Composition and protein profile analysis of rice protein ingredients

Journal of Food Composition and Analysis 59 (2017) 18–26

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Composition and protein profile analysis of rice protein ingredients Luca Amagliania , Jonathan O’Reganb , Alan L. Kellya , James A. O’Mahonya,* a b

School of Food and Nutritional Sciences, University College Cork, Cork, Ireland Nestlé R&D Center, Wyeth Nutritionals Ireland, Askeaton, Co. Limerick, Ireland

A R T I C L E I N F O

Article history: Received 11 March 2016 Received in revised form 29 December 2016 Accepted 29 December 2016 Available online 31 December 2016 Keywords: Rice protein ingredients Dairy protein ingredients Macronutrient composition Mineral composition Amino acid composition Protein profile analysis Rice protein fractions Food analysis Food composition

A B S T R A C T

The objective of this study was to investigate the nutrient composition and protein profile of a range of rice protein ingredients containing 32–78% total protein. Rice protein ingredients had significantly (P < 0.05) lower levels of calcium and total essential amino acids compared to selected dairy protein ingredients, i.e., skim milk powder, whey protein isolate and whey protein hydrolysate. Protein profiles of the ingredients were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and size exclusion-high pressure liquid chromatography (SE-HPLC). Since the dominant rice protein fraction (i.e., glutelin) is extensively aggregated and crosslinked through disulfide bonds, a strong reducing buffer was developed in order to solubilise the rice protein ingredients prior to analysis by SDSPAGE. Intact rice protein ingredients (n = 3) contained proteins with molecular weight (MW) ranging from
11 to >250 kDa, while rice protein hydrolysates (n = 4) were composed mainly of low MW peptides. In parallel, enriched protein fractions were extracted from defatted rice flour based on their solubility and characterised by SDS-PAGE to facilitate the identification of protein bands in the rice protein ingredients. The results of this study underpin the understanding, prediction and control of physicochemical functionality of rice protein ingredients. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Rice (Oryza sativa L.) is a monocotyledon plant which belongs to the family Poaceae (Champagne et al., 2004) and is a major food crop, with global annual production estimated at about 480 million metric tons (expressed on a milled rice basis) (USDA, 2015). It is grown today in more than 100 countries (Muthayya et al., 2014), with China and India alone accounting for more than 50% of global rice production (USDA, 2015). The mature rice grain is harvested in the form of rough rice (or paddy rice), in which the caryopsis (or brown rice) is encased in a tough siliceous hull (or husk) (Juliano and Bechtel, 1985), and needs to be processed before being consumed by humans, as only the caryopsis is edible (Juliano, 1993). After cleaning of rough rice from impurities, the hull (20% of paddy rice weight) is broken loose and separated from the rice caryopsis. Thereafter, the bran (8–10% of paddy rice weight), consisting of pericarp, seed coat, nucellus, aleurone, pulverized embryo, and some starchy endosperm and hull fragments, is removed by either abrasive or friction methods, or a combination

* Corresponding author. E-mail address: [email protected] (J.A. O’Mahony). http://dx.doi.org/10.1016/j.jfca.2016.12.026 0889-1575/© 2017 Elsevier Inc. All rights reserved.

of the two, to obtain milled or white rice, composed entirely of endosperm (Bond, 2004; Orthoefer and Eastman, 2004). Varietal, environmental and processing variability all influence the composition and properties of the rice grain and its milling fractions (Abdul-Hamid et al., 2007; Monks et al., 2013). Milled rice consists of about 78% starch, while protein represents the second most abundant constituent (6–7%). Conversely, rice bran contains high levels of fat (15–20%) and protein (11–15%), as well as being a good source of fibre (7–11%). Also, rice bran displays much higher levels of minerals and vitamins than the other rice milling fractions (Juliano, 1985). The protein component of rice is generally regarded as hypoallergenic (Helm and Burks, 1996) and its nutritional quality is estimated to be equivalent or higher than that of other cereals but considerably lower compared to proteins derived from animal sources, legumes and oilseed crops (Day, 2013). Rice proteins are categorized according to the solubility-based classification described by Osborne (1924). The four protein fractions of rice are albumin (water-soluble), globulin (salt-soluble), glutelin (alkali/ acid-soluble), and prolamin (alcohol-soluble) (Shih, 2003). Rice grain milling fractions differ in terms of protein composition, and data reported in the literature regarding rice protein fractions vary widely, depending on the rice variety and the extraction procedures. A singular feature of rice is that prolamin, which

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

represents the major endosperm storage protein in other cereals except, oats (Shewry and Halford, 2002), is a minor protein in all rice grain milling fractions, whereas glutelin is the dominant protein in brown and milled rice. The proportion of albumin, globulin, glutelin and prolamin has been reported to be 5–10, 7–17, 75–81 and 3–6%, respectively, in brown rice, 4–6, 6–13, 79–83 and 2–7%, respectively, in milled rice, and 24–43, 13–36, 22–45 and 1– 5%, respectively, in rice bran (Adebiyi et al., 2009; Agboola et al., 2005; Cao et al., 2009; Ju et al., 2001; Juliano, 1985; Zhao et al., 2012). The interest of food researchers and industry in rice protein ingredients has been limited to date, due to the relatively low protein content of rice and the low solubility of rice proteins in water, which makes them difficult to recover and incorporate into formulated foods (Shih, 2003). However, the awareness of the nutritional and health properties of plant proteins has been increasing in recent years. Currently, alkaline, enzymatic and physical treatments for the extraction of proteins from sources such as rice flour and rice bran are being studied, refined and applied industrially (Fabian and Ju, 2011; Shih, 2003) and rice proteins are used as value-added ingredients in nutritional products, including sport nutrition supplements, as an alternative to the more commonly used milk and soy proteins, and infant formulas (Agostoni et al., 2007; Fiocchi et al., 2006; Lasekan et al., 2006; Reche et al., 2010). The purpose of this study was to characterise the macronutrient composition of a range of rice protein ingredients and to compare their mineral and amino acid profiles to that of selected dairy protein ingredients. A method for studying the protein profile of intact rice protein-based ingredients was developed, consisting of the solubilisation of proteins in a reducing buffer prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; the method allowed the characterisation of enriched rice protein fractions, extracted from defatted rice flour based on their solubility, and their profiles were used to identify the protein subunits of the rice protein ingredients analysed in this study. 2. Materials and methods 2.1. Materials The ingredients analysed included seven rice protein ingredients, i.e., three rice protein concentrates (RPC 1, RPC 2 and RPC 3), two rice endosperm protein hydrolysates (RPH 1 and RPH 2) and two rice bran protein hydrolysates (RBPH 1 and RBPH 2). Rice flour (RF) and rice bran (RB) obtained from Beneo (Tienen, Belgium) were analysed as commodity rice ingredients. The nutrient composition of three dairy protein ingredients, i.e., low heat skim milk powder (SMP) (33.7% protein) obtained from the Irish Dairy Board (Dublin, Ireland), whey protein isolate (WPI) (89.7% protein) obtained from Davisco Foods International (Le Sueur, MN, US) and whey protein hydrolysate (WPH) (78.3% protein, DH 12.9%) obtained from Kerry Group (Tralee, Co. Kerry, Ireland) was also analysed. 2.2. Compositional analysis Total nitrogen was determined by the Kjeldahl method according to AOAC Official Method 930.29 (AOAC, 2005); nitrogen-protein conversion factors of 5.95 and 6.38 were used to calculate total protein content of rice and dairy ingredients, respectively. Fat content of RPHs and RBPHs was determined using the Röse-Gottlieb method according to AOAC Official Method 932.06 (AOAC, 2005). Fat content of RF, RB and RPCs was determined by the Soxhlet method using a Soxtec 2055 (Foss, Ballymount, Co. Dublin, Ireland). Moisture content was determined

19

by drying the ingredients in an oven at 103  C for 5 h. Ash content was determined by ashing the ingredients in a muffle furnace at 500  C for 5 h. Total carbohydrate was calculated by difference (100 – sum of protein, fat, ash and moisture). Total starch, damaged starch and total dietary fibre were determined using enzymatic kits K-TSTA, K-SDAM and K-TDFR, respectively (Megazyme, Bray, Co. Wicklow, Ireland). The degree of hydrolysis of the hydrolysed protein ingredients (RPHs, RBPHs and WPH) was determined by the o-phthalaldehyde (OPA) method described by Nielsen et al. (2001); the value htot, defined as the number of peptide bonds in the protein substrate (meq/g protein), was determined from the total amino acid content of the protein ingredients. Assuming the average molecular weight of amino acids to be 125 g/mol, htot values for RPH 1, RPH 2, RBPH 1, RBPH 2 and WPH were calculated to be 7.4, 7.7, 6.7, 7.3 and 8.6 meq/g protein, respectively. pH was measured for 5% (w/v) protein dispersions/solutions of the ingredients using a H260G benchtop pH meter (Hach Company, Loveland, CO, US). The mineral profile of the ingredients was determined using inductively coupled plasma-emission spectroscopy (ICP-ES) according to AOAC Official Methods 984.27 and 971.27 (AOAC, 2005). The amino acid profile was determined according to the method of Schuster (1988). Tryptophan concentration was determined according to AOAC Official Method 988.15 (AOAC, 2005). 2.3. Extraction of rice protein fractions Extraction of rice protein fractions was performed according to the method of Ju et al. (2001) with modifications. RF (100 g) was defatted with 400 mL of hexane for 30 min, and the defatted RF was dried under a hood at 20  C for 24 h. RF was then subjected to protein extraction by mixing using an overhead stirrer (300 rpm) with 400 mL of deionised water at 20  C for 4 h and centrifuged at 3000g for 30 min to obtain albumin. After albumin extraction, the residue was extracted with 400 mL of 5% NaCl at 20  C for 4 h and centrifuged at 3000g for 30 min to obtain globulin. The residue was subsequently extracted with 400 mL of 0.02 M NaOH (pH adjusted to 11) at 20  C for 30 min and centrifuged at 3000g for 30 min to obtain glutelin. Prolamin was extracted separately, as extracting this rice protein fraction sequentially resulted in extensive crosscontamination with other rice protein fractions. In order to obtain the prolamin fraction, RF (100 g) was defatted as described above, extracted with 300 mL of 70% ethanol at 20  C for 4 h and centrifuged at 3000g for 30 min. Each extraction was repeated twice. Albumin, globulin and glutelin were precipitated from the relevant supernatants by adjusting the pH to their isoelectric points of 4.1, 4.3 and 4.8, respectively. Prolamin was precipitated by adding a threefold volume of acetone to the supernatant. The precipitated proteins (albumin, globulin, glutelin and prolamin) were washed twice with deionised water, adjusted to pH 7.0, frozen (20  C) for 24 h, freeze-dried, and then stored at 4  C for subsequent analysis. 2.4. Protein profile analysis 2.4.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The protein profile of all samples was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The sample powders were solubilised using a modified version of a reducing buffer used in the study of Van Der Borght et al. (2006), consisting of 1% dithiothreitol (DTT), 2% SDS, 2 M thiourea and 6 M urea, by shaking at 500 rpm for 2 h at 20  C, followed by centrifugation at 10,000 rpm for 15 min at 20  C in order to remove any insoluble material. An aliquot of the supernatant was mixed with Lane Marker Reducing Sample Buffer (5) (Pierce Biotechnology, Rockford, IL, US), consisting of 0.3 M

20

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

Tris-HCl, 5% SDS, 50% glycerol and 100 mM DTT, before being heated at 95  C for 5 min while mixing at 300 rpm using an Eppendorf Thermomixer compact (Eppendorf, Hamburg, Germany). Solutions were allowed to cool and sample volumes corresponding to 32 mg of protein (assuming that the solubilisation buffer had solubilised all of the protein) were loaded onto pre-cast 4–20% acrylamide, 10  10 cm, tris-glycine gels (Pierce Biotechnology, Rockford, IL, US) in an AcquaTank mini gel unit (Acquascience, Uckfield, UK). PageRuler Plus prestained protein ladder (Pierce Biotechnology, Rockford, IL, US) with molecular weight standards ranging from 10 to 250 kDa was used as molecular weight marker. Electrophoresis was performed at 150 V for 2 h. Following electrophoresis, the gels were stained with Bio-Safe Coomassie Brilliant Blue G-250 Stain (Bio-Rad Laboratories, Inc., Hercules, CA, US). The gels were destained by washing with ultrapure water until a clear background was obtained. Subsequently, they were scanned using a Scanjet G4010 desktop flatbed scanner (Hewlett-Packard, Palo Alto, CA, US) into a JPEG image format, and the molecular weight of the protein bands were estimated by densitometric analysis of the scanned images using TotalLab Quant software version 13.2 (TotalLab Ltd., Newcastle upon Tyne, UK). 2.4.2. Size exclusion-high pressure liquid chromatography and measurement of solubility The hydrolysed rice protein ingredients were reconstituted in ultrapure water (0.25%, w/v, protein), and centrifuged at 10,000 rpm for 15 min at 20  C in order to remove any insoluble material, and an aliquot (20 mL) of the supernatants was injected onto the column. The analysis was carried out using a TSK Gel G2000SW, 7.8 mm  600 mm column (Tosoh Bioscience GmbH, Stuttgart, Germany) and an isocratic gradient of 30% MeCN containing 0.1% TFA, with a run time of 60 min. All solvents were filtered under vacuum through 0.45 mm high velocity filters (Millipore Ltd., Durham, UK). Commercial a-lactalbumin, b-lactoglobulin B, bovine serum albumin, Cytochrome C, Bacitracin (Sigma-Aldrich, Dublin, Ireland), Asp-Glu and Leu-Trp-Met-Arg (Bachem AG, Bubendorf, Switzerland) were used as molecular weight standards. Standards were prefiltered through 0.22 mm low protein binding membrane filters (Sartorius Stedim, Surrey, UK) prior to application to the column. Solubility of the ingredients in water was measured by means of a protein solubility assay; the ingredients were reconstituted in ultrapure water (0.25%, w/v, protein), and centrifuged at 10,000 rpm for 15 min at 20  C and the supernatants were analysed for their protein content by the Kjeldahl method according to AOAC Official Method 930.29 (AOAC, 2005). Solubility was calculated as the protein content of each

supernatant expressed as a percentage of the total protein content of the initial solution. 2.5. Statistical data analysis Analysis of the mineral composition of the samples was performed in duplicate, with all other analyses performed in triplicate. Analysis of variance (ANOVA; Tukey’s HSD test) was carried out using Minitab1 16 (Minitab Ltd, Coventry, UK) statistical analysis package. The level of statistical significance was determined at P < 0.05. 3. Results and discussion 3.1. Macronutrient composition The macronutrient composition of the rice-based ingredients is shown in Table 1. RB displayed a protein content (14.7%) two-fold higher than RF (7.32%), these values being in line with those typically reported in the literature, which show rice bran to have the highest protein content among rice grain milling fractions (Juliano, 1993). According to Schramm et al. (2007), protein in rice bran is mainly concentrated in its outer portion, as indicated by the fact that the protein content of bran samples from two long grain rice varieties decreased with increasing milling time. Structural, functional and nutritional properties of rice are significantly influenced by its protein component (Shih, 2004). Among the rice protein ingredients, RPC 3 had the highest protein content (78.2%), while RBPHs (32.0–34.6%) displayed the lowest values (Table 1). Carbohydrates represent the major constituents of cereals, including rice. Available carbohydrates have been reported to be in the range 34–62% in rice bran, 73–87% in brown rice and 77–89% in milled rice (Juliano, 1993). RF displayed the highest total starch content (75.5%), while RB contained only 15.1% starch. Starch is the most abundant component of milled rice (Juliano, 1993). Lamberts et al. (2007) reported the distribution of starch in brown rice to be 84.6% in the core endosperm, 6.2% in the middle endosperm, 2.8% in the outer endosperm, and the remaining 6.4% in bran. Physical and cooking properties of rice are determined by starch and its interactions with other constituents of the rice endosperm, such as lipids, proteins and water, or with other ingredients used in the processing of rice (Fitzgerald, 2004). As regards the rice protein ingredients, total starch content ranged from 0.39% (RPC 2) to 16.0% (RBPH 2) (Table 1). Milling or grinding of cereal grains to obtain flour can cause damage to starch granules, disruption of starch crystalline

Table 1 Macronutrient composition (%, w/w), degree of hydrolysis (DH) (%) and pH of rice flour (RF), rice bran (RB), rice protein concentrates (RPCs), rice endosperm protein hydrolysates (RPHs) and rice bran protein hydrolysates (RBPHs). Values are means  standard deviations of data from triplicate analysis.

Protein Carbohydrate Total starch Damaged starch Fibre Fat Ash Moisture DH pH

RF

RB

RPC 1

RPC 2

RPC 3

RPH 1

RPH 2

RBPH 1

RBPH 2

7.32  0.04g 81.1  0.15a 75.5  0.46a 9.55  0.04b 0.44  0.04ef 0.78  0.01ef 1.30  0.18f 9.53  0.02a NA NA

14.7  0.03f 52.3  0.26c 15.1  0.21b 3.84  0.21f 6.66  0.05a 20.9  0.20a 8.01  0.22b 4.08  0.04d NA NA

75.0  0.38b 14.6  0.26e 6.50  0.71de 5.66  0.10e 0.72  0.06def 0.79  0.00ef 3.42  0.24d 6.24  0.08b NA 4.71  0.00g

75.5  0.46b 9.03  0.36f 0.39  0.14f 0.32  0.08g 0.96  0.19cd 1.97  0.04d 9.55  0.50a 3.95  0.03d NA 5.87  0.00d

78.2  0.16a 16.2  0.39d 4.88  0.42e 4.46  0.18ef 0.84  0.18de 0.95  0.04e 2.35  0.38e 2.25  0.05f NA 5.30  0.00f

75.6  0.08b 13.7  0.20e 7.45  0.82cd 7.28  0.18d 0.30  0.15f 0.26  0.07fg 6.16  0.07c 4.32  0.06c 17.8  0.41b 5.84  0.01e

70.5  0.38c 15.8  0.39d 9.54  0.76c 9.27  0.85bc 0.27  0.14f 0.25  0.05g 10.0  0.04a 3.52  0.05e 22.5  1.48a 6.78  0.01a

34.6  0.08d 52.3  0.20c 9.42  0.77c 8.16  0.14cd 1.35  0.17c 2.73  0.27c 8.09  0.05b 2.27  0.07f 17.7  0.37b 6.21  0.00c

32.0  0.04e 54.7  0.39b 16.0  0.63b 14.5  0.53a 2.16  0.14b 7.23  0.28b 3.87  0.12d 2.14  0.06f 24.2  1.10a 6.36  0.01b

Values followed by different superscript letters in the same row are significantly different (P < 0.05). NA = not applicable.

616  3.50b 32.5  0.50i 1325  5.00e 62.7  0.25d 279  1.50g 267  0.00f 0.13  0.00g 0.96  0.01f 0.04  0.00h 0.28  0.00j Values followed by different superscript letters in the same row are significantly different (P < 0.05).

WPH WPI

73.2  0.15c 20.0  0.00j 27.7  0.15f 4.45  0.10e 692  1.50c 62.9  0.75i 0.06  0.00g 0.46  0.00h 0.04  0.00h 0.05  0.00j 1245  5.00a 941  6.00b 1675  5.00c 111  0.50c 367  2.50f 961  0.50c 0.06  0.00g 0.25  0.00h 0.03  0.00h 3.74  0.03e

SMP RBPH 2

54.1  0.55d 303  0.50d 1715  15.0c 236  2.50b 104  1.00h 855  5.00d 1.09  0.01e 4.17  0.05e 6.73  0.03c 2.14  0.01g 33.3  0.30ef 301  1.00d 2455  15.0b 251  4.00b 786  6.50b 841  7.50d 3.02  0.03b 5.27  0.10d 8.36  0.10b 2.82  0.04f

RBPH 1 RPH 2

9.90  0.04ghi 274  1.00e 2720  70.0a 10.7  0.00e 1160  0.00a 306  1.00e 2.11  0.00c 0.71  0.00g 0.59  0.00fg 2.63  0.00f 29.5  1.25f 1990  0.00a 1635  75.0cd 14.1  0.60e 1090  50.0a 219  9.50g 0.34  0.03f 1.08  0.05f 0.59  0.03fg 1.52  0.06h

RPH 1 RPC 3

18.2  0.05g 562  0.50c 26.8  0.35f 15.1  0.00e 466  0.50e 302  0.00e 1.48  0.08d 17.7  0.05b 0.73  0.00f 9.40  0.00c 17.4  0.05gh 95.0  2.00g 17.8  2.40f 9.56  0.10e 601  2.00d 1200  0.00b 4.30  0.00a 24.8  0.05a 2.74  0.00d 10.8  0.05b

RPC 2 RPC 1

3.79  0.01i 133  1.00f 20.3  0.10f 4.54  0.02e 94.7  0.40h 318  1.50e 2.07  0.00c 5.07  0.01d 0.43  0.00g 11.7  0.10a

RB

43.3  0.30e 82.0  1.00h 1500  10.0d 768  16.0a 12.4  0.05i 1590  10.0a 0.96  0.01e 7.63  0.04c 21.1  0.15a 6.25  0.04d

RF

7.32  0.01hi 37.5  0.50i 113  0.50f 47.1  0.30d 12.4  0.00i 146  0.50h 0.17  0.01g 0.46  0.01h 1.07  0.01e 1.17  0.01i Ca Cl K Mg Na P Cu Fe Mn Zn

Table 2 Mineral composition (mg/100 g fresh weight) of rice flour (RF), rice bran (RB), rice protein concentrates (RPCs), rice endosperm protein hydrolysates (RPHs), rice bran protein hydrolysates (RBPHs), skim milk powder (SMP), whey protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from duplicate analysis.

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

21

lamellae, and degradation of starch molecules, which are associated with alteration of starch gelatinisation and pasting properties (Hasjim et al., 2013). In this study, the percentage of damaged starch (as % of total starch) was 12.6% and 25.4% in RF and RB, respectively, whereas it ranged from 82.1% (RPC 2) to 97.7% (RPH 1) among the rice protein ingredients (Table 1). In addition to starch, rice contains dietary fibre, defined as “the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine” (AACC, 2001). Polysaccharides (cellulose, hemicellulose, pectins, gums, mucilages, b-glucans, inulin, arabinoxylans, arabinogalactans), oligosaccharides, lignin and associated plant substances (waxes, cutin, suberin) are all included in the definition of dietary fibre (AACC, 2001). RF contained only 0.44% fibre, whereas RB showed the highest dietary fibre content (6.66%). These results were expected based on comparison with literature data, which show fibre to be mainly concentrated in the outer layers of the rice caryopsis (Juliano, 1993). Also, dietary fibre content was higher for RBPHs (1.35–2.16%) compared to the other rice protein ingredients (0.27–0.96%) (Table 1). It has been demonstrated that consumption of fibre provides health benefits, such as prevention of constipation, lowering of blood cholesterol and increased satiety. Diets rich in fibre are also linked with reduced risk of diabetes, coronary heart disease, obesity and colorectal cancer (Buttriss and Stokes, 2008). Although lipid is usually a minor component in rice, it is significant in terms of nutritional, sensory and functional properties (Godber and Juliano, 2004). Lipids in rice are classified into starch lipids, which account for a relatively small proportion of the total lipid composition of rice and are found mainly in the endosperm, where they are thought to play a role in starch synthesis and significantly influence starch functionality (Morrison, 1995), and nonstarch lipids, the most abundant form of lipids in rice, which are found in the aleurone, subaleurone and embryo of brown rice (Godber and Juliano, 2004). The fat content followed the same trend as dietary fibre, being highest for RB (20.9%) and significantly (P < 0.05) higher for RBPHs (2.73–7.23%) than the rest of the rice protein ingredients (0.25–1.97%) (Table 1). Besides being abundant, rice bran oil contains bioactive components such as tocopherols and tocotrienols (vitamin E), and g-oryzanol, a mixture of trans-ferulic acid esters of triterpene alcohols and sterols. A number of studies have suggested that these components possess antioxidant, hypocholesterolemic and antidiabetic activities (Burlando and Cornara, 2014; Esa et al., 2013). Ash is also concentrated in the outer portions of the rice caryopsis, with its distribution being reported to be 61% in bran, 23.7% in the outer endosperm, 3.7% in the middle endosperm and 11.6% in the core endosperm (Lamberts et al., 2007). RB held a significantly (P < 0.05) higher amount of ash (8.01%) than RF (1.30%). The ash content measured for RB was within the range of values previously reported in the literature (6.6–9.9%), whereas RF displayed a higher value compared to those reported for milled rice (0.3–0.8%) (Juliano, 1993). Ash content of rice protein ingredients ranged from 2.35% (RPC 3) to 10.0% (RPH 2) (Table 1). The functional properties of food proteins can be enhanced by partial enzymatic hydrolysis, with the most marked improvement being the increase in solubility (Panyam and Kilara, 1996). Solubility is an important requirement for proteins which are to act as functional ingredients in food systems (Nielsen, 1997). Enzymatic hydrolysis is thus essential in order to extend the range of applications of rice protein ingredients in food products; the hydrolysed rice protein ingredients had degree of hydrolysis ranging from 17.7% (RBPH 1) to 24.2% (RBPH 2).

5.28  0.17 2.62  0.04f 12.1  0.51a 3.24  0.10a 17.5  0.74bc 1.85  0.05d 4.56  0.12c 4.10  0.10f 3.98  0.12b

5.42  0.07bc 2.83  0.05f 11.2  0.33a 2.54  0.03b 18.3  0.62b 1.87  0.02d 6.41  0.19b 5.31  0.11a 3.17  0.06cd

3.2. Mineral composition

3.36  0.05 3.64  0.07e 7.56  0.17d 0.77  0.05gh 21.0  0.69a 1.94  0.06d 9.76  0.27a 5.31  0.17a 5.12  0.09a 6.77  0.10 8.22  0.04abc 9.29  0.22bc 1.40  0.03f 13.1  0.48d 5.35  0.09a 4.45  0.12c 4.85  0.08bcd 3.47  0.03c 6.50  0.20 7.37  0.07d 8.41  0.19cd 1.33  0.01f 12.7  0.40d 5.29  0.10a 3.74  0.10d 3.99  0.06f 3.05  0.01d 5.37  0.13 8.65  0.18a 9.49  0.29bc 0.97  0.03g 17.8  0.71bc 4.40  0.11b 4.14  0.14cd 5.26  0.16ab 5.21  0.12a 5.33  0.21 8.37  0.16ab 9.90  0.72b 0.62  0.01h 17.7  0.79bc 4.22  0.10bc 4.36  0.14c 5.03  0.16abc 5.04  0.10a Values followed by different superscript letters in the same row are significantly different (P < 0.05). y Conditionally essential amino acids.

5.26  0.02 7.84  0.10bcd 8.65  0.16cd 1.69  0.10e 16.9  0.05bc 4.12  0.05bc 4.36  0.02c 4.77  0.12cd 5.16  0.05a 5.85  0.19 8.67  0.04a 8.00  0.22d 1.39  0.10f 13.1  0.00d 5.25  0.06a 4.18  0.08cd 4.58  0.05de 3.82  0.04b 5.06  0.07 7.81  0.10cd 8.47  0.20cd 1.77  0.03de 16.2  0.50c 4.04  0.08c 4.30  0.11c 4.76  0.07cd 4.99  0.05a 6.44  0.20 8.18  0.38abc 8.61  0.08cd 2.04  0.17cd 12.7  0.12d 5.47  0.10a 4.10  0.04cd 4.23  0.04ef 3.17  0.12cd

5.30  0.14 8.06  0.23bc 8.41  0.27cd 2.14  0.03c 16.7  0.61bc 4.20  0.12bc 4.17  0.19cd 4.65  0.15cde 5.14  0.18a Alanine Argininey Aspartic acid Cystiney Glutamic acid Glyciney Proliney Seriney Tyrosiney

RF

c

RB

a

RPC 1

c

RPC 2

b

RPC 3

c

RPH 1

c

RPH 2

bc

RBPH 1

a

RBPH 2

a

SMP

d

WPI

c

WPH

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26 Table 3 Non essential amino acid composition (g/100 g protein) of rice flour (RF), rice bran (RB), rice protein concentrates (RPCs), rice endosperm protein hydrolysates (RPHs), rice bran protein hydrolysates (RBPHs), skim milk powder (SMP), whey protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from triplicate analysis.

22

Table 2 shows the mineral composition of the ingredients. Minerals exert several essential physiological functions in the human body; they are classified as major minerals (Ca, Cl, K, Mg, Na, P) or trace elements (including Cu, Fe, Mn, Zn). The classification of minerals depends on the amount required in the diet (>100 mg/day and <100 mg/day for major minerals and trace elements, respectively) and the amount present in the body, and not on their biological importance (Insel et al., 2013). When compared to RF, RB contained significantly (P < 0.05) higher levels of all the minerals listed, except Na. This observation is in agreement with the results of Itani et al. (2002), who reported the concentrations of specific minerals (Ca, K, Mg, P and Mn) to be higher in the outer than in the inner portions of dehulled rice grains, as determined in samples from eleven rice cultivars. Ca concentration was highest in SMP (1245 mg/100 g) followed by WPH (616 mg/100 g); however, WPI had only 73.2 mg/100 g of Ca. Even lower levels of Ca were found within the rice protein ingredients (3.79–54.1 mg/100 g). On the other hand, rice protein ingredients contained significantly (P < 0.05) higher concentrations of Cl (95.0–1990 mg/100 g) compared to whey protein ingredients (20.0–32.5 mg/100 g). Also, among the protein ingredients, the highest concentrations of the major minerals Cl, K, Mg, Na and P were found within the rice protein ingredients. The relatively high levels of Cl, K and Na displayed by some of the rice protein hydrolysates might be due to the addition of hydrochloric acid (HCl) or alkaline salts such as potassium hydroxide (KOH) or sodium hydroxide (NaOH) required for pH adjustment during the enzymatic hydrolysis reaction. As regards the trace elements, rice protein ingredients had considerably higher Cu (0.34–4.30 mg/ 100 g) and Mn (0.43–8.36 mg/100 g) levels compared to dairy protein ingredients (0.06–0.13 and 0.03–0.04 mg/100 g, respectively). The concentration of Fe was significantly (P < 0.05) higher in RPC 2 (24.8 mg/100 g) and RPC 3 (17.7 mg/100 g) than in any of the other protein ingredients. Furthermore, rice protein ingredients had significantly (P < 0.05) higher concentrations of Zn (1.52–11.7 mg/100 g) compared to whey protein ingredients (0.05– 0.28 mg/100 g), with RPCs displaying the highest values (9.40– 11.7 mg/100 g) among the protein ingredients analysed. 3.3. Amino acid composition The amino acid composition of the ingredients is presented in Tables 3 and 4 and 4. RF and RB had similar levels of total essential amino acids, with values being 34.4 and 32.7 g/100 g protein, respectively. However, their essential amino acid profiles differed, with RF having significantly (P < 0.05) higher levels of isoleucine, leucine, methionine and phenylalanine, whereas the concentration of histidine, lysine and tryptophan was significantly (P < 0.05) higher in RB. Lysine is the first limiting amino acid among cereal proteins (Young and Pellett, 1994). However, rice has a higher content of lysine compared to several other cereal grains, which explains its higher BV, as a direct correlation exists between the BV and the concentration of the first limiting essential amino acid (i.e., lysine) (Eggum, 1979). The higher concentration of lysine found in RB (4.65 g/100 g protein) compared to RF (3.16 g/100 g protein) is due to the fact that rice bran contains a considerably higher proportion of albumin compared to milled rice, with albumin having the highest concentration of lysine among rice protein fractions (Juliano, 1985). Rice protein ingredients had significantly (P < 0.05) higher levels of the conditionally essential amino acids (i.e., amino acids whose synthesis can be carried out by mammals but may be limited by several factors; Reeds, 2000) arginine and glycine compared to dairy protein ingredients (Table 3). On the other hand,

1.74  0.04g 6.54  0.12a 10.9  0.31b 10.2  0.14b 2.37  0.10b 3.41  0.13g 7.40  0.22a 2.04  0.10b 5.71  0.14ab 50.3  0.75a Values followed by different superscript letters in the same row are significantly different (P < 0.05).

WPH WPI

1.91  0.02fg 5.89  0.18b 13.2  0.66a 11.6  0.14a 2.43  0.22ab 3.80  0.19fg 5.09  0.19b 2.72  0.10a 5.03  0.14def 51.7  1.37a 2.49  0.08b 5.14  0.15c 9.53  0.22c 7.65  0.11c 2.77  0.09a 4.81  0.08cd 4.30  0.10c 1.48  0.05cd 5.94  0.13a 44.1  0.48b 2.32  0.02bc 3.69  0.12de 6.88  0.11ef 4.63  0.11e 1.86  0.00c 4.38  0.07de 3.96  0.05cd 1.28  0.03de 5.46  0.13abcd 34.5  0.33de 2.49  0.04b 3.02  0.07f 5.73  0.10g 4.66  0.12e 1.71  0.01c 3.62  0.13fg 3.78  0.06de 1.26  0.05e 4.74  0.13f 31.0  0.22f

SMP RBPH 2 RBPH 1

3.4. Protein profile analysis

2.33  0.01bc 3.93  0.17d 7.59  0.23de 3.85  0.03f 1.68  0.06c 5.45  0.14a 3.50  0.07e 1.17  0.02e 5.16  0.18cdef 34.7  0.77de

RPH 2 RPH 1

23

the total essential amino acid content was significantly (P < 0.05) higher in dairy protein ingredients (44.1–51.7 g/100 g protein) than in rice protein ingredients (31.0–37.6 g/100 g protein). Specifically, dairy protein ingredients had significantly (P < 0.05) higher levels of the essential amino acids isoleucine (5.14–6.54 g/100 g protein), leucine (9.53–13.2 g/100 g protein) and lysine (7.65–11.6 g/100 g protein) than rice protein ingredients. Among the rice protein ingredients, RPC 2 had the highest concentration of lysine (5.62 g/ 100 g protein), followed by the RBPHs (4.63–4.66 g/100 g protein), with the remaining rice protein ingredients having lysine concentrations in the range 3.42–3.85 g/100 g protein. Furthermore, whey protein ingredients displayed significantly (P < 0.05) higher levels of the essential amino acids threonine (5.09–7.40 g/ 100 g protein) and tryptophan (2.04–2.72 g/100 g protein) compared to the other protein ingredients. The concentration of tryptophan was particularly low in RPH 1 (0.60 g/100 g protein). As regards some of the other essential amino acids, among the protein ingredients analysed, RPC 2 and RPH 2 displayed the highest concentrations of histidine (2.89 g/100 g protein) and phenylalanine (5.45 g/100 g protein), respectively.

2.00  0.04ef 3.73  0.14de 7.37  0.21de 3.42  0.02fg 1.96  0.07c 4.93  0.13bc 3.55  0.10e 0.60  0.02f 4.91  0.15ef 32.5  0.79ef 2.18  0.03cde 4.16  0.12d 7.90  0.17d 3.44  0.06fg 2.43  0.10ab 5.31  0.05ab 3.48  0.07e 1.24  0.01e 5.32  0.13bcde 35.5  0.52cd

RPC 3 RPC 2

2.89  0.10a 3.87  0.12d 7.53  0.19de 5.62  0.18d 1.96  0.04c 4.74  0.18cd 3.77  0.06de 1.59  0.04c 5.62  0.19abc 37.6  0.38c 2.26  0.05bcd 3.99  0.14d 7.73  0.22de 3.56  0.01fg 2.52  0.06ab 5.34  0.15ab 3.39  0.01e 1.32  0.02de 5.21  0.21bcdef 35.3  0.82cd 2.45  0.15b 3.32  0.04ef 6.36  0.11fg 4.65  0.42e 1.74  0.09c 4.04  0.12ef 3.71  0.07de 1.47  0.06cd 4.98  0.11def 32.7  1.02def

RPC 1 RB RF

2.05  0.04def 3.86  0.14d 7.72  0.25de 3.16  0.07g 2.72  0.18ab 4.94  0.15bc 3.39  0.15e 1.19  0.10e 5.34  0.17bcde 34.4  1.09de Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Total EAAs

Table 4 Essential amino acid (EAA) composition (g/100 g protein) of rice flour (RF), rice bran (RB), rice protein concentrates (RPCs), rice endosperm protein hydrolysates (RPHs), rice bran protein hydrolysates (RBPHs), skim milk powder (SMP), whey protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from triplicate analysis.

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

The electrophoretic profiles of the four rice protein fractions are shown in Fig. 1a. Albumin was resolved into a wide range of subunits, with molecular weight (MW) ranging from about 13 to 110 kDa. However, some unresolved protein material (MW > 250 kDa) was also present in the gel loading wells. Data reported in the literature confirm that rice albumin is a highly heterogeneous protein. MW of rice albumin subunits has been reported to range from 10 to 200 kDa, as determined by gel filtration on Sephadex G100 (Iwasaki et al., 1982). Size exclusion-high pressure liquid chromatography (SE-HPLC) showed fully dissociated rice albumin polypeptides to have MW ranging from 10 to 100 kDa (Hamada, 1997). Rice albumin was resolved into 6 bands with MW in the range 15–56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two major albumin subunits, having MW of 40 and 55 kDa, were determined by SDS-capillary electrophoresis (SDS-CE) (Agboola et al., 2005). In the current study, two main polypeptide subunits with MW of about 19–22 and 53–56 kDa, with the former being predominant, and two minor subunits of about 11 and 13 kDa were seen for globulin. SDS-PAGE of rice endosperm albumin revealed two abundant proteins having MW of 16 kDa and 25 kDa (Krishnan et al., 1992) while, in the study of Agboola et al. (2005), rice globulin was resolved by SDS-CE into 6 subunits with MW ranging from 23 to 105 kDa, with the 54 kDa subunit being predominant. Two major polypeptide subunits with MW of 30–40 (a or acidic) and 19–23 kDa (ß or basic) have been reported for rice glutelin (Agboola et al., 2005; Chrastil and Zarins, 1992; Krishnan and Okita, 1986; Luthe, 1983; Robert et al., 1985; Sarker et al., 1986; Yamagata et al., 1982). Glutelin is synthesized first as a polypeptide with a MW reported to be in the range 51–57 kDa (Krishnan and Okita, 1986; Luthe, 1983; Sarker et al., 1986; Yamagata et al., 1982). The glutelin precursor is then enzymatically hydrolysed to yield a and b subunits. The subsequent polymerization of the glutelin subunits through disulfide bonds results in the formation of macromolecular complexes (Sugimoto et al., 1986). In the current study, glutelin displayed major polypeptide subunits with MW of about 28–33 kDa (a-glutelin), 17–21 (b-glutelin) and 52 kDa (glutelin precursor). Minor subunits were also observed at about 11, 13, 100 and 110 kDa, possibly due to cross-contamination with albumin and globulin. Furthermore, some unresolved protein material (MW > 250 kDa) was present in this sample. Rice prolamin has been reported to be composed of three polypeptide groups having MW of 10, 13 and 16 kDa, with the

24

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

Fig. 1. Representative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of (a) enriched rice protein fractions (1: Molecular weight marker; 2: Albumin; 3: Globulin; 4: Glutelin; 5: Prolamin); (b) rice flour (RF), rice bran (RB), and rice protein concentrates (RPCs) (1: Molecular weight marker; 2: RF; 3: RB; 4: RPC 1; 5: RPC 2; 6: RPC 3) and (c) rice endosperm protein hydrolysates (RPHs) and rice bran protein hydrolysates (RBPHs) (1: Molecular weight marker; 2: RPH 1; 3: RPH 2; 4: RBPH 1; 5: RBPH 2).

13 kDa prolamin being predominant, as determined by SDS-PAGE (Hibino et al., 1989; Ogawa et al., 1987). In the current study, prolamin showed one major band with MW of about 10 kDa. Also, two minor subunits of about 18 kDa and 31–32 kDa were present, most likely due to cross-contamination with glutelin. The method used to extract the rice protein fractions from RF coupled with their solubilisation in the strong reducing buffer prior to SDS-PAGE analysis, provided good resolution of the proteins characterising

the different fractions, which allowed the identification of the protein subunits of the intact rice protein ingredients. Fig. 1b shows the protein profile of RF, RB and the intact rice protein ingredients. RF displayed three main bands at about 13 (globulin), 18–20 (b-glutelin) and 31–33 kDa (a-glutelin), while RB displayed a wide range of subunits, with the major subunit having MW of about 58 kDa, which most likely represents a globulin subunit. As regards the rice protein ingredients, RPC 1 showed a

100%

Protein distribution

80%

<0.5 kDa 60%

0.5-1 kDa 1-2 kDa 2-5 kDa 5-10 kDa

40%

10-20 kDa >20 kDa

20%

0%

RPH 1

RPH 2

RBPH 1

RBPH 2

Fig. 2. Molecular weight distribution of the soluble fraction of rice endosperm protein hydrolysates (RPHs) and rice bran protein hydrolysates (RBPHs) as determined by size exclusion-high pressure liquid chromatography (SE-HPLC).

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

profile similar to that of RF, although the intensity of the bands was higher for the former and a band at about 52 kDa (glutelin precursor) was also evident. RPC 2 showed one main band at about 58 kDa (globulin subunit), whereas RPC 3 proved difficult to resolve and showed only one faint band at about 13 kDa (globulin subunit). Unresolved high MW protein material (MW > 250 kDa) was always present among the rice protein concentrates. This result indicates that the intact rice protein ingredients could not be completely solubilised, despite the use of a strong reducing buffer prior to SDS-PAGE analysis. Rice proteins have low solubility in water, mainly because of extensive aggregation and crosslinking through disulfide bonds which characterise rice glutelin (Hamada, 1997; Sugimoto et al., 1986), the dominant protein fraction in brown and milled rice. The processing that the intact rice protein ingredients may have undergone (e.g., heat treatment), could have caused protein denaturation and further aggregation, which would have prevented their complete solubilisation. Electrophoretic profiles of the rice protein hydrolysates are shown in Fig. 1c. RPH 1 and RPH 2 ingredients were composed of protein/peptide material with MW less than 10 kDa. Conversely, RBPH 1 showed one distinct band at about 58 kDa which, according to the electrophoretic profiles of the enriched rice protein fractions, may correspond to a globulin subunit. Also, both RBPH 1 and RBPH 2 showed unresolved high MW protein material (MW > 250 kDa) and protein material with MW < 15 kDa. The protein profile of the hydrolysed protein ingredients was further investigated by SE-HPLC, as represented in Fig. 2. RBPH 1 and RBPH 2 were composed of 20.8 and 13.5% insoluble protein material, respectively, whereas RPH 1 and RPH 2 were completely soluble (results not shown). About 3 and 2% of the soluble protein material of RBPH 1 and RBPH 2, respectively, had MW greater than 20 kDa, indicating the presence of intact proteins in these ingredients, which was in agreement with the results obtained by SDS-PAGE. However, the greatest proportion of the soluble protein material of the hydrolysed rice protein ingredients had MW less than 1 kDa. Furthermore, about 52, 49, 46 and 38% of the soluble material of RPH 1, RPH 2, RBPH 1 and RBPH 2, respectively, was composed of peptides with MW less than 0.5 kDa. The low levels of intact proteins and the high levels of low MW peptides which characterised these ingredients are consistent with their high measured DH values. The protein profile of the hydrolysates shows that aggregated rice proteins can be, at least partially, cleaved through enzymatic hydrolysis. A decrease in the molecular weight of proteins is among the effects of enzymatic hydrolysis, which coupled with the increase in the number of ionisable groups and the exposure of hydrophobic groups otherwise buried within the protein core, may improve some functional properties of proteins. Factors such as specificity of the enzyme, environmental conditions and degree of hydrolysis influence the type of peptides generated and thus the extent of enhancement of the functional properties of the hydrolysed protein ingredients (Nielsen, 1997; Panyam and Kilara, 1996). Hydrolysis of rice proteins has been shown to yield improvements in terms of solubility, emulsifying and foaming properties, making the resulting ingredients suitable for a wider range of food applications compared to their intact counterparts (Anderson et al., 2001; Bandyopadhyay et al., 2008; Guo et al., 2013; Li et al., 2012; Paraman et al., 2007). 4. Conclusions In this study, the nutrient composition of a range of intact and hydrolysed rice protein ingredients was determined and compared with that of commercially available dairy protein ingredients. According to the results obtained, supplementation of rice protein ingredients with calcium and some essential amino acids,

25

including isoleucine, leucine, lysine, threonine and tryptophan is necessary in order for these ingredients to match, or exceed, the nutritional value of dairy protein ingredients. On the other hand, novel plant protein ingredients with a complete and well balanced amino acid profile may be obtained by combining rice with other plant proteins such as those derived from legumes (e.g., peas, lentils, beans and lupins), the latter being characterised by low levels of sulphur-containing amino acids (i.e., cystine and methionine) but considerably higher levels of lysine compared to rice proteins. Dairy protein ingredients are currently used in a wide range of food applications, including desserts, baked goods, toppings, soups, sauces, salad dressings, ice cream, infant formulae, medical and clinical nutrition products, protein bars, and sports and nutritional beverages. In order to assess whether rice protein ingredients could partially or totally replace dairy protein ingredients in the aforementioned food products, investigation of their physicochemical properties is required; partial or total replacement of animal proteins with plant proteins in food products is a strategy that is nowadays being applied commercially for reasons of cost and sustainability. In addition, a strong reducing buffer was developed, which allowed the solubilisation and characterisation of the protein profile of the enriched rice protein fractions and the rice protein ingredients. This study provides essential knowledge for the interpretation of the physicochemical properties of rice protein ingredients. Acknowledgement The authors would like to acknowledge Dr André Brodkorb (Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland) for his assistance in performing size exclusion-high pressure liquid chromatography analysis. The authors would like to acknowledge Nestle for providing financial support for this study. References AACC (American Association of Cereal Chemists), 2001. The definition of dietary fiber. Cereal Foods World 46, 112–126. AOAC, 2005. Official Methods of Analysis, 18th ed. Association of Official Analytical Chemists, Gaithersburg, MD. Abdul-Hamid, A., Sulaiman, R.R.R., Osman, A., Saari, N., 2007. Preliminary study of the chemical composition of rice milling fractions stabilized by microwave heating. J. Food Compos. Anal. 20, 627–637. Adebiyi, A.P., Adebiyi, A.O., Hasegawa, Y., Ogawa, T., Muramoto, K., 2009. Isolation and characterization of protein fractions from deoiled rice bran. Eur. Food Res. Technol. 228, 391–401. Agboola, S., Ng, D., Mills, D., 2005. Characterisation and functional properties of Australian rice protein isolates. J. Cereal Sci. 41, 283–290. Agostoni, C., Fiocchi, A., Riva, E., Terracciano, L., Sarratud, T., Martelli, A., Lodi, F., D’Auria, E., Zuccotti, G., Giovannini, M., 2007. Growth of infants with IgEmediated cow’s milk allergy fed different formulas in the complementary feeding period. Pediatr. Allergy Immunol. 18, 599–606. Anderson, A., Hettiarachchy, N.S., Ju, Z.Y., 2001. Physicochemical properties of pronase-treated rice glutelin. J. Am. Oil Chem. Soc. 78, 1–6. Bandyopadhyay, K., Misra, G., Ghosh, S., 2008. Preparation and characterisation of protein hydrolysates from Indian defatted rice bran meal. J. Oleo Sci. 57, 47–52. Bond, N., 2004. Rice milling, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 283–300. Burlando, B., Cornara, L., 2014. Therapeutic properties of rice constituents and derivatives (Oryza sativa L.): a review update. Trends Food Sci. Technol. 40, 82– 98. Buttriss, J.L., Stokes, C.S., 2008. Dietary fiber and health: an overview. Nutr. Bull. 33, 186–200. Cao, X., Wen, H., Li, C., Gu, Z., 2009. Differences in functional properties and biochemical characteristics of congenetic rice proteins. J. Cereal Sci. 50, 184– 189. Champagne, E.T., Wood, D.F., Juliano, B.O., Bechtel, D.B., 2004. The rice grain and its gross composition, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 77–107. Chrastil, J., Zarins, Z.M., 1992. Influence of storage on peptide subunit composition of rice oryzenin. J. Agric. Food Chem. 40, 927–930. Day, L., 2013. Proteins from land plants – potential resources for human nutrition and food security. Trends Food Sci. Technol. 32, 25–42.

26

L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26

Eggum, B.O., 1979. The nutritional value of rice in comparison with other cereals. Proceedings of the Workshop on Chemical Aspects of Rice Grain Quality, Los Baños, Laguana, Philippines: International Rice Research Institute, pp. 91–111. Esa, N.M., Ling, T.B., Peng, L.S., 2013. By-products of rice processing: an overview of health benefits and applications. J. Rice Res. 1, 107. Fabian, C., Ju, Y.H., 2011. A review on rice bran protein: its properties and extraction methods. Crit. Rev. Food Sci. Nutr. 51, 816–827. Fiocchi, A., Restani, P., Bernardini, R., Lucarelli, S., Lombardi, G., Magazzù, G., Marseglia, G.L., Pittschieler, K., Tripodi, S., Troncone, R., Ranzini, C., 2006. A hydrolysed rice-based formula is tolerated by children with cow’s milk allergy: a multi-centre study. Clin. Exp. Allergy 36, 311–316. Fitzgerald, M., 2004. Starch, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 109–141. Godber, J.S., Juliano, B.O., 2004. Rice lipids, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 163–190. Guo, X., Zhang, J., Ma, Y., Tian, S., 2013. Optimization of limited hydrolysis of proteins in rice residue and characterization of the functional properties of the products. J. Food Process. Preserv. 37, 245–253. Hamada, J.S., 1997. Characterization of protein fractions of rice bran to devise effective methods of protein solubilization. Cereal Chem. 74, 662–668. Hasjim, J., Li, E., Dhital, S., 2013. Milling of rice grains: effects of starch/flour structures on gelatinization and pasting properties. Carbohydr. Polym. 92, 682– 690. Helm, R.M., Burks, A.W., 1996. Hypoallergenicity of rice protein. Cereal Foods World 41, 839–843. Hibino, T., Kidzu, K., Masumura, T., Ohtsuki, K., Tanaka, K., Kawabata, M., Fujii, S., 1989. Amino acid composition of rice prolamin polypeptides. Agric. Biol. Chem. 53, 513–518. Insel, P., Ross, D., McMahon, K., Bernstein, M., 2013. Water and major minerals, Nutrition. 4th ed. Jones & Bartlett Learning, Burlington, MA, pp. 467–506. Itani, T., Tamaki, M., Arai, E., Horino, T., 2002. Distribution of amylose, nitrogen: and minerals in rice kernels with various characters. J. Agric. Food Chem. 50, 5326– 5332. Iwasaki, T., Shibuya, N., Suzuki, T., Chikubu, S., 1982. Gel filtration and electrophoresis of soluble rice proteins extracted from long, medium: and short grain varieties. Cereal Chem. 59, 192–195. Ju, Z.Y., Hettiarachchy, N.S., Rath, N., 2001. Extraction: denaturation and hydrophobic properties of rice flour proteins. J. Food Sci. 66, 229–232. Juliano, B.O., Bechtel, D.B., 1985. The rice grain and its gross composition, In: Juliano, B.O. (Ed.), Rice: Chemistry and Technology. 2nd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 17–57. Juliano, B.O., 1985. Polysaccharides, proteins, and lipids of rice, In: Juliano, B.O. (Ed.), Rice: Chemistry and Technology. 2nd ed. American Association of Cereal Chemists, St. Paul,MN, pp. 59–174. Juliano, B.O., 1993. Rice in human nutrition. food and agriculture organization of the united nations. Food Nutr. Ser.. Krishnan, H.B., Okita, T.W., 1986. Structural relationship among the rice glutelin polypeptides. Plant Physiol. 81, 748–753. Krishnan, H.B., White, J.A., Pueppke, S.G., 1992. Characterization and localization of rice (Oryza sativa L.) seed globulins. Plant Sci. 81, 1–11. Lamberts, L., De Bie, E., Vandeputte, G.E., Veraverbeke, W.S., Derycke, V., De Man, W., Delcour, J.A., 2007. Effect of milling on colour and nutritional properties of rice. Food Chem. 100, 1496–1503. Lasekan, J.B., Koo, W.W.K., Walters, J., Neylan, M., Luebbers, S., 2006. Growth, tolerance and biochemical measures in healthy infants fed a partially hydrolysed rice protein-based formula: a randomized, blinded, prospective trial. J. Am. Coll. Nutr. 25, 12–19. Li, X., Xiong, H., Yang, K., Peng, D., Peng, H., Zhao, Q., 2012. Optimization of the biological processing of rice dregs into nutritional peptides with the aid of trypsin. J. Food Sci. Technol. 49, 537–546. Luthe, D.S., 1983. Storage protein accumulation in developing rice (Oryza sativa L.) seeds. Plant Sci. Lett. 32, 147–158.

Monks, J.L.F., Vanier, N.L., Casaril, J., Berto, R.M., de Oliveira, M., Gomes, C.B., de Carvalho, M.P., Dias, A.R.G., Elias, M.C., 2013. Effects of milling on proximate composition, folic acid: fatty acids and technological properties of rice. J. Food Compos. Anal. 30, 73–79. Morrison, W.R., 1995. Starch lipids and how they relate to starch granule structure and functionality. Cereal Foods World 40, 437–446. Muthayya, S., Sugimoto, J.D., Montgomery, S., Maberly, G.F., 2014. An overview of global rice production, supply, trade: and consumption. Ann. N. Y. Acad. Sci. 1324, 7–14. Nielsen, P.M., Petersen, D., Dambmann, C., 2001. Improved method for determining food protein degree of hydrolysis. J. Food Sci. 66, 642–646. Nielsen, P.M., 1997. Functionality of protein hydrolysates. In: Damodaran, S., Paraf, A. (Eds.), Food Proteins and Their Applications. Marcel Dekker, Inc., New York, NY, pp. 443–472. Ogawa, M., Kumamaru, T., Satoh, H., Iwata, N., Omura, T., Kasai, Z., Tanaka, K., 1987. Purification of protein body-I of rice seed and its polypeptide composition. Plant Cell Physiol. 28, 1517–1527. Orthoefer, F.T., Eastman, J., 2004. Rice bran and oil, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul,MN, pp. 569–593. Osborne, T.B., 1924. The Vegetable Proteins. Longmans, Green and Co, London, UK. Panyam, D., Kilara, A., 1996. Enhancing the functionality of food proteins by enzymatic modification. Trends Food Sci. Technol. 7, 120–125. Paraman, I., Hettiarachchy, N.S., Schaefer, C., Beck, M.I., 2007. Hydrophobicity, solubility: and emulsifying properties of enzyme-modified rice endosperm protein. Cereal Chem. 84, 343–349. Reche, M., Pascual, C., Fiandor, A., Polanco, I., Rivero-Urgell, M., Chifre, R., Johnston, S., Martin-Esteban, M., 2010. The effect of a partially hydrolysed formula based on rice protein in the treatment of infants with cow’s milk protein allergy. Pediatr. Allergy Immunol. 21, 577–585. Reeds, P.J., 2000. Dispensable and indispensable amino acids for humans. J. Nutr. 130, 1835S–1840S. Robert, L.S., Nozzolillo, C., Altosaar, I., 1985. Homology between rice glutelin and oat 12 S globulin. Biochim. Biophys. Acta 829, 19–26. Sarker, S.C., Ogawa, M., Takahashi, M., Asada, K., 1986. The processing of a 57-kDa precursor peptide to subunits of rice glutelin. Plant Cell Physiol. 27, 1579–1586. Schramm, R., Abadie, A., Hua, N., Xu, Z., Lima, M., 2007. Fractionation of the rice bran layer and quantification of vitamin E, oryzanol, protein: and rice bran saccharide. J. Biol. Eng. 1, 1–9. Schuster, R., 1988. Determination of amino acids in biological, pharmaceutical: plant and food samples by automated precolumn derivatization and highperformance liquid chromatography. J. Chromatogr. B 431, 271–284. Shewry, P.R., Halford, N.G., 2002. Cereal seed storage proteins: structures: properties and role in grain utilization. J. Exp. Bot. 53, 947–958. Shih, F.F., 2003. An update on the processing of high-protein rice products. Nahrung/ Food 47, 420–424. Shih, F.F., 2004. Rice proteins, In: Champagne, E.T. (Ed.), Rice: Chemistry and Technology. 3rd ed. American Association of Cereal Chemists, St. Paul, MN, pp. 143–162. Sugimoto, T., Tanaka, K., Kasai, Z., 1986. Molecular species in the protein body II (PBII) of developing rice endosperm. Agric. Biol. Chem. 50, 3031–3035. USDA, 2015. Grain: World Markets and Trade. United States Department of Agriculture, Foreign Agricultural Service, Washington, DC August 2015. Van Der Borght, A., Vandeputte, G.E., Derycke, V., Brijs, K., Daenen, G., Delcour, J.A., 2006. Extractability and chromatographic separation of rice endosperm proteins. J. Cereal Sci. 44, 68–74. Yamagata, H., Sugimoto, T., Tanaka, K., Kasai, Z., 1982. Biosynthesis of storage proteins in developing rice seeds. Plant Physiol. 70, 1094–1100. Young, V.R., Pellett, P.L., 1994. Plant proteins in relation to human protein and amino acid nutrition. Am. J. Clin. Nutr. 59, 1203S–1212S. Zhao, Q., Selomulya, C., Xiong, H., Chen, X.D., Ruan, X., Wang, S., Xie, J., Peng, H., Sun, W., Zhou, Q., 2012. Comparison of functional and structural properties of native and industrial process-modified proteins from long-grain indica rice. J. Cereal Sci. 56, 568–575.