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

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Journal of Food Composition and Analysis


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

A R T I C L E I N F O A B S T R A C T

Article history:
Received 11 March 2016 The objective of this study was to investigate the nutrient composition and protein profile of a range of
Received in revised form 29 December 2016 rice protein ingredients containing 32–78% total protein. Rice protein ingredients had significantly
Accepted 29 December 2016 (P < 0.05) lower levels of calcium and total essential amino acids compared to selected dairy protein
Available online 31 December 2016 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 (SDS-
Keywords: PAGE) and size exclusion-high pressure liquid chromatography (SE-HPLC). Since the dominant rice
Rice protein ingredients protein fraction (i.e., glutelin) is extensively aggregated and crosslinked through disulfide bonds, a strong
Dairy protein ingredients
reducing buffer was developed in order to solubilise the rice protein ingredients prior to analysis by SDS-
Macronutrient composition
PAGE. Intact rice protein ingredients (n = 3) contained proteins with molecular weight (MW) ranging
Mineral composition
Amino acid composition from 11 to >250 kDa, while rice protein hydrolysates (n = 4) were composed mainly of low MW peptides.
Protein profile analysis In parallel, enriched protein fractions were extracted from defatted rice flour based on their solubility and
Rice protein fractions characterised by SDS-PAGE to facilitate the identification of protein bands in the rice protein ingredients.
Food analysis The results of this study underpin the understanding, prediction and control of physicochemical
Food composition functionality of rice protein ingredients.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction of the two, to obtain milled or white rice, composed entirely of


endosperm (Bond, 2004; Orthoefer and Eastman, 2004).
Rice (Oryza sativa L.) is a monocotyledon plant which belongs to Varietal, environmental and processing variability all influence
the family Poaceae (Champagne et al., 2004) and is a major food the composition and properties of the rice grain and its milling
crop, with global annual production estimated at about 480 million fractions (Abdul-Hamid et al., 2007; Monks et al., 2013). Milled rice
metric tons (expressed on a milled rice basis) (USDA, 2015). It is consists of about 78% starch, while protein represents the second
grown today in more than 100 countries (Muthayya et al., 2014), most abundant constituent (6–7%). Conversely, rice bran contains
with China and India alone accounting for more than 50% of global high levels of fat (15–20%) and protein (11–15%), as well as being a
rice production (USDA, 2015). The mature rice grain is harvested in good source of fibre (7–11%). Also, rice bran displays much higher
the form of rough rice (or paddy rice), in which the caryopsis (or levels of minerals and vitamins than the other rice milling fractions
brown rice) is encased in a tough siliceous hull (or husk) (Juliano (Juliano, 1985).
and Bechtel, 1985), and needs to be processed before being The protein component of rice is generally regarded as
consumed by humans, as only the caryopsis is edible (Juliano, hypoallergenic (Helm and Burks, 1996) and its nutritional quality
1993). After cleaning of rough rice from impurities, the hull (20% of is estimated to be equivalent or higher than that of other cereals
paddy rice weight) is broken loose and separated from the rice but considerably lower compared to proteins derived from animal
caryopsis. Thereafter, the bran (8–10% of paddy rice weight), sources, legumes and oilseed crops (Day, 2013). Rice proteins are
consisting of pericarp, seed coat, nucellus, aleurone, pulverized categorized according to the solubility-based classification de-
embryo, and some starchy endosperm and hull fragments, is scribed by Osborne (1924). The four protein fractions of rice are
removed by either abrasive or friction methods, or a combination 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
* Corresponding author.
widely, depending on the rice variety and the extraction
E-mail address: sa.omahony@ucc.ie (J.A. O’Mahony). procedures. A singular feature of rice is that prolamin, which

http://dx.doi.org/10.1016/j.jfca.2016.12.026
0889-1575/© 2017 Elsevier Inc. All rights reserved.
L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26 19

represents the major endosperm storage protein in other cereals by drying the ingredients in an oven at 103  C for 5 h. Ash content
except, oats (Shewry and Halford, 2002), is a minor protein in all was determined by ashing the ingredients in a muffle furnace at
rice grain milling fractions, whereas glutelin is the dominant 500  C for 5 h. Total carbohydrate was calculated by difference (100
protein in brown and milled rice. The proportion of albumin, – sum of protein, fat, ash and moisture). Total starch, damaged
globulin, glutelin and prolamin has been reported to be 5–10, 7–17, starch and total dietary fibre were determined using enzymatic kits
75–81 and 3–6%, respectively, in brown rice, 4–6, 6–13, 79–83 and K-TSTA, K-SDAM and K-TDFR, respectively (Megazyme, Bray, Co.
2–7%, respectively, in milled rice, and 24–43, 13–36, 22–45 and 1– Wicklow, Ireland). The degree of hydrolysis of the hydrolysed
5%, respectively, in rice bran (Adebiyi et al., 2009; Agboola et al., protein ingredients (RPHs, RBPHs and WPH) was determined by
2005; Cao et al., 2009; Ju et al., 2001; Juliano, 1985; Zhao et al., the o-phthalaldehyde (OPA) method described by Nielsen et al.
2012). (2001); the value htot, defined as the number of peptide bonds in
The interest of food researchers and industry in rice protein the protein substrate (meq/g protein), was determined from the
ingredients has been limited to date, due to the relatively low total amino acid content of the protein ingredients. Assuming the
protein content of rice and the low solubility of rice proteins in average molecular weight of amino acids to be 125 g/mol, htot
water, which makes them difficult to recover and incorporate into values for RPH 1, RPH 2, RBPH 1, RBPH 2 and WPH were calculated
formulated foods (Shih, 2003). However, the awareness of the to be 7.4, 7.7, 6.7, 7.3 and 8.6 meq/g protein, respectively. pH was
nutritional and health properties of plant proteins has been measured for 5% (w/v) protein dispersions/solutions of the
increasing in recent years. Currently, alkaline, enzymatic and ingredients using a H260G benchtop pH meter (Hach Company,
physical treatments for the extraction of proteins from sources Loveland, CO, US). The mineral profile of the ingredients was
such as rice flour and rice bran are being studied, refined and determined using inductively coupled plasma-emission spectros-
applied industrially (Fabian and Ju, 2011; Shih, 2003) and rice copy (ICP-ES) according to AOAC Official Methods 984.27 and
proteins are used as value-added ingredients in nutritional 971.27 (AOAC, 2005). The amino acid profile was determined
products, including sport nutrition supplements, as an alternative according to the method of Schuster (1988). Tryptophan concen-
to the more commonly used milk and soy proteins, and infant tration was determined according to AOAC Official Method 988.15
formulas (Agostoni et al., 2007; Fiocchi et al., 2006; Lasekan et al., (AOAC, 2005).
2006; Reche et al., 2010).
The purpose of this study was to characterise the macronutrient 2.3. Extraction of rice protein fractions
composition of a range of rice protein ingredients and to compare
their mineral and amino acid profiles to that of selected dairy Extraction of rice protein fractions was performed according to
protein ingredients. A method for studying the protein profile of the method of Ju et al. (2001) with modifications. RF (100 g) was
intact rice protein-based ingredients was developed, consisting of defatted with 400 mL of hexane for 30 min, and the defatted RF was
the solubilisation of proteins in a reducing buffer prior to sodium dried under a hood at 20  C for 24 h. RF was then subjected to
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein extraction by mixing using an overhead stirrer (300 rpm)
analysis; the method allowed the characterisation of enriched rice with 400 mL of deionised water at 20  C for 4 h and centrifuged at
protein fractions, extracted from defatted rice flour based on their 3000g for 30 min to obtain albumin. After albumin extraction, the
solubility, and their profiles were used to identify the protein residue was extracted with 400 mL of 5% NaCl at 20  C for 4 h and
subunits of the rice protein ingredients analysed in this study. centrifuged at 3000g for 30 min to obtain globulin. The residue was
subsequently extracted with 400 mL of 0.02 M NaOH (pH adjusted
2. Materials and methods to 11) at 20  C for 30 min and centrifuged at 3000g for 30 min to
obtain glutelin. Prolamin was extracted separately, as extracting
2.1. Materials this rice protein fraction sequentially resulted in extensive cross-
contamination with other rice protein fractions. In order to obtain
The ingredients analysed included seven rice protein ingre- the prolamin fraction, RF (100 g) was defatted as described above,
dients, i.e., three rice protein concentrates (RPC 1, RPC 2 and RPC 3), extracted with 300 mL of 70% ethanol at 20  C for 4 h and
two rice endosperm protein hydrolysates (RPH 1 and RPH 2) and centrifuged at 3000g for 30 min. Each extraction was repeated
two rice bran protein hydrolysates (RBPH 1 and RBPH 2). Rice flour twice. Albumin, globulin and glutelin were precipitated from the
(RF) and rice bran (RB) obtained from Beneo (Tienen, Belgium) relevant supernatants by adjusting the pH to their isoelectric
were analysed as commodity rice ingredients. The nutrient points of 4.1, 4.3 and 4.8, respectively. Prolamin was precipitated by
composition of three dairy protein ingredients, i.e., low heat skim adding a threefold volume of acetone to the supernatant. The
milk powder (SMP) (33.7% protein) obtained from the Irish Dairy precipitated proteins (albumin, globulin, glutelin and prolamin)
Board (Dublin, Ireland), whey protein isolate (WPI) (89.7% protein) were washed twice with deionised water, adjusted to pH 7.0,
obtained from Davisco Foods International (Le Sueur, MN, US) and frozen (20  C) for 24 h, freeze-dried, and then stored at 4  C for
whey protein hydrolysate (WPH) (78.3% protein, DH 12.9%) subsequent analysis.
obtained from Kerry Group (Tralee, Co. Kerry, Ireland) was also
analysed. 2.4. Protein profile analysis

2.2. Compositional analysis 2.4.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


The protein profile of all samples was determined by sodium
Total nitrogen was determined by the Kjeldahl method dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
according to AOAC Official Method 930.29 (AOAC, 2005); nitro- under reducing conditions. The sample powders were solubilised
gen-protein conversion factors of 5.95 and 6.38 were used to using a modified version of a reducing buffer used in the study of
calculate total protein content of rice and dairy ingredients, Van Der Borght et al. (2006), consisting of 1% dithiothreitol (DTT),
respectively. Fat content of RPHs and RBPHs was determined using 2% SDS, 2 M thiourea and 6 M urea, by shaking at 500 rpm for 2 h at
the Röse-Gottlieb method according to AOAC Official Method 20  C, followed by centrifugation at 10,000 rpm for 15 min at 20  C
932.06 (AOAC, 2005). Fat content of RF, RB and RPCs was in order to remove any insoluble material. An aliquot of the
determined by the Soxhlet method using a Soxtec 2055 (Foss, supernatant was mixed with Lane Marker Reducing Sample Buffer
Ballymount, Co. Dublin, Ireland). Moisture content was determined (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 supernatant expressed as a percentage of the total protein content
heated at 95  C for 5 min while mixing at 300 rpm using an of the initial solution.
Eppendorf Thermomixer compact (Eppendorf, Hamburg,
Germany). Solutions were allowed to cool and sample volumes 2.5. Statistical data analysis
corresponding to 32 mg of protein (assuming that the solubilisation
buffer had solubilised all of the protein) were loaded onto pre-cast Analysis of the mineral composition of the samples was
4–20% acrylamide, 10  10 cm, tris-glycine gels (Pierce Biotechnol- performed in duplicate, with all other analyses performed in
ogy, Rockford, IL, US) in an AcquaTank mini gel unit (Acquascience, triplicate. Analysis of variance (ANOVA; Tukey’s HSD test) was
Uckfield, UK). PageRuler Plus prestained protein ladder (Pierce carried out using Minitab1 16 (Minitab Ltd, Coventry, UK)
Biotechnology, Rockford, IL, US) with molecular weight standards statistical analysis package. The level of statistical significance
ranging from 10 to 250 kDa was used as molecular weight marker. was determined at P < 0.05.
Electrophoresis was performed at 150 V for 2 h. Following
electrophoresis, the gels were stained with Bio-Safe Coomassie 3. Results and discussion
Brilliant Blue G-250 Stain (Bio-Rad Laboratories, Inc., Hercules, CA,
US). The gels were destained by washing with ultrapure water until 3.1. Macronutrient composition
a clear background was obtained. Subsequently, they were scanned
using a Scanjet G4010 desktop flatbed scanner (Hewlett-Packard, The macronutrient composition of the rice-based ingredients is
Palo Alto, CA, US) into a JPEG image format, and the molecular shown in Table 1. RB displayed a protein content (14.7%) two-fold
weight of the protein bands were estimated by densitometric higher than RF (7.32%), these values being in line with those
analysis of the scanned images using TotalLab Quant software typically reported in the literature, which show rice bran to have
version 13.2 (TotalLab Ltd., Newcastle upon Tyne, UK). the highest protein content among rice grain milling fractions
(Juliano, 1993). According to Schramm et al. (2007), protein in rice
2.4.2. Size exclusion-high pressure liquid chromatography and bran is mainly concentrated in its outer portion, as indicated by the
measurement of solubility fact that the protein content of bran samples from two long grain
The hydrolysed rice protein ingredients were reconstituted in rice varieties decreased with increasing milling time. Structural,
ultrapure water (0.25%, w/v, protein), and centrifuged at functional and nutritional properties of rice are significantly
10,000 rpm for 15 min at 20  C in order to remove any insoluble influenced by its protein component (Shih, 2004). Among the rice
material, and an aliquot (20 mL) of the supernatants was injected protein ingredients, RPC 3 had the highest protein content (78.2%),
onto the column. The analysis was carried out using a TSK Gel while RBPHs (32.0–34.6%) displayed the lowest values (Table 1).
G2000SW, 7.8 mm  600 mm column (Tosoh Bioscience GmbH, Carbohydrates represent the major constituents of cereals,
Stuttgart, Germany) and an isocratic gradient of 30% MeCN including rice. Available carbohydrates have been reported to be in
containing 0.1% TFA, with a run time of 60 min. All solvents were the range 34–62% in rice bran, 73–87% in brown rice and 77–89% in
filtered under vacuum through 0.45 mm high velocity filters milled rice (Juliano, 1993). RF displayed the highest total starch
(Millipore Ltd., Durham, UK). Commercial a-lactalbumin, b-lacto- content (75.5%), while RB contained only 15.1% starch. Starch is the
globulin B, bovine serum albumin, Cytochrome C, Bacitracin most abundant component of milled rice (Juliano, 1993). Lamberts
(Sigma-Aldrich, Dublin, Ireland), Asp-Glu and Leu-Trp-Met-Arg et al. (2007) reported the distribution of starch in brown rice to be
(Bachem AG, Bubendorf, Switzerland) were used as molecular 84.6% in the core endosperm, 6.2% in the middle endosperm, 2.8%
weight standards. Standards were prefiltered through 0.22 mm low in the outer endosperm, and the remaining 6.4% in bran. Physical
protein binding membrane filters (Sartorius Stedim, Surrey, UK) and cooking properties of rice are determined by starch and its
prior to application to the column. Solubility of the ingredients in interactions with other constituents of the rice endosperm, such as
water was measured by means of a protein solubility assay; the lipids, proteins and water, or with other ingredients used in the
ingredients were reconstituted in ultrapure water (0.25%, w/v, processing of rice (Fitzgerald, 2004). As regards the rice protein
protein), and centrifuged at 10,000 rpm for 15 min at 20  C and the ingredients, total starch content ranged from 0.39% (RPC 2) to
supernatants were analysed for their protein content by the 16.0% (RBPH 2) (Table 1).
Kjeldahl method according to AOAC Official Method 930.29 (AOAC, Milling or grinding of cereal grains to obtain flour can cause
2005). Solubility was calculated as the protein content of each 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.

RF RB RPC 1 RPC 2 RPC 3 RPH 1 RPH 2 RBPH 1 RBPH 2


Protein 7.32  0.04g 14.7  0.03f 75.0  0.38b 75.5  0.46b 78.2  0.16a 75.6  0.08b 70.5  0.38c 34.6  0.08d 32.0  0.04e
Carbohydrate 81.1  0.15a 52.3  0.26c 14.6  0.26e 9.03  0.36f 16.2  0.39d 13.7  0.20e 15.8  0.39d 52.3  0.20c 54.7  0.39b
Total starch 75.5  0.46a 15.1  0.21b 6.50  0.71de 0.39  0.14f 4.88  0.42e 7.45  0.82cd 9.54  0.76c 9.42  0.77c 16.0  0.63b
Damaged starch 9.55  0.04b 3.84  0.21f 5.66  0.10e 0.32  0.08g 4.46  0.18ef 7.28  0.18d 9.27  0.85bc 8.16  0.14cd 14.5  0.53a
Fibre 0.44  0.04ef 6.66  0.05a 0.72  0.06def 0.96  0.19cd 0.84  0.18de 0.30  0.15f 0.27  0.14f 1.35  0.17c 2.16  0.14b
Fat 0.78  0.01ef 20.9  0.20a 0.79  0.00ef 1.97  0.04d 0.95  0.04e 0.26  0.07fg 0.25  0.05g 2.73  0.27c 7.23  0.28b
Ash 1.30  0.18f 8.01  0.22b 3.42  0.24d 9.55  0.50a 2.35  0.38e 6.16  0.07c 10.0  0.04a 8.09  0.05b 3.87  0.12d
Moisture 9.53  0.02a 4.08  0.04d 6.24  0.08b 3.95  0.03d 2.25  0.05f 4.32  0.06c 3.52  0.05e 2.27  0.07f 2.14  0.06f
DH NA NA NA NA NA 17.8  0.41b 22.5  1.48a 17.7  0.37b 24.2  1.10a
pH NA NA 4.71  0.00g 5.87  0.00d 5.30  0.00f 5.84  0.01e 6.78  0.01a 6.21  0.00c 6.36  0.01b

Values followed by different superscript letters in the same row are significantly different (P < 0.05).
NA = not applicable.
L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26 21

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
lamellae, and degradation of starch molecules, which are

1325  5.00e

0.04  0.00h
62.7  0.25d

0.13  0.00g

0.28  0.00j
32.5  0.50i

0.96  0.01f
616  3.50b

279  1.50g
267  0.00f
associated with alteration of starch gelatinisation and pasting
properties (Hasjim et al., 2013). In this study, the percentage of

WPH
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).

0.04  0.00h
0.46  0.00h
0.06  0.00g
4.45  0.10e
20.0  0.00j

0.05  0.00j
62.9  0.75i
73.2  0.15c

692  1.50c
27.7  0.15f
In addition to starch, rice contains dietary fibre, defined as “the
WPI 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,
1245  5.00a

1675  5.00c

mucilages, b-glucans, inulin, arabinoxylans, arabinogalactans),

0.25  0.00h
0.03  0.00h
0.06  0.00g

3.74  0.03e
941  6.00b

961  0.50c
367  2.50f
111  0.50c

oligosaccharides, lignin and associated plant substances (waxes,


cutin, suberin) are all included in the definition of dietary fibre
SMP

(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
1715  15.0c
54.1  0.55d

6.73  0.03c
1.09  0.01e

2.14  0.01g
855  5.00d

4.17  0.05e
303  0.50d

236  2.50b
104  1.00h

fibre to be mainly concentrated in the outer layers of the rice


RBPH 2

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 constipa-
2455  15.0b
33.3  0.30ef

3.02  0.03b

8.36  0.10b
5.27  0.10d

2.82  0.04f
786  6.50b
251  4.00b
301  1.00d

841  7.50d

tion, lowering of blood cholesterol and increased satiety. Diets rich


RBPH 1

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
9.90  0.04ghi
protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from duplicate analysis.

0.59  0.00fg
2720  70.0a

1160  0.00a

0.71  0.00g

significant in terms of nutritional, sensory and functional


10.7  0.00e

2.63  0.00f
2.11  0.00c
306  1.00e
274  1.00e

properties (Godber and Juliano, 2004). Lipids in rice are classified


RPH 2

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 (Morri-
1635  75.0cd
1990  0.00a

1090  50.0a

0.59  0.03fg
1.52  0.06h
14.1  0.60e

0.34  0.03f
29.5  1.25f

1.08  0.05f
219  9.50g

son, 1995), and nonstarch lipids, the most abundant form of lipids
in rice, which are found in the aleurone, subaleurone and embryo
RPH 1

of brown rice (Godber and Juliano, 2004). The fat content followed
Values followed by different superscript letters in the same row are significantly different (P < 0.05).

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
9.40  0.00c
1.48  0.08d
18.2  0.05g

15.1  0.00e

0.73  0.00f
17.7  0.05b
26.8  0.35f

302  0.00e
466  0.50e
562  0.50c

of the rice protein ingredients (0.25–1.97%) (Table 1). Besides being


abundant, rice bran oil contains bioactive components such as
RPC 3

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
1200  0.00b
17.4  0.05gh

possess antioxidant, hypocholesterolemic and antidiabetic activi-


95.0  2.00g

4.30  0.00a

2.74  0.00d
24.8  0.05a

10.8  0.05b
9.56  0.10e
601  2.00d
17.8  2.40f

ties (Burlando and Cornara, 2014; Esa et al., 2013).


RPC 2

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
94.7  0.40h

0.43  0.00g
5.07  0.01d
4.54  0.02e

2.07  0.00c
3.79  0.01i

20.3  0.10f

11.7  0.10a
318  1.50e
133  1.00f

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
RPC 1

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
1500  10.0d

1590  10.0a

6.25  0.04d
43.3  0.30e
82.0  1.00h

0.96  0.01e
7.63  0.04c
12.4  0.05i
768  16.0a

21.1  0.15a

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
RB

being the increase in solubility (Panyam and Kilara, 1996).


Solubility is an important requirement for proteins which are to
7.32  0.01hi

0.46  0.01h
1.07  0.01e
47.1  0.30d

act as functional ingredients in food systems (Nielsen, 1997).


12.4  0.00i

0.17  0.01g
146  0.50h
37.5  0.50i

1.17  0.01i
113  0.50f

Enzymatic hydrolysis is thus essential in order to extend the range


of applications of rice protein ingredients in food products; the
RF

hydrolysed rice protein ingredients had degree of hydrolysis


ranging from 17.7% (RBPH 1) to 24.2% (RBPH 2).
Table 2

Mn
Mg
Na

Cu

Zn
Ca

Fe
Cl
K

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

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),
3.2. Mineral composition

5.42  0.07bc

3.17  0.06cd
2.54  0.03b
18.3  0.62b
1.87  0.02d
6.41  0.19b
2.83  0.05f
11.2  0.33a

5.31  0.11a
Table 2 shows the mineral composition of the ingredients.

WPH
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

17.5  0.74bc
1.85  0.05d

3.98  0.12b
2.62  0.04f

4.56  0.12c
3.24  0.10a
12.1  0.51a
c

4.10  0.10f
5.28  0.17
classification of minerals depends on the amount required in
the diet (>100 mg/day and <100 mg/day for major minerals and
WPI

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
0.77  0.05gh
d

3.64  0.07e

1.94  0.06d
9.76  0.27a

5.12  0.09a
21.0  0.69a
7.56  0.17d

5.31  0.17a
3.36  0.05

of all the minerals listed, except Na. This observation is in


agreement with the results of Itani et al. (2002), who reported the
SMP

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.
4.85  0.08bcd
8.22  0.04abc
9.29  0.22bc

5.35  0.09a
13.1  0.48d

3.47  0.03c
4.45  0.12c
a

1.40  0.03f
6.77  0.10

Ca concentration was highest in SMP (1245 mg/100 g) followed


RBPH 2

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 concentra-
8.41  0.19cd

3.05  0.01d
12.7  0.40d
a

7.37  0.07d

3.99  0.06f
5.29  0.10a
3.74  0.10d
1.33  0.01f
6.50  0.20

tions of Cl (95.0–1990 mg/100 g) compared to whey protein


RBPH 1

ingredients (20.0–32.5 mg/100 g). Also, among the protein ingre-


dients, the highest concentrations of the major minerals Cl, K, Mg,
Na and P were found within the rice protein ingredients. The
whey protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from triplicate analysis.

relatively high levels of Cl, K and Na displayed by some of the rice


9.49  0.29bc

5.26  0.16ab
bc

17.8  0.71bc

4.14  0.14cd
0.97  0.03g

4.40  0.11b
8.65  0.18a

5.21  0.12a
5.37  0.13

protein hydrolysates might be due to the addition of hydrochloric


acid (HCl) or alkaline salts such as potassium hydroxide (KOH) or
RPH 2

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/
5.03  0.16abc
4.22  0.10bc
8.37  0.16ab

17.7  0.79bc
0.62  0.01h
9.90  0.72b
c

5.04  0.10a
4.36  0.14c
5.33  0.21

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, respec-
RPH 1

tively). 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 ingre-
7.84  0.10bcd

16.9  0.05bc

4.77  0.12cd
4.12  0.05bc
8.65  0.16cd
c

4.36  0.02c

Values followed by different superscript letters in the same row are significantly different (P < 0.05).

dients had significantly (P < 0.05) higher concentrations of Zn


5.16  0.05a
1.69  0.10e
5.26  0.02

(1.52–11.7 mg/100 g) compared to whey protein ingredients (0.05–


RPC 3

0.28 mg/100 g), with RPCs displaying the highest values (9.40–
11.7 mg/100 g) among the protein ingredients analysed.
4.58  0.05de
4.18  0.08cd
8.00  0.22d

3.82  0.04b
5.25  0.06a
8.67  0.04a

13.1  0.00d
b

3.3. Amino acid composition


1.39  0.10f
5.85  0.19
RPC 2

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,
8.47  0.20cd
1.77  0.03de

4.76  0.07cd
7.81  0.10cd

4.99  0.05a
4.04  0.08c
c

16.2  0.50c

4.30  0.11c

respectively. However, their essential amino acid profiles differed,


5.06  0.07

with RF having significantly (P < 0.05) higher levels of isoleucine,


RPC 1

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
8.18  0.38abc
8.61  0.08cd

4.10  0.04cd
2.04  0.17cd

4.23  0.04ef
3.17  0.12cd

proteins (Young and Pellett, 1994). However, rice has a higher


a

12.7  0.12d
5.47  0.10a
6.44  0.20

content of lysine compared to several other cereal grains, which


explains its higher BV, as a direct correlation exists between the BV
RB

and the concentration of the first limiting essential amino acid (i.e.,
Conditionally essential amino acids.

lysine) (Eggum, 1979). The higher concentration of lysine found in


4.65  0.15cde
8.06  0.23bc
8.41  0.27cd

16.7  0.61bc

RB (4.65 g/100 g protein) compared to RF (3.16 g/100 g protein) is


4.20  0.12bc
4.17  0.19cd
c

2.14  0.03c

5.14  0.18a
5.30  0.14

due to the fact that rice bran contains a considerably higher


proportion of albumin compared to milled rice, with albumin
RF

having the highest concentration of lysine among rice protein


fractions (Juliano, 1985).
Rice protein ingredients had significantly (P < 0.05) higher
Glutamic acid
Aspartic acid

levels of the conditionally essential amino acids (i.e., amino acids


Argininey

Tyrosiney
Glyciney
Cystiney

Proliney
Alanine

Seriney

whose synthesis can be carried out by mammals but may be


Table 3

limited by several factors; Reeds, 2000) arginine and glycine


y

compared to dairy protein ingredients (Table 3). On the other hand,


L. Amagliani et al. / Journal of Food Composition and Analysis 59 (2017) 18–26 23

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
the total essential amino acid content was significantly (P < 0.05)

5.71  0.14ab
50.3  0.75a
10.9  0.31b

2.04  0.10b
7.40  0.22a
6.54  0.12a

2.37  0.10b
1.74  0.04g

10.2  0.14b

3.41  0.13g
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,

WPH
dairy protein ingredients had significantly (P < 0.05) higher levels
of the essential amino acids isoleucine (5.14–6.54 g/100 g protein),

5.03  0.14def
2.43  0.22ab
leucine (9.53–13.2 g/100 g protein) and lysine (7.65–11.6 g/100 g

1.91  0.02fg

3.80  0.19fg
13.2  0.66a

5.09  0.19b
5.89  0.18b

2.72  0.10a

51.7  1.37a
11.6  0.14a
protein) than rice protein ingredients. Among the rice protein
ingredients, RPC 2 had the highest concentration of lysine (5.62 g/
WPI

100 g protein), followed by the RBPHs (4.63–4.66 g/100 g protein),


with the remaining rice protein ingredients having lysine

4.81  0.08cd

1.48  0.05cd
concentrations in the range 3.42–3.85 g/100 g protein. Further-
2.49  0.08b

44.1  0.48b
2.77  0.09a
9.53  0.22c

5.94  0.13a
4.30  0.10c
7.65  0.11c
5.14  0.15c

more, whey protein ingredients displayed significantly (P < 0.05)


higher levels of the essential amino acids threonine (5.09–7.40 g/
SMP

100 g protein) and tryptophan (2.04–2.72 g/100 g protein) com-


pared to the other protein ingredients. The concentration of
5.46  0.13abcd

tryptophan was particularly low in RPH 1 (0.60 g/100 g protein). As


4.38  0.07de

34.5  0.33de
3.96  0.05cd
2.32  0.02bc

1.28  0.03de
3.69  0.12de
6.88  0.11ef

1.86  0.00c
4.63  0.11e

regards some of the other essential amino acids, among the protein
RBPH 2

ingredients analysed, RPC 2 and RPH 2 displayed the highest


concentrations of histidine (2.89 g/100 g protein) and phenylala-
nine (5.45 g/100 g protein), respectively.
3.78  0.06de
3.62  0.13fg
2.49  0.04b

1.26  0.05e
4.66  0.12e
5.73  0.10g
3.02  0.07f

1.71  0.01c

31.0  0.22f
4.74  0.13f

3.4. Protein profile analysis


RBPH 1
(SMP), whey protein isolate (WPI) and whey protein hydrolysate (WPH). Values are means  standard deviations of data from triplicate analysis.

The electrophoretic profiles of the four rice protein fractions are


shown in Fig. 1a. Albumin was resolved into a wide range of
5.16  0.18cdef

subunits, with molecular weight (MW) ranging from about 13 to


34.7  0.77de
7.59  0.23de
2.33  0.01bc

3.50  0.07e
1.68  0.06c
3.93  0.17d

5.45  0.14a
3.85  0.03f

1.17  0.02e

110 kDa. However, some unresolved protein material (MW > 250
kDa) was also present in the gel loading wells. Data reported in the
RPH 2

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 G-
3.73  0.14de
7.37  0.21de
2.00  0.04ef

3.42  0.02fg

4.93  0.13bc

32.5  0.79ef
4.91  0.15ef
1.96  0.07c

3.55  0.10e
0.60  0.02f

100 (Iwasaki et al., 1982). Size exclusion-high pressure liquid


RPH 1

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
5.32  0.13bcde

range 15–56 kDa by sodium dodecyl sulfate-polyacrylamide gel


2.18  0.03cde

35.5  0.52cd
5.31  0.05ab
3.44  0.06fg
2.43  0.10ab

3.48  0.07e
1.24  0.01e
4.16  0.12d
7.90  0.17d

Values followed by different superscript letters in the same row are significantly different (P < 0.05).

electrophoresis (SDS-PAGE), whereas two major albumin subunits,


having MW of 40 and 55 kDa, were determined by SDS-capillary
RPC 3

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 predomi-
5.62  0.19abc
3.77  0.06de
7.53  0.19de

4.74  0.18cd
3.87  0.12d

5.62  0.18d
1.96  0.04c

1.59  0.04c
2.89  0.10a

37.6  0.38c

nant, and two minor subunits of about 11 and 13 kDa were seen for
globulin. SDS-PAGE of rice endosperm albumin revealed two
RPC 2

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
5.21  0.21bcdef
2.26  0.05bcd

2.52  0.06ab

35.3  0.82cd
1.32  0.02de
7.73  0.22de

5.34  0.15ab
3.56  0.01fg

from 23 to 105 kDa, with the 54 kDa subunit being predominant.


3.39  0.01e
3.99  0.14d

Two major polypeptide subunits with MW of 30–40 (a or


RPC 1

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;
32.7  1.02def
4.98  0.11def
3.71  0.07de
1.47  0.06cd
3.32  0.04ef

Yamagata et al., 1982). Glutelin is synthesized first as a polypeptide


4.04  0.12ef
6.36  0.11fg
4.65  0.42e
2.45  0.15b

1.74  0.09c

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).
RB

The glutelin precursor is then enzymatically hydrolysed to yield a


and b subunits. The subsequent polymerization of the glutelin
5.34  0.17bcde
2.05  0.04def

subunits through disulfide bonds results in the formation of


34.4  1.09de
7.72  0.25de

4.94  0.15bc
2.72  0.18ab
3.86  0.14d

3.16  0.07g

3.39  0.15e
1.19  0.10e

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
RF

(glutelin precursor). Minor subunits were also observed at about


Phenylalanine

11, 13, 100 and 110 kDa, possibly due to cross-contamination with
Methionine

Tryptophan

Total EAAs

albumin and globulin. Furthermore, some unresolved protein


Threonine
Isoleucine
Histidine

Leucine

material (MW > 250 kDa) was present in this sample.


Valine
Lysine
Table 4

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 the different fractions, which allowed the identification of the
(Hibino et al., 1989; Ogawa et al., 1987). In the current study, protein subunits of the intact rice protein ingredients.
prolamin showed one major band with MW of about 10 kDa. Also, Fig. 1b shows the protein profile of RF, RB and the intact rice
two minor subunits of about 18 kDa and 31–32 kDa were present, protein ingredients. RF displayed three main bands at about 13
most likely due to cross-contamination with glutelin. The method (globulin), 18–20 (b-glutelin) and 31–33 kDa (a-glutelin), while RB
used to extract the rice protein fractions from RF coupled with their displayed a wide range of subunits, with the major subunit having
solubilisation in the strong reducing buffer prior to SDS-PAGE MW of about 58 kDa, which most likely represents a globulin
analysis, provided good resolution of the proteins characterising subunit. As regards the rice protein ingredients, RPC 1 showed a

100%

80%

<0.5 kDa
Protein distribution

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 25

profile similar to that of RF, although the intensity of the bands was including isoleucine, leucine, lysine, threonine and tryptophan is
higher for the former and a band at about 52 kDa (glutelin necessary in order for these ingredients to match, or exceed, the
precursor) was also evident. RPC 2 showed one main band at about nutritional value of dairy protein ingredients. On the other hand,
58 kDa (globulin subunit), whereas RPC 3 proved difficult to novel plant protein ingredients with a complete and well balanced
resolve and showed only one faint band at about 13 kDa (globulin amino acid profile may be obtained by combining rice with other
subunit). Unresolved high MW protein material (MW > 250 kDa) plant proteins such as those derived from legumes (e.g., peas,
was always present among the rice protein concentrates. This lentils, beans and lupins), the latter being characterised by low
result indicates that the intact rice protein ingredients could not be levels of sulphur-containing amino acids (i.e., cystine and
completely solubilised, despite the use of a strong reducing buffer methionine) but considerably higher levels of lysine compared
prior to SDS-PAGE analysis. Rice proteins have low solubility in to rice proteins. Dairy protein ingredients are currently used in a
water, mainly because of extensive aggregation and crosslinking wide range of food applications, including desserts, baked goods,
through disulfide bonds which characterise rice glutelin (Hamada, toppings, soups, sauces, salad dressings, ice cream, infant
1997; Sugimoto et al., 1986), the dominant protein fraction in formulae, medical and clinical nutrition products, protein bars,
brown and milled rice. The processing that the intact rice protein and sports and nutritional beverages. In order to assess whether
ingredients may have undergone (e.g., heat treatment), could have rice protein ingredients could partially or totally replace dairy
caused protein denaturation and further aggregation, which would protein ingredients in the aforementioned food products, investi-
have prevented their complete solubilisation. gation of their physicochemical properties is required; partial or
Electrophoretic profiles of the rice protein hydrolysates are total replacement of animal proteins with plant proteins in food
shown in Fig. 1c. RPH 1 and RPH 2 ingredients were composed of products is a strategy that is nowadays being applied commercially
protein/peptide material with MW less than 10 kDa. Conversely, for reasons of cost and sustainability. In addition, a strong reducing
RBPH 1 showed one distinct band at about 58 kDa which, according buffer was developed, which allowed the solubilisation and
to the electrophoretic profiles of the enriched rice protein characterisation of the protein profile of the enriched rice protein
fractions, may correspond to a globulin subunit. Also, both RBPH fractions and the rice protein ingredients. This study provides
1 and RBPH 2 showed unresolved high MW protein material essential knowledge for the interpretation of the physicochemical
(MW > 250 kDa) and protein material with MW < 15 kDa. properties of rice protein ingredients.
The protein profile of the hydrolysed protein ingredients was
further investigated by SE-HPLC, as represented in Fig. 2. RBPH 1 Acknowledgement
and RBPH 2 were composed of 20.8 and 13.5% insoluble protein
material, respectively, whereas RPH 1 and RPH 2 were completely The authors would like to acknowledge Dr André Brodkorb
soluble (results not shown). About 3 and 2% of the soluble protein (Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork,
material of RBPH 1 and RBPH 2, respectively, had MW greater than Ireland) for his assistance in performing size exclusion-high
20 kDa, indicating the presence of intact proteins in these pressure liquid chromatography analysis. The authors would like
ingredients, which was in agreement with the results obtained to acknowledge Nestle for providing financial support for this
by SDS-PAGE. However, the greatest proportion of the soluble study.
protein material of the hydrolysed rice protein ingredients had
MW less than 1 kDa. Furthermore, about 52, 49, 46 and 38% of the References
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