Proteins of Amaranth (Amaranthus spp.

),
Buckwheat (Fagopyrum spp.), and Quinoa
(Chenopodium spp.): A Food Science and
Technology Perspective
Frederik Janssen, Anneleen Pauly, Ine Rombouts, Koen J.A. Jansens, Lomme J. Deleu, and Jan A. Delcour

Abstract: There is currently much interest in the use of pseudocereals for developing nutritious food products.
Amaranth, buckwheat, and quinoa are the 3 major pseudocereals in terms of world production. They contain high levels
of starch, proteins, dietary fiber, minerals, vitamins, and other bioactives. Their proteins have well-balanced amino acid
compositions, are more sustainable than those from animal sources, and can be consumed by patients suffering from
celiac disease. While pseudocereal proteins mainly consist of albumins and globulins, the predominant cereal proteins are
prolamins and glutelins. We here discuss the structural properties, denaturation and aggregation behaviors, and solubility,
as well as the foaming, emulsifying, and gelling properties of amaranth, buckwheat, and quinoa proteins. In addition,
the technological impact of incorporating amaranth, buckwheat, and quinoa in bread, pasta, noodles, and cookies and
strategies to affect the functionality of pseudocereal flour proteins are discussed. Literature concerning pseudocereal
proteins is often inconsistent and contradictory, particularly in the methods used to obtain globulins and glutelins. Also,
most studies on protein denaturation and techno-functional properties have focused on isolates obtained by alkaline
extraction and subsequent isoelectric precipitation at acidic pH, even if the outcome of such studies is not necessarily
relevant for understanding the role of the native proteins in food processing. Finally, even though establishing in-depth
structure–function relationships seems challenging, it would undoubtedly be of major help in the design of tailor-made
pseudocereal foods.
Keywords: gluten-free, Osborne classification, protein denaturation and aggregation, pseudocereals, techno-functional
properties

Introduction
The growth of the world population (United Nations Dept.
of Economic and Social Affairs 2015), the increasing concerns
over sustainability issues as well as the changes in dietary pat-
terns, which are driven by health or disease outcomes (El
Nehir and Simsek 2012) necessitate the development of efficient
food systems from vegetable (underutilized) sources (El Nehir
and Simsek 2012; Raikos and others 2014; Alemayehu and oth-
ers 2015). Animal proteins such as those in eggs and milk have
high nutritional value and important techno-functional proper-
ties, but they are less sustainable than vegetable proteins (Pimentel
and Pimentel 2003; Nijdam and others 2012). Celiac disease, an
autoimmune-mediated disorder, is related to consumption of foods
MS 20161064 Submitted 5/7/2016, Accepted 26/9/2016. Authors are
with Laboratory of Food Chemistry and Biochemistry and Leuven Food Sci-
ence and Nutrition, Research Centre (LFoRCe), KU Leuven, Kasteelpark
Arenberg 20, B-3001, Leuven, Belgium. Direct inquiries to author Janssen (E-mail:
frederik.janssen@kuleuven.be).
containing gluten from mainly wheat, barley, and rye. Its reported
prevalence has increased over the years (Lohi and others 2007). In
addition, the dependence on cereal-based diets in underdeveloped
and developing countries contributes to protein malnutrition as
most cereal protein sources, such as wheat or maize, have too low
levels of 1 or more of the essential amino acids (Hoffman and Falvo
2004).
While pseudocereals are dicotyledonous, true cereals such as
wheat are monocotyledonous. The seeds of both pseudocereals
and cereals themselves are rich in starch. Amaranth (Amaranthus
spp.), buckwheat (Fagopyrum spp.), and quinoa (Chenopodium spp.)
are globally the best-known pseudocereals. 2 buckwheat species
are cultivated worldwide: common buckwheat (Fagopyrum esculen-
tum Moench) and tartary buckwheat (Fagopyrum tataricum). Both
buckwheat types are closely related to one another but have small
differences in growth conditions and leaf and flower anatomy (Cai
and others 2016). Figure 1 shows the phylogeny of some true
cereals, pseudocereals, edible flowering plants, and legumes. Ama-
ranth and quinoa share a more recent common ancestor and are
thus more closely related to one another than to buckwheat. Both


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doi: 10.1111/1541-4337.12240 Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 1
Proteins of amaranth, buckwheat, and quinoa . . .
Figure 1–Phylogenetic tree for several true cereals, pseudocereals, edible flowering plants, and legumes (Stevens 2001 onwards).
belong to the Amaranthaceae, while buckwheat is a member of the
Polygonaceae (Bremer and others 2009). In 2014, the world-wide
productions of buckwheat and quinoa were estimated at approxi-
mately 2 million and 200000 metric tons, respectively (FAOSTAT
2014). Information on the global production of amaranth is not
available in the database of the Food and Agriculture Organiza-
tion of the United Nations (FAO), but in 2004 was estimated to
approximate between 600000 and 1.7 million metric tons (Corke
and others 2016). In contrast, the world production of common
cereals such as maize, rice, and wheat in 2014 was estimated at
1 billion, 740 million, and 728 million metric tons, respectively
(FAOSTAT 2014).
Proteins from amaranth (Venskutonis and Kraujalis 2013;
Mota and others 2016), buckwheat (Steadman and others 2001;
Bonafaccia and others 2003; Wei and others 2003; Mota and others
2016), and quinoa (Vega-Gálvez and others 2010; Mota and oth-
ers 2016) have better balanced amino acid compositions and thus
higher biological values than those of most cereals. Furthermore,
they can safely be consumed by people suffering from celiac dis-
ease (Bai and others 2013; Colgrave and others 2015) and are more
sustainable than those from animal sources (Pimentel and Pimentel
2003; Alemayehu and others 2015). Amaranth, buckwheat, and
quinoa not only have unique amino acid compositions, they are
also rich in other constituents important for human health, such
as dietary fiber (Steadman and others 2001; Bonafaccia and oth-
ers 2003; Vega-Gálvez and others 2010; Venskutonis and Kraujalis
2013), antioxidants (Paśko and others 2009; Zhu 2016), minerals
(Wei and others 2003; Vega-Gálvez and others 2010; Venskutonis
and Kraujalis 2013), and vitamins (Bonafaccia and others 2003;
Vega-Gálvez and others 2010; Venskutonis and Kraujalis 2013).
Because of the above-mentioned aspects, it is not a surprise that
numerous studies have been published on the general structure,
denaturation, and aggregation behavior of pseudocereal proteins,
and also on their techno-functional properties (such as solubility,
2 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
foaming, emulsifying, and gelling). We here provide the 1st com-
prehensive food science and technology perspective of the cur-
rent knowledge on these subjects for amaranth, buckwheat, and
quinoa. In addition, an overview is given of the technological im-
pact of pseudocereals on quality attributes of bread, pasta, noodles,
and cookies. Finally, crucial knowledge gaps and/or misconcep-
tions are highlighted and perspectives for further research that
could benefit the general use of pseudocereals in the food industry
are suggested. It is expected that a better understanding of the
process-induced structural changes of pseudocereal proteins, their
aggregation, and their techno-functional properties will provide a
solid scientific basis for the design of high-quality food products
based on pseudocereals.
Osborne-Type Fractionation and Structural Properties
Osborne (1907) developed a fractionation scheme which has
stood the test of time. Indeed, it is still frequently used to classify
cereal grain proteins into different fractions based on their sequen-
tial extractability or lack thereof in different media. Albumins are
extractable in water, globulins in aqueous salt solutions (such as
0.4 M NaCl), and prolamins in aqueous alcohol solutions (such
as 60% (v/v) ethanol). The remaining insoluble fraction contains
the glutelins, which are partly extractable in dilute acids or bases
(Belitz and others 2009). The relative distribution of amaranth,
buckwheat, and quinoa proteins in Osborne-type fractionation
schemes reveals that they are more closely related to legume than
to cereal proteins (Table 1) (Segura-Nieto and others 1999).
In wheat and many other cereals, prolamins and glutelins are
the most abundant protein fractions (Table 1). Especially in wheat,
they play a prominent techno-functional role (Delcour and others
2012). In contrast, most legume proteins such as those from soy and
pea are extractable in water or salt solutions and thus belong to the
albumin or globulin fractions. Literature on protein distributions
in Osborne fractions of pseudocereals is inconsistent (Table 1).

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Proteins of amaranth, buckwheat, and quinoa . . .

Table 1–Average protein distributions (%) in Osborne fractions of several pseudocereals, cereals, and legumes.

Reference Albumins Globulins Prolamins Glutelins Remaining

Amaranth 1 19 38 13 21 9
2 51 16 2 31 –
3 47 19 1 29 4
4 44 20 36
5 11 51 0 7 31
6 52 16 2 31 –
Common buckwheat 7 22 15 5 14 44
8 25 70 0 4 1
9 18 43 1 23 15
Tartary buckwheat 7 17 5 3 13 62
10 44 8 11 37 –
Quinoa 11 60 4 36 –
12 29 34 4 16 18
5 13 56 0 5 26
Wheat 15 7 33 46 –
Barley 12 8 25 55 –
Rice 11 10 12 77 –
13
Maize 4 3 48 45 –
Soy 10 90 0 0 –
Pea 21 66 0 12 –
“—” Indicates not determined or not quantifiable.
Considerable adaptations to the Osborne-type fractionation process as described in section “Osborne-type fractionation and structural properties” are listed below in detail:
1 Zheleznov and others (1997). Average values of 24 wild and cultivated amaranth species. Only the proteins from the pellet after the extraction in aqueous ethanol that were extractable in dilute alkali were
classified as glutelins.
2 Segura-Nieto and others (1992); Venskutonis and Kraujalis (2013). Protein distribution for Amaranthus hypochondriacus L. Albumins were not extracted with water, but with 0.01 M potassium phosphate
buffer (pH 7.5) containing 0.1 M NaCl, 1 mM ethylenediaminetetraacetic acid, 0.1 mM phenylmethanesulfonyl fluoride, and 0.02 % (w/v) sodium azide. Globulins were extracted with the same buffer containing
1.0 instead of 0.1 M NaCl.
3 Barba De La Rosa and others (1992a). Albumins and globulins were extracted with 0.1 M potassium phosphate buffer (pH 7.0). Albumins were then enriched by dialysis in water. Prolamins were extracted with
70% (v/v) 2-propanol and glutelins with potassium borate buffer (pH 10.0) containing 1.0% (w/v) sodium dodecyl sulfate (SDS) and 0.6 % (v/v) 2-mercaptoethanol.
4 Gorinstein and others (2001). Values are based on an extraction procedure involving both water and aqueous salt solution. A small part (4%) of the globulins was falsely classified as albumin-2. Albumin,
globulin, prolamin, and glutelin were sequentially extracted in water, 0.5 M NaCl, 70% (v/v) 2-propanol, and 0.01 M sodium carbonate buffer (pH 10.0) containing 0.6% (v/v) 2-mercaptoethanol and 0.5%
(w/v) SDS.
5 Thanapornpoonpong and others (2008). Average values for 2 amaranth varieties and 2 quinoa cultivars Values were based on an extraction procedure involving both water and aqueous salt solution. A part
(4%) of the globulins was falsely classified as albumin-2. Albumin, globulin, prolamin, and glutelin were sequentially extractable in water, 0.5 M NaCl, 70% (v/v) 2-propanol, and 10 mM sodium carbonate buffer
(pH 10.0) containing 0.6% (v/v) 2-mercaptoethanol and 0.5% (w/v) SDS.
6 Corke and others (2016).
7 Wei and others (2003). Average values for 3 common buckwheat cultivars and 1 tartary buckwheat cultivar. Albumin, globulin, prolamin, and glutelin were extracted with distilled water, 0.5 M NaCl, 50% (v/v)
2-propanol, and 0.1 M KOH, respectively. The remaining protein fraction was not analyzed.
8 Radovic and others (1996, 1999); Milisavljevic and others (2004).
9 Javornik and Kreft (1984). Protein distribution of Fagopyrum esculentum (cultivar Siva dolenjska). Albumin, globulin, prolamin, and glutelin were sequentially extracted with distilled water, 0.5 M NaCl, 70%
(v/v) ethanol, and borate buffer (pH 0.9) containing 1.0% (w/v) SDS.
10 Guo and others (2006). Albumin, globulin, prolamin, and glutelin were extracted with distilled water, 1.0 M NaCl, 55% (v/v) 1-propanol, and 0.05 M KOH, respectively. The remaining protein fraction was not
analyzed.
11 Jancurová and others (2009).
12 Watanabe and others (2003). Protein distribution for Chenopodium quinoa Willd. (cultivar Real). Only the proteins from the pellet after the extraction in aqueous ethanol that were extractable in dilute alkali
were classified as glutelins.
13 Belitz and others (2009).

Still, the most dominant pseudocereal Osborne fractions are albu-
mins and globulins. It is of note here that the traditional Osborne
fractionation scheme is not always rigorously applied to pseudoce-
reals. For instance, there are important differences in the methods
used to obtain pseudocereal globulin fractions (Segura-Nieto and
others 1999). Various levels and types of salts in extraction media,
and with various extraction temperatures and times, likely affect
both the quality and relative quantity of the extracted proteins.
In addition, some proteins in amaranth are classified as albumins,
even if they are not extracted from flour with water but enriched
in a complex extraction procedure involving both water and salt
solution. Finally, while it is general practice to classify all proteins
in the residue after the 3rd extraction (as with aqueous alcohol)
as glutelin (Belitz and others 2009), some authors only classify the
part of these proteins that are extractable in dilute acids or bases as
glutelin. Table 2 lists some characteristics of albumins, globulins,


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Proteins of amaranth, buckwheat, and quinoa . . .

Table 2–Structural properties reported for Osborne protein fractions from amaranth, common and tartary buckwheat, and quinoa.

Amaranth Buckwheat Quinoa

Albumins
General structure
One major protein with MM of
133 kDa, which consists of 12
noncovalently, linked subunits
of approximately 10 kDa.1
In addition, 1 major protein with
MM around 34 kDa and several
smaller (10 to 18 kDa) and
larger peptides (>78 kDa)
have been detected by
SDS–PAGE.2
pI 7.57
Secondary structure8
4% α-helices
37% β-sheets
55% aperiodic structure
Globulins
General structure
Major 11S globulin
“Amarantin” with MM of 300 kDa
and consisting of 6 similar
subunits (52 to 59 kDa), which
each consist of an acidic (34 to
36 kDa) and a basic (22 to 24
kDa) protein linked by an
SS-bond.9
Minor 7S globulin
Vicilin-like proteins with apparent
MM of 186.0 kDa made up of 8
noncovalently linked subunits:
90.1, 70.9, 40.0, 37.4, 35.2,
31.2, 23.6, and 15.6 kDa.10
pI 11S, acidic: 5.69
11S, basic: 9.29
7S: between 5.2 and 5.810
Common buckwheat:
Mostly 2S albumins with MM One major 2S albumin with MM
between 8 and 12 kDa. A around 12 kDa, with 2 SS-linked
minority of the 2S albumins has subunits of 3 and 8 kDa.5
a MM of 16 kDa. The proteins
were not linked by an SS-bond.3
Tartary buckwheat:
Proteins with MMs of 57 and 64 In addition, various proteins within
kDa which consist of SS-linked the range of 25 to 83 kDa have
subunits with MMs between 14 been detected.6
and 22 kDa, and proteins with
MMs around 38 and 41 kDa.4
? ?
Common buckwheat:
2% α-helices 4% α-helices
46% β-sheets 50% β-sheets
52% aperiodic structure 46% aperiodic structure
Common buckwheat:
Major 13S globulin Major 11S globulin
Legume-like proteins with MM of “Chenopodin” with MM of 320 kDa
280 kDa and consisting of 6 and consisting of 6 similar subunits
nonidentical subunits (43 to 68 (52 to 59 kDa), which each consist
kDa) which interact of an acidic (30 to 40 kDa) and a
noncovalently and each consist basic (20 to 25 kDa) protein linked
of an acidic (30 to 38 kDa) and by an SS-bond.15
a basic (23 to 25 kDa) protein
linked by an SS-bond.11
Minor 8S globulin ?
Vicilin-like proteins constituted
by subunits with high (57 to 58
kDa) and low (26 to 36 kDa)
MM.12
Minor 2S globulin
Closely related to the 2S
albumins13
Tartary buckwheat:
Proteins with MMs of 64, 57, 41,
38, 34, 28, 23, 21, 19, 15 kDa.
The 2 largest proteins consist
of SS-linked subunits.14
13S, acidic: 6.816 11S, acidic: between pH 5.0 and
13S, basic: 8.517 6.518
8S: 6.816 11S, basic: ?
2S: ? 2S: ?

Secondary structure 11S globulin10 13S globulin8

7.8% α-helices 16.0% α-helices
57.6% β-sheets 34.5% β-sheets
17.6% β-turns 20.0% β-turns
16.9% random coils 14.4% random coils
7S Globulin10
12.6% α-helices
49.6% β-sheets
12.2% β-turns
24.9% random coils
Globulin8 Globulin8 Globulin8
31% α-helices 25% α-helices 20% α-helices
27% β-sheets 30% β-sheets 35% β-sheets
42% aperiodic structure 45% aperiodic structure 45% aperiodic structure
Albumin-28 Albumin-28 Albumin-28
16% α-helices 14% α-helices 10% α-helices
41% β-sheets 29% β-sheets 37% β-sheets
53% aperiodic structure 57% aperiodic structure 53% aperiodic structure
Prolamins
General structure Common buckwheat: ?
Proteins with MMs around 53, 34 Proteins with MMs around 22, 32,
and 25 kDa, linked by SS 39, 50 and 59 kDa.20
bonds.19
(Continued)

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Proteins of amaranth, buckwheat, and quinoa . . .

Table 2–Continued.

Amaranth Buckwheat Quinoa

Tartary buckwheat:
Major protein with MM 15 and
17 kDa that were not impacted
by a reducing agent. In
addition, some minor proteins
with a MM of 14 and 20 kDa
and some SS-linked proteins
with MMs of 26 and 29 kDa
have been observed.14
pI ? 5.5 to 7.319 ?

Secondary structure ? ? ?
Glutelins
General structure Common buckwheat: ?
Major SS-linked proteins with SS-linked proteins with MMs
MMs around 22 and 32 kDa, between 43 and 66 kDa.22
minor SS-linked proteins with
MMs around 16 and 39 kDa, Tartary buckwheat:
and few SS-linked proteins with SS-linked proteins with MMs
MMs between 50 and 65 between 12 and 66 kDa.23
kDa.21
pI Acidic proteins: ? ?
between 5.7 and 6.3.24
Basic proteins: ?
Secondary structure ? ? ?
References:
1 Marcone and others (1994).
2 Gorinstein and others (1991bb, 2001); Barba de la Rosa and others (1992aa, 2009); Drzewiecki and others (2003).
3 Radovic and others (1999).
4 Guo and Yao (2006).
5 Brinegar and others (1996).
6 Thanapornpoonpong and others (2008).
7 Marcone and others (1994).
8 Drzewiecki and others (2003).
9 Konishi and others (1985); Barba de la Rosa and others (1992aa, 1992bb); Valdez-Ortiz and others (2005).
10 Marcone (1999); Quiroga and others (2010).
11 Radovic and others (1996); Milisavljevic and others (2004); Choi and Ma (2006bb); Tang (2007aa).
12 Radovic and others (1996); Milisavljevic and others (2004);
13 Radovic and others (1999); Biacs and others (2002).
14 Guo and Yao (2006).
15 Brinegar and Goundan (1993).
16 Radovic and others (1996).
17 Choi and Ma (2006bb).
18 Mäkinen and others (2016).
19 Barba de la Rosa and others (1992aa).
20 Nał˛ecz and others (2009).
21 Gorinstein and others (1991aa); Barba de la Rosa and others (1992aa); Thanapornpoonpong and others (2008).
22 Gao and others (2008).
23 Javornik and Kreft (1984); Guo and Yao (2006).
24 Vasco-Méndez and Paredes-López (1994).
MM, molecular mass; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SS, disulfide; ?, yet to be determined by future researchers.

prolamins, and glutelins extracted from amaranth, buckwheat, and Aphalo and others 2004; Quiroga and others 2007; Tandang-Silvas
quinoa. and others 2012). Already at low salt concentrations (ࣘ0.01 M),
the structure of the so-called albumin-2 evolves into a partially
Albumins unfolded conformation, which may trigger aggregate formation
Amaranth. Several authors (Konishi and others 1991; Gorin- (Castellani and others 1998). Based on tandem mass spectrometry
stein and others 2001; Drzewiecki and others 2003) have de- of purified albumin-2 single proteins, of albumin-2 aggregates,
scribed 2 amaranth albumin types, which they obtained by a 2-step and of partially purified globulin, Quiroga and others (2009) con-
extraction procedure involving both water and a salt solution. cluded that albumin-2 aggregates are isoforms of 11S globulin, in-
A major protein fraction, correctly classified as albumin-1, is dicating that albumin-2 and 11S globulin are encoded by the same
extracted from flour with salt solutions followed by dialysis against gene. This way, amaranth albumin-2 is most likely a polymeriza-
water and subsequent removal of the globulins (as pellet) by cen- tion product of 11S globulin, which may explain its distinctive ex-
trifugation. A minor protein fraction can then be extracted with tractability in the Osborne fractionation process. Structural charac-
water from the flour residue. The literature often refers to this teristics of the albumin-2 are discussed in the “Globulins” section.
fraction as “albumin-2,” “minor albumin,” or “globulin-P.” How- For the analysis of amaranth albumins, classical sodium do-
ever, even if these proteins are extracted with water, salt is present decyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is
in the flour as a result of the 1st extraction with salt solution. preferred over chip electrophoresis, which compresses the bands
These proteins are thus most likely globulins, especially because of the very abundant proteins of low molecular mass (MM)
they share several features with other amaranth globulins such as (Džunková and others 2011). However, SDS–PAGE also has some
molecular size and surface reactivity (Drzewiecki and others 2003; limitations. For instance, the bands in an SDS–PAGE profile only


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Proteins of amaranth, buckwheat, and quinoa . . .
provide an estimation of the MM of a protein. Hence, the term ap-
parent MM should be used in this context. The albumin-1 fraction
in amaranth, extracted either with a salt solution followed by dial-
ysis against water and removal of the globulins or simply extracted
with water, has been frequently analyzed by SDS–PAGE. Most
authors observed a major band corresponding to albumin-1 around
34 kDa (which withstands reducing conditions), and minor bands
with apparent MMs in the range of 10 to 18 kDa (Gorinstein and
others 1991b; Barba de la Rosa and others 1992a; Drzewiecki and
others 2003). A high MM (apparent MM >78 kDa) protein has
also been described (Gorinstein and others 2001; Barba de la Rosa
and others 2009). Marcone and others (1994) described amaranth
albumin-1 as a 133-kDa homo-dodecamer with 12, low-MM sub-
units held together by noncovalent (for example, hydrogen and hy-
drophobic) bonds. They found no evidence for covalent disulfide
(SS) linkages between the subunits. According to Shewry (2016),
the albumin-1 fraction migrates with a 2S sedimentation coeffi-
cient. If so, the homododecameric protein described by Marcone
and others (1994) may be an aggregation product of the 2S al-
bumin stabilized by noncovalent interactions. If not, it remains to
be investigated whether the higher apparent MM bands observed
by Barba de la Rosa and others (2009) and Gorinstein and others
(2001) are derived from globulin impurities or whether some albu-
mins were not present in the albumin fraction purified by Marcone
and others (1994). Based on SDS–PAGE, Segura-Nieto and others
(1992) concluded that, of the Osborne protein fractions, albumins
are the most polymorphous. Analysis of amaranth albumin-1 by
electrophoretic isoelectric focusing (IEF), electrophoretic titration,
and zeta potential analysis indicated an isoelectric point (pI, the pH
at which the net charge of the proteins equals zero) of 7.5 (Mar-
cone and others 1994). Secondary structure analysis revealed that
it has relatively high levels of β-sheets but a very little discernable
tertiary structure, as evidenced by relatively low near-ultraviolet
(UV) (240 to 320 nm) circular dichroic (CD) intensities (Marcone
and others 1994). The secondary structure of amaranth albumins
has been estimated to consist of 4% α-helices, 37% β-sheets, and
55% aperiodic structure (Drzewiecki and others 2003).
Buckwheat. Most common buckwheat albumins migrate with
a 2S sedimentation coefficient and have apparent MMs between
8 and 12 kDa as determined by SDS–PAGE (Radovic and oth-
ers 1999). A minor fraction of the 2S albumins has an apparent
MM close to 16 kDa. These proteins are not linked by SS-bonds
(Radovic and others 1999). The 2S albumins are widely distributed
in dicotyledonous seeds. They are related to the 2S albumins of
wheat and inhibitors of trypsin and α-amylase from cereals (Kreis
and others 1985). However, the 2S albumins in legumes are present
at higher levels and do contain SS-bonds (Shewry and Pandya
1999). The albumins account for about 25% of total proteins ex-
tractable in salt solution, but this content is drastically reduced
under sulfur-deficiency conditions during growth (Radovic and
others 1999). Javornik and Kreft (1984) separated albumins of
common buckwheat by SDS–PAGE into 8 electrophoretic bands
with apparent MMs between 17 and 67 kDa. Their results seem
in line with those obtained for tartary buckwheat of which the
nonreduced albumin fraction showed major bands at 64, 57, 52,
41, and 38 kDa (Guo and Yao 2006). Under reducing conditions,
some minor bands at 14 to 22 kDa appeared at the expense of
the 57 and 64 kDa albumins, while the 38 and 41 kDa protein
bands remained. The secondary structure of common buckwheat
albumins has been estimated to consist of 2% α-helices, 46%
β-sheets, and 52% aperiodic structure (Drzewiecki and others
2003).
6 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
Quinoa. The major seed storage protein of quinoa is the 2S
albumin. Under reducing conditions, an electrophoretically het-
erogeneous collection of proteins with apparent MMs between 8
and 9 kDa is observed (Brinegar and others 1996). Since native 2S
albumins in castor bean, rapeseed, and Brazil nut are dimers com-
posed of a 7- to 9-kDa subunit SS linked to a 3- to 4-kDa subunit,
it has been suggested that quinoa also contains the smaller 2S sub-
units, even if they are not resolved under the electrophoretic con-
ditions employed (Brinegar and others 1996). Very different results
were presented by Thanapornpoonpong and others (2008) when
applying a fractionation scheme for amaranth proteins (Gorinstein
and others 1991b). In their hands, it yielded 2 albumin fractions:
the correctly classified albumin-1 and the alleged albumin-2 (cfr.
supra). When extracted from flour with salt solution followed by
dialysis against water to precipitate the globulins, the albumin-1
is separated by SDS–PAGE into a wide range of protein subunits
with apparent MMs between 25 and 83 kDa. The secondary struc-
ture of quinoa albumins consists of 4% α-helices, 50% β-sheets,
and 46% aperiodic structure (Drzewiecki and others 2003).
Globulins
Amaranth. The main amaranth protein is a globulin, the 11S
globulin or amarantin. It consists of 3 subunits in their pro-form
(proglobulin). Cotranslational signal peptide cleavage of the sub-
unit in the endoplasmic reticulum induces association of the sub-
units in a head-to-tail pattern. The proglobulin is subsequently
transported to the protein storage vacuoles. Herein, its post-
translational cleavage by an endopeptidase releases acidic and basic
chains and triggers the assembly of 2 trimers into a homohex-
amer (Tandang-Silvas and others 2012; Carrazco-Pena and others
2013). This homohexamer is made up of subunits with MMs be-
tween 52 and 59 kDa, each consisting of an acidic (34 to 36 kDa,
pI 5.6) and a basic (22 to 24 kDa, pI 9.2) protein linked by an
SS-bond (Konishi and others 1985; Barba de la Rosa and others
1992a; Barba de la Rosa and others 1992b; Valdez-Ortiz and oth-
ers 2005). Martı́nez and others (1997) described the same or at
least a very similar, protein, but falsely classified it as albumin-2
(see section “Albumins”), based on data by Konishi and others
(1991). Much as the 11S globulin, this protein has an apparent
MM of 300 kDa and contains subunits with apparent MMs of 52,
54, and 56 kDa. However, only the 52 and 56 kDa subunits seem
to consist of a larger acidic peptide (31 to 38 kDa) SS-linked to a
smaller basic (19 to 23 kDa) peptide. Gel filtration chromatogra-
phy of amaranth proteins yielded 6 main fractions of around 300,
180, 120, 45, 25, and below 10 kDa (Zheleznov and others 1997;
Juan and others 2007). The fraction with the highest apparent
MM may correspond to the intact hexameric 11S globulin. We
speculate that the fraction with apparent MM of 180 kDa repre-
sents 11S globulin trimers and/or the minor amaranth globulin.
The smaller and larger proteins, as well as the 52 to 56 kDa sub-
units, were also detected by SDS–PAGE. The α- and β-peptides
of 11S globulin appear in the fractions with apparent MMs of
45 and 25 kDa (Thanapornpoonpong and others 2008). Elec-
trophoretic patterns under denaturing and reducing conditions
showed 3 main fractions with apparent MMs between 50 and
64 kDa, 33 and 37 kDa, and 18 and 25 kDa, respectively (Juan
and others 2007). Chip electrophoresis analysis of reduced ama-
ranth globulins yielded the same 3 fractions (Džunková and others
2011). Again, it was suggested that they represent the 52 to 56 kDa
subunit and the constituting α- and β-peptides respectively, of
the 11S globulin (Juan and others 2007). Marcone and Yada

C 2016 Institute of Food Technologists®
Proteins of amaranth, buckwheat, and quinoa . . .
(1997) showed that 11S amaranth globulin is both glycosylated and
phosphorylated.
The minor amaranth globulin, the 7S vicilin-like globulin, is a
hetero-oligomer with an apparent MM estimated by gel filtration
chromatography of 186 kDa made up of a variety of 8 noncova-
lently linked subunits (with SDS–PAGE MMs of 90, 71, 40, 37,
35, 31, 24, and 16 kDa) (Marcone 1999). Barba de la Rosa and
others (1992b) described a very similar protein containing 7 non-
covalently linked subunits with MMs of 72, 67, 52, 38, 34, 32, and
25 kDa. More recently, Quiroga and others (2010) reported on the
amaranth 7S globulin subunit structure and observed main subunits
with MMs of 66, 52, 38, and 16 kDa, in reasonable agreement
with Barba de la Rosa and others (1992b) and Marcone (1999).
Electrophoretic IEF of the 7S amaranth globulin indicated a pI
between 5.2 and 5.8. At lower pH, the 7S globulin was subjected
to (i) a large surface charge density change and (ii) an acid-induced
dissociation of its subunits (Marcone 1999). Near-UV (240 to
320 nm) CD intensities revealed that the purified 7S globulin
secondary structure is relatively poor in α-helices, but possesses
high levels of β-sheets (Marcone 1999). The secondary structure
of the amaranth globulins has been estimated to consist of 31%
α-helices, 27% β-sheets, and 42% aperiodic structure (Drzewiecki
and others 2003), while that of the albumin-2 fraction, which
most likely also contains globulins, has been estimated as 16%
α-helices, 41% β-sheets, and 53% aperiodic structure (Tandang-
Silvas and others 2012) and less hydrophobic surfaces than the
albumins, as follows from 8-anilinonaphthalene-1-sulfonic acid
fluorescence measurements (Gorinstein and others 2001).
Buckwheat. The major storage protein of common buckwheat
is the 13S legume-like globulin. Konishi and others (1985) re-
ported that its apparent MM is 440 kDa and that it is composed
of at least 4 different subunit types (MMs of 18, 20, 32, and 36
kDa). It is now generally accepted that the 13S globulins ex-
ist as proteins with an apparent MM between 280 and 390 kDa
(Maksimovic 1995; Marcone and others 1998; Fujino and others
2001; Nair and Adachi 2002; Samardžić and others 2004; Choi
and Ma 2006a; Choi and others 2006) made up of 6 nonidenti-
cal monomers (Maksimovic and others 1995; Choi and Ma 2005;
Choi and others 2006). The monomers interact noncovalently and
each consists of a smaller (16 to 29 kDa) basic subunit SS-linked
to a larger (30 to 47 kDa) acidic subunit (Maksimovic and others
1995; Radovic and others 1996; Rout and others 1997; Bharali
and Chrungoo 2003; Choi and Ma 2005; Choi and others 2006;
Tang 2007a). In IEF, all larger proteins of the 13S globulin are vis-
ible in the acidic region of the gel. While a minor fraction of the
small proteins has pIs of about 6.8 (Radovic and others 1996), the
majority shows pIs of about 8.5 (Choi and Ma 2006b). Choi and
others estimated that the secondary structure of the 13S globulin
consists of 34.5% β-sheets, 20.0% β-turns, 16.0% α-helices, and
14.4% random coils.
The minor buckwheat globulin, the 8S vicilin-like globulin, is
mainly made up of proteins with apparent MMs between 57 and
58 kDa, and to a lesser extent of proteins with apparent MMs
between 26 and 36 kDa (Radovic and others 1996; Milisavljevic
and others 2004). The 8S globulin is not affected by reducing
conditions. It has a pI of approximately 6.8 (Radovic and others
1996). Separation of buckwheat globulin proteins in a sucrose
gradient shows a 3rd distinct 2S globulin fraction, which is closely
related to the 2S albumin fraction (Radovic and others 1999;
Biacs and others 2002). The globulins in tartary buckwheat
have been less well characterized and the reports found they are
contradictory. First, Doi and others (1995), based on SDS–PAGE,

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 7
concluded that the globulins in tartary buckwheat consist of a ma-
jor globulin with apparent MM of 443 kDa globulin, composed
of 2 or 3 proteins of 179 kDa linked by hydrogen bonds, and
a minor 679 kDa globulin. A more recent report mentions that
SDS–PAGE shows bands at 15, 19, 21, 23, 28, 34, 38, 41, 57, and
64 kDa, the 2 largest proteins of which are no longer visible under
reducing conditions. This indicates that they consist of SS-linked
subunits. The same report mentions that reducing conditions
did not induce notable changes in the 38 and 41 kDa proteins
(Guo and Yao 2006). Drzewiecki and others (2003) reported
25% α-helices, 30% β-sheets, and 45% aperiodic structure for the
common buckwheat globulin fraction, and 14% α-helices, 29%
β-sheets, and 57% aperiodic structure for the so-called albumin-2
fraction, which probably also contains globulins
Quinoa. The major seed storage protein of quinoa is the 11S
globulin chenopodin, a hexameric protein with a MM of 320
kDa, of which each subunit consists of a basic (20 to 25 kDa)
and an acidic (30 to 40 kDa) peptide covalently linked by an
SS-bond (Brinegar and Goundan 1993; Thanapornpoonpong
and others 2008). It makes up 37% of the total protein content
in quinoa seeds (Abugoch James 2009). SDS–PAGE, N-terminal
sequencing, and amino acid analysis have allowed to conclude
that it is a member of the highly conserved 11S storage globulin
family (Brinegar and Goundan 1993; Balzotti 2006). The alleged
albumin-2 fraction is also present in quinoa. It consists of 4
major subunits with apparent MMs of 23, 31, 35, and 52 kDa
(Thanapornpoonpong and others 2008). All observed protein
bands correspond to the subunits or monomers in the globulin
fraction, which again indicates that the albumin-2 fraction, in fact,
contains globulins. The secondary structure of quinoa globulin
and the alleged albumin-2 fraction have been estimated to consist
of 20% α-helices, 35% β-sheets, and 45% aperiodic structure
and 10% α-helices, 37% β-sheets, and 53% aperiodic structure,
respectively (Drzewiecki and others 2003).
Prolamins
A common observation for all pseudocereals is that they contain
low levels of prolamins.
Amaranth. The prolamins of amaranth are made up of fewer
and less abundant components than all other protein fractions
(Segura-Nieto and others 1992). They cannot be readily ana-
lyzed by chip electrophoresis because of their low concentration
(Džunková and others 2011). Nevertheless, SDS–PAGE under re-
ducing conditions showed that they have apparent MMs of 52 to
54, 33 to 34, and 22 to 27 kDa and are linked by SS-bonds (Barba
de la Rosa and others 1992a). They share common subunits with
amaranth glutelins, and the differences in solubility between these
proteins and amaranth prolamins have been ascribed to differences
in levels and locations of SS-bonds (Barba de la Rosa and others
1992a).
Buckwheat. Skerritt (1986) separated a common buckwheat
prolamin fraction by SDS–PAGE and observed a dominant range
of low MM (10 to 28 kDa) proteins without sharp resolution and
some minor bands at 32, 35, 68, 75, and 80 kDa. Nałecz˛ and others
(2009) fractionated buckwheat prolamins into 5 two-dimensional
(2D) SDS–PAGE subregions with proteins with apparent MMs
around (i) 50 kDa and pIs between 6.0 and 7.3, (ii) 39 kDa and
pIs between 6.2 and 6.8, (iii) 32 kDa and pIs around 5.9, (iv)
between 31 and 59 kDa and pIs around 5.5, and (v) around
22 kDa and pIs between 5.9 and 6.6. 2D electrophoresis is
very useful for detecting buckwheat prolamins, especially since
the lack of their sequences in databases makes it impossible
Proteins of amaranth, buckwheat, and quinoa . . .
to identify them directly with peptide-based mass spectrometry
(Nałecz
˛ and others 2009). Tartary buckwheat prolamin has 2 mi-
nor and 2 major protein bands at 14 and 20 kDa and 15 and
17 kDa, respectively, which are not influenced by a reducing agent.
Under reducing conditions, 2 additional bands at 26 and 29 kDa
are observed, possibly derived from the high MM proteins which
do not enter the gel (Guo and Yao 2006).
Quinoa. The prolamins are an insignificant fraction of quinoa
seed proteins (Fairbanks and others 1990; Thanapornpoonpong
and others 2008) and information on their MM distribution is
scarce.
Glutelins
Amaranth. 2D gel electrophoresis reveals some glutelins as ma-
jor proteins in amaranth seeds (Segura-Nieto and others 1992).
SDS–PAGE shows some proteins with low apparent MM (ap-
proximately 24 kDa), but more proteins of intermediate (32 to 38
kDa) and high apparent MM (48 to 57 kDa) (Gorinstein and others
1991b; Barba de la Rosa and others 1992a; Thanapornpoonpong
and others 2008). Gorinstein and others (1991a) reported some-
what contradictory results. They found that the majority (83%) of
the proteins of amaranth extracted in 55% (v/v) 2-propanol, con-
taining 5% (v/v) mercaptoethanol, supposedly mostly are glutelins
and have MMs around 10 to 14 kDa, that a 2nd fraction (7%) con-
sists of proteins of 20 kDa, and that the remaining 10% is made up
of minor fractions. Chip electrophoresis provided more detailed
information. Of all the protein fractions, amaranth glutelins form
the clearest patterns under reducing conditions with several clearly
separated proteins which can be used to identify amaranth hybrid
accessions and wild species (Džunková and others 2011). Glutelins
of various amaranth species show polymorphism not only in the
position of the bands in the patterns, but also in their intensity.
Amaranth glutelins have 2 bands of medium intensity (15 and 17
kDa), 3 intense bands (21 to 23 kDa), 2 intense bands (31 and
33 kDa), 1 polymorphic low-intensity band (38 or 39 kDa) and
a polymorphic area (50 to 65 kDa) (Džunková and others 2011).
Part of the amaranth glutelin subunits (pI 5.7 to 6.3) show struc-
tural homology to the acidic globulin subunits (Vasco-Méndez and
Paredes-López 1994).
Buckwheat. There is high glutelin polymorphism among dif-
ferent common buckwheat cultivars. Their glutelins consist of
3 to 5 SS-linked subunits with apparent MMs between 43 and
66 kDa (Gao and others 2008). Glutelins from tartary buckwheat
in many cases only show minor and poorly defined SDS–PAGE
bands. Some of these proteins do not enter the gel and/or are not
soluble in the gel sample buffer (Guo and Yao 2006). Neverthe-
less, 9 electrophoretic subfractions with MMs ranging from 12 to
66 kDa, with little polymorphism, can be distinguished (Javornik
and Kreft 1984; Guo and Yao 2006).
Quinoa. SDS–PAGE of quinoa glutelins yields few protein
bands and great differences are noted between cultivars. Apparent
MMs of the subunits range from 16 to 94 kDa (Thanapornpoon-
pong and others 2008). All proteins in the glutelin fraction have
electrophoretic mobility identical to that of albumins and globulins
(Fairbanks and others 1990).
Denaturation and Aggregation Behavior
In the studies cited below on protein denaturation and ag-
gregation, the studied protein isolates were obtained by alkaline
extraction and subsequent isoelectric precipitation (IEP) at acidic
pH, unless otherwise stated. Table 3 lists the pH values of the ex-
8 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
traction media, during IEP, and analysis of amaranth, buckwheat,
and quinoa protein isolates.
Protein denaturation as evaluated by differential scanning
calorimetry
Amaranth. Differential scanning calorimetry (DSC) of ama-
ranth protein isolates revealed 2 endotherms with denaturation
temperatures (Td ) readings in the ranges of 70 to 73 °C and 94 to
100 °C, respectively (Table 4). The 2nd endotherm was ascribed
to denaturation of the alleged albumin-2 and globulins, whereas
albumin-1, glutelins, and the minor components of globulins led
to the 1st endotherm (Martinez and Anon 1996; Gorinstein and
others 2001; Bejarano-Lujan and Netto 2010). A small decrease in
Td was noted when the extraction pH increased from 8.0 to 11.0.
It should be stressed that Td values of proteins may be influenced
by specific prior extraction conditions since alkaline extraction can
induce conformational changes (Martinez and Anon 1996; Castel-
lani and others 1998; Salcedo-Chávez and others 2002; Abugoch
James and others 2003; Cordero-De-Los-Santos and others 2005;
Bejarano-Lujan and Netto 2010). Several authors (Ventureira and
others 2012b; Shevkani and others 2014a; Shevkani and Singh
2015) reported similar Td readings for such amaranth protein
isolates. While 2 distinct denaturation peaks were visible at pH 8.0,
no such peaks were observed at pH 2.0. This indicates that low pH
induces extensive unfolding of amaranth proteins extracted under
alkaline conditions (Abugoch James and others 2010; Ventureira
and others 2012b; Bolontrade and others 2013a). Furthermore,
Salcedo-Chávez and others (2002) noted that 10 different com-
binations of extraction pH and pH of IEP of amaranth protein
isolates (Table 3) had no significant effect on their Td readings.
DSC of amaranth protein isolates obtained by extraction at pH
7.0 and subsequent precipitation by micellization revealed 2 en-
dothermic transitions of which the Td readings were 75.6 and 97.2,
respectively (Cordero-De-Los-Santos and others 2005). Martinez
and Anon (1996) reported on the Td of amaranth protein frac-
tions purified under relatively mild conditions. DSC Td readings of
64 °C for albumin-1 and 94 °C for the falsely classified albumin-
2 and most of the globulins were noted. The glutelin fraction
showed endothermic peaks at 70 °C and 96 °C. Amaranth glob-
ulins also extracted under mild conditions showed endothermic
transitions at 80.2 and 94.3 °C. These were attributed to the 7S
and 11S globulins, respectively (Table 4) (Marcone 1999). The Td
of extracted proteins depends on pH. The so-called albumin-2
fraction has the highest Td between pH 6.0 and 8.0.
High-pressure treatment (200 to 600 MPa) also provokes de-
naturation of amaranth proteins. The protein fractions with the
lower Td readings (70 to 73 °C range, see above) have greater
sensitivity to high-pressure treatment than proteins characterized
by Td readings in a 94 to 100 °C range. Whereas the proteins
with lower Td are almost completely denatured after treatment at
200 MPa, those of higher thermal stability partially retain their
native structures. Furthermore, the proteins with low and high Td
that resist high-pressure treatment have lower and higher thermal
stability, respectively, than the untreated protein fractions (Condes
and others 2012; Condes and others 2015).
Buckwheat. Common buckwheat proteins show a minor and a
major denaturation peak at about 80 °C and about 102 °C, which
have been ascribed to the 8S and 13S globulins, respectively (Table
4) (Tang 2007a). The Td values were not affected by prior lipid
extraction, indicating that endogenous lipids do not affect the
thermal stability of buckwheat proteins (Tang 2007c). Extreme
acidic and alkaline pH conditions induce a decrease in Td of the

C 2016 Institute of Food Technologists®
Proteins of amaranth, buckwheat, and quinoa . . .

Table 3–Overview of pH of extraction, pH of isoelectric precipitation, and pH of analysis reported for protein isolates used in studies on denaturation,
aggregation, and solubility as well as foaming, emulsifying, and gelling properties of amaranth, buckwheat, and quinoa proteins.

Pseudocereal pH of extraction pH of IEP pH of analysis Reference

Protein denaturation Amaranth 8.0 to 11.0 5.0 7.0 1
9.0 3.0 to 7.0 3.0 to 7.0 1
9.0 and 11.0 5.0 7.0 2
9.0 5.0 7.0 3, 4, 5, 6, 7
8.0 4.5 4.5 8
9.0 4.5 4.5
8.0 5.5 5.5
9.0 5.5 5.5
7.8 5.0 5.0
9.2 5.0 5.0
8.5 4.3 4.3
8.5 5.7 5.7
8.0 5.0 5.0
8.5 5.0 5.0
9.0 – 7.0 9
11.0 5.0 7.0 10, 11
Buckwheat 8.0 4.5 7.0 12
quinoa 9.0 and 11.0 5.0 7.0 13
9.0 5.0 7.0 14
8.0 to 11.0 4.5 7.0 15

Protein aggregation Amaranth 9.0 5.0 7.0 4, 16

9.0 to 11.0 5.0 7.0 17
11.0 5.0 7.0 10, 11, 18
Quinoa 8.0 to 12.0 – 8.0 to 12.0 19
9.0 5.0 7.0 20, 21

Solubility Amaranth 0.25% NaOHa – 3.0 to 9.0 22

9.0 5.0 7.0 and 8.5 23
9.0 5.0 7.0 5
9.0 – 3.0, 4.5, and 7.0 9
Buckwheat 0.25% NaOHa – 3.0 to 9.0 22
8.0 4.5 7.0 24, 25
Quinoa 0.015 M NaOHa 4.5 3.0 to 8.0 26
9.0 and 11.0 5.0 3.0 to 11.0 13
5.0 to 10.0 3.0 to 6.0 1.0 to 10.0 27
9.0 5.0 3.0 to 11.0 14

Foaming properties Amaranth 0.25% NaOHa Ȅ 7.0 22

9.0 and 11.0 5.0 4.0, 7.0, and 9.0 2
9.0 5.0 7.5 28
9.0 5.0 2.0 and 8.0 4, 29
9.0 5.0 2.0 to 9.0 5
Buckwheat 0.25% NaOHa – 7.0 22
Quinoa 0.015 M NaOHa 4.7 7.0 30
0.015 M NaOHa 4.5 3.0 to 8.0 26

Emulsifying properties Amaranth – – 7.0 22

9.0 5.0 2.0, 6.3, and 8.0 31
9.0 5.0 2.0 and 8.0 3
9.0 5.0 2.0 to 9.0 5

Buckwheat – – 7.0 32
8.0 4.5 2.0 to 10.0 24, 33
Quinoa 0.015 M NaOHa 4.7 7.0 30
0.015 M NaOHa 4.5 3.0 to 8.0 26
5.0 to 10.0 3.0 to 6.0 10.0 27

Gelling properties Amaranth 9.0 4.5 7.0 34

9.0 5.0 7.0 35, 36

Quinoa 9.0 5.0 8.5 and 10.5 20

8.0 to 11.0 4.5 6.5 15
a The authors mention NaOH concentration instead of pH.
“—” Indicates not specified.
References: 1. Martinez and Anon (1996), 2. Abugoch James and others (2010), 3. Ventureira and others (2012b), 4. Bolontrade and others (2013a), 5. Shevkani and others (2014a), 6. Shevkani and Singh
(2015), 7. Cordero-De-Los-Santos and others (2005), 8. Salcedo-Chávez and others (2002), 9. Bejarano-Lujan and Netto (2010), 10. Condes and others (2012), 11. Condes and others (2015), 12. Tang (2007cc),
13. Abugoch James and others (2008), 14. Steffolani and others (2015), 15. Ruiz and others (2016), 16. Scilingo and others (2002), 17. Avanza and Anon (2007), 18. Condes and others (2013), 19. Valenzuela
and others (2013), 20. Mäkinen and others (2015), 21. Mäkinen and others (2016), 22. Bejosano and Corke (1999), 23. Condes and others (2009), 24. Tomotake and others (2002), 25. Tang and others (2009),
26. Aluko and Monu (2003), 27. Elsohaimy and others (2015), 28. Ventureira and others (2012a), 29. Bolontrade and others (2013b), 30. Chauhan and others (1999), 31. Ventureira and others (2010), 32.
Zheng and others (1997), 33. Tang (2007b), 34. Bejarano-Lujan and others (2010), 35. Avanza and others (2005a), 36. Avanza and others (2005b).
IEP, isoelectric precipitation.


C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 9
Proteins of amaranth, buckwheat, and quinoa . . .
Table 4–Differential scanning calorimetry (DSC) protein denaturation temperatures reported for Osborne fractions from amaranth, common and tartary
buckwheat, and quinoa.
Reference
Amaranth Marnez and Anon (1996)
Marcone (1999)
Common buckwheat Tang (2007a)
Tartary buckwheat
Quinoa Abugoch James and others (2009)
DSC, differential scanning calorimetry; Td , denaturation temperature; ?, yet to be determined by future researchers.
buckwheat globulin fraction, thus indicating lower protein thermal
stability (Choi and Ma 2005).
Quinoa. Heat-induced denaturation of quinoa proteins has only
sporadically been investigated. Quinoa protein isolate denatures at
about 98 °C (Abugoch James and others 2008; Steffolani and oth-
ers 2015; Ruiz and others 2016). Protein isolates extracted at pH
11.0 did not show any endotherm, indicating that such alkaline
extraction resulted in complete denaturation (Abugoch James and
others 2008; Ruiz and others 2016). Gorinstein and others (1996)
reported a Td of 58 °C for quinoa globulins. However, the main
quinoa globulin, namely the 11S globulin (see section “Globu-
lins”), and 11S globulins of other protein sources (such as soy)
typically denature at much higher temperatures (> 85 °C) (Mar-
tinez and Anon 1996; Tarone and others 2013). Abugoch James
and others (2009) reported a Td of 99 °C for quinoa proteins in
flour (Table 4). Since quinoa starch gelatinizes at around 60 °C
(Li and others 2016), it cannot be excluded that the gelatinization
endotherm overlaps with an endotherm of proteins with lower
Td .
Effect of salt, reducing agents, and protein denaturants. Other
constituents present can affect protein denaturation. Salt increases
the thermal stability of amaranth proteins (Bolontrade and oth-
ers 2013a), amaranth albumin-2 (Castellani and others 1998), and
buckwheat globulins (Choi and Ma 2005; Tang 2007a), as evi-
denced by higher Td readings. At ionic strength of 0.54 M, the
Td of amaranth albumin-2 reaches a maximum at pH 6.0, but it is
much lower below pH 5.0 and above pH 8.5. Thus, both acid and
alkaline pH conditions induce conformational changes in its struc-
ture, which lower its thermal stability. Furthermore, at alkaline pH
values and ionic strength of 0.54 M, a 2nd endotherm appears at
about 73 °C. This points to the formation of new molecules of
lower thermal stability than that of the undivided albumin-2 (Td of
102 °C, pH 7.5). In this context, sedimentation and gel filtration
elution patterns have shown that alkaline pH triggers the dissocia-
tion of the so-called albumin-2 into its trimeric subunits (see sec-
tions “Albumins” and “Globulins”). These subunits are probably
less thermostable than the undivided molecules (Castellani
and others 1998). The Td of common buckwheat globulins de-
pends on the type of salt and decreases in the lyotropic series of
anions (thus in the order Cl– , Br– , I– ,and SCN– ) (Choi and Ma
10 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
Denaturaon
Protein fracon
temperature (°C)
Albumin-1, minor components of
70 to 73
globulins, and glutelins
94 to 100 Albumin-2, and 11S globulin
80.2 7S globulin
80 8S globulin
102 13S globulin
? ?
98 to 99 11S globulin
2005; Tang 2007a). While Cl− and Br− ions stabilize the protein,
I− and SCN− ions destabilize it (Tang 2007a). The presence of
the protein denaturant urea led to a decrease in Td of amaranth
globulins, whereas 2 mercaptoethanol, an SS-reducing agent, did
not affect it (Gorinstein and others 1996; Bolontrade and others
2013a). Hence, hydrogen bonds are more important for the ther-
mal stability of amaranth globulins than SS-bonds (Gorinstein and
others 1991b; Gorinstein and others 1996). Much as it is the case
for amaranth proteins, the Td readings of common buckwheat
globulins decrease with increasing levels of protein denaturants
(including urea and SDS), confirming the importance of hydro-
gen bonds and hydrophobic interactions for their thermal stability
(Tang 2007a). It is of note here that sufficiently high concentra-
tions of 2-mercaptoethanol are necessary to decrease the Td of
buckwheat 13S globulin (Choi and Ma 2005; Tang 2007c).
Protein aggregation
Little information is available on heat-induced aggregation of
pseudocereal proteins, especially so for proteins extracted under
mild conditions.
Amaranth. Thermal treatment (10 min at 90 °C) of amaranth
proteins suspended in a potassium phosphate buffer (pH 8.5) causes
a decrease in protein solubility. This is mainly due to aggrega-
tion of the globulin fraction (Scilingo and others 2002). Moder-
ate heating (50 °C) of amaranth proteins during alkaline extrac-
tion induces partial protein unfolding and, as a consequence, the
formation of high MM aggregates, as evidenced by DSC, size-
exclusion high-performance liquid chromatography (SE-HPLC),
a lower free thiol (SH) content, and a reduction in protein sol-
ubility (Bejarano-Lujan and Netto 2010). Mild heating (70 °C)
of aqueous dispersions of amaranth proteins decreases their sur-
face hydrophobicity. The effect of longer heating times and higher
temperatures on surface hydrophobicity depends on the pH (9.0
compared with 11.0) at which the proteins are extracted. Both
protein denaturation and aggregation likely affect their surface hy-
drophobicity. High MM aggregates are formed during heating,
which are stabilized by both SS-bonds and noncovalent bonds.
A protein isolate obtained at pH 9.0 is more sensitive to thermal
treatment than one obtained at pH 11.0 (Avanza and Anon 2007;
Condes and others 2013).

C 2016 Institute of Food Technologists®
Proteins of amaranth, buckwheat, and quinoa . . .
High-pressure treatment of amaranth protein isolates affects the
degree of dissociation or association of amaranth proteins ob-
tained by alkaline isolation. High-pressure treatment induces the
formation of insoluble aggregates, triggers the dissociation of sol-
uble aggregates, and increases surface hydrophobicity (Condes and
others 2012, 2015).
Protein aggregation can also occur at room temperature when
altering the pH and/or ionic strength of the solution, which evi-
dently affects protein solubility (see section “Solubility”). Higher
ionic strength in a pronounced way reduces the solubility of
amaranth proteins, especially for proteins 1st isolated at pH 9.0
and subsequently dispersed in a pH 2.0 buffer, indicating the
importance of charge-shielding for protein aggregation and sub-
sequent precipitation (Bolontrade and others 2013a).
Buckwheat. DSC, SDS–PAGE, and SE-HPLC analyses have
shown that heat treatment of common buckwheat globulin dis-
solved in 10.0 mM phosphate buffer (pH 7.4) containing 1.0 M
NaCl induces (i) the dissociation of proteins with apparent MM
in a 34 to 36 kDa and a 21 to 22 kDa range and (ii) association
of proteins with subsequent aggregate formation (Choi and Ma
2006a; Choi and others 2006). During thermal aggregation, in-
termolecular SS-bonds are formed (Choi and others 2006). In the
presence of N-ethylmaleimide, an SH-blocking agent, aggregates
of higher MM and hydrodynamic radius are formed than in its ab-
sence (Choi and Ma 2006a). Transmission electron microscopy
demonstrated the formation of strand-like small aggregates as
well as that of large compact globular soluble macroaggregates
(Choi and Ma 2006a; Choi and others 2006). Protein aggregation
also occurs during puffing (Mariotti and others 2008) or auto-
clave treatment (Tomotake and others 2012) of dehulled common
buckwheat seeds. Puffing reduces the surface hydrophobicity of
common buckwheat flour proteins. Protein aggregation presum-
ably makes its surfaces less accessible for the hydrophobic fluores-
cent probe used in the analysis due to increasing mutual interac-
tions between hydrophobic protein surfaces (Mariotti and others
2008).
Quinoa. The effect of heat treatment (0 to 15 min, 100 °C)
on quinoa proteins isolated at pH 9.0 depends on the pH during
heating (Mäkinen and others 2015, 2016). Heating at pH 8.5 or
10.5 disrupts the SS-bond connecting the acidic and basic subunits
of 11S globulin (Table 2), while the covalent bond remains intact
when heating is at pH 6.5. Large aggregates with intermolecular
SS-bonds are formed by heating at pH 6.5 and 8.5, while no
aggregates are formed at pH 10.5, which are extractable in SDS-
containing medium. The results also indicate that a low degree
of protein hydrolysis occurs during heating at pH 10.5 (Mäkinen
and others 2016). The free SH-content of the globulin fraction
increases during the 1st 5 min of heating at pH 8.5 and 10.5, but it
decreases with prolonged heating to a level lower than that of the
nonheat-treated globulin fraction. Heating at pH 6.5 results in a
slight increase in the level of free SH. The surface hydrophobicity
increased during the 1st 5 min of heating (100 °C) at all tested
pH values. Upon prolonged heating, a small increase in surface
hydrophobicity was observed at pH 6.5 and 8.5, while at pH
10.5 it decreased almost to the reading observed for the nonheat-
treated globulins (Mäkinen and others 2016). Heating of quinoa
proteins obtained under alkaline conditions (pH 8.5 or pH 10.5)
may trigger deamidation of glutamine and asparagine residues and
thereby drastically affect protein surface properties. The degree of
deamidation after heating was about 30% at both pH 8.5 and 10.5
(Mäkinen and others 2015).

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 11
Techno-functional Properties
The techno-functional properties of amaranth proteins have
been more extensively studied than those of their buckwheat and
quinoa counterparts. As above, protein isolates in the studies de-
scribed below were obtained by alkaline extraction and subsequent
IEP at lower pH (Table 3), unless otherwise stated. We again stress
that the techno-functional properties of the extracted proteins are
likely impacted by the specific extraction conditions since these
can trigger conformational changes.
Solubility
Protein isolates. Protein solubility is important for its techno-
functional properties in different (food) applications. In general,
the solubility of amaranth, buckwheat, and quinoa proteins is
poor (range of 2 to 35% range) in a pH ranging from 3.0
to 5.0. Maximum solubility, with percentages ranging from 50
to almost 100%, is found at alkaline pH values (Bejosano and
Corke 1999; Tomotake and others 2002; Aluko and Monu 2003;
Silva-Sanchez and others 2004; Cordero-De-Los-Santos and oth-
ers 2005; Tömösközi and others 2008; Tang and others 2009;
Bejarano-Lujan and Netto 2010; Elsohaimy and others 2015; Stef-
folani and others 2015; Ruiz and others 2016). Depending on the
particular study, very good solubility has also been observed at pH
2.0 (Tomotake and others 2002; Tang and others 2009; Shevkani
and others 2014a). Part of the differences in optimal pH values
and solubility percentages can be explained by differences in cul-
tivar (Silva-Sanchez and others 2004; Shevkani and others 2014a;
Steffolani and others 2015) and extraction procedure (such as the
extraction pH). However, the latter has only been demonstrated
for quinoa proteins. In this case, proteins extracted at pH 9.0 are
more soluble at pH values exceeding 5.0 than those extracted at
pH 11.0 (Abugoch James and others 2008; Ruiz and others 2016).
Finally, no significant differences in solubility were observed be-
tween amaranth, buckwheat, or quinoa proteins over a pH range of
3.0 to 11.0. Hydrolysis of amaranth proteins with trypsin (Condes
and others 2009), of amaranth and buckwheat proteins with pepsin
(Bejosano and Corke 1999), and of quinoa proteins with Alcalase
(Aluko and Monu 2003) improved solubility. The solubility of
quinoa protein hydrolysates (degree of hydrolysis [DH] of 48%)
hardly depends on pH, in contrast to what has been observed for
their nonhydrolyzed counterparts (Aluko and Monu 2003).
Osborne-type fractions. Amaranth albumins and globulins and
proteins extracted under alkaline conditions show similar low sol-
ubility at pH values between 3.0 and 5.0. Above pH 5.0, its albu-
mins (maximum solubility of 90% at pH 11.0) are more soluble
than its globulins (maximum solubility around 50% at pH 11.0
(Tömösközi and others 2008). According to Carrazco-Pena and
others (2013), the amaranth 11S globulins, which are the major
proteins of amaranth (Table 2), show minimal solubility between
pH 2.6 and 4.6 (around 10% and 20% at low and high ionic
strength, respectively). Approximately 90% of the 11S globulin
fraction is soluble at pH values exceeding 6.1. The introduction of
4 successive methionine residues in the variable region V of recom-
binant amaranth 11S proglobulin drastically reduces its solubility
due to an increase in surface hydrophobicity. Here, it is of benefit
to mention that the properties of the recombinant unmodified 11S
proglobulin differ from those of the native protein purified from
amaranth seeds, most likely due to the lack of co- and/or post-
translational cleavages of the recombinant unmodified protein (see
section “Globulins”) (Carrazco-Pena and others 2013).
Proteins of amaranth, buckwheat, and quinoa . . .
Foaming properties
Protein isolates. The pH affects both amaranth protein foam
formation and stability. At pH 2.0, amaranth proteins foam better
than at pH 8.0 due to faster diffusion to and/or faster adsorp-
tion at the air/water interface. The higher flexibility of amaranth
proteins at acid than at alkaline pH facilitates their diffusion to
and adsorption at the air/water interface, thereby reducing the
interfacial tension (Bolontrade and others 2013a). Ventureira and
others (2012a) further showed that acid-treated amaranth extract
contains an endogenous peptidase which is active at pH 2.0. (Par-
tial) hydrolysis of amaranth proteins into smaller fragments im-
proves their diffusion to the air/water interface. Amaranth protein
foams are more stable at pH 2.0 than at pH 8.0, due to their interfa-
cial protein films being more viscoelastic and flexible at the former
pH (Bolontrade and others 2013b). However, the pH at which
foaming is optimal seems to be cultivar-dependent (Shevkani and
others 2014a; Steffolani and others 2015). In addition, differences
in foaming may also be explained by differences in protein ex-
traction procedures. Abugoch James and others (2010) showed
that whether amaranth proteins are isolated from flour at pH 9.0
or 11.0 influences their conformation and surface hydrophobicity
and, as a consequence, their foaming properties. While their foam-
ing capacity was similar, foams prepared from extracts obtained at
pH 11.0 were more stable than those from extracts obtained at
pH 9.0, especially under acidic conditions (Abugoch James and
others 2010). High ionic strength favors amaranth protein foam
formation at pH 2.0 and pH 8.0 (Bolontrade and others 2013a).
However, the obtained foams are less stable than those produced
at low ionic strength (Bolontrade and others 2013b). At low ionic
strength, the protein film at the interface is thicker. This retards
gas diffusion and, hence, bubble disproportionation and coales-
cence. Furthermore, there is more electrostatic repulsion at low
than at high ionic strength (Bolontrade and others 2013b). Ama-
ranth proteins foam better than soy protein isolates obtained under
similar conditions, due to a faster reduction in interfacial tension
in the former case (Bejosano and Corke 1999; Ventureira and oth-
ers 2012a). Bejosano and Corke (1999) reported better stability of
amaranth than of soy protein foams, while Ventureira and others
(2012a) observed amaranth protein foams to have lower stability
than those of soy proteins. Buckwheat (Bejosano and Corke 1999)
and quinoa (Aluko and Monu 2003) protein isolates show optimal
foaming properties at higher pH than do amaranth proteins.
Osborne-type fractions. Papers dealing with foaming of pro-
teins isolated in a mild way are rather limited. Amaranth albu-
mins foam better and yield foams with better stability than ama-
ranth proteins extracted under alkaline conditions. The latter, in
turn, foam better than amaranth globulins (Tömösközi and oth-
ers 2008). The foaming properties of amaranth albumins are even
similar to those of casein (Tömösközi and others 2008) and egg al-
bumins at acidic pH values (Silva-Sanchez and others 2004). Both
protein types are frequently used in industrial applications for their
foaming behavior.
Effect of (pre)treatments. (Pre)treatments of pseudocereal pro-
tein isolates have been applied for improving their foaming prop-
erties. Amaranth proteins 1st treated with acid (pH 2.0 for 3 h)
and then neutralized to pH 7.5 reduce the interfacial tension faster
than their untreated counterparts. This results in improved foam
formation and stability. The acid-treated proteins are partially un-
folded and undergo limited hydrolysis due to the presence of an en-
dogenous peptidase (see above). Limited hydrolysis by the endoge-
nous peptidase thus favors migration of proteins to the air/water
interface and renders them more flexible, thereby enhancing
12 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
conformational rearrangements at the interface (Ventureira and
others 2012a). Protein hydrolysis is indeed a frequently used pre-
treatment to improve foam formation (Bejosano and Corke 1999;
Aluko and Monu 2003; Condes and others 2009). Limited hy-
drolysis of amaranth proteins (DH of 6.4%) results in foams that
are more stable than those produced with their nonhydrolyzed
counterparts, while foams prepared from moderately hydrolyzed
amaranth (DH varying between 11.6% and 17.0%) or buckwheat
(DH of 11.8%) (Bejosano and Corke 1999) proteins, or from
severely hydrolyzed quinoa (DH of 48%) (Aluko and Monu 2003)
proteins, are less stable than those from their native counterparts.
The latter proteins are probably too small to form viscoelastic
interfacial layers with sufficient mechanical strength.
Effect of endogenous constituents. Other constituents in pseu-
docereal protein isolates can also affect foaming properties. For
amaranth, Fidantsi and Doxastakis (2001) found that interactions
between proteins and polysaccharides can enhance both foam for-
mation and stabilization. Prior defatting of amaranth flour has,
depending on the cultivar, no or a positive impact on foam for-
mation, while foam stabilization clearly improves as a result of
flour-defatting (Shevkani and others 2014b). Mixed protein-lipid
interfaces are inherently unstable as they compete for the air/water
interface and weaken each other’s ability to stabilize it (Wilde
2000). Depending on the cultivar, quinoa may contain saponins,
which are a complex mixture of triterpene glycosides with surface-
active properties. Removing saponins from alkali-extracted pro-
teins reduces foam formation, but makes the obtained foams more
stable (Chauhan and others 1999).
Emulsifying properties
Protein isolates. Similar to what is described above for foaming
properties (see section “Foaming properties”), the pH also affects
emulsification and stabilization by amaranth proteins. In general,
emulsification by amaranth protein isolates is low at their pI, as
under such conditions proteins cannot diffuse rapidly to the in-
terface (Shevkani and others 2014a). While Bejosano and Corke
(1999) reported better emulsifying properties for amaranth than
for soy proteins, Cordero-De-Los-Santos and others (2005) and
Shevkani and others (2014a) found both protein types to behave
similarly in this respect. In addition, amaranth proteins obtained
via alkaline extraction and subsequent IEP produce a more stable
emulsion than do proteins obtained by alkaline extraction at the
same pH and subsequently dialyzed against water. This is likely due
to a lower coalescence rate as shown by monitoring the oil droplet
size distribution with light microscopy (Fidantsi and Doxastakis
2001). The emulsion stability between pH 7.0 and 10.0 of buck-
wheat proteins is lower than observed for soy protein isolate and
casein (Tomotake and others 2002). In contrast, while Bejosano
and Corke (1999) reported that commercial soy protein isolates
have emulsifying properties inferior to those of buckwheat pro-
teins, Zheng and others (1997) reported both protein emulsions to
behave in a similar way. In addition, below pH 4.0 and above pH
6.0, emulsification and stability of spray-dried buckwheat protein
emulsions are comparable to those formed from spray-dried soy
protein isolates (Tang 2007b). With regard to quinoa protein emul-
sions, Elsohaimy and others (2015) studied the effect of protein
concentration (0.1% to 3.0%). At higher protein concentrations,
emulsification increases, but the obtained emulsions are less stable.
Osborne-type fractions. Amaranth albumins show optimal
emulsification around pH 5.0 (Silva-Sanchez and others 2004).
At pH 8.0, Tömösközi and others (2008) reported superior emul-
sifying properties for amaranth albumins than for its globulins.

C 2016 Institute of Food Technologists®
Proteins of amaranth, buckwheat, and quinoa . . .
However, the purity of the albumin fraction was much lower
than that of the globulins (49.0 compared with 87.0%). It may
have been contaminated with polysaccharides, which positively
affect both the formation and stability of interfaces (Patino and
Pilosof 2011). The overall emulsification by amaranth globulins is
strongly pH-dependent. Emulsification and stabilization are poor
at pH < 5.0, but they improve substantially between pH 5.0 and
7.0 (Konishi and Yoshimoto 1989; Marcone and Kakuda 1999).
Under mildly alkaline conditions, they are inferior to those mea-
sured at pH > 9.0 (Konishi and Yoshimoto 1989). Between pH
3.0 and 9.0, amaranth globulins show better emulsification than
their soy counterparts (Marcone and Kakuda 1999). This may be
due to the high degree of glycosylation and phosphorylation of
amaranth globulins (see section “Globulins”). Furthermore, the
protein-stabilizing layer, which encloses the oil droplets, is highly
charged when the amaranth globulins are further from their pI,
which may enhance charge repulsion between emulsion particles.
In addition, high levels of glycosylation and phosphorylation also
generate a more hydrated layer, which retards droplet coalescence
(Marcone and Kakuda 1999). At pH 7.0 or 8.0, the emulsification
by amaranth globulins is lower than that by casein protein isolate
(Konishi and Yoshimoto 1989; Tömösközi and others 2008).
Effect of (pre)treatments. The emulsifying properties of pseu-
docereal protein isolates can be improved by (pre)treatments. Heat
treatment of a solution of amaranth globulins (50 to 100 °C) at
pH 7.5 improves emulsification due to (partial) denaturation of
the globulins (Table 3), which goes in hand with the exposure
of hydrophobic groups, as evidenced by 8-anilinonaphthalene-1-
sulfonic acid fluorescence measurements (Konishi and Yoshimoto
1989). Acid treatment (pH 2.0 for 3 h) and subsequent neutral-
ization to pH 7.5 of amaranth proteins obtained under alkaline
conditions improves their adsorption kinetics to and the elastic-
ity of the oil/water interface obtained. CD intensities and DSC
data have revealed that acid treatment induces extensive unfold-
ing of amaranth proteins. This may stabilize the interface. More-
over, higher elasticity of the interfacial oil/water layer results in
higher resistance of the prepared emulsions against coalescence
and, hence, in improved emulsion stability (Ventureira and oth-
ers 2012b). At a pH of 2.0, enzymatic hydrolysis of amaranth
proteins with trypsin to a DH of 2.2% or with Alcalase to DHs
of 1.7 or 9.5% slightly improves their emulsification (Ventureira
and others 2010). Severe enzymatic hydrolysis of quinoa proteins
with Alcalase (DH of 48%) lowers emulsification between pH
3.0 and 8.0, but improves the stability of the formed emulsion
(Aluko and Monu 2003). This indicates that some peptides were
still large enough to form a stable viscoelastic layer at the oil/water
interface.
Effect of endogenous constituents. Other constituents in pseu-
docereal protein isolates can also affect emulsifying properties.
Polysaccharides contribute to emulsion stability by crosslinking
proteins that are adsorbed at the interface (Fidantsi and Doxas-
takis 2001). Removing saponins from alkaline protein extracts
of quinoa reduces their emulsification, while the obtained emul-
sions are more stable (Chauhan and others 1999). The presence of
lipids in buckwheat protein isolates negatively affects the protein
emulsifying properties, especially at pH > 6.0 (Tang 2007c). As
observed for foaming (see section “Foaming properties”), mixed
protein-lipid interfaces negatively affect the emulsifying properties,
as proteins and lipids interfere with each other in the stabilization
of the interface (Wilde 2000).

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 13
Gelling properties
Although protein gelation is important in food processing, the
literature on pseudocereal protein gelation is scarce and limited to
a few studies on amaranth and quinoa proteins.
Protein isolates. Avanza and others (2005a) found a protein
concentration of 7.0% (w/v) and a temperature of 70 °C to be
the minimal requirements to induce gel formation by amaranth
proteins. At high protein concentrations, amaranth proteins form
ordered gel structures, which are stabilized by intermolecular SS-
bonds formed as a result of SH/SS-interchange reactions between
denatured 11S globulins and the alleged albumin-2 (see section
“Albumins”). Noncovalent bonds (primarily hydrogen bonds and
hydrophobic interactions), mainly between monomeric proteins
of apparent MM 42 kDa, stabilize the gel structure, but to a lower
extent than do SS-bonds. Proteins with an apparent MM below
20 kDa are present in the interstitial spaces of the gel matrix
(Avanza and others 2005b), which is elastic, hard, and displays
high fracturability and cohesiveness (Avanza and others 2005a). At
lower protein concentrations (< 15% w/v), temperatures close to
70 °C and short heating times, more disordered gel structures are
formed in which the proportion of noncovalent bonds is higher
than in gels formed at high protein concentration, higher temper-
atures, or longer heating times (Avanza and others 2005a, 2005b).
Whether amaranth proteins are isolated under acid or alkaline con-
ditions has a strong impact on the structure and rheology of the
formed gels. Gels from proteins extracted at pH 4.5 are inferior
to those formed from proteins extracted at pH 9.0 due to lower
globulin levels in the former extract (Bejarano-Lujan and others
2010). Higher temperatures during extraction at pH 9.0 further
improve amaranth protein gelation. This likely results from partial
protein denaturation and aggregation (Bejarano-Lujan and others
2010).
Gels prepared from quinoa proteins isolated under alkaline con-
ditions and heated (15 min at 100 °C) at pH 10.5 are more elastic
and show a finer and more regular structure than when produced
by heating at pH 8.5. Heating at pH 10.5 forms small soluble ag-
gregates, while at pH 8.5 it produces larger aggregates, which then
leads to coarse gel structures (Mäkinen and others 2015). Very re-
cently, Ruiz and others (2016) demonstrated that the (alkaline) pH
at which quinoa proteins are isolated has a strong impact on their
gelation. Proteins isolated at pH 8.0 or 9.0 (followed by IEP at
pH 4.5) formed dense semisolid gels already during heating (from
20 to 90 °C at 1 °C/min), while under such conditions no gel
was formed from proteins isolated at pH 10.0 or 11.0. A soft gel
was formed only during cooling (from 90 to 20 °C at 3 °/min)
when quinoa protein extraction was done at pH 10.0 or 11.0.
More protein is denatured during extraction under very alkaline
conditions, which likely results in aggregation and sedimentation
of large particles and reduced aggregation of smaller particles.
Osborne-type fractions. In contrast to gel formation by ama-
ranth protein isolates (see above), native amaranth 11S globulin
forms ordered gels when its concentration exceeds 21.8% and
when heat-treated at a temperature of at least 100 °C (Carrazco-
Pena and others 2013). The introduction of 4 methionine residues
in the variable region V of the 11S amaranth proglobulin decreases
the elastic (G ) and viscous (G ) moduli of the obtained gel. Here
again, differences between gelling properties of native and re-
combinant 11S globulins are ascribed to the lack of co- and/or
post-translational cleavages of the recombinant protein (see section
“Globulins”) (Carrazco-Pena and others 2013).
Proteins of amaranth, buckwheat, and quinoa . . .
Pseudocereal-Based Cereal End Products
The growing demand for high-quality and nutrient-dense
gluten-free products has increased the incorporation of pseu-
docereals in wheat and gluten-free food formulations (Capriles
and Arêas 2014; Pellegrini and Agostoni 2015). Even though
wheat and gluten-free products supplemented with pseudocereals
are more attractive from a nutritional point of view, mimicking
the unique functionality of wheat gluten remains technologically
challenging. We here provide an overview of the technological
impact of including pseudocereal flour in wheat and gluten-free
bread, pasta, noodles, and cookies. Recipes used in the literature
to study the, often empirical, effect of incorporating pseudoce-
reals in wheat and gluten-free foods greatly differ in the studies
cited below and often contain many ingredients. As synergistic
effects cannot be excluded, it is challenging to unravel the func-
tional role of pseudocereal proteins in such complex food matrices.
Strategies to affect the functionality of pseudocereal flour proteins
and concomitant, the quality of the end product, include the use
of enzymes aiming at cross-linking (transglutaminases and glu-
cose oxidases) or proteolysis (proteases), high-pressure treatments,
and sourdough fermentation (Taylor and others 2016). Strategies
with an explicit direct effect on flour proteins (enzymes and high-
pressure treatment) will be discussed in detail (see section “Strate-
gies to improve the functionality of pseudocereal proteins in dough
systems”).
Wheat and gluten-free bread
Pseudocereals have been incorporated in wheat bread formula-
tions in order to improve its nutritional profile (Schoenlechner and
others 2008). Partial substitution of wheat by pseudocereal flour
weakens the strength of the viscoelastic gluten network and, con-
comitant, its gas-holding capacity. This results in bread of lower
volume and a coarse and heterogeneous crumb structure (Lorenz
and Coulter 1991; Rosell and others 2009; Garcı́a-Mantrana and
others 2014; Bilgiçli and İbanoğlu 2015).
Flour from amaranth or buckwheat and flour or bran from
quinoa have been incorporated in gluten-free bread formulations
(Masure and others 2016). The inclusion of amaranth, buckwheat,
or quinoa flour in gluten-free bread recipes containing rice flour
and potato starch improves bread specific volume and crumb soft-
ness (Alvarez-Jubete and others 2010). Schoenlechner and others
(2010a) used a response surface methodology to study the effect of
water (60%,70%, and 80% flour basis), egg white albumen (0.0%,
2.5%, and 5.0% flour basis), and (vegetable) fat ((0.0%)0.0%, 2.0%,
and 4.0% flour basis) on quality characteristics of gluten-free bread
made from 40% amaranth flour and 60% gluten-free flour (consist-
ing of maize and potato starch, rice flour, and locust bean gum).
Increasing levels of egg white albumen and fat decreased bread
crumb firmness. Torbica and others (2010) substituted 10% to
30% of rice flour by 90% to 70% of hulled or dehulled buckwheat
flour. Using dynamic oscillations, they observed that the dough
elastic modulus is optimal at substitution levels of 10% and 20% for
hulled and dehulled buckwheat flour, respectively. As the authors
did not report on the elasticity of 100% rice dough, the effect
of buckwheat flour on the elasticity of gluten-free control dough
cannot be considered. Replacing up to 40% of a commercially
available gluten-free bread mixture by dehulled buckwheat flour
increased the net amount of carbon dioxide released, the maxi-
mum dough height (Mariotti and others 2013), and loaf specific
volume (Wronkowska and others 2013). Elgeti and others (2014)
reported that the specific volume of gluten-free bread from 25%
maize starch and 75% quinoa white flour was 33% higher than
14 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
that of control bread from 25% maize starch, 25% maize flour,
and 50% rice flour. The authors suggested that surface-active con-
stituents such as proteins and/or polar lipids might be essential
for gas cell stabilization. Inclusion of quinoa bran at levels be-
low 30% in a gluten-free control formulation based on maize
starch, and maize and rice flour increased gluten-free bread spe-
cific volume and decreased crumb mean pore size (Föste and others
2014).
Wheat and gluten-free pasta and noodles
Pasta and noodles are produced from nonfermented dough
(Delcour and Hoseney 2010) either by lamination or extrusion.
Their quality attributes such as appearance, texture, and cook-
ing quality to a dominant degree rely on the unique ability of
wheat gluten to provide strong and viscoelastic dough (Bruneel
and others 2010; Delcour and Hoseney 2010; Hou and others
2013).
As already mentioned for bread making, the incorporation of
pseudocereals in pasta and noodle recipes aims at improving their
nutritional profile. Protein extracts obtained under alkaline con-
ditions from amaranth or buckwheat flour were incorporated in
a wheat noodle formulation (Bejosano and Corke 1998). Even
though the overall effect on noodle quality was limited, inclusion
of buckwheat protein isolate strengthened the dough, as observed
with mixography and dynamic oscillatory measurements. This was
mainly due to the water-insoluble fraction of the protein extract.
Increasing levels of whole meal amaranth in a wheat pasta formu-
lation resulted in pasta properties which were inferior in terms of
firmness and cooking losses (Martinez and others 2014). Substi-
tution of 50% durum wheat semolina by 30% buckwheat flour
and 20% durum wheat bran in a spaghetti recipe did not signif-
icantly affect cooking losses or overall acceptability (Chillo and
others 2008a). Han and others (2013b) studied the effect of incor-
porating Tartary buckwheat flour in a wheat noodle formulation.
Higher substitution levels (10% to 30%) resulted in less firm noo-
dles, while tensile strength and cooking losses were comparable to
observations on ‘control’ wheat noodles made without buckwheat
flour.
Gluten-free pasta made from amaranth flour is less firm and
needs shorter cooking time than such pasta based on quinoa or
buckwheat flour (Schoenlechner and others 2010b). The inclu-
sion of amaranth flour in gluten-free rice pasta increases pro-
tein solubility, but does not affect cooking losses. Extrusion-
cooking of both rice and amaranth flour prior to production of
the amaranth-enriched rice pasta has beneficial effects on pasta
firmness and, in contrast to their nonextrusion-cooked counter-
parts, reduces cooking losses (Cabrera-Chávez and others 2012).
Recently, D’Amico and others (2015) observed higher cooking
losses and inferior elasticity for pasta made from a 20/20/60 blend
of amaranth flour, quinoa flour, and whole meal buckwheat, re-
spectively, rather than from durum wheat. They also studied the
impact of predrying and/or high-temperature drying on the qual-
ity of such pasta. Longer predrying times and higher drying tem-
peratures have beneficial effects on gluten-free pasta firmness, elas-
ticity, and cooking losses. However, cooking such pasta resulted in
more cooking losses than did durum pasta, which resulted in infe-
rior elasticity readings. Protein solubility values of the pasta indi-
cated that protein aggregates are only formed when drying above
100 °C. The authors speculate that the effect of predrying and
high-temperature drying on the formation of a firm and elastic
protein network during the production of pasta supplemented
with pseudocereals results from the high levels of accessible

C 2016 Institute of Food Technologists®
Proteins of amaranth, buckwheat, and quinoa . . .
cysteine residues present in their proteins (D’Amico and others
2015). As illustrated in the section “Denaturation and aggregation
behavior,” high-temperature treatment provokes the denaturation
of amaranth, buckwheat, and quinoa proteins and, as a conse-
quence, triggers the formation of high MM aggregates stabilized
by intermolecular SS-bonds. Hatcher and others (2011) studied
textural attributes of soba noodles [that is, noodles that are com-
monly made from a blend of wheat flour and at least 60% buck-
wheat flour and water (Giménez-Bastida and others 2015)] made
from 80% whole meal buckwheat from different cultivars. The
inclusion of dehulled, whole meal brown Tartary buckwheat re-
sulted in noodles of improved texture compared to those produced
from whole meal green testa or from common buckwheat flour
(Hatcher and others 2011). The incorporation of quinoa flour
(16, 50, 100, 150, and 184 g/kg flour basis) in a maize gluten-
free formulation resulted in a product which, when cooked, had
acceptable stickiness (Caperuto and others 2001). In addition, in-
clusion of whole meal quinoa in an amaranth spaghetti recipe
showed beneficial effects on overall cooking quality and texture
parameters (Chillo and others 2008b).
Wheat and gluten-free cookies
Only few studies deal with the incorporation of amaranth or
quinoa in cookie systems. Sindhuja and others (2005) included
increasing levels of whole meal amaranth in short dough wheat
cookies and noticed that, even at substitution levels of 25%, cookie
dimensions and texture were substantially better than those of
the wheat control. Incorporation of amaranth or quinoa flour in
oat (Inglett and others 2015) or wheat cookies (Chauhan and
others 2016) increased dough elasticity. Higher substitution levels
of amaranth whole meal increased cookie diameter and spread
ratio, but decreased cookie hardness (Chauhan and others 2016).
More has been published on the production of cookies containing
buckwheat. Addition of increasing levels of buckwheat flour to a
wheat flour recipe resulted in inferior properties of short dough
cookies (Baljeet and others 2010; Jan and others 2015) and ginger
nut biscuits (Filipčev and others 2011).
Substitution of increasing levels of dehulled buckwheat flour
in a gluten-free cookie formulation based on rice flour provided
more viscous and less extensible dough. Furthermore, the overall
cookie quality decreases when the buckwheat flour substitution
level was raised from 10 to 30% (Hadnađev and others 2013).
The incorporation of buckwheat flour in gluten-free sugar-snap
cookies resulted in cookies with an oven spread comparable to that
of sugar-snap cookies produced from maize flour or whole-meal
teff (Mancebo and others 2015).
Strategies to improve the functionality of pseudocereal
proteins in dough systems
Enzymes. Transglutaminase (EC 2.3.2.13), a glutamine-γ -
glutamyl transferase, catalyzes the formation of iso-peptide
bonds between ε-amino groups of lysine residues and
the γ -carboxyamide functional group of glutamine residues
(Reinikainen and others 2003). Renzetti and others (2008a,b)
prove that a transglutaminase promotes covalent cross-links be-
tween common buckwheat proteins. In addition, confocal laser
scanning micrographs showed that the application of transglutam-
inase improves the homogeneity of the protein network. This
results in more elastic buckwheat dough (Han and others 2013a)
and in buckwheat bread of improved crumb softness and elasticity
(Renzetti and others 2008b). Transglutaminase treatment trig-
gers the formation of high-MM polymers in all Osborne protein

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 15
fractions (Renzetti and others 2008a). SE-HPLC chromatograms
confirmed that high MM protein structures are formed in the
common buckwheat albumin and globulin fraction when treated
with transglutaminase, but not in the prolamin and glutelin frac-
tions. The positive effect of transglutaminase treatment on net-
work formation between buckwheat proteins most likely results
from the high levels of accessible lysine and glutamine residues.
In the presence of molecular oxygen, glucose oxidase (EC
1.1.3.4) acts as a catalyst for the oxidation of β-d-glucose to hy-
drogen peroxide and d-gluconic acid. The hydrogen peroxide,
an oxidizing agent, interacts with the free SH-groups of cysteine
residues to give rise to the formation of SS-bonds (Joye and others
2009). Only 1 study deals with the effect of a glucose oxidase
treatment on the formation of cross-links between proteins from
pseudocereal flour (Renzetti and Arendt 2009). The addition of
glucose oxidase did not significantly affect buckwheat batter vis-
cosity or buckwheat bread specific volume and crumb texture.
This is likely due to the low availability of free SH-groups in
the major globulin fraction, as their subunits are intramolecularly
linked via an SS-bond (Table 2) (Renzetti and Arendt 2009; Taylor
and others 2016).
Peptidases (EC 3.4), act by hydrolyzing the peptide bonds link-
ing amino acids in proteins. Treatment with a commercial protease
lowered buckwheat batter viscosity as a result of protein hydrol-
ysis, but significantly increased buckwheat loaf specific volume
(Renzetti and Arendt 2009). In addition, the center of the crumb
showed a very large void.
High-pressure treatment. In general, high-pressure treatment
reduces the large number of noncovalent bonds, resulting in (par-
tial) unfolding of the protein. This way, the SH-groups of cys-
teine residues become more accessible to SH-interchange reactions
(Vallons and others 2011). Hence, high levels of free SH-
groups are a prerequisite to induce structure formation be-
tween proteins by high pressure, which is not the case for
buckwheat flour proteins. Consequently, the impact of high-
pressure treatment on these proteins is rather limited (Vallons
and others 2011). This observation is in agreement with the
negative effect of glucose oxidase (see subsection “Enzymes”)
on the formation of covalent bonds between buckwheat flour
proteins.
Conclusions and Perspectives
The well-balanced amino acid composition and the high levels
of dietary fiber, antioxidants, vitamins, and minerals make pseudo-
cereals interesting raw materials for highly nutritious cereal-based
food systems. An asset is that people with celiac disease can con-
sume pseudocereals. While prolamins and glutelins are the most
abundant protein fractions of true cereals (such as wheat), the
proteins of pseudocereals are predominantly albumins and globu-
lins. Although it is general practice to classify cereal grain proteins
by using an Osborne fractionation scheme, such scheme is not
always rigorously followed for pseudocereals. Especially pseudo-
cereal globulins and glutelins are in most instances not extracted in
a standard Osborne protocol. Furthermore, research outcomes on
structural properties of pseudocereal proteins are often contradic-
tory. Here, the application of mass spectrometry for characterizing
pseudocereal protein sequences (Qian and others 2008; Gao and
others 2010; Lagrain and others 2013) would be helpful. Pseudo-
cereals are classified as nonmodel plant species since their genome
sequence is currently not available. Hence, only scarce protein
sequences are available in online databases. A more thorough un-
derstanding of the primary, secondary, and tertiary structures of
Proteins of amaranth, buckwheat, and quinoa . . .
pseudocereal proteins is required to establish in-depth structure–
function relationships.
As demonstrated in this review paper, the basic foaming, emulsi-
fying, and gelling properties of amaranth, buckwheat, and quinoa
proteins have been investigated. These techno-functional proper-
ties are often similar to or even better than those of industrially
important protein sources such as soy protein and casein, which
makes them a promising alternative in many food applications.
In most cases, in-depth studies on, among others, the kinetics of
protein adsorption to the interface and properties of the film at
the interface, are missing. Moreover, the majority of the studies
on pseudocereal protein functionality cited in this paper focus on
protein isolates obtained by alkaline extraction and subsequent
IEP at acidic pH. The extraction is, in fact, a pretreatment that
can induce changes in protein conformation and/or structure and
in some cases can cause (partial) denaturation and thereby affect
techno-functional properties, as already demonstrated for foaming
and gelling properties. Hence, the outcomes of such studies are not
necessarily predictive for the properties of proteins in pseudocereal
flour.
Strategies to affect the functionality of pseudocereal flour pro-
teins and, concomitantly, to improve the quality of the end prod-
ucts include the use of enzymes (such as transglutaminases, glucose
oxidases, and peptidases), high-pressure treatments, and sourdough
fermentation. The most appropriate strategy depends on the raw
material used and the end product considered. For instance, the
native structure of the major buckwheat globulins seems responsi-
ble for the only limited effect of glucose oxidase and high-pressure
treatments. In contrast, transglutaminase mediates the formation of
cross-links between buckwheat flour albumins and globulins, likely
due to high levels of accessible lysine and glutamine residues in the
soluble protein fraction. Hence, among the strategies discussed in
section “Wheat and gluten-free bread,” transglutaminases appear
to have the largest potential to improve the rheological behavior
of pseudocereal dough systems.
In conclusion, only very few studies exist on the properties of
native pseudocereal proteins, and only limited fundamental stud-
ies on pseudocereal proteins (in cereal-based end products) are
available. As a result, information on their functionality in estab-
lishing end product characteristics is scarce. Food products often
consist of foam, an emulsion, a gel, or a combination thereof.
Hence, elucidation of the denaturation and aggregation behavior,
and of the techno-functional properties of amaranth, buckwheat,
and quinoa proteins, can form a basis for improving overall prod-
uct quality. In addition, the techno-functional properties of both
animal and vegetable proteins can be improved by enzyme treat-
ments (Gaspar and de Góes-Favoni 2015; Wouters and others
2016), mild heating, or phosphorylation (Du and others 2002;
Li and others 2004; Hayashi and others 2009). Whether, or to
what extent, similar techniques can further improve the techno-
functional properties of pseudocereal proteins remains largely
unknown.
Acknowledgments
Frederik Janssen, Anneleen Pauly, and Ine Rombouts grate-
fully acknowledge the Research Foundation – Flanders (FWO –
Vlaanderen, Brussels, Belgium) for positions as doctoral (FJ) and
postdoctoral (AP and IR) researchers. Jan A. Delcour is W.K. Kel-
logg Chair in Cereal Science and Nutrition at KU Leuven. This
work is part of the Methusalem program “Food for the Future” at
KU Leuven.
16 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
Author Contributions
Frederik Janssen, Anneleen Pauly, Ine Rombouts, Koen J.A.
Jansens, and Lomme J. Deleu wrote the manuscript with critical
input and corrections by Jan A. Delcour. Frederik Janssen did the
final editing. All authors contributed to locating and to interpret-
ing the literature sources.
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