Trends in Food Science & Technology 64 (2017) 1e12

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Trends in Food Science & Technology

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Review

The composition, extraction, functionality and applications of rice

proteins: A review
Luca Amagliani a, Jonathan O'Regan b, Alan L. Kelly a, James A. O'Mahony a, *
a
School of Food and Nutritional Sciences, University College Cork, Cork, Ireland
b
Nestl

e 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: Background: Rice is a staple food and an important source of proteins in many regions worldwide. Its
Received 22 February 2016 protein component is generally regarded as hypoallergenic, and a number of studies have highlighted the
Received in revised form nutritional and health benefits associated with consumption of rice proteins. In recent years, the pro-
20 January 2017
cessing of plant proteins has drawn scientific and industrial interest, and rice protein-enriched in-
Accepted 30 January 2017
Available online 27 February 2017
gredients have become commercially available.
Scope and Approach: The aim of this study was to provide a critical review of the state of the art
regarding the composition, extraction methods, functional properties and applications of rice proteins.
Keywords:
Rice proteins
Key Findings and Conclusions: Rice is composed of four protein fractions, namely albumin (water-solu-
Composition ble), globulin (salt-soluble), glutelin (alkali-soluble), which represents the dominant protein in brown
Rice protein fractions and milled rice, and prolamin (alcohol-soluble), a minor protein in all rice milling fractions. Different
Extraction methods methods to extract proteins from rice, including alkaline, enzymatic and physical methods, have been,
Functional properties and continue to be, evaluated for their efficacy, and some have been applied industrially. However, only a
Applications limited amount of studies have described the functional properties of rice proteins and how these can be
improved by means of enzymatic hydrolysis. Applications of intact rice proteins are limited due to their
poor solubility in water. However, hydrolysed rice proteins are used in the formulation of hypoallergenic
infant formulas and may potentially be used in a variety of food systems due to their improved func-
tionality. Despite the advances in the characterisation of rice proteins, a greater understanding of their
physicochemical properties and how to modify their functionality is required in order to expand their
range of food applications.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction 2004). Proteins significantly influence structural, functional and

Rice (Oryza sativa L.) represents one of the leading food crops in
the world, with a global annual production estimated at about 480
million metric tons (milled rice basis) (USDA, 2015), and it is
cultivated today in more than 100 countries, on every continent
except Antarctica. It is the staple food for over half the world's
population, mainly in Asian countries, where it provides a consid-
erable proportion of the protein intake for millions of people
(Muthayya, Sugimoto, Montgomery, & Maberly, 2014). Its total food
protein production per hectare is second only to that of wheat,
although the yield of utilisable protein is actually higher for rice
than for wheat, due to the superior quality of rice proteins (Childs,
* Corresponding author.
E-mail address: sa.omahony@ucc.ie (J.A. O'Mahony).
nutritional properties of rice, and their characterisation has been
the subject of extensive research (see previous reviews by Fabian &
Ju, 2011; Hamaker, 1994; Juliano, 1985; Lasztity, 1995; Shih, 2003,
2004). The protein digestibility and biological value of rice have
been reported to be higher than those of the other major cereals
(i.e., wheat, corn and barley) (Eggum, 1979). Also, rice proteins are
generally regarded as hypoallergenic (Helm & Burks, 1996), with
several studies suggesting that rice proteins (in many cases, rice
protein-based hydrolysates and specific peptide fractions there-
from) have anti-oxidative (Adebiyi, Adebiyi, Yamashita, Ogawa, &
Muramoto, 2009b; Burris et al., 2010; Chanput, Theerakulkait, &
Nakai, 2009; Yang et al., 2012b; Zhang et al., 2010, 2009; Zhao
et al., 2012b), anti-hypertensive (Li, Qu, Wan, & You, 2007), anti-
cancer (Kannan, Hettiarachchy, Johnson, & Nannapaneni, 2008;
Kannan, Hettiarachchy, & Narayan, 2009; Kannan, Hettiarachchy,
Lay, & Liyanage, 2010) and anti-obesity (Yang et al., 2011, 2012a,

http://dx.doi.org/10.1016/j.tifs.2017.01.008
0924-2244/© 2017 Elsevier Ltd. All rights reserved.
2 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
2007; Kannan, Hettiarachchy, & Mahedevan, 2012; Zhang, Bartley,
Mitchell, Zhang, & Yokoyama, 2011) activities. Therefore, rice rep-
resents an interesting source of proteins for the development of
protein-enriched ingredients for the formulation and manufacture
of nutritional products.
2. Distribution and composition of proteins in rice
The protein content of rice is usually calculated from Kjeldahl
nitrogen multiplied by the nitrogen-to-protein conversion factor of
5.95, which is based on the nitrogen content (16.8%) of glutelin, the
major rice protein, although a nitrogen-to-protein conversion fac-
tor of 6.25 is used in nutritional studies in order to maintain a
common nitrogen-based calculation for all proteins (Shih, 2004).
Rice grain milling fractions differ in terms of protein content, with
that of brown rice, i.e., the rice caryopsis, being higher compared to
milled or white rice (composed entirely of endosperm) due to the
removal of the protein-rich bran during milling (Table 1). Rice bran
consists of a fine, floury material, which includes pericarp, seed
coat, nucellus, aleurone, pulverized embryo, and some hull and
endosperm fragments (Orthoefer & Eastman, 2004) (Fig. 1). The
protein content of rice is influenced by factors such as management
and cultural practices, climate and genotype (Champagne, Wood,
Juliano, & Bechtel, 2004).
2.1. Rice protein fractions
Rice proteins are categorized according to the solubility-based
classification described by Osborne (1924); albumin (water-solu-
ble), globulin (salt-soluble), glutelin (alkali/acid-soluble), and pro-
lamin (alcohol-soluble) are the four main rice protein fractions.
Rice proteins are mainly observed in the form of storage or-
ganelles called protein bodies (PBs). Two types of morphologically
distinct PBs have been identified in rice endosperm, namely type I
(PB-I) and type II (PB-II) (Fig. 2). PB-I has a lamellar structure, is
spherical in shape and is rich in prolamin, whereas PB-II has a
crystalline structure, displays an irregular shape and contains
predominantly glutelin (Bechtel & Juliano, 1980; Bechtel &
Pomeranz, 1978; Ogawa et al., 1989, 1987; Tanaka, Sugimoto,
Ogawa, & Kasai, 1980). According to Ogawa et al. (1987), endo-
sperm storage proteins comprise 60e65% PB-II, 20e25% PB-I and
10e15% albumin and globulin in the cytoplasm.
Rice grain milling fractions differ in terms of protein composi-
tion (Table 2). Data reported in the literature regarding rice protein
solubility fractions vary widely, depending on the rice variety and
the extraction procedures. Albumin, globulin, glutelin and prolamin
have been reported to account for 5e10, 7e17, 75e81 and 3e6%,
respectively, of total protein in brown rice, 4e6, 6e13, 79e83 and
2e7%, respectively, of total protein in milled rice, and 24e43,
13e36, 22e45 and 1e5%, respectively, of total protein in rice bran
(Adebiyi, Adebiyi, Hasegawa, Ogawa, & Muramoto, 2009a; Agboola,
Ng, & Mills, 2005; Cao, Wen, Li, & Gu, 2009; Ju, Hettiarachchy, &
Rath, 2001; Juliano, 1985; Zhao et al., 2012a). Glutelin is the main
Table 1
Chemical composition (%) of rice grain milling fractions at 14% moisture (adapted
from Juliano & Bechtel, 1985).
Brown Milled
Protein (N
5.95) 7.1e8.3 6.3e7.1
Crude fat 1.6e2.8 0.3e0.5
Available carbohydrates 73e76 77e78
Starch 66 78
Crude fiber 0.6e1.0 0.2e0.5
Crude ash 1.0e1.5 0.3e0.8
protein fraction in brown and milled rice, whereas prolamin, which
represents the major endosperm storage protein in all other cereals
except oats (Shewry & Halford, 2002), is a minor protein in all rice
grain milling fractions.
Differences in protein composition of the rice milling fractions
result in differences in the nutritional quality of their protein
component. Han, Chee, and Cho (2015) showed that rice bran
protein has a higher nutritional quality than rice endosperm pro-
tein; protein efficiency ratio (PER), net protein retention (NPR) and
net protein utilisation (NPU) were 2.39, 3.77 and 70.7, respectively,
for rice bran protein, and 1.96, 3.26 and 61.4, respectively, for rice
endosperm protein. PER and NPR were higher for both rice protein
ingredients compared to soy protein isolate (SPI) (1.71 and 3.04,
respectively, for SPI), but lower compared to casein (2.58 and 4.02,
respectively, for casein) and whey protein isolate (WPI) (3.26 and
4.54, respectively, for WPI). Of those protein ingredients analysed
by Han et al. (2015), WPI was the only ingredient with a higher NPU
(74.0) than rice bran protein. Also, rice bran protein had true di-
gestibility (TD), biological value (BV) and protein digestibility-
corrected amino acid score (PDCAAS) of 94.8%, 72.6% and 0.90,
respectively, whereas the same indices for rice endosperm protein
were 90.8%, 66.7% and 0.63, respectively. PDCAAS of rice endo-
sperm protein, but not that of rice bran protein, was considerably
lower compared to that of the other ingredients analysed, which
had values ranging from 0.95 (SPI) to 1.00 (casein and WPI).
However, for both rice protein ingredients, TD was comparable to
that of SPI (91.7%) and dairy protein ingredients (92.8e94.8%),
whereas BV was lower than only that of WPI (78.8%). Furthermore,
rice endosperm and rice bran proteins had a similar content of total
essential amino acids (41.1e41.7 g/100 g protein) to that of SPI
(41.2 g/100 g protein), but these values were significantly lower
compared to those observed for casein and WPI (50.6e55.2 g/100 g
protein) (Table 3).
An analysis of the amino acid profile of individual rice proteins
(Table 4) shows that prolamin has the lowest content of lysine, the
first limiting amino acid among cereal proteins (Young & Pellett,
1994). The highest lysine content is found in albumin, followed
by glutelin and globulin. Being rich in albumin, rice bran displays a
higher content of lysine compared to brown or milled rice. Also, due
to the low content of prolamin, rice has a higher lysine content than
other cereal grains (Day, 2013). As regards the other essential amino
acids, albumin shows the highest content of histidine and threo-
nine, whereas prolamin has the highest proportions of isoleucine,
leucine and phenylalanine. Furthermore, globulin has the highest
content of the sulfur-containing amino acids cysteine and methi-
onine, while prolamin has the lowest. Based on their amino acid
composition, albumin has been estimated to have the highest and
prolamin the lowest BV among the rice protein fractions (Padhye &
Salunkhe, 1979).
2.1.1. Albumin
Albumin is readily soluble in water due to the presence of suf-
ficient net charge and the lack of any extensive disulfide cross-
linking or aggregation (Hamada, 1997). However, globulin
contamination occurs when albumin is extracted with water,
because of minerals present in the rice grain that dissolve in water,
thus increasing the solubility of globulin (Villareal & Juliano, 1981).
Therefore, in order to obtain purified albumin at laboratory scale,
Bran additional steps have to be carried out, which include repeated
precipitation, centrifugation and dialysis (Hamada, 1997; Iwasaki,
11e15
15e20 Shibuya, Suzuki, & Chikubu, 1982; Mawal, Mawal, & Ranjekar,
34e52 1987). Application of isoelectric focusing (IEF) (pH 3.5e10) to mil-
14 led rice albumin revealed 16e18 bands, 10e11 of which had iso-
7.0e11 electric point (pI) in the range 6.0e7.5 (Villareal & Juliano, 1981).
6.6e9.9
However, only one band, with pI 6.4, was found in the study of
 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12 3

Fig. 1. Structure of rice grains (from Blakeney, 1984).

Fig. 2. Schematic structure of rice protein bodies and compound starch granule in the endosperm subaleurone layer (from Coffman & Juliano, 1987).

Table 2 Padhye and Salunkhe (1979), whereas Mawal et al. (1987) reported
Distribution (%) of rice proteins among rice grain milling fractions (data collated a major fraction of rice albumin to be a 60 kDa glycoprotein with pI
from Adebiyi et al., 2009a; Agboola et al., 2005; Cao et al., 2009; Ju et al., 2001;
6.5. Following extraction from defatted rice flour, turbidity mea-
Juliano, 1985; Zhao et al., 2012a).
surement of the supernatant indicated that albumin had pI of pH
Brown Milled Bran 4.1 (Ju et al., 2001).
Albumin 5e10 4e6 24e43 Gel filtration on Sephadex G-100 showed the molecular weight
Globulin 7e17 6e13 13e36 (MW) of milled rice albumin to range from 10 to 200 kDa (Iwasaki
Glutelin 75e81 79e83 22e45
et al., 1982). According to Hamada (1997), fully dissociated rice bran
Prolamin 3e6 2e7 1e5
4 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12

Table 3
Essential amino acid (EAA) composition (g/100 g protein) of rice endosperm protein
(REP), rice bran protein (RBP), soy protein isolate (SPI), casein and whey protein
isolate (WPI) (adapted from Han et al., 2015).
10e150 kDa (Hamada, 1997). Brown rice globulin was resolved by
SDS-CE into 6 subunits with MW ranging from 23 to 105 kDa, with
the 54 kDa one being predominant (Agboola et al., 2005).

Amino acid REP RBP SPI Casein WPI 2.1.3. Glutelin

Histidine 2.46 4.48 2.58 2.97 1.97 Native rice glutelin has low solubility in water but is readily
Isoleucine 3.80 3.61 4.30 4.88 6.24 soluble under alkaline (pH > 10) or acidic (pH < 3) conditions; it has
Leucine 8.15 7.69 7.84 9.73 10.9
a high MW, and is heterogeneous (Juliano, 1985). Glutelin is
Lysine 3.31 4.55 6.14 8.15 9.96
Methionine þ Cysteine 3.88 2.70 2.55 2.94 5.36 composed of two major polypeptide subunits, with MW of 30e40
Phenylalanine þ Tyrosine 10.1 8.24 8.65 10.7 5.94 (a or acidic) and 19e23 kDa (b or basic) (Agboola et al., 2005;
Threonine 3.46 3.68 3.67 4.30 7.52 Chrastil & Zarins, 1992; Krishnan & Okita, 1986; Luthe, 1983;
Tryptophan 0.82 1.17 1.28 1.05 1.72
Robert, Nozzolillo, & Altosaar, 1985; Sarker, Ogawa, Takahashi, &
Valine 5.12 5.53 4.16 5.88 5.57
Total EAAs 41.1 41.7 41.2 50.6 55.2
Asada, 1986; Sugimoto, Tanaka, & Kasai, 1986; Wen & Luthe,
1985; Yamagata, Sugimoto, Tanaka, & Kasai, 1982; Zhao,
Gatehouse, & Boulter, 1983). Padhye and Salunkhe (1979) re-
ported the presence of 7 acidic (pI 5.7e6.9) and 5 basic (pI 8.0e8.7)
Table 4
glutelin bands in milled rice, as determined by IEF. In the study of
Amino acid composition (g/16.8 g nitrogen) of rice protein fractions (adapted from
Juliano, 1985). Wen and Luthe (1985), a and b glutelins were resolved into at least
12 (pI 6.5e7.5) and 9 (pI 9.4e10.3) bands, respectively. According to
Amino acid Albumin Globulin Glutelin Prolamin
Zarins and Chrastil (1992), purified acid (33 kDa) and basic (22 kDa)
Alanine 7.1e8.5 5.6e6.3 5.6e5.9 6.7e7.6 glutelin subunits can be separated into 9 (pI 5.0e8.0) and 5 (pI
Arginine 7.9e10 7.2e14 9.0e11 6.1e6.9
8.0e11) bands, respectively. Ju et al. (2001) reported glutelin to
Aspartic acid 10e11 7.1e14 10e11 8.3e8.7
Cysteine 1.9e2.3 3.3e4.0 1.2e1.8 trace-0.8
have a pI of pH 4.8, as determined by turbidity measurement of the
Glutamic acid 13e18 17e19 19e21 23e33 supernatant following extraction from defatted rice flour.
Glycine 6.3e8.4 5.8e6.4 4.3e5.3 3.0e3.7 Glutelin is synthesized first as a polypeptide with a MW re-
Histidine 2.9e3.4 1.7e2.7 2.6e2.7 1.3e2.1 ported to be in the range 51e57 kDa (Krishnan & Okita, 1986; Luthe,
Isoleucine 3.5e3.8 2.4e4.1 4.3e4.7 4.6e5.2
1983; Sarker et al., 1986; Yamagata et al., 1982); the glutelin pre-
Leucine 6.6e8.0 6.6e6.8 7.3e9.3 13e15
Lysine 5.1e6.4 1.9e3.7 2.7e4.5 0.3e1.2 cursor is then enzymatically hydrolysed to yield a and b subunits of
Methionine 1.9e2.1 3.0e5.4 2.0e3.1 0.5e0.9 the protein. The subsequent polymerization of the glutelin subunits
Phenylalanine 3.7e4.6 3.3e4.8 5.4e6.0 5.8e6.7 through disulfide bonds results in the formation of macromolecular
Proline 4.5e7.1 3.8e7.5 4.9e6.2 5.0e6.7
complexes (Sugimoto et al., 1986). The lack of functional properties
Serine 4.2e5.4 5.5e6.5 4.5e6.2 4.2e6.1
Threonine 4.2e5.2 2.5e2.7 2.8e5.1 2.5e2.8
of native rice glutelin may be explained by the formation of high
Tryptophan 1.5e1.8 1.4e1.5 1.0e1.6 0.5e2.6 MW water-insoluble protein structures.
Tyrosine 4.4e5.1 5.5e6.3 5.3e5.5 9.2e9.9
Valine 5.9e7.8 5.4e6.5 6.3e6.9 6.5e7.1 2.1.4. Prolamin
Prolamin is soluble in alcohol solutions (e.g., 60e70% (v/v)
ethanol) but essentially insoluble in water (Shewry & Casey, 1999).
albumin polypeptides have MW ranging from 10 to 100 kDa, as IEF (pH 3.5e10) of milled rice prolamin revealed 5 bands with pI
determined by size exclusion-high pressure liquid chromatography ranging from 6.0 to 6.5 (Padhye & Salunkhe, 1979). SDS-PAGE
(SE-HPLC). Brown rice albumin was resolved into 6 bands with MW showed that prolamin consists of three polypeptide subunits hav-
in the range 15e56 kDa by sodium dodecyl sulfate-polyacrylamide ing MW of 10, 13 and 16 kDa, with the 13 kDa prolamin being
gel electrophoresis (SDS-PAGE), whereas SDS-capillary electro- predominant (Hibino et al., 1989; Ogawa et al., 1987). The 13 kDa
phoresis (SDS-CE) revealed the presence of two major albumin prolamin was found to be composed of at least 7 subunits with pI
subunits having MWs of 40 and 55 kDa (Agboola et al., 2005); in- values in the range 5.0e9.0, whereas the 10 kDa prolamin consisted
consistencies in results obtained in the reported studies may be of two subunits with pI 8.9 and 10, and the 16 kDa prolamin was a
ascribed to differences in rice varieties, extraction procedures and single polypeptide, as determined by IEF (Hibino et al., 1989).
analytical tools used. Analysis of the amino acid composition of rice bran prolamin
polypeptide subunits showed the 13 kDa prolamin to have a high
2.1.2. Globulin content of glutamic acid and leucine, but a low content of lysine and
Globulin is easily solubilised by salt solutions mainly because of sulfur-containing amino acids; on the other hand, sulfur-containing
its net electrical charge (Hamada, 1997). IEF (pH 3.5e10) of milled amino acids were abundant in the 10 and 16 kDa prolamins, with
rice globulin showed 9 bands with pI in the range 5.9e7.3 (Padhye the 10 kDa prolamin being particularly rich in methionine (Hibino
& Salunkhe, 1979). However, Ju et al. (2001) reported two pI values et al., 1989).
for rice globulin, at pH 4.3 and pH 7.9, as determined by turbidity
measurement of the supernatant after extraction from defatted rice 3. Extraction of proteins from rice
flour.
According to Iwasaki et al. (1982), milled rice globulins have In the past, there was little interest in the processing of rice
MW ranging from 16 to 130 kDa, as determined by gel filtration on proteins, mainly because of the relatively low protein content of
Sephadex G-100. Two rice endosperm globulin polypeptides, hav- rice, the low solubility of rice proteins in water, and the limited
ing MW of 16 kDa and 25 kDa, have been identified by SDS-PAGE; commercial opportunities for such protein-enriched ingredients.
double-label immunogold electron-microscopic localization indi- Rice flour from milled rice or broken rice kernels has been typically
cated that both globulin polypeptides are localized in discrete processed industrially to extract the starch, while the residual
zones within the irregularly shaped protein bodies (Krishnan, protein-containing fractions have traditionally been regarded as
White, & Pueppke, 1992). SE-HPLC showed the MW of fully- co-products of limited value. Also, despite being relatively rich in
dissociated rice bran globulin polypeptides to be in the range proteins, rice bran has been under-utilised as a food material given
 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
its low starch content, and has mainly been used as an ingredient in
animal feed or even as boiler fuel (Fabian & Ju, 2011; Shih, 2003).
However, the nutritional and health properties of plant proteins in
general, and rice proteins in particular, are widely recognized today,
and rice protein-containing products have become commercially
available in recent years. The increasing interest of industry and the
academic research communities in these ingredients enabled the
development of processes for the extraction, enrichment, purifi-
cation and functionalisation of rice proteins. Alkaline, enzymatic
and physical treatments have all been, and continue to be, evalu-
ated for their potential in the extraction of proteins from rice, and
some have been applied industrially.
The processing of rice bran proteins represents a challenge. Rice
bran contains high amounts of fat, i.e., 15e20% by weight (Table 1),
which provides a substrate for the activity of lipase, lipoxygenase
and peroxidase enzymes, resulting in the development of rancidity
and off-flavours (Orthoefer & Eastman, 2004). Therefore, rice bran
needs to be defatted (by hexane extraction, wet extrusion or me-
chanical pressing) or stabilised by heat treatment (e.g., retorting,
extrusion cooking, fluidized bed drying, microwave heating, ohmic
heating, pan roasting or parboiling) or reduction of pH in order to
inhibit lipase and oxidative enzymes (Khan et al., 2011; Lakkakula,
Lima, & Walker, 2004; Orthoefer & Eastman, 2004; Ramezanzadeh,
Rao, Prinyawiwatkul, Marshall, & Windhauser, 2000; Sayre,
Saunders, Enochian, Schultz, & Beagle, 1982). However, several of
these treatments have the potential to induce protein denaturation
and/or aggregation, thus reducing protein solubility and making
the processing of rice bran proteins even more challenging.
Furthermore, rice bran contains fiber (7e11%) and phytate
(0.9e2.2%) (Orthoefer & Eastman, 2004), which are extensively
associated with/bound to proteins, making the separation of pro-
tein bodies from these components difficult to achieve (Tang,
Hettiarachchy, & Shellhammer, 2002). On the other hand, rice
flour contains endosperm storage proteins, which appear to be
more readily extractable than rice bran proteins (Shih, 2003).
3.1. Alkaline extraction
In the food industry, alkaline treatment of rice flour is generally
applied to facilitate removal of the endosperm proteins for the
production of purified starch, as opposed to direct extraction of
proteins, with the protein fraction being traditionally discarded or
used as an ingredient in animal feed formulations (Shih, 2003).
However, alkaline conditions are particularly effective in extracting
proteins from rice flour, where glutelin is the dominant protein
fraction. Cagampang, Cruz, Espiritu, Santiago, and Juliano (1966)
reported that dilute alkaline solvents (e.g., 0.1 N potassium or so-
dium hydroxide) extracted up to 97% of total proteins from milled
rice flour. According to Paraman, Hettiarachchy, and Schaefer
(2008), alkaline extraction (pH 11, 40  C) of milled rice flour fol-
lowed by either ultrafiltration or isoelectric precipitation of the
supernatant provided products with 71 and 86% protein, respec-
tively, with proteins recovered by ultrafiltration having superior
functional properties.
Alkaline treatment followed by isoelectric precipitation (pH
4e4.5) has also been used for the extraction of proteins from rice
bran, resulting in products with protein contents ranging from
about 40 to >80% (Bandyopadhyay, Misra, & Ghosh, 2008; Chandi &
Sogi, 2007; Gnanasambandam & Hettiarachchy, 1995;
Jiamyangyuen, Srijesdaruk, & Harper, 2005; Prakash &
Ramanatham, 1994; Yadav, Yadav, & Chaudhary, 2011). Rice
cultivar, degree of milling, specific alkaline conditions, the use of
unstabilised or stabilised rice bran, and the stabilisation technique
employed may account for differences in terms of protein content,
5
with thermal stabilisation reducing the extractability of proteins
and altering the protein profile and amino acid composition of the
extracted proteins (Gnanasambandam & Hettiarachchy, 1995; Khan
et al., 2011; Prakash & Ramanatham, 1994).
In vitro and in vivo studies demonstrated that rice proteins
prepared by alkaline extraction have higher digestibility and
bioavailability compared to those prepared by degradation of starch
by a-amylase as a result of the structural changes in the poorly
digestible PB-I caused by alkaline extraction (Kubota et al., 2010;
Kumagai et al., 2006, 2009). However, alkaline extraction pre-
sents some drawbacks, as alkaline conditions could lead to (i)
denaturation and hydrolysis of proteins, (ii) increased extraction of
non-protein components that co-precipitate with proteins,
lowering the protein purity of the resultant ingredient, (iii)
increased Maillard reactions (due to high pH) resulting in dark
coloured (i.e., melanoidin-based) products, and (iv) the formation
of toxic compounds such as lysinoalanine (Otterburn, 1989; Wang,
Hettiarachchy, Qi, Burks, & Siebenmorgen, 1999).
3.2. Enzymatic extraction
Extraction of proteins from rice flour and rice bran for food
applications is normally achieved by means of enzymatic methods.
Depending on rice cultivar, degree of milling and enzymatic treat-
ment, products with protein content higher than 90% (i.e., protein
isolates) can be obtained by the processing of rice flour with starch-
hydrolysing enzymes. Since starch represents the major constituent
of rice endosperm, enzymes such as a-amylase, glucoamylase and
pullulanase, which solubilise starch, are often used to separate
proteins in rice flour (Shih, 2003). However, because of the high
cost and low protein content of milled rice and rice flour, rice
proteins can be more conveniently extracted from a low-cost
source such as rice dregs, a by-product from the processing of
rice syrups, which has protein content higher than 50% (Zhao et al.,
2013). In the study of Shih and Daigle (2000), treatment of a
protein-enriched rice flour (brown rice protein concentrate) with
Termamyl 120L (an a-amylase) (at pH 6.5, 90  C) followed by Dia-
zyme L200 (a glucoamylase; at pH 4, 60e62  C), resulted in a
product (insoluble fraction) with 85% protein, which was increased
to 91% protein by a subsequent treatment with Viscozyme L (a
multi-enzyme complex containing a range of carbohydrate-
digesting enzymes, including arabanase, cellulase, b-glucanase,
hemicellulase and xylanase).
Carbohydrate-digesting enzymes (e.g., cellulase, hemicellulase,
pectinase and xylanase), which hydrolyse high MW cell wall
components, have mainly been employed to enhance protein
extractability from rice bran, by liberating proteins from the
polysaccharide-based structures. Ansharullah, Hourigan, and
Chesterman (1997) reported that treatment of full-fat extruded
rice bran with Viscozyme L at pH 3.8 and 50  C would result in the
extraction of 58% nitrogen, as predicted by response surface
methodology. A rice bran protein isolate with a protein content of
92% and a protein yield of 75% was prepared from defatted unsta-
bilised rice bran using a combination of phytase and xylanase (pH 5,
55  C) (Wang et al., 1999); however, the alkaline treatment applied
(pH 10) to inactivate the enzymes prior to the recovery of proteins
may have caused an increase in protein extractability. Using the
same combination of enzymes and a similar extraction method as
Wang et al. (1999), Khan et al. (2011) obtained protein concentrates
from defatted unstabilised and heat-stabilised (microwave, dry
heat, parboiling) rice bran having protein contents ranging from
61% (parboiled rice bran) to 85% (unstabilised rice bran).
Proteases have also been used for extracting proteins from rice;
a number of food-grade commercially available proteases of animal,
6 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
bacterial, fungal or plant origin have been evaluated for this pur-
pose. However, the protein extracted with the aid of protease
treatment is hydrolysed (i.e., the product is a hydrolysate), with
different structural and physicochemical properties to the intact
proteins. Treatment of broken rice flour with an alkaline protease
(pH 10, 45  C) or papain (pH 7, 50  C) generated protein hydroly-
sates with protein contents (calculated from nitrogen levels, and
thus including intact proteins and fragments thereof) of 56 and
65%, respectively, with protein recoveries being 75 and 46%,
respectively (Hou, Zhu, & Li, 2010). In the study of Li et al. (2012), a
hydrolysate with a protein content of 81%, protein recovery of 76%,
and degree of hydrolysis (DH) of 7% was obtained by treating rice
dregs with trypsin under optimised conditions (pH 7.6, 52.8  C).
Nitrogen yields of rice dregs-based protein hydrolysates generated
using Neutrase (pH 7.6, 50.4  C), Alcalase (pH 9.2, 50.2  C),
bromelain (pH 7.3, 56.5  C) or papain (pH 6.9, 51.5  C) under
optimal conditions were 49, 57, 31 and 17%, respectively, with the
protein contents of the products being 79, 63, 58 and 55%,
respectively (Guo, Zhang, Ma, & Tian, 2013).
Hamada (1999) reported that about 74e92% of the proteins of
defatted rice bran could be extracted by treatment with Opticlean
(an alkaline protease) (pH 8 or 10, 40  C) to 2e10% DH values.
Alcalase and Flavourzyme (pH 8, 50  C), extracted 81 and 88% of
proteins from defatted rice bran, respectively, resulting in protein
hydrolysates having protein contents of 28% (DH 7.5%) and 30% (DH
8.8%), respectively (Hamada, 2000).
3.3. Physical extraction
Physical methods are usually preferred to chemical or enzymatic
methods in the processing of food, as they induce less modifica-
tions (Shih, 2003), as well as generally being more economical and
easier to be adapted and utilised in industry. Freeze-thaw, high-
speed blending, sonication, hydrothermal cooking (HTC), micro-
fluidization and the use of subcritical water (SW) are among the
physical approaches tested to extract proteins from rice.
Several physical treatments (i.e., freeze-thaw, high-speed
blending, high pressure and sonication) have been investigated for
their effectiveness in facilitating the extraction of proteins from
heat-stabilised defatted rice bran (Tang et al., 2002). Among the
physical treatments tested, sonication gave the highest protein
extraction yield, but this was only 15%. However, protein extract-
ability increased significantly when these physical treatments were
coupled with the action of enzymes such as a-amylase and Protease
P. According to Xia et al. (2012a), colloid milling of broken rice
followed by microfluidization and density-based separation pro-
vided protein yield of 82% and protein purity of 44%, with protein
purity increasing further to 88% when subsequently treated with
amylase and glucoamylase enzymes. The proteins obtained using
this method had higher in vitro digestibility but considerably lower
solubility in the pH range 6e10 than those obtained by alkaline
extraction. Xia et al. (2012b) employed HTC, a steam-infusion
treatment characterised by high temperature and shearing, to
extract proteins from heat-stabilised rice bran. Protein yield and
protein purity were 37 and 28%, respectively, at 120  C, and 50% and
53%, respectively, at 150  C. When combining HTC processing with
amylase pre-treatment at 120  C or 150  C, protein purity levels
increased from 28-53% to 72e74%, whereas protein yield improved
only for the 120  C treatment (from 37 to 45%). In contrast, alkaline
extraction followed by acid precipitation resulted in lower protein
yield (15%) at a protein purity of 68%. Depending on HTC temper-
ature and amylase pre-treatment, HTC-prepared protein isolates
exhibited higher surface hydrophobicity, sulfhydryl and disulfide
bond contents, and superior emulsifying properties, compared to
alkali-extracted proteins.
An emerging method for the extraction of proteins from rice is
that based on the use of water at subcritical conditions. Subcritical
water (SW) extraction employs water at temperatures between 100
and 374  C (the latter being the critical temperature of water) and
pressure high enough to maintain its liquid state. When the tem-
perature of the water is increased from ambient to 250  C, its
relative dielectric constant decreases from around 80 to values
close to 27, which is similar to that of ethanol at ambient temper-
ature (Herrero, Cifuentes, & Iban ~ ez, 2006); therefore, SW has the
ability to dissolve hydrophobic material. Also, because at subcritical
conditions the dissociation constant of water (Kw) to hydrogen and
hydroxyl ions is much greater than that of ambient water, SW can
catalyse chemical reactions such as degradation and hydrolysis
(Hata, Wiboonsirikul, Maeda, Kimura, & Adachi, 2008;
Wiboonsirikul et al., 2007); therefore, biomolecules such as car-
bohydrates (Haghighat Khajavi, Ota, Kimura, & Adachi, 2006) and
proteins (Sereewatthanawut et al., 2008; Sunphorka, Chavasiri,
Oshima, & Ngamprasertsith, 2012; Watchararuji, Goto, Sasaki, &
Shotipruk, 2008) can be hydrolysed in SW without any additional
catalyst.
SW has been utilised to extract various functional compounds
(proteins, amino acids, carbohydrates and phenolics) from rice bran
(Hata et al., 2008; Sereewatthanawut et al., 2008; Watchararuji
et al., 2008; Wiboonsirikul et al., 2007). Treatment of rice bran
with water at subcritical conditions has provided high protein
yields. In the study of Watchararuji et al. (2008), 84% of protein was
extracted from defatted rice bran using SW at 220  C for 30 min.
Sereewatthanawut et al. (2008) reported that the amount of pro-
tein recovered from defatted rice bran in the extract solubilised by
SW treatment at 200  C for 30 min was comparable to the amount
in the original bran. This was mainly attributed to the breakdown of
proteins into soluble peptides or amino acids, due to the increase of
Kw at high temperatures. Sunphorka et al. (2012) proposed a
mechanism to explain the changes occurring in rice bran proteins
during SW treatment, which includes three steps: (i) protein ag-
gregation, (ii) protein disaggregation by hydrolysis to polypeptides
and (iii) amino acid liberation; these steps follow first-, second- and
zero-order reaction kinetics, respectively. Soluble rice bran extracts
resulting from SW extraction displayed radical scavenging and
antioxidant activity (Hata et al., 2008; Sereewatthanawut et al.,
2008; Watchararuji et al., 2008; Wiboonsirikul et al., 2007;
Wiboonsirikul, Kimura, Kanaya, Tsuno, & Adachi, 2008). Although
SW treatment appears to be a promising technique for the
extraction of proteins from rice, so far it has only been used as a
batch process at pre-commercial scale and further research is
required to evaluate the quality, stability and functionality of the
extracted proteins.
4. Functional properties of rice proteins
The utilisation of proteins as food ingredients depends on their
functional properties. These properties, which determine the
behaviour and performance of proteins in food systems (i.e., func-
tionality) during preparation, processing, storage and consumption,
are influenced by the nature and extent of interactions of the
proteins with themselves, with other components, and with water
in the food system (Panyam & Kilara, 1996). The functional prop-
erties required depend on the application of the protein in-
gredients. Since many formulated foods are physically presented as
beverages, emulsions or foams, the protein ingredients used in
their formulation have to possess good solubility, heat stability and
surface-active properties. Water and oil absorption ability of pro-
teins are also essential properties for the formulation of a range of
processed foods.
 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
4.1. Solubility
Solubility of proteins is, under a given set of conditions, the
thermodynamic manifestation of the equilibrium between protein-
protein and protein-solvent interactions, and is directly related to
the physicochemical nature of the protein surface (Damodaran,
1994). This property represents an important prerequisite for
proteins which are to act as functional ingredients in food systems
(Nielsen, 1997). Factors such as temperature, pH, salt concentration
and dielectric constant of the solvent alter protein solubility, as
variations in these conditions lead to changes in protein structural
conformations, which in turn affect protein functionality (Adebiyi,
Adebiyi, Ogawa, & Muramoto, 2007). Rice proteins display mini-
mum solubility in water in the pH range 4e5, while solubility in-
creases with increasing alkalinity or acidity (Cao et al., 2009;
Chittapalo & Noomhorm, 2009; Gnanasambandam & ditions of pH (5e9), NaCl (0.5e1.5%) and sucrose (5e15%) concen-
Hettiarachchy, 1995; Khan et al., 2011; Romero et al., 2012; Shih
& Daigle, 2000; Shih, Champagne, Daigle, & Zarins, 1999; Wang
et al., 1999; Xia et al., 2012b; Zhao et al., 2012a, 2013). The pH-
dependent aqueous solubility of rice proteins is mainly influ-
enced by the properties of glutelin, as it represents the dominant
protein fraction in the endosperm and accounts for a significant
proportion of total proteins in rice bran (Table 2). The high insol-
ubility of rice glutelin in water is mainly due to its extensive ag-
gregation and crosslinking through disulfide bonds (Hamada, 1997;
Sugimoto et al., 1986). Extreme acidic and alkaline conditions
promote dissociation of glutelin aggregates and thus increase its
solubility. In the study of Cao et al. (2009) solubility of rice bran
proteins was found to be higher than that of proteins from brown
and white rice in the pH range 4e7, whereas white rice proteins had
higher solubility than rice bran and brown rice proteins at pH
values below 4 and above 7, being >80% at pH 10. A rice protein
concentrate, mainly composed of glutelin, displayed poor solubility
(25e55%) in the pH range 2e10, with a minimum at pH 5 (Romero
et al., 2012). According to Zhao et al. (2012a), rice endosperm and
rice dregs-based protein isolates had maximum solubility of 64 and
73%, respectively, at pH 11, and minimum solubility at pH 5 (<1%).
Solubility values of 53, 8, 62, 78, 82 and 80% at pH 2, 4, 6, 8, 10 and
12, respectively, were reported for a rice bran protein isolate ob-
tained by enzymatic extraction (Wang et al., 1999). In the study of
Chittapalo and Noomhorm (2009), rice bran protein concentrates
obtained by alkaline extraction and ultrasonic-assisted alkaline
extraction exhibited minimum solubility (2e4%) in the pH range
4e6 and maximum solubility (85e95%) at pH 12.
4.2. Foaming properties
Foaming properties depend on the ability of proteins to (1)
rapidly adsorb at the air-water interface, (2) undergo rapid
conformational change and rearrangement at the interface, and (3)
form a cohesive viscoelastic film at the interface via intermolecular
interactions (Damodaran, 1994). Several methods are available for
the preparation of foams, including gas injection, agitation (e.g., by
stirring, whipping or beating) and supersaturation (based on the
dissolution of a gas in a liquid under pressure) (Huppertz, 2010).
Foaming capacity (FC) describes the amount of interfacial area that
proteins are able to stabilise per unit weight or concentration, and
is related to high molecular flexibility, charge density, and hydro-
phobicity. Conversely, rheological properties of protein films, such
as viscosity and shear resistance, film elasticity, and the magnitude
of disjoining pressure between the protein layers, affect foam sta-
bility (FS), which refers to the ability of proteins to stabilise the
foam against gravitational and mechanical stresses (Damodaran,
1994). In the study of Cao et al. (2009), FC of brown rice, white
rice and rice bran proteins was 3.65-, 5.52- and 4.21-fold higher,
7
respectively, at pH 11 than at pH 5. FC of rice bran proteins was
enhanced significantly with an increase in sodium chloride (NaCl)
concentration from 0.4 to 2%, while FC of white and brown rice
proteins increased significantly in the NaCl concentration range
0.4e0.8%, but higher concentrations of NaCl had no effect; FS of the
three rice proteins increased with increasing NaCl concentration
(0.4e2.0%). Sucrose concentrations above 12% significantly
decreased FC of the three rice proteins (Cao et al., 2009). Rice
endosperm and rice dregs-based protein isolates had FC of 116 and
119%, respectively, and FS of about 98% after 20 min (Zhao et al.,
2012a). The rice bran protein isolate obtained in the study of
Wang et al. (1999) displayed similar FC (18.9 vs. 20.5 mL) but
significantly lower FS (108 vs. 120 min) compared to egg white.
Chandi and Sogi (2007) reported lower FC but higher FS for rice
bran protein concentrates compared to casein under varying con-
trations. In particular, rice bran protein concentrates of Basmati 370
displayed good FS at a sucrose concentration of 15% (pH 7), with a
half-life of 42.6 h, which was attributed to a more flexible random
coiled structure of proteins in the presence of high sucrose
concentrations.
4.3. Emulsifying properties
Emulsifying properties of proteins are affected by factors similar
to those that affect their foaming properties, including the rate of
adsorption of protein at the oil-water interface, the amount of
protein adsorbed, conformational rearrangement at the interface,
the extent of reduction in interfacial tension, and formation of a
cohesive film (Damodaran, 1994). Emulsifying activity (EA), emul-
sifying capacity (EC) and emulsion stability (ES) are indices
commonly used to evaluate the emulsifying properties of proteins.
EA represents the maximum interfacial area per unit weight of
protein of a stabilised solution, EC refers to the maximum amount
of oil that is emulsified under specified conditions by a standard
amount of protein, and ES expresses the ability of a protein to form
an emulsion that resists changes in its properties during a certain
period of time, at a specific temperature and gravitational force
(Pearce & Kinsella, 1978). Also, the emulsifying properties of pro-
teins are evaluated by measuring droplet size distribution of
emulsions after homogenisation and during storage using light
scattering approaches (Piorkowski & McClements, 2014). According
to Shih and Daigle (2000), the EA of a rice protein isolate obtained
by enzymatic extraction was relatively low, especially at pH < 6, but
was significantly enhanced in the presence of xanthan gum (due to
increased viscosity of the continuous phase). In the study of
Agboola et al. (2005), 20% oil-in-water emulsions prepared using
soya oil and stabilised by rice glutelin (1 and 2% protein) exhibited
surface-weighted mean droplet diameter (D[3,2]) values ranging
from 0.73 to 1.94 mm. Increasing protein content from 1 to 2%
resulted in a higher proportion of fat globules in the low particle
size range (0.1e10 mm), as did increasing homogenisation pressure
from 14.3 to 42.9 MPa at both protein concentrations. Cao et al.
(2009) reported that the EC of white rice, brown rice and rice
bran proteins was minimal at pH 5 and increased significantly at
both lower and higher pH values. Brown rice, white rice and rice
bran proteins showed maximum emulsifying volumes of 44, 47 and
43%, respectively, at pH 11. ES followed the same pattern as EC, but
was maximum at pH 3. For the three rice proteins, increasing NaCl
concentration in the range 0.4e2% impaired emulsifying proper-
ties. EC was maximal at 4% sucrose and slowly decreased with
increasing sucrose concentrations (Cao et al., 2009). Oil-in-water
emulsions containing 50% sunflower oil and stabilised by a rice
protein concentrate (3% protein) displayed lower volume-weighted
mean droplet diameter (D[4,3]) at both 1 and 20 d at pH 2 (D[4,3] of
8 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
0.78 and 0.84 mm at 1 and 20 d, respectively) compared to pH 8 (D
[4,3] of 1.16 and 1.50 mm at 1 and 20 d, respectively); the emulsions
were diluted in a buffer (0.05 M Tris-HCl, pH 8) containing SDS (1%)
to facilitate disruption of flocculated oil droplets prior to the
measurement of droplet size distribution (Romero et al., 2012). Rice
endosperm and rice dregs-based protein isolates displayed EA of 4
and 7 m2/g, and ES of 31 and 77%, respectively (Zhao et al., 2012a).
According to Wang et al. (1999), the emulsifying properties of
rice bran protein isolate were significantly poorer than those of
bovine serum albumin. This result was attributed to the low surface
hydrophobicity of rice bran protein isolate, which led to weaker
interactions between proteins and oil. Oil-in water emulsions
containing 10% corn oil and stabilised by rice bran proteins (0.5%)
extracted by HTC at 120  C and 150  C, with or without amylase
pre-treatment, exhibited enhanced emulsifying properties
compared to rice bran proteins obtained by alkaline extraction, as
shown by lower D[3,2] (0.45e1.25 vs. 2.48 mm) and D[4,3]
(0.64e2.15 vs. 3.27 mm) values and higher absolute values of z-
potential (Xia et al., 2012b). This improvement was attributed to a
more disordered conformation of the HTC-extracted proteins,
which resulted in the exposure of more hydrophobic groups, with
unfolded proteins adsorbing more readily at the oil-water interface
compared to native protein molecules and then rearranging to form
a viscoelastic film (Xia et al., 2012b).
4.4. Water absorption capacity
High water absorption capacity (WAC) of proteins is required in
order to reduce moisture loss in packed bakery goods, to maintain
freshness and moist mouthfeel of baked foods, and to provide
various food systems with an optimal texture (Chandi & Sogi,
2007). Rice endosperm- and rice dregs-based protein isolates
have been reported to have WAC of 2.81 and 3.02 g/g, respectively
(Zhao et al., 2012a). Cao et al. (2009) reported that the WAC of
proteins extracted from rice bran (3.54 mL/g) was higher than that
of proteins extracted from brown (1.96 mL/g) and white rice
(1.78 mL/g). In the study of Chandi and Sogi (2007), rice bran pro-
tein concentrates displayed WAC ranging from 3.87 to 5.60 g/g.
WAC of proteins extracted from regular and heat-stabilised rice
bran was in the range 2.6e3.8 mL/g (Khan et al., 2011). According to
Aletor, Oshodi, and Ipinmoroti (2002), WAC in the range
1.49e4.72 g/g are considered critical in viscous foods, such as soups
and gravies. Therefore, based on the data reported above, rice
proteins could represent important components of value-added
ingredients for use in such products.
4.5. Oil absorption capacity
Oil absorption capacity (OAC) of proteins is essential in order to
improve mouthfeel and flavor retention of certain food products
(Khan et al., 2011). In the study of Zhao et al. (2012a), protein iso-
lates obtained from rice endosperm and rice dregs had OAC of 2.14
and 2.38 g/g, respectively. OAC of brown rice, white rice and rice
bran proteins were 2.93, 2.56 and 3.83 mL/g, respectively (Cao et al.,
2009). According to Chandi and Sogi (2007), rice bran protein
concentrates possessed higher OAC than casein, with values for the
rice-based ingredients ranging from 3.74 to 9.18 g/g. Proteins
extracted from unstabilised and heat-stabilised rice bran had OAC
in the range 2.4e3.3 mL/g (Khan et al., 2011). Protein ingredients
with high OAC can be used in the formulation of food matrices like
cake batters, mayonnaise, salad dressings and sausages (Chandi &
Sogi, 2007).
4.6. Thermal properties
Differential scanning calorimetry (DSC) is often employed to
analyse the thermal properties of proteins. The denaturation peak
temperature (Tp) can be used as an indicator of the thermal stability
of proteins, whereas the enthalpy of denaturation (DH) is related to
the proportion of undenatured proteins or the extent of ordered
protein structure (Arntfield & Murray, 1981). In the study of Ju et al.
(2001), rice endosperm albumin, globulin and glutelin each dis-
played a single enthalpy peak, while prolamin showed no peak. The
Tp and DH of glutelin (82.2  C, 3.79 J/g) were significantly higher
than those of globulin (78.9  C, 3.14 J/g) and albumin (73.3  C,
2.88 J/g). Furthermore, Zhao et al. (2012a) reported that proteins
isolated from rice endosperm and rice dregs had Tp of 85.6 and
96.3  C, and DH of 27.3 and 87.0 J/g, respectively, whereas the rice
bran protein isolate obtained in the study of Wang et al. (1999) had
Tp of 83.4  C, and a much lower DH value, of 0.96 J/g. However,
comparison of data obtained from different DSC studies of protein
denaturation/aggregation is often complex, as many aspects of
sample preparation and instrument operation influence the results.
4.7. Enhancing the functionality of rice proteins by enzymatic
hydrolysis
It is widely acknowledged that the functionality of food proteins
can be altered by enzymatic hydrolysis. The action of proteases
causes a decrease in MW, an increase in the number of ionisable
groups, and the exposure of hydrophobic groups otherwise buried
within the protein core. Specificity of the enzyme, environmental
conditions and degree of hydrolysis influence the extent of
enhancement of protein functionality. Limited hydrolysis is gener-
ally recommended in order to obtain improvements in function-
ality. Conversely, while extensive hydrolysis generally improves
solubility, it may impair other functional properties, such as
foaming and emulsifying properties, and it may potentially result in
the development of bitterness, due to the accumulation of low MW
hydrophobic peptides (Nielsen, 1997; Panyam & Kilara, 1996;
Schwenke, 1997). Enzymatic hydrolysis of rice proteins has been
shown to improve some functional properties, making these
hydrolysate-based ingredients suitable for use in a wider range of
food applications, compared with the intact protein ingredients.
In the study of Anderson, Hettiarachchy, and Ju (2001), the
solubility of rice glutelin in the pH range 2e12 was improved by
treatment with Pronase E (pH 7.5, 37  C). The solubility increased
from 16 to over 36% at pH 2 and from 30 to 62% at pH 12 during
60 min of hydrolysis; minimum solubility was reported at pH 5 (8%)
for native rice glutelin and at pH 4 (19%) for the glutelin hydroly-
sate. Foaming and emulsifying properties of rice glutelin were also
enhanced by hydrolysis with Pronase E. Paraman, Hettiarachchy,
Schaefer, and Beck (2007) reported that solubility of a rice endo-
sperm protein isolate (alkali-extracted) improved upon hydrolysis
with Alcalase (pH 9, 60  C), Liquipanol (pH 7, 50  C) or pepsin (pH 3,
37  C); solubility increased linearly with increasing DH, although a
large proportion of the protein remained insoluble even at the
highest DH values obtained for each enzyme. Overall, Alcalase
treatment gave the greatest improvement, with solubility of the
resulting hydrolysate being about 27% at 13.5% DH. Depending on
the enzyme used, optimum DH for obtaining good EA and ES was in
the range 6e10%. The rice dregs-based protein hydrolysate ob-
tained in the study of Li et al. (2012) following treatment with
trypsin showed a considerably higher solubility compared to native
rice dregs proteins, with values being above 75% in the pH range
2e11. According to Guo et al. (2013), treatment of rice dregs with
Alcalase, bromelain, Neutrase or papain generated protein hydro-
lysates which were highly soluble at pH 7, with values in the range
 L. Amagliani et al. / Trends in Food Science & Technology 64 (2017) 1e12
96e98%, and about 90% being soluble at pH 5. Conversely, alkali-
extracted rice dregs proteins had solubilities of 62% at pH 7 and
only 8% at pH 5. Also, EA and ES of the hydrolysates at pH 7 were in
the range 30e45% and 11e17%, respectively, whereas those of
alkali-extracted proteins were 17 and 12%, respectively. Functional
properties of a defatted rice bran protein isolate (alkali-extracted)
improved upon papain treatment (pH 8, 50  C) for 10e60 min
(Bandyopadhyay et al., 2008). Solubility of the protein isolate was
29, 54, 80 and 85% at pH 3, 5, 7 and 9, respectively, whereas solu-
bility of the hydrolysates at the same pH values was in the range
30e45, 60e62, 84e87 and 86e95%, respectively. Modification by
papain-based hydrolysis improved EA of the protein isolate from
109 m2/g to values in the range 150e185 m2/g, whereas the ES
increased from 37 to 48 min upon papain treatment for 60 min.
Also, hydrolysis of the protein isolate using papain improved FC
from 70% to values in the range 118e151%, but decreased FS.
5. Applications of rice proteins
Rice is generally recognized as a hypoallergenic food, being one
of the first solid foods to be introduced into the diet of infants, and
being used in most elimination diets for food allergy diagnostic
programs in children and adults (Gastana ~duy, Cordano, & Graham,
1990). Therefore, rice protein-based formulas represent a suitable
alternative for children allergic to cow's milk. Infants diagnosed
with cow's milk allergy (CMA) are usually fed extensively hydro-
lysed cow's milk formulas, soy protein-based formulas or amino
acid-based formulas. However, these products present some
drawbacks. Despite being generally well tolerated, extensively
hydrolysed cow's milk formulas (Committee on Nutrition of the
American Academy of Pediatrics, 2000; Giampietro, Kjellman,
Oldaeus, Wouters-Wesseling, & Businco, 2001; Kaczmarski, Wasi-
lewska, & Lasota, 2005; Klemola et al., 2002; Vandenplas et al.,
2007) and soy formulas (Klemola et al., 2002; Zeiger et al., 1999),
have the ability to cause allergic reactions in infants with CMA,
whereas poor palatability and high cost limit the use of amino acid-
based formulas, although they are non-allergic (Caffarelli et al.,
2010). A number of studies have shown that hydrolysed rice
protein-based formulas are tolerated by and nutritionally adequate
for infants with CMA (Agostoni et al., 2007; D'Auria et al., 2003;
Fiocchi et al., 2006, 2003; Reche et al., 2010) and thus represent
suitable alternatives to the aforementioned formulas. Furthermore,
a partially-hydrolysed rice protein-based formula fortified with
lysine and threonine has been shown to support normal growth
and plasma biochemistry when used as the exclusive source of
nutrition in healthy term infants during the first four months of life
(Lasekan, Koo, Walters, Neylan, & Luebbers, 2006).
The use of rice protein ingredients as supplements in sports
nutrition, as an alternative to the more commonly used casein,
whey and soy proteins, is also increasing, and some studies have
shown that rice protein concentrates can be used as value-added
ingredients in the production of bread (Jiamyangyuen et al.,
2005), biscuits (Yadav et al., 2011) and edible films (Adebiyi,
Adebiyi, Jin, Ogawa, & Muramoto, 2008; Shih, 1996) with
improved nutritional and functional properties.
The generally low solubility in water of intact rice protein in-
gredients limits their potential applications to products that do not
require high solubility of the protein ingredients, e.g., meat ana-
logues, baked goods, breakfast cereals, protein bars and pet food.
However, the functionality of rice proteins may be improved by
means of enzymatic hydrolysis, allowing for the use of these pro-
tein ingredients in a wider range of food products including soups,
sauces, salad dressings, whipped toppings, coffee creamers, forti-
fied beverages, enteral and clinical nutrition products, as well as
pharmaceuticals.
9
In addition to the applications of isolated rice protein in-
gredients, it is worth mentioning that rice flour currently repre-
sents the most commonly used ingredient in the production of
gluten-free products on the market, such as bread and pasta,
with rice proteins contributing significantly to the quality and
technological functionality of these products (Hager, Wolter, Jacob,
Zannini, & Arendt, 2012; Moroni, Iametti, Bonomi, Arendt, & Dal
Bello, 2010). Despite the technological difficulties related to the
use of gluten-free flours in bread-making, due to the fact that, after
hydration and mixing, proteins do not develop into a viscoelastic
network, as occurs with the use of wheat proteins, the textural
quality of brown rice breads was shown to improve following
transglutaminase treatment (Renzetti, Dal Bello, & Arendt, 2008).
According to Renzetti et al. (2012) this effect is to be ascribed to the
formation of large protein complexes, resulting from glutelin
polymerization, and the new and stronger hydrophobic in-
teractions among proteins induced by transglutaminase treatment.
6. Conclusions
Research has highlighted the nutritional properties and health
benefits of proteins extracted from rice, a relatively low cost natural
food. Since the interest of consumers worldwide for ingredients
derived from natural sources such as plant proteins is ever-
growing, increasing utilisation of rice proteins in food and phar-
maceutical applications is also expected. However, although prog-
ress has been made in understanding the composition, distribution,
processing and functional properties of rice proteins, more research
is needed to thoroughly characterise and modify the functionality
of rice protein-based ingredients in order to widen the range of
applications of such ingredients and meet the needs of the food
processing and market sectors.
Acknowledgement
The authors would like to thank Nestle for providing financial
support for this study.
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