus from waste and extracting it from unused resources have attracted a great deal of attention because of this difficulty with producing phosphorus predicted in the future. Considering the potential of rice bran as protein and phosphorus sources, the development of safe and lowcost methods to recover these nutrients may partly contribute to establishment of sustainable use of renewable resources. However, simultaneous recovery of proteins and phosphorus compounds from rice bran by using solvent-free process has not been demonstrated. We developed a novel process in this study that could produce highly concentrated proteins and phosphorus compounds simultaneously without organic solvents that can be applied to commercial use. We analyzed their concentrations, recovery ratios, and chemical compositions. In addition, a technique of isoelectric precipitation combined with electrolyzed-water treatment (IP-EWT)
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.07.009
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was intensively focused on in terms of protein content and efficiency of desalination and protein purification. MATERIALS AND METHODS Materials Raw (full-fat) rice bran (fat content: 20 w/w%) was prepared from brown rice (Oryza sativa, cv. Koshihikari, harvested in Niigata prefecture, Japan) by using a rice polishing machine (NCP100B; Satake Co., Ltd., Higashi-Hiroshima, Japan) (10). The raw rice bran (supply rate of 120 kg/h) was machine defatted to prepare defatted rice bran (approximately 50% defatted) by using an oil press (V-05W;
207
Suehiro EPM Co. Ltd., Yokkaichi, Japan) after pre-heating the raw rice bran at 110e125 C. Experimental procedures Rice protein and phosphorus concentrates were prepared using acid and alkaline extraction, followed by the isoelectric precipitation (IP) and a washing process with electrolyzed/distilled water, as shown in Fig. 1. Thirty grams of raw rice bran or a defatted rice bran sample and 300 ml of deionized water (less than 2 mS cm1) were placed into a vessel, and pH was adjusted to 3.5 by using a 6 N HCl solution to elute the phosphorus compounds from the rice bran. After that, the vessel was centrifuged at 5000 g for 10 min at ambient temperature to separate eluted phosphorus compounds (supernatant) from rice bran residue (precipitate). The supernatant was adjusted to pH 7.5 with 6 N NaOH to
FIG. 1. Flow diagram for recoveries of phosphorus compound and protein concentrates from rice bran.
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precipitate the phosphorus compounds and centrifuged at 5000 g for 10 min at ambient temperature. The precipitated fraction containing the phosphorus compounds was heat dried at 80 C and stored in a desiccator for later analysis. The supernatant obtained after the phosphorus compounds had been precipitated was mixed with the rice bran residue at a setting of 500 rpm on a high torque stirrer (SWRS077, Nisshin Rika Co., Ltd., Tokyo, Japan) for an arbitrary time. NaOH was added to the mixture to dissolve and restore rice bran protein into the supernatant. The mixture was then centrifuged at 5000 g for 10 min at ambient temperature and the supernatant was collected as protein extracts and used to quantitative analyze the extracted protein. Isoelectric precipitation of protein from the protein extracts was done by adjusting the pH with acetic acid within a pH range of 3e6. Rice crude protein was precipitated by centrifugation at 5000 g for 10 min at ambient temperature, and washed with pH-adjusted electrolyzed water (EW) or distilled deionized water (DW) to remove water-soluble impurities. EW was prepared by using an electrolyzed water ionizer (TK8051, Panasonic Electric Works Co., Ltd., Shiga, Japan). This apparatus generated different pHs for EW by electrolysis with a tap water system that had a volume flow rate 2.5 l min1. Four kinds of EW were used in the experiment: EW with pH 3, oxidative-reduction potential (ORP) þ748 mV; EW with pH 4, ORP þ909 mV; EW with pH 5, ORP þ964 mV; and EW with pH 6, ORP þ1025 mV. Element analysis of protein and phosphorus concentrates The elements in the rice protein and phosphorus concentrates were detected and semi-quantitatively analyzed with a scanning electron microscope (T-3000, Hitachi Co., Ltd, Japan) equipped with an energy dispersive X-ray spectroscopy (EDX) detector (Quantax 70, Bruker AXS Microanalysis GmbH, Germany). A working distance of 25 mm, an accelerating voltage of 20 kV, and a beam current of approximately 1 mA (about 1300 cps) were used. A time of 400 s for each measurement was used for identification. Samples were dried by using the tert-butyl alcohol freeze-drying before the measurements (11). Heavy metal analyses of both concentrates were performed with an inductively coupled plasma atomic emission spectrometer (ICPS-7510, Shimadzu Co., Ltd, Japan). Samples were prepared by using open digestion with nitric acid and hydrochloric acid (12). Quantification of protein and phytic acid The protein content in rice bran and extracted rice protein concentrates was determined by using a total nitrogen analyzer (variMAX CN; Elementar Analysensysteme GmbH, Denmark) in compliance with the improved Dumas method. The percentages of total protein content were read from the output of the instrument by using a conversion factor of 5.95 (13). The protein recovery ratio (%) from both types of rice bran was calculated as: Protein recovery ratio (%) ¼ 100 (Cf A)/(Cb B)
(1)
where A is the value of the dry weight of the recovered protein fraction (see in Fig. 1) derived from 30 g of both type of rice bran, B is the value of the dry weight of total protein of 30 g of both type of rice bran, Cf is the percentages of total protein content (w/w%) of recovered protein as a crude or purified protein fraction and Cb is the percentages of total protein content (w/w%) of rice bran. The phytic acid content in the rice phosphorus concentrates was determined by colorimetry according the method by Haug et al. (14).
RESULTS AND DISCUSSION Effect of NaOH concentration and extraction time on solubilization of protein First, we examined what effects sodium hydroxide concentration had on solubilization of the rice bran protein after the phosphorus compounds had been removed when the extraction time was fixed to 2 h. As can be seen in Fig. 2A, the concentrations of protein extracted from full-fat and defatted rice bran attained nearly a saturated level under sodium hydroxide concentrations of 0.5 w/v% for the former and 1.0 w/v% for the latter. In addition, pH values of both extracts were 12.1 for the former and 12.8 for the latter. Fig. 2B shows the concentrations of extracted protein from full-fat and defatted rice bran with the changes in extraction time under sodium hydroxide concentration of 0.5 and 1.0 w/v%, respectively. A minimum extraction time of 1 h was required for full-fat rice bran and 2 h was required for defatted rice bran. The protein extracted from defatted rice bran in this time of 2 h was about 90% of that extracted in 4 h. Hence, 2 h was selected as a standard time for protein extraction throughout the experiment from the viewpoint of cost benefits such as reducing process energy. Effect of removing phosphorus compounds from rice bran on extracted protein content Rice bran contains inorganic and organic forms of phosphorus compounds (2). Separation of
FIG. 2. Effect of NaOH concentration (A) and mixing time (B) on solubilization of rice bran protein. Mixing time was fixed to 2 h (A). Bars indicate standard deviations (n ¼ 3). Incubation was carried out at 20 C in 500-ml conical flask containing 30 g of rice bran and 300-ml NaOH solution. Symbols: closed circles, machine defatted rice bran; open circles, full-fat rice bran.
such phosphorus compounds from rice bran was considered as they are impurities in recovering high quality protein from it and they are also new phosphorus resources that have remained unused in the past. We examined what effect removing phosphorus compounds from full-fat and machine defatted rice bran would have on the protein content and recovery ratio. Higher protein content was observed in the phosphorus-removed protein fractions (PhosR þ IP) than in the protein fractions without phosphorus removal (IP) within a pH range of 3e5, as is summarized in Table 1. However, phosphorus removal had no effect on increasing the protein content at pH 6. An increasing protein recovery ratio was also observed within a pH range of 3e5 in the PhosR þ IP of defatted rice bran. This suggests that the phosphorus removal process before recovering protein was recovered was effective to decrease the insoluble form of phosphorus compounds in purified protein fractions, as the result, extraction of high quality protein might be attained. Effect of electrolyzed water treatment on extracted protein content and protein recovery ratio EW has been regarded as a new sanitizer in recent years. It possesses antimicrobial properties against a variety of microorganisms (15). EW is generated by the electrolysis of deionized or tap water containing a low concentration of sodium chloride or potassium chloride in an electrolysis chamber where anode and cathode electrodes are separated by an ion-permeable exchange diaphragm. Acidic EW with low pH is produced from the anode and alkali EW with high pH is produced from the cathode (16). The pH of EW can be simply adjusted by using the quantitative ratios of acidic to alkali EW. Therefore, electrolytes in the protein concentrate except for protein can be removed by using EW adjusted to pH values used in the IP process without having to add acids or alkalis while keeping the precipitated protein resuspended. We clarified the ability of isoelectric precipitation and electrolyzed water treatment (IP-EWT) when used for protein recovery and purification after the IP process. The highest protein content (52.3 w/w%) of the protein fraction derived from machine defatted rice bran was attained by using a simultaneous recovery process of protein and phosphorus compounds that included IPEWT at pH 5 (Table 2). In addition, the protein recovery ratios obtained with IP-EWT were efficiently improved within the pH range of 3e5, compared to those with IP-distilled deionized water treatment (IP-DWT) (Table 2). The protein content and recovery ratios exceed those archived with reported physical
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TABLE 1. Effect of removing phosphorus compounds from full-fat and machine defatted rice bran on extracted protein content (w/w%) and protein recovery ratio (%). Material
Procedure
IP pH value 3
FFRB FFRB MDRB MDRB
IP PhosR þ IP IP PhosR þ IP
32.4 40.8 40.9 44.0
1.8 2.3 1.9 2.1
(42.3 (45.9 (38.8 (50.4
4
3.6) 2.9) 2.2) 1.4)
35.6 39.8 36.2 40.7
1.5 1.3 3.5 3.2
(41.4 (43.5 (34.4 (51.7
5
2.8) 3.4) 1.7) 2.1)
38.2 40.0 34.6 35.9
0.8 1.5 2.6 1.8
(40.1 (41.2 (32.8 (45.3
6
2.2) 1.3) 3.1) 0.9)
37.4 37.7 31.7 31.1
3.0 1.7 1.0 0.4
(41.7 (40.9 (25.1 (19.5
4.3) 3.8) 0.8) 0.3)
Content (w/w%) together with recovery ratio (%) in parenthesis are shown. FFRB, full-fat rice bran; MDRB, machine defatted rice bran; IP, isoelectric precipitation; PhosR, phosphorus removal.
Stoichiometry of the phosphorus fractions in Table 5 indicated large differences from the molar ratio of Mg, N, and P in magnesium ammonium phosphate (MAP: MgNH4PO4$6H2O) (17). The formation of MAP also requires higher pH (pH8e9) than the pH values of our phosphorus compound recovery process (pH7.5, see Fig. 1) and hydroxyapatite (HAP: Ca5OH(PO4)3) formation under aquatic conditions (18,19). In addition, phytic acid content in the phosphorus fractions derived from full-fat and defatted rice bran was equal at 46 w/w%. Moreover, the phosphorus fractions indicated relatively high contents of C and O (Table 5), which are assumed to be parts of the carbon cyclic skeleton and phosphate ions of phytic acid. Thus, phosphorus fractions derived from rice bran might be composed of both organic and inorganic forms of phosphorus such as phytic acid and insoluble phosphate salt except for MAP. In relation to that, phytase treatment increases the extracting yield of rice bran protein to about 57% (10). When phytase is used in combination with xylanase, the yield reaches 74.6%. These positive effects of phytase on improving the extracting yield are caused by its catalytic activity to digest phytic acid in an insoluble proteinphytate complex (20). The phosphorus removal process reported here may have almost the same effect on the cleavage of the bonds considered in phytase treatment. Additionally, at pH values above the isoelectric point (approximately 5) of rice bran protein, both proteins and phytic acid have net negative charge, according to de Rham et al. (21). However, phytic acid at this pH can still bind with proteins in the presence of multivalent cations such as calcium through forming a ternary complex of protein-cation-phytic acid. The formation of complex may hinder the effects of DWT and EWT on decreasing the phosphorus weight ratios in protein fraction (Tables 3 and 4). We studied the simultaneous recovery process of protein and phosphorus compounds based on combinations of the recovery (removal) of phosphorus compounds and the IP-EWT method for protein recovery and purification. The highest protein content (52.3 w/w%) was attained in the protein fraction derived from machine defatted rice bran by using this process. In addition, higher values for phosphorus content (15.1 and 16.4 w/w% P) were also obtained in the phosphorus fractions compared with that of commercial phosphate rock. These results suggested that this process is suitable in practice for recovering highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants.
treatment processes, which have included colloid milling (recovery yield: 15% and purity: 7.5%), colloid milling combined with homogenization (recovery yield: 16% and purity: 3.2%), freezethawing (recovery yield: 12%), high pressure (recovery yield: 11%), and sonication (recovery yield: 11%), and seem to be similar to those of the enzyme treatment process using amylase and protease (1). Contrary to the increases in the protein content and recovery ratio by IP-EWT obtained with defatted rice bran, its effects were limited when full-fat rice bran was used (Table 2). Machine defatted rice bran contains approximately 50 w/w% of rice bran fat (11 w/w% in rice bran (data not shown)). Consequently, due to the reduction in the remaining amount of rice bran fat, the protein content in the protein fractions derived from the defatted rice bran had higher values than those obtained from the full-fat rice bran. These results suggest that IP-EWT can effectively recover protein concentrates with higher content and recover protein with higher efficiency from machine defatted rice bran. Element analysis of protein and phosphorus fractions The compositions and weight ratios of elements derived from full-fat and defatted rice bran were analyzed with respect to the phosphorus removed protein fraction (Tables 3 and 4) and the phosphorus fractions (Table 5). Decreases in the weight ratios of Na, K, and Cl in both protein fractions obtained with IP-DWT and IP-EWT were observed under all pH conditions compared with those obtained with IP, as listed in Tables 3 and 4. DWT and EWT did not reduce the phosphorus ratios. These results suggest that the IP-EWT process not only increased the protein content and recovery ratio but also contributed to desalination. The composition and weight ratio of elements in the phosphorus fractions (phosphorus fraction derived from full-fat rice bran (PFFRB) and phosphorus containing fraction derived from machine defatted rice bran (P-MDRB)) are summarized in Table 5. We found 15.1 w/w% of phosphorus in P-FFRB and 16.4 w/w% in PMDRB. These values are higher than the average phosphorus content of commercial phosphate ore (less than 13 w/w% P) (9). In addition no heavy metal ions were not detected (less than 0.005 w/w%) from the results of ICP-AES analysis from either fractions (P-FFRB and P-MDRB) throughout our study (data not shown). Thus, P-FFRB and P-MDRB might be used as an alternative ingredient or nutritional supplements to promote phosphorus intake.
TABLE 2. Effect of the electrolyzed water treatments (EWT) for the phosphorus compound removed protein recovery ratio (%) of protein fraction derived from full-fat and machine defatted rice bran. Material
Procedure
IP pH value 3
FFRB FFRB MDRB MDRB
PhosR-DWT PhosR-EWT PhosR-DWT PhosR-EWT
40.3 41.1 44.1 44.6
1.0 2.8 2.6 0.9
(37.3 (43.0 (40.2 (51.1
4
2.1) 1.7) 1.6) 3.3)
43.5 44.7 45.0 50.1
1.4 3.7 1.8 2.2
(39.4 (43.3 (37.6 (50.3
5
2.3) 2.5) 0.4) 2.4)
45.2 45.8 46.2 52.3
2.5 1.9 3.3 1.2
(38.1 (41.4 (35.7 (44.2
6
3.6) 2.2) 2.3) 2.5)
44.3 44.7 46.0 46.3
2.3 0.7 1.9 1.3
(31.6 (35.1 (17.1 (17.0
5.3) 3.9) 4.8) 3.1)
Content (w/w%) together with recovery ratio (%) in parenthesis are shown. FFRB, full-fat rice bran; MDRB, machine defatted rice bran; IP, isoelectric precipitation; PhosR, phosphorus removal; DWT, distilled deionized water treatment.
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TABLE 3. Composition and weight ratios of elements in the protein fractions recovered after phosphorus and protein extraction from full-fat rice bran. Element (w/w%)
Na Mg Al Si P S Cl K Ca Fe
pH 3
pH 4
pH 5
pH 6
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
1.1 0.1 N.D. N.D. 2.0 0.4 0.5 0.4 N.D. N.D.
0.9 0.1 N.D. N.D. 2.3 0.6 0.3 0.2 N.D. N.D.
0.7 0.2 N.D. N.D. 2.5 0.6 0.2 0.2 N.D. N.D.
2.2 0.2. N.D. N.D. 1.5 0.4 1.1 0.6 N.D. N.D.
1.1 0.2 N.D. N.D. 1.7 0.5 0.5 0.3 N.D. N.D.
0.9 0.2 N.D. 0.1 1.9 0.6 0.6 0.2 N.D. 0.1
2.2 0.2 N.D. 0.1 1.0 0.5 1.4 0.2 N.D. N.D.
1.2 0.2 N.D. 0.1 1.0 0.5 0.5 0.2 N.D. N.D.
0.9 0.2 N.D. N.D. 1.0 0.5 0.4 0.2 N.D. N.D.
2.7 0.3 N.D. 0.1 1.0 0.4 1.4 0.8 N.D. N.D.
1.2 0.2 N.D. 0.1 0.7 0.5 0.6 0.2 N.D. 0.1
0.9 0.1 N.D. N.D. 0.5 0.5 0.4 0.3 N.D. N.D.
N.D., not detectable; IP, isoelectric precipitation.
TABLE 4. Composition and weight ratios of elements in the protein fractions recovered after phosphorus and protein extraction from machine defatted rice bran. Element (w/w%)
Na Mg Al Si P S Cl K Ca Fe
pH 3
pH 4
pH 5
pH 6
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
IP
IP-DWT
IP-EWT
2.2 0.1 N.D. 0.1. 2.6 0.7 1.0 0.5 0.1 0.1
0.9 N.D. N.D. N.D. 3.4 0.8 0.5 0.3 N.D. N.D.
0.7 N.D. N.D. 0.2. 3.3 0.7 0.4 0.2 0.2 0.1
5.2 0.3. N.D. 0.1 1.6 0.5 3.7 0.7 N.D. N.D.
1.4 0.2 N.D. 0.1 1.6 0.6 0.4 0.3 N.D. 0.1
1.4 0.2 N.D. 0.1 1.6 0.5 0.4 0.2 N.D. 0.2
6.4 0.3 N.D. 0.1 0.8 0.4 3.2 0.5 N.D. N.D.
1.9 0.1 N.D. 0.1 1.1 0.7 0.6 0.3 N.D. N.D.
2.2 0.2 N.D. 0.1 1.1 0.6 0.7 0.4 N.D. 0.2
6.8 0.3 N.D. 0.1 1.2 0.6 3.0 1.0 N.D. N.D.
1.9 0.2 N.D. 0.2 0.3 0.5 0.7 0.3 N.D. N.D.
1.9 0.1 N.D. 0.1 0.4 0.6 0.6 0.4 N.D. 0.1
N.D., not detectable; IP, isoelectric precipitation.
TABLE 5. Composition and weight ratios of elements in the phosphorus fractions extracted from full-fat rice bran (P-FFRB) and machine defatted rice bran (P-MDRB). Element (w/w%) C N O Na Mg Al Si P S Cl K Ca Fe
P-FFRB
P-MDRB
16.6 0.1 49.0 2.7 9.5 N.D. 0.1 15.1 0.1 1.7 4.8 0.5 N.D.
13.6 0.2 49.4 2.8 9.8 N.D. 0.1 16.4 N.D. 1.7 5.4 0.4 0.1
N.D., not detectable.
From the viewpoints of nutritional value and food safety, detailed information such as protein composition and rice allergens would be necessary for practical application of protein recovered with this process. These approaches are currently being investigated.
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20. Wang, M., Hettiarachchy, Qi. M., Burks, M., and Siebenmorgen, T.: Preparation and functional properties of rice bran protein isolate, J. Agric. Food Chem., 47, 411e416 (1999). 21. de Rham, O. and Jost, T.: Phytate-protein interactions in soybean extracts and low phytate soy protein products, J. Food Sci., 44, 596e600 (1979).