Food Chemistry Advances 1 (2022) 100001

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Food Chemistry Advances

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Pseudocereals proteins- A comprehensive review on its isolation,

composition and quality evaluation techniques
Amanda Manoj Malik a,∗, Awanish Singh a,b
a
Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, IN (148106)
b
Department of Food Engineering and Technology, Tezpur University, Tezpur, Assam, IN (784028)

a r t i c l e i n f o a b s t r a c t

Keywords: In the recent years, there has been immense focus on the underutilized pseudocereals due to their high nutritional
Pseudocereals value, potential health benefits, bioactive properties and also as ideal candidate for gluten free products. The ma-
Protein-isolation jor focused pseudocereals are Amaranth, Buckwheat, Quinoa, Chia, Wattle and Album. The immense potential of
Amino acid score
these grains has led to the development of techniques for the isolation of important compositional components.
Protein digestibility
Being rich in the proteins, different isolation techniques have been employed such as the physical, enzymatic
and chemical methods. The high value of the pseudocereals extracted proteins in comparison to the conventional
cereals was established by essential amino acid index, biological value and nutritional index. Based on the above,
a clear perspective towards the isolation techniques was critically reviewed in this article. This review also docu-
ments amino acid composition, amino acid scores, in vitro digestibility values and protein efficiency ratio of the
isolated proteins.

1. Introduction have been given in Fig. 1. Pseudocereals are dicotyledonous according to

Pseudocereals grains are edible seeds belonging to dicotyledonous
species that are known as such due to their similar physical appearance
and high starch content similar to true cereals (monocotyledonous of
the Poaceae family) (Alvarez-Jubete et al., 2010a). Pseudocereals are
promising crops of future due to their high genetic variability that is ad-
vantageous for them to be adapted to different environments from trop-
ical to temperate climatic conditions (Joshi et al., 2018, 2019). “Sub-
exploited foods” could be interpreted as the foods that were replaced
in the 20th centenary by more foods that succeed in the diets of the
world population. The pseudocereals as a whole and their constituents
such as proteins can be used for various novel food applications like
developing food for gluten intolerant people, replacing foods derived
from animal-based proteins, development of novel food packaging ma-
terials, edible and biodegradable containers, etc. The different process-
ing technologies for pseudocereal proteins and their effective utilization
for development of novel food products and as novel food ingredients
botanic characteristics (Schoenlechner, Siebenhandl, & Berghofer, 2008,
Ciudad-Mulero et al., 2019). There are several major pseudocereals that
are produced in the world, including amaranth, buckwheat, quinoa,
chia, wattleseed, and album (Table 1). Studies have examined various
non-conventional sources of protein in recent years, such as the ama-
ranth (Amaranthum), quinoa (Chenopodium quinoa Wild), chia (Salvia
hispanica L.), wattleseed (Acacia auriculiformis) and album (Chenopodium
album). These seeds resemble true cereals in composition and function,
so they are referred to as pseudocereals (Alvarez-Jubete et al., 2010).
The majority of protein isolates are obtained by solubilizing the protein
source in high pH environment, away from its isoelectric point, further
precipitating the solubilized proteins towards the isoelectric point. Most
of the protein isolation from pseudocereals have been done by the use
of alkalis like NaOH, which quickly dissolve seed proteins. Alkali solu-
tions with varied pH ranges are used to dissolve the proteins, followed
by a mild acid such as dilute (Agboola & Aluko, 2009; Australian Agri-
cultural Consensus, 2018, FAO Expert Consultation, 2011, FAO/WHO’

Abbreviations: AA, amino acid; AAS, amino acid score; AIPE, alkaline isoelectric precipitation extraction; AOT, aerosol OT sodium bis (2-ethylhexyl) sulfosuccinate;
API, album protein isolate; A/TE, essential amino acid to sum of essential amino acids in total; BCAA, branched chain amino acids; BV, biological value; CE, catechin
equivalent; CNS, central nervous system; CS, chemical score; DH, degree of hydrolysis; E/T, essential to total; EAA, essential amino acids; EAAI, essential amino acid
index; EAI, emulsifying activity index; FAO, food and agriculture organization; GI, glycemic index; IPC, isoelectric protein concentrates; IVD, in-vitro digestibility;
IVPD, in-vitro protein digestibility; LA, lysine to arginine; NPU, net protein utilization; NRC, national research council; PDCAAS, protein digestibility corrected
amino acid score; PER, protein efficiency ratio; PI, isoelectric point; PS, protein solubility; TR, retention time; TVP, texturized vegetable protein; USDA, united states
department of agriculture; UUAAIP, Ultrasound-Ultrafiltration-Assisted Alkaline Isoelectric Precipitation; WHO, world health organization.
∗
Corresponding author.
E-mail address: ammymalik11694@gmail.com (A.M. Malik).

https://doi.org/10.1016/j.focha.2021.100001
Received 11 September 2021; Received in revised form 1 November 2021; Accepted 8 November 2021
2772-753X/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
A.M. Malik and A. Singh Food Chemistry Advances 1 (2022) 100001

Fig. 1. Protein isolation from pseudocereals, their characterization and utilization for creating future foods.

Table 1 of lysine, arginine, tryptophan and sulphur containing amino-acids. The

Production volume and top producers of pseudocereals in the world. lysine content of amaranth is twice that of wheat and three times that of
Total Production (in tonnes) maize. The nutritive value is also very high in comparison with barley,
maize and wheat (Gorinstein et al., 2002) (). Buckwheat has high protein
World
Pseudocereal quality as its biological value is above 90%, probably due to the high
Production volume Top producer concentration of essential amino acids and sulphur-containing amino
Album NA∗ NA∗ acids (Sytar et al., 2016). The amino acid composition of buckwheat re-
Amaranth NA∗ Mexico (2019) sembles that of cereals with low content of lysine and the concentration
Buckwheat 16,12,235 Russia (2019) of proline and glutamic acid represents about half of the amino acids.
Chia NA∗ China (2019)
Chia seeds also contain a high percentage of glutamic acid, arginine and
Quinoa 1,61,415 Peru (2019)
Wattleseed 92,87,000# Australia (2018) aspartic acid and the values are higher than most of the cereal grains.
The protein of quinoa seed is rich in essential amino acids, particularly
∗
NA- not available methionine, threonine and lysine, which are the limiting amino acids in
#
Approximate value.Ref: Australian Agricultural Consensus most cereal grains(González, Konishi, Bruno, Valoy, & Prado, 2012).
2018FAO/Food and Agriculture Organization of the United Nations
An essential amino acid (EAA) is considered the most important fea-
(FAO)- FAOSTAT, 2019.
ture of a protein as it cannot be synthesized by humans because of which
it must be obtained through the diet. The essential amino acids thus play
a greater role in growth and maintenance of metabolic processes than
Expert Group, 1965, National Research Council, 1987, United States the rest of the amino acids. Additionally, there is also a third category of
Department of Agriculture, 2007; Branlard & Bancel, 2007; Cordero-De- ’conditionally essential’ amino acids, meaning that the body cannot pro-
Los-Santos, Osuna-Castro, Borodanenko, & Paredes-López, 2005; García, duce them under specific physiological or pathological conditions, and
Puchalska, Esteve, & Marina, 2013; González, Konishi, Bruno, Valoy, & their intake is therefore essential in this situation (WHO, 2007). The
Prado, 2012; Gorinstein et al., 2002a, 2002b; Hadnađev et al., 2017; amino acid score of the pseudocereals is almost comparable with EAAI
Salcedo-Chávez, Osuna-Castro, Guevara-Lara, Domínguez-Domínguez, values for pseudocereals of amaranth, buckwheat and quinoa. Buck-
& Paredes-López, 2002; Schoenlechner, Siebenhandl, & Berghofer, 2008; wheat and chia stand out among all the pseudocereals for having high
Shevkani & Singh, 2015)HCl solution to precipitate the proteins. The sol- quantity of essential amino-acids. The aromatic amino-acids and Sul-
ubilization of protein can also be achieved by using saline solutions fol- phur containing amino-acids are high in chia seeds in comparison with
lowed by protein precipitation via ultrafiltration and diafiltration. Prior other pseudocereals.
to being dried, micellar proteins are produced, and the native state of For protein to be nutritionally valuable, digestibility needs to be con-
the protein is maintained (Hadnađev et al., 2017). It has also been used sidered. By estimating the Protein Digestibility Corrected Amino Acid
to increase yields and make products of higher quality and purity by Score (PDCAAS), which represents the protein quality in human nutri-
using ultrafiltration membranes. In recent years, the use of enzymes has tion during different life stages (WHO, 2007), EAA content can be esti-
also emerged as a promising technique for protein isolation as this tech- mated. The in-vitro protein digestibility (IVPD) of the pseudocereals is
nology offers high specificity in addition to better yields and purity. relatively high (>70%) with the exception of chia seeds which have a
The protein content of the pseudocereals lies in the range of 10- low value for IVPD (about 50%). The present review lays out a compar-
20%. The nutritional quality of pseudocereal proteins is also very high ative study on various characteristics of Amaranth, buckwheat, quinoa,
in comparison to cereals and some legumes. Amaranth has high contents chia, wattle and album proteins based on current research. Furthermore,

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A.M. Malik and A. Singh Food Chemistry Advances 1 (2022) 100001

it points out the different protein isolation techniques, amino acid com- different extraction pH (alkaline media pH scale 8 to 11) and precip-
position and protein quality evaluation of all the pseudocereals. itation pH (acidic media pH 4 to 6) combos and analyzing the isolate
properties. There are specific disadvantages of the isoelectric methods
like the loss of some useful properties and production of certain antinu-
2. Protein isolation from pseudocereals using different methods
tritional factors like phytic acid and lysinoalanine giving way to reduc-
tion of the nutrient quality of proteins (Rahma et al., 2000). A different
Extraction of proteins from grains is becoming an important subject
method used alkali wet milling to steep the grains in NaOH. The pro-
nowadays as they are used for many food formulations that require pro-
tein liquor was set apart from the starch fraction by centrifuging the
tein isolates as their main ingredient like fitness products, TVP, etc. As
filtrate. Wet curd was obtained when acid precipitate was frozen and
they are not a complete source of proteins like meat, they need to be sup-
thawed to disrupt the emulsion and water separation, followed by dry-
plemented with other grains or legumes to fulfill the lacking nutritional
ing and crushing into a powdery substance. Partial breakdown of the
characteristics. Table 2 states more precisely about the different meth-
protein concentrates was done by the use of pepsin enzyme. Incuba-
ods used for isolation of proteins, their processing conditions, their yield
tion was done through moderate shaking and pH adjustments and then
and purity. Further, in order to extract proteins from pseudo-cereals
freeze drying and degree of hydrolysis (DH) was calculated (Bejosano &
and/or to impact the color, flavor and/or texture of their finish product,
Corke, 1999). When we talk about sources of high-quality proteins, only
alkalescent conditions are used. Protein extraction yields increase by us-
certain pseudocereals fulfill the desired nutritional requirements and
ing alkali by (1) matrix breakdown in which proteins are present, and (2)
quinoa falls in the category of such pseudocereal grains. From quinoa,
increasing protein solubility. Proteins possess higher charge once they
proteins can be isolated by setting defatted quinoa flour in deionized wa-
are beyond their pI (generally 4.5 to 5.0 range), herewith their solubility
ter at alkaline pH. The suspension was stirred and centrifuged to give
in aqueous compound media will increase. Acid precipitation may also
supernatants that were acidified and centrifuged again to get pellets;
motivate certain changes subsequently. There are techno-functional and
which were again suspended in deionized water. High pH values cre-
biological process implications, typically caused by changes in struc-
ated proteins of 90–93% purity whereas lower pH values yielded pro-
ture and/or composition, together with isomerization, crosslinking and
teinaceous compounds of 82–88% purity. The results showed a bigger
degradation (Deleu et al., 2019). Physical techniques are used as they
impact of process temperature on the DH of quinoa protein compounds
are less complicated to adapt and highly economical when compared
as compared to extraction pH (Ruiz et al., 2016). The proteinaceous
with other techniques of protein isolation. General overview of protein
concentrate had considerably higher emulsifying property (EAI) than
isolation by different methods like chemical method, physical method
the hydrolyzed products in all hydrogen ion concentration values. The
or enzymatic methods have been given in Fig. 2. Physical methods are
poor foaming capability could also be because of the globular nature
more preferrable than enzymatic or chemical techniques in processing
of the proteins, that reduces the power to make interfacial membranes
of foods as they cause lesser alterations within the food. Cell disruption
round the air bubbles (Branlard and Bancel, 2007). Among the factors,
by physical processes will increase protein extraction. Some physical
temperature and NaCl concentration conferred a negative result dur-
strategies used for protein extraction are colloid milling, freeze-thaw,
ing protein extraction and this principally happens with hydrophobic
high speed mixing, blending and homogenization. Disruption of cells is
globular proteinaceous compounds (Chi et al., 2003). The ascertained
due to shear forces generated during high-speed mixing and homoge-
negative result is probably because of the decrease in solubility due to
nization. Cell walls will be broken owing to sonication and molecular
the non-polar nature of the surface of quinoa proteinaceous compounds,
bonds, due to the resulting high temperature and shock waves that cause
a development called salty precipitation and conjointly ascertained with
cavitational collapsing of the bubbles due to ultrasound energy (Fabian
proteins from soybean.
& Ju 2011).
As we go on to discuss protein extraction from pseudocereals, there
An alternative methodology that enables the extraction of proteins
are plenty of them that are nutritionally rich, yet they remain neglected
at neutral and slightly basic pH levels involves the use of enzymes. Pro-
or underutilized. Buckwheat is a nutrition dense pseudocereal which
teins are not exposed to alkalescent conditions during these techniques,
can be utilized to fulfill protein requirements in the diet. We can ex-
which usually lead to the formation of undesirable elements and loss of
tract the albumin and globulin protein fractions (containing the bulk of
nutritional attributes. Enzymes facilitate in the protein extraction in sev-
buckwheat protein isolate) from buckwheat flour by the alkali method
eral ways. 𝛼-amylase and phytase enzymes better the protein extraction
(e.g., pH 8.0–8.5). When using enzymes for protein extraction, enzyme
by breaking the protein’s interactions with starch and phytate within
specificity, degree of hydrolysis (DH) and nature of free peptides (e.g.,
the bran, that interferes with the protein extraction (Tang et al., 2002).
molecular mass and amino acid composition) are principally liable for
Greater recovery of proteins happen once enzymes are utilized in com-
the free radical scavenging properties of the hydrolysates (Tang et al.,
parison to basic extraction. However, because of their high prices, usage
2009). When the methodologies for protein isolation are employed, cer-
of enzymes remains restricted to make the method a lot more econom-
tain nutritional properties of the proteins are altered. To counter these
ical, henceforth immobilized enzymes are usually used for protein ex-
alterations in protein nutritional properties, certain modifications need
traction.
to be introduced within the wet alkaline method (Grossman et al., 1980).
A number of studies are conducted in an attempt to scale-back aller-
2.1. Chemical methods of protein isolation from pseudocereals genicity of buckwheat (Hadidi et al., 2020). Buckwheat seed fraction
when undergoing Maillard-type glycosylation and salting technique re-
The use of chemicals for isolating proteins from cereal grains is one sulted in partial reduction of its allergenicity. Treatment with heat, acid,
of the most widely used methods due to the hidden benefits it offers. alkali, and reduced carboxymethylation additionally caused positive ef-
Apart from getting proteinaceous components in pure form, the method- fects on the reduction of allergenicity. Further, the proteins from quinoa
ology also proves to be a time saving approach. The process started with seeds can be separated by various techniques. Mixtures of various chemi-
the step of suspending defatted amaranth flour in water at a particular cals can also be utilized for the purpose of protein segregation (Bejarano-
hydrogen ion concentration, stirred and centrifuged thereafter. The su- Luján et al., 2010). One such technique operated by suspending the flour
pernatant was precipitated and resuspended in water, neutralized, and in HCl and NaCl at alkaline pH, stirring and centrifuging next, followed
freeze dried. With reference to food functionality of amaranth protein by collection and filtration of the supernatants. The proteinaceous ex-
isolates, Fidantsi and Doxastakis (2001) found that their isolates could tract was used as a feed stream for ceramic membrane ultrafiltration.
be exploited for effective foaming, emulsification and stabilization ap- Limitation with this technique is the issue in maintaining the structure
plications. In another methodology, (Salcedo-Chávez et al., 2002) opti- and functionality of proteins. Results showed that each ultrafiltration
mized the isoelectric method of protein isolates from A. cruentus, using and pH scale of extracts affected amino acid composition, thermal prop-

3
 Table 2
Different methods of isolation of protein from pseudocereals.

A.M. Malik and A. Singh

Isolation Conditions Yield Purity References
Pseudocereal method
protein

Alkali Extractant: pH pH pH pH pH 8(4.0) pH 8(5.0) pH 12(3.0) pH 12(6.0) Acosta

Amaranth method + Substrate=1:10(w/v); 8(4.0) 8(4.0) 12(4.0) 12(6.0) Aqua NaCl Aqua NaCl et al., 2016
isolate Salting Extraction pH= pH 8-12; Aqua NaCl Aqua NaCl
Extraction time=20 min.
82.33% 37.66% 83.42% 30.32%
20.52% 53.77% 19.31% 53.01%

Alkali Extractant: - 45.6% Shevkani and Singh,

Amaranth method Substrate=1:10(w/v); 2015
isolate Extraction pH- 9 &
time-20 min; Precipitation
pH-5 & time-20 min;
Centrifugation-8000g & 20
min
Extraction pH=7. 13.72% 44.5% Cordero-De-Los-
Amaranth Micellization Extraction time=2h. Santos, Osuna-Castro,
isolate technique Extraction temp=25°C. Borodanenko, &
Paredes-López, 2005
Dual Solid/solvent ratio =1:8 57.93% 49.07% Chen et al., 2019
Buckwheat enzymatic (w/w), Colloid mill
isolate process speed= 600rpm. Enzyme
incubation time & temp.
=3h at 70°C. Enzyme
deactivation temp. &
time= 100°C for 5 min.
Alkaline Extractant: Solvent=1:10. 8.1% 69.7% Tang et al., 2009
4

Buckwheat method Extraction time & temp=1

isolate h at RT, Extraction
pH=8.5.
Alkaline Extractant: Solvent=1:10. BPI- BPI- BPI- BPI- BPI-C BPI-M BPI-P Tang et al., 2009
Buckwheat method Extraction time & temp=1 C M E P BPI-
isolate h at RT, Extraction E
pH=8.0
46.9% (0.6) 49.4% (0.2) 49.1% (0.1)
21.9 21.4 21.9 20.6 48.7%
% % % % (0.1)
(0.2) (0.3) (0.7) (0.9)
Isoelectric Extraction time-4h, Temp- - SM- SU-BWP DSU-BWP Tang, 2007
Buckwheat precipita- Ambient, pH-8.0 BWP FM- FU- DFU-
protein tion with Ultrasonic freq -15 kHz, BWP BWP BWP
prod- ultrasonic Power- 500 W,
ucts aid Time-30min.
65.1% 80.6%

Food Chemistry Advances 1 (2022) 100001

80.5% 81.4% 66.1% 82.3%
Quinoa Alkali Extractant: Solvent =1:9 52.0 % 82% Ruiz et al., 2016
isolate method (10 % w/w), pH- 8-11,
Extraction time - 4h.
Temp-RT. Incubation-4°C,
16h.
Chia Alkali Extraction time -20min - Purity Coelho et al., 2018
protein method Extractant:
concen- Solvent=1:20(%w/w),
trate pH-10.0,
Stirring-1500rpm,
Temp-25°C.
CPC1 CPC2
70.9% 74.1%
A.M. Malik and A. Singh Food Chemistry Advances 1 (2022) 100001

Fig. 2. Generic overview of protein isolation from pseudocereals by different methods.

5
A.M. Malik and A. Singh
erties and therefore the spacial conformation of protein. The addition of
isoelectric precipitation step as a part of AIPE and UUAAIP raised the
purity by selective precipitation of proteins from alternative polymers.
Furthermore, the ultrasound in UUAAIP method is able to bring
about bubble cavitation facilitating the disruption of biological matri-
ces and resulting in the freeing of proteins. Ultrasonic-assisted alkali ex-
traction followed by ultrafiltration yields considerably higher proteina-
ceous content (60 g/100 g and 89 g/100 g) for quinoa than for alfalfa
leaves proteinaceous concentrate (70 g/100 g) (Hadidi et al., 2020). In
the case of certain pseudocereals, extracting proteins is not that easy
due to strong binding of different components with one another and re-
quires a completely different approach. One such methodology utilized
the addition of quinoa powder to reverse micelle solution and putting in
a conical bottle. The water bath oscillator was used for extraction and
then centrifugation was done. The system was then divided into 2 layers
and the protein content was calculated. Results showed that higher tem-
perature, smaller particle size and better reverse micelle concentration
were helpful to the internal diffusion reaction. Throughout the extrac-
tion method of the AOT/isooctane system, the diffusion of protein was
the preceding step of mass transfer (Wu et al., 2018).
Chemical technique was additionally economical for the extraction
of proteins than the dry fractionation technique, owing to the lower
fat content and better protein content. For membrane assisted salting
out technique, an ultra-centrifugal mill was accustomed to grind wat-
tleseed and sieve them and henceforth stir in tris–HCl buffer for ex-
traction. Obtained suspension was centrifuged to obtain supernatant so
as to fractionate it using ammonium sulphate in increasing concentra-
tions. Dialysis was done on the precipitates using tris–HCl buffer doing
three changes. The extract was freeze dried finally and held until more
use. Stable secondary structures of wattleseed proteinaceous fractions
were pictured from these results, even at high temperatures, particu-
larly at completely different pH values than those from PI (Agboola &
Aluko, 2009).
2.2. Physical methods of protein isolation from pseudocereals
The physical processes are capable of producing products with
greater ease and safety regards however, the yield of the desired prod-
ucts is a bit lower. They are also time and resource consuming when
compared with other methods of isolation, but they offer more econom-
ical stability to the process than other methods. When used to obtain the
required proteinaceous mass, they are a viable substitute of wet extrica-
tion, so we obtain protein-embellished fragments possessing native char-
acteristics, which is possible only through dry fractionation owing to it
being highly energy efficient (Schutyser & Van der Goot, 2011). These
methods of conventional dry fractionation are divided into 2 steps: seg-
regation of starch particles from smaller protein-rich bodies by milling
during successive grouping, components isolation by turbo-segregation
or electrostatic segregation depending on the dissimilarities in physico-
chemical characteristics subsequently. A replacement pathway is possi-
ble through two-step dry fractionation for constituent production which
circumvents the addition of water and keeps characteristic activity of
components (Laguna et al., 2018). Obtaining protein rich mass from
buckwheat flour through physical processes requires the flour mass to
go through roller mills to get bran flour. Further, the bran flour was
dispersed in distilled water and processed in a colloid mill. The pH of
the dispersion was adjusted, and incubation was done after adding 𝛼-
Amylase and shaking to make slurry followed by addition of another en-
zyme and incubation. Heating was done to deactivate enzymes and then
cooking to obtain the enzyme hydrolysate solution. In order to get the
retentate, extract segregation was performed. Lyophilization was done
to get protein powder of buckwheat. The powder was high in polyphe-
nols. This process was able to yield 1.29–1.45 times better protein con-
centration. Moreover, polyphenol concentration was positively related
with inflated protein content. The polyphenol concentration was bet-
tered with the protein component and slowly went up in order from in-
6
Food Chemistry Advances 1 (2022) 100001
ner to the outer fractions, as affirmed in an identical study Tang, 2007.
Therefore, polyphenols and protein concentrated components of buck-
wheat could be conveyed through surface excoriation methodology as a
utility. Nevertheless, highly pure protein concentrates of isoelectric pre-
cipitate of 75.02% and salt-based separation process of 66.49% could be
availed by dry segregation of surface excoriation. The disadvantage of-
fered by physical processes is the lack of obtaining high yields. In order
to improve the yield, certain physical operations could be employed to
breakdown the food matrix and release the desired components. Ultra-
sound is used to fulfill such demands and could be used in conjunction
with other methods. Like with the case of Ultrasound aided membrane
separation, buckwheat seeds were ground in a disintegrator and sieved
by a 60-mesh sieve in order to bring about thin flour from whole grain.
Buckwheat flour was dispersed in a pH-8 buffer of Trisaminomethane–
Hydrochloric Acid and normal automated mixed or given ultrasound
treatment (frequency 15kHz, power 500W) for extraction. The super-
natant was extracted from the given sludge and leftovers by application
of high-speed mixing. A precipitate of proteins formed at the bottom
as a result of adjusting pH to near isoelectric points and segregating
them by mixing. The precipitates were scattered and counterbalanced
by sodium hydroxide after washing with saltless H2O, and spray-dried,
or lyophilized to give isolates. Protein solubility (PS) of Buckwheat pro-
teins proved better when compared to other cereal proteins at low pH. At
pH lower than seven, the solubility of Buckwheat proteins prepared us-
ing high frequency acoustic cavitation extraction was better than those
of automated isolation or by using high frequency sound waves followed
by auxiliary fat removal operation (Tang, 2007). Literature on physical
methods for Amaranth, Chia and Buckwheat protein isolation are scarce.
As quinoa is a pseudocereal with high nutritional characteristics, pro-
cessing it requires certain measures to be in place in order to reduce nu-
trient losses. Milling fractionation method fulfills the requirements when
used in conjunction with a solvent extraction system. In the method of
milling fractionation and solvent extraction, the proteins in the bran
fraction were enriched using different techniques and further concen-
tration was enhanced using solvent extraction with optional purifica-
tion. Proteins were solubilized in a suspension of quinoa-bran and wa-
ter. The pH was maintained in range of 7-12, and the suspensions were
stirred. The soluble proteins were separated from the insoluble proteins
through centrifugation. The purification of the isolates from the epicarp
of quinoa was done at varying ranges of pH for precipitate and segrega-
tion. For purification of protein, supernatants after solubilization steps
were adjusted to a certain pH with acid. The precipitated proteins were
centrifuged, resuspended in water, neutralized and freeze-dried. Results
pointed out that pH-values greater than nine and less than five brought
down thermal stability and brought up revamping of protein structure
(Scilingo et al., 2002).
2.3. Enzymatic methods of protein isolation from pseudocereals
Whenever we look for ways to obtain a protein isolate of high pu-
rity and with selective modification of the ingredients, enzymes have
the characteristics to fulfill such requirements. As we design processes
based on enzymatic methods, possibilities for separating proteins and
starch of amaranth flour were examined. In the selected procedure, en-
zymatic breakdown of polysaccharides to liquefy starch by hydrolyz-
ing it into soluble glucose and enriching the solid phase with vegetable
protein was carried out. The procedure consisted in liquefying starch
into soluble glucose by an aggregate of 3 specific enzymes and cellulase
mixed together and dosed into dry matter of flour to carry out enzyme-
assisted breakdown of polysaccharides. Starch chains were broken down
by Amylases and cross-linked starch segments were destroyed by glu-
coamylases. Studies on enzyme kinetics of polysaccharide breakdown
of amaranth flour was also carried out. When the enzymatic breakdown
was completed, the mixture was centrifuged and liquid and solid frac-
tions were separated and dehydrated to estimate the protein content
(Mokrejs et al., 2011). Reviews on the protein isolation using enzymes
A.M. Malik and A. Singh Food Chemistry Advances 1 (2022) 100001

Table 3
Comparison of Amino-acid profile of Amaranth, Buckwheat, Quinoa, Chia, Wattleseed and Album.

Amino acid (mg per 100 Amaranth Buckwheat Quinoa Chia Wattle-seed Album
g of dry weight basis)

Ala 459 573 531 940 405 4320

Arg 1222 1496 1096 2000 583 8700
Asp 899 1301 1079 1280 1210 9100
Cys 145 179 114 420 149 1270
Glu 2170 2844 2032 2870 1570 15120
Gly 1056 881 671 910 810 6300
Ser 834 754 596 940 706 5370
Pro 554 556 497 1280 448 3180
Tyr 588 582 577 610 258 3520
His 398 422 431 610 307 3100
Ile 406 470 420 740 321 3180
Leu 731 902 795 1420 687 6720
Lys 559 624 546 930 701 5900
Met 335 316 361 670 160 2100
Phe 684 813 633 1160 359 5730
Thr 452 510 446 540 404 4000
Val 453 595 486 790 708 4100
EAA 4,606 5,234 4,695 7,470 3,905 3,483
NEAA 7,339 8,584 6,618 10,640 5,881 5,688
Sulphur containing AA 480 495 475 1,090 309 3370
Aromatic AA 1,272 1,395 1,210 1,770 617 9250
Refs Motta et al., 2019 Motta et al., 2019 Motta et al., 2019 Nitrayová et al., 2014 Agboola and Aluko, 2009 Mir et al., 2019

for Buckwheat, Chia, Quinoa, album and Wattleseed could not be pos-
sible in this review work owing to the scarcity of findings for the same.
As explained by Hamada (1999), there are 2 qualities of protein break-
ing enzymes: outer and inner, which could be availed for experiments.
Enzymes which break outer structures generally divide any one end of
the amino acid chain by one unit of amino acid, whereas inner pro-
tein breaking enzymes break bonds in the intramural region of the pro-
tein chain. Flavorzyme, a combination of inner and outer bond break-
ing enzymes, yielded 87.6% of proteins while 81.4% of retrieval was
possible with alcalase, an inner side bond breaking enzyme when ap-
plied for isolation using H2O with eight pH and temperature of 50°C
(Hamada, 1999). When using biomolecules, high amounts of proteins
can be recovered in contrast to segregation by alkalis. Yet, cost is the
main limiting factor. Application of immobilization techniques could
enhance recovery and result in dropping of usage fare as biomolecules
can be recycled and reused.
3. Amino acid composition of different pseudocereals
AA composition analysis is a classical protein analysis technique,
finding a huge number of usages in medical and food science research
and is indispensable for protein profiling. This is a complex methodol-
ogy involving two steps, hydrolysis of the substrate and application of
chromatographic techniques for segregation and discovery of the left-
overs (Fountoulakis & Lahm, 1998). The AA profile of almost all dietary
proteins encompasses important roles in day-to-day chores of humans
and affects by keeping homeostasis in any possible way. AAs are impor-
tant for making an array of proteins with significant roles like carrying
oxygen, vitamins, carbon dioxide, biocatalysts and structural proteins
(Chalamaiah et al., 2012). Table 3 shows a Comparison of AA profile of
Amaranth, Buckwheat, Quinoa, Chia, Wattleseed and Album.
3.1. Amino acid composition of Amaranth (Amaranthum)
Amaranth proteins are laced with important amino acids depending
upon plant species and cultivar. Amino acid profile of amaranth does
not compare well with cereals as to legumes, with sulfur AAs being the
exception as they are available in large quantities in amaranth as com-
pared to legumes. Amaranth protein essential amino acid index (90.4%)
is in line with proteins of egg and decreases to 85.4% after thermal rem-
edy at 170-190°C for 30s. Muscle & bone meal may be replaced by us-
ing amaranth because of its surprisingly relatable content of important
amino acids (Písaříková et al., 2005). Amaranth flour with bran included
from A. hypochondriacus has better lysine content (5.95 in comparison
of 2.90 g/16 g N), also an excess of certain vital AAs (Dodok et al.,
1997) in comparison with wheat flour. To check the quality of proteins
and perform the quantification of AAs in amaranth, a number of tests
need to be performed. Since we are concerned with qualitative anal-
ysis, the selection of test methods should be as such that they do not
affect the of AAs under consideration in any way. The amaranth pro-
tein concentrate, segregated at 11 pH and precipitated at 4.5 pH, was
having good quantity of the amino acids which are limited in other ce-
reals and legumes, whilst leucine was present in the flour with threo-
nine, and valine, with 78, 87, and 90, respectively, as their chemical
scores (FAO Expert Consultation 2011). Good amounts of lysine could
be found in the flour and the protein enriched fraction, and as a result
of this, these materials can be an important accessory for cereal flour.
Inadequate amounts of leucine had been found in the whole-meals and
lysine was barely deficient. Moreover, leucine quantity was also low, a
meagre 0.54g/100g for amaranth but still higher than most of the ce-
real grains. Lysine constituted 5% of the amino acids (and up to 6.9%)
and sulfur amino acids constituting 4.4%, as from the composition. All
grains contain those amino acids in them but mostly as restricting ones.
Owing to them containing high concentration of lysine and other essen-
tial amino acids, amaranth was rated above others pseudocereals grains
for its nutritional capacity (Coelho et al., 2018) (Písaříková et al., 2005).
3.2. Amino acid composition of Buckwheat (Fagopyrum esculentum)
Buckwheat is a source of high-quality proteins which possess func-
tional properties. Besides this, a good amount of lysine (avg-6.1%) is the
main characteristic of buckwheat proteins which is higher and beats all
other cereal grains. Meagre amounts of Glutamic acid and proline are
found in buckwheat proteins, but they are rich in arginine and aspar-
tic acid. In order to predict the restricting AAs or rough estimate of AA
stability of meals, diverse scoring techniques have been recommended
as initial screening steps. Different predictions regarding dietary ben-
efit of the proteins (NRC-1963; FAO/WHO’ Expert Group 1965) have
been given by these procedures. Egg-protein was used as a reference for
evaluating dietary values of the buckwheat proteins (avg score of spec-
imens referred). Aspartic acid, lysine, and arginine are found in higher
amounts in buckwheat than they are in cereals, and glutamic acid and

7
A.M. Malik and A. Singh
proline are absent. The high nutrition value of buckwheat comes from
its high content of lysine, which is the first limiting amino acid for many
cereal protein sources and as a result, buckwheat is a healthier, higher
quality alternative to other cereals. In order of decreasing lysine con-
tent, albumin was followed by glutelin, then globulin, then prolamin.
Besides lysine, threonine exhibits a similar tendency. A decrease in thre-
onine content from prolamin > glutelin > albumin > globulin has been
demonstrated for the proteins from common Buckwheat varieties (F. es-
culentum) and Tartary Buckwheat varieties (F. tataricum) (Sytar et al.,
2016). Successful balance and higher nutritional capacity of buckwheat
compared with other grains had been indicated by means of chemical
amino acid assays when they had to be blended with meals which had
been low in lysine. The effects were compatible with feeding tests of
Sure (1955) (Pomeranz & Robbins, 1972).
3.3. Amino acid composition of Quinoa (Chenopodium quinoa Wild)
When we talk about pseudocereals, Quinoa emerges as a source
of high-quality proteins among them all. It also contains almost all-
important amino acids making it a complete source of protein. In ad-
dition, Quinoa contains unrefined protein in the range of 17.4g-18.9g
out of 100g Dry Matter. Similar results of quinoa have been formerly
disclosed by Wright et al. (2002) and Comai et al. (2007). Another ex-
periment by (Abderrahim et al., 2012) showed that a lower amount was
observed for quinoa. Spearman rank correlation analysis was used for
comparing disparity between amino acids. The AAs recognized in the
quinoa samples like proline with alanine, isoleucine with valine, tyro-
sine with phenylalanine showed on par correlation coefficients (with
coefficients in excess of 0.9) and their maxima and minima values and
their standard deviation were used for representing the contents of AAs
of specimen quinoa for tapering as per the standard chemical protocols.
Amino acids were found in all the samples studied. Variabilities were
observed in the AA composition of the quinoa, with glutamic acid and
threonine as having utmost presence, with mean values of 8.79g/100g
protein and 6.47g/100g protein, respectively. Aspartic acid, glycine and
arginine were next in line, with average values of 3g/100g protein mat-
ter. The concentrations of different AAs were much less than 2.5g/100g
protein. Using variable genotypes of quinoa over the years prompted this
variability, for marking and confirmation; as they are critical for devel-
oping standardization formulas that could be helpful for further estima-
tions (González-Martín et al., 2006). Similar degrees of values of the AAs
had been put up for the glutamic acid, methionine, histidine and tryp-
tophan. Higher contents of threonine and glycine had been pronounced
in a lot of assignments (Stikic et al., 2012); whilst lowering in content
material was witnessed for the remaining AAs. Cereals are the most con-
sumed food materials in the world and are a staple diet in almost all
corners of the world and fulfill protein requirements of many people
who are dependent on them for protein needs as they are cheap sources
of proteins. Also, quinoa consists of important AAs in larger quantities
compared to different starchy grains, specifically lysine fraction which is
captivating for being a crucial promoter of growth (Pérez-Álvarez et al.,
2014). Since cereals are often incomplete sources of proteins, they fail
to provide us with required crucial AAs and so is the case of quinoa.
The two aromatic AAs had been the restricting AAs for quinoa which
had 86 as its chemical rating (score) for uncooked and 85 for washed
one. Threonine was the 2nd restricting AA and lysine closely followed it
up next. Quinoa protein has a score like that from soybeans. Amino acid
scores acquired in the present research (besides tryptophan) contain val-
ues downward to those mentioned by Gross et al. (1989), Comparable
outcomes of amino acid analysis had been reported by Mahoney et al.,
(1975) which were lesser than the ones acquired in this research. Sa-
jama type contained much less methionine than determined in other
varieties while less lysine was reported in the Blanca variety. Methion-
ine and tryptophan were the primary restricting important AAs in most
samples. Tryptophan and lysine have been present three times more in
quinoa protein than that of complete wheat. Also, greater methionine
8
Food Chemistry Advances 1 (2022) 100001
is present in quinoa than in wheat. A treatment with high temperature
for extraction of saponin was developed to look at its impact on pro-
tein character, however no changes inside the amino acid content were
present in 4 kinds of quinoa specimens after processing with H2 O at 50,
70 and 87°C (Ruales and Nair, 1992).
3.4. Amino acid composition of Chia (Salvia hispanica L.)
Chia seeds are a very powerful nutrient source as they are packed
with all required nutrients in ample quantities. They are often called
high-quality protein sources as they have all crucial AAs inside their
grains. Amino acid composition of the isolated globulin fraction con-
tains excessive amounts of sulfur and aromatic AAs and also threonine
and histidine. Most parts of chia proteins showcase enhanced amounts of
glutamic acid and aspartic acids, characteristic of seeds containing large
amounts of globulins. Low degrees of lysine were assessed in both spec-
imens. When AAs containing Sulphur are present in abundance, they
will be deeply engaged in keeping the third order and fourth order ar-
rangement of the proteins; also, capability of glutamic acid to revive
the CNS (system of nerves) and functions of immunization in people has
been of prime importance for the food industry. The capability of foods
high in aspartic acid for functioning of the hormonal flows for properly
maintaining CNS has been studied. In trend, the protein-quality of chia
has been showcased to be of better quality than other grains and seeds
producing oils. The dietary value of proteins of chia seeds has been ex-
hibited to be superior to most of the cereal grains and oil producing
crops (Nitrayová et al., 2014) as shown in Table 3.
3.5. Amino acid composition of Wattleseeds (Acacia auriculiformis)
Wattleseeds are pseudocereals with relatively high protein content
when compared with other cereal grains. But many of the AAs are
present in meagre amounts or absent and as a result, it is a poor source
of quality proteins. AA characterization for Wattleseed flour has been
put up for the first time and is discussed thereafter. Large amounts of
AAs characterized from the protein fractions were mainly Glutamate,
aspartate and lysine at 14.4%, 11.1% and 9.13% respectively, whilst
the sulfur AAs namely cysteine (0.67%) and methionine (0.99%), as
well as tryptophan (0.75%) were present in meagre amounts. The rel-
atively greater quantity of amide (Glu–glutamine, Asp–asparagine, Arg
- arginine) is much like 7S globular proteins present and most prob-
ably arising due to storage related functions in the plant (Agboola &
Aluko, 2009).
3.6. Amino acid composition of Album (Chenopodium album)
The grains of album are a good source of proteins and other dietary
components. They also supply the diet with certain good quality amino
acids which are lacking in most of the grains of our staple diet. Table-3
shows the AA profile of Chenopodium Album protein isolates. It was
witnessed that Chenopodium Album proves to be great source of EAAs,
while low amounts of Sulphur AAs are present in both types. As albu-
mins are hydrophilic and densely contain Sulphur AAs, they get leached
out partially upon washing of flour with H2 O before extraction with al-
kalis. Acidic properties of the API are due to the presence of aspartic acid
and glutamic acid AAs. AAs such as proline and arginine act as precur-
sors for aspartate and most of the other biocatalysts while glutamic acid
acts as a substrate. The increase in the aspartic acid can be explained
by a reduction in the proline content. AAs proposed to build muscle
tissue are isoleucine, leucine and valine and they belong to the group
of Essential BCAAs. With regard to album protein isolates, the study
shows a higher value of leucine and valine in comparison to isoleucine.
Basal nitrogen surplus is maintained by amino acids like threonine as it
helps in reducing nitrogen removal out of the system (Mir et al., 2019;
Sibian et al., 2017).
A.M. Malik and A. Singh Food Chemistry Advances 1 (2022) 100001

Table 4
Amino acid scores of different pseudocereals with reference to FAO standards.

Amino Acid FAO/WHO/UNU-2007 Amaranth Buckwheat Quinoa Album

His 15 1.79 2.97 2.14 193.75

Ile 30 0.91 1.10 1.03 106
Leu 59 0.83 1.07 0.99 110.16
Lys 45 0.83 0.96 0.90 122.91
Thr 23 1.32 1.55 1.43 160
Val 39 0.77 1.07 0.91 102.5
SAA (Meth+Cys) 22 1.47 1.57 1.69 146.52
AAA (Phe+Tyr) 38 2.25 2.57 2.49 225.60
EAAI 45.5 52.1 50.4 77.55
Refs Motta et al., 2019 Motta et al., 2019 Motta et al., 2019 Mir et al., 2019

Table 5
In-Vitro Protein Digestibility and PER values of pseudocereals.

Pseudocereal IVPD (%) PER References

Amaranth 76.03 1.52 – 2.57 Hejazi et al., 2016, Bankole et al., 2019.
Buckwheat 73.56 2.55 Deng et al., 2015, Nosworthy et al., 2017.
Quinoa 81.46 <2.0 Martinez et al., 2020.
Chia 49.4 1.82 da-Silva et al., 2016, Olivos-Lugo et al., 2010.
Album NA∗ PER1: 2.23PER2: 2.21PER3: 1.67 Mir et al., 2019.

NA∗ = not available.

4. Protein quality evaluation of pseudocereal proteins
Protein-quality aims to estimate the extent of dietary protein sources
and diets to fulfill the proteins and vital amino-nitrogen demands, i.e.,
to meet the metabolic needs for AAs and nitrogen. Protein demands
at present are dictated in terms of intakes desired to fulfill metabolic
demands for maintenance as displayed by nitrogen balance in corre-
sponding age bracket plus those involved with the protein requirements
for general growth of toddlers and children, pregnant and milk produc-
ing women. In diets related to protein-quality evaluation, dietary AAs
should be counted as a single nutrient and possible data for digestible
or biologically available AAs should be mentioned in tables on sepa-
rate AA basis for women. Thus, the real quantification of protein-quality
for humans is to assess directly the efficacy of various protein sources
to support normal growth and, or different functions relying on proper
protein nutrition in areas representing the population under study. How-
ever, not holding out this definition with the ideal situation, the eval-
uation of protein quality in large human groups over the past decades
has assurance on indirect methods involving in-vitro assays, and ani-
mal/human metabolic reporting which could be utilized on a regular
basis and to ensure safety in order to guess human protein and AA us-
ages. To confirm the aptness and broad-spectrum usability, the daily
procedures necessarily require to contain routine methods for check-
ing all of the general variables which together estimate the quality of
a protein: pure and parallel quantities of essential Amino Acids (EAA),
digestibility of proteins, and the biological availability of Amino Acids
(Consultation, 2011). With regards to this, Tables 4 and 5 show the dif-
ferent Amino acid scores, EAAI, IVPD and PER values of pseudocereals.
4.1. Protein quality of Amaranth (Amaranthum)
Amaranth grains are the most valuable for having a great deal of AAs
in them. For this reason, the grains of amaranth can be supplemented
with other cereal grains so as to produce a more balanced diet which
supplies all the important AAs. To evaluate the quality of protein inside
the grains of amaranth seeds, a number of qualitative and quantitative
tests are carried out and evaluation of AAs characteristics was done and
various attributes noted down. Leucine was having the least AA score
(0.86) in the entire meal of amaranth whilst in protein concentrates,
lysine was having a score of 0.88. Protein digestibility in terms of PD-
CAAS could then be 0.86∗ 0.74 = sixty-four for whole meals, and it would
be 0.88∗ 0.81 (protein digestibility) =0.71 for protein concentrates. The
scores were then calculated for wholewheat as 0.4, wheat protein as 14 ,
oat as 12 , dietary meal of peanut as 0.52 and canned lentil proteins as
0.52 (Bejosano & Corke, 1998).
The EAAI of amaranth proteins came out as 45.5 which was good tak-
ing in account the provision of important AAs but the amount is lowest
of all the pseudocereals (Motta et al., 2019). Raw amaranth whole meal
flour confirmed average protein digestibility of 74%. Similar values for
A. cruentus L (73.85%) and A. hypochondriacus L (71.93%) in terms of
protein digestibility were reported by Muyonga et al. (2014). A. cruen-
tus L raw had better digestibility (73.85%) than A. hypochondriacus L
among all the variations and treatments in the study. Protein digestibil-
ity changed significantly after heating. Digestibility values reduced sig-
nificantly after heating for all the varieties but such differences between
genotypes for digestibility of proteins were fairly small, but significant.
This could be attributed to dry heat processing, as a result of which
amino acid degradation, formation of disulfide bonds within the molecu-
lar structure and Maillard reactions might occur (Muyonga et al., 2014).
Even though in-vitro checks are faster and convenient approaches, they
were not proper for observation of protein breakdown. Outcomes from
the two strategies were compared by Mitchell and Grundel (1986) and
discovered that in-vitro check tends to underestimate protein digestibil-
ity. Small changes inside the enzyme protein arrangement in response
to relative humidity associated damages brings about the change owing
to its ultra-sensitivity. Lowering down of dye-associated lysine and en-
hancement of enzyme-based colour changes were discovered that had
zero impact on IVPD. Genuine digestibility of Amaranthus proteins was
more according to data. Amaranth protein has an in-vitro digestibility
of 75.40%, which indeed is a significantly good value among different
proteins of the identical values. It showed that the nutritional value and
digestibility might be enhanced by means of inferior value malting and
grain germination pre-processing as proven by Hejazi et al. (2016). Fur-
ther, the higher protein digestibility (84%) was determined after grow-
ing grains at 28°C through 2 days. Reduction in values of phytate and ox-
alate was observed throughout the study, and tannin content increased.
Lemos et al. (2012) investigated IVD of amaranth starch and revealed
about excessive GI (glycemic index) of foods derived from amaranth.
Protein digestibility of the amaranth specimens were examined after
pancreatin digestion as per starting and end value, the results were pre-
sented numerically. Raw amaranth samples were associated with IVPD
of about 76%. Following germination, the IVPD results went up as much
as around 84% showcasing an approximate 8% jump in the protein di-

9
A.M. Malik and A. Singh
gestibility. The value changed in relation with the time of germination
as indicated by the changes in IVPD values. Additionally, the number
(76%) shows a superior digestibility value because the sample in consid-
eration is in uncooked form and the value will clearly go up after cook-
ing or processing step. Anti-nutrient elements reduced after germination
like phytates and oxalates but growth in the level of coloured compo-
nents (tannin) was witnessed. Kanensi et al. (2011) studied the impact
of seed growth on tannins and found its initial value (8% CE) reduced
to untraceable values after 3 days of growth in amaranth seed, unlike
the current study. Anti-nutritional components plus IVPD ranges show-
cased comparable (tannin) and opposite (phytate/oxalate) relations in-
dicating possible correlations among those quantities. Since amaranth
flours contain the most digested protein (∼53.0%), there is a possibility
that some of their proteins have been hydrolysed (∼2.5%). The exten-
sive digestion phase of raw and heat-treated amaranth grains, and the
milling process, likely contributed to the high protein digestibility. The
process of heating amaranth grains appears to improve the digestibil-
ity of the nutrients contained in them, as it occurs with many other
plant-based foods. There are however, different effects of various heat
treatments on the structure and composition of macronutrients and mi-
cronutrients. The unfolding or denaturation of proteins, hydrolysis, or
formation of aggregates and complexes caused by processing can al-
ter their properties and digestibility (Capuano et al., 2018). Another
factor responsible for increasing protein digestibility was demonstrated
to be the degradation of antinutrients, especially phytates and oxalate.
Also, grinding (milling) plays a significant role in particle size reduc-
tion, which was the determining factor in this part of the study, since
the flours had higher digestibility than the whole grains or the puffed
amaranth (Grundy et al., 2020). The predicted-PER and predicted-BV
was between 1.52-2.57 and 14.78-77.56 respectively. Lysine became
readily available in the seed flour and hydrolysed samples, as it is a re-
stricting essential AA in cereals. The primary and secondary restricting
AA in seed flour of amaranth are isoleucine along with Sulphur contain-
ing AAs. After taking in consideration the FAO/WHO (2007) standards,
protein isolates are limited in Sulphur amino acids while the protein hy-
drolysates samples contain all in excess. Further, the 1st and 2nd restrict-
ing AAs in flour of amaranth seed are the Sulphur AAs and isoleucine.
Only Sulphur AAs groups are limited in protein isolate and the sample
hydrolysed with porcine pancreatin pepsin is limited only in valine and
other hydrolysed samples with 3 enzymes available in excess. Plenty of
critical AAs are needed for the growth of youngsters and others could
be supplied through seed flours, protein isolate and hydrolysates. It can
also be used in cereal based complementary diets as a protein supple-
ment (Bankole et al., 2019).
4.2. Protein quality of Buckwheat (Fagopyrum esculentum)
The dietary value of buckwheat is higher than many grains as it con-
tains most of the accessory nutrients. The proteins of buckwheat seeds
are one of the best sources of high-quality protein owing to the exis-
tence of many of the essential AAs inside them. Many essential AAs
are abundant in buckwheat than in wheat grain like Lys & Thr along
with Sulphur AAs as the examples. The true digestibility of proteina-
ceous compounds found in buckwheat were very little for having read-
ings less than 88%. The net protein utilization values (NPU) higher than
70% resulted in abundant BV of the seeds of buckwheat when put next
to grains of wheat because the NPU of wheat was solely 53%. As all
the 3 samples had just about identical proteinaceous content, the utiliz-
able proteinaceous compound (NPU multiplied by percent protein per
100g) was more for the seeds of buckwheat. There is less lysine content
in the normal cereal grains. Therefore, abundant effort has been placed
by big agencies across different borders, plant-biologists, biochemists
and many more professionals for the job of increasing lysine content
in proteins from starchy grains. Majority of the essential amino acids,
as well as lysine at 5.0 and 5.2g/16g N, are available in high amounts
in buckwheat. Within the 2 samples; these numbers are close to no-
10
Food Chemistry Advances 1 (2022) 100001
table lysine availability for babies (=5.2g/16 g N) that is stated by the
Food and Agriculture organization. The BV data within the specimens of
buckwheat grain were matching even for the foremost rich variety with
high-lysine content of the cereal grains, but the large portion of unfil-
tered crude fiber could create less dense environment for energy, and
therefore, comparatively phenol-enriched product would be produced
which could cause low protein digestibility. The nutrient protein qual-
ity of processed and raw pseudocereals was determined. AAS and EAAI
were determined as indicators for the protein quality. Essential amino
acid and aromatic amino acids (AAA-essential amino-acid and tyrosine)
bestowed the very high values of AAS considering all pseudo-cereals
evaluated. The limiting amino acid was the lysine in raw buckwheat
and bestowed with 0.96% value which may be thought as non-limiting.
As stated by Klose et al. (2009), decrease or increase of amino acids be-
cause of totally different metabolic activity of protein compounds will
be discovered in all fractions of the protein. In many of the cereal grains,
lysine is taken into account as the limiting amino acid. Studies on nutri-
tional protein quality within the literature does not seem to incorporate
processed pseudo-cereals. In raw buckwheat, the limiting amino acid is
lysine and provides a value of 0.96. High values of AAS shows that the
grain is extremely nutritious, and lysine is thought as non-limiting. Re-
garding EAAI index, raw buckwheat samples provide values of the order
of 52.1 that could be a sensible amount considering the protein is from
plant supply, Motta et al. (2019). The IVPD values of buckwheat flour
proteinaceous compound comes to be 78.32% and also the digestibility
is considerably on top of other grains. Improvement in IVPD values is
attributed to the denaturation of the proteinaceous compounds, destruc-
tion of the trypsin inhibitor, and reduction of phenols and phytic acid
content as they are very closely associated with the process extraction.
The protein quality is determined by amino acid composition and di-
gestibility. This is often called the protein efficiency ratio. PER depends
upon growth, that is the quantity of weight gained on the premise of pro-
tein consumption over a time period of a month. An estimated value of
2.89 was taken as the PER value for untreated buckwheat. The untreated
PER goes through adjustments related to the casein so as to standardize
PER determination across different laboratories. These findings indicate
that untreated buckwheat is pretty much as good as extruded buckwheat
in terms of nutrient potential (Nosworthy et al., 2017).
4.3. Protein quality of Quinoa (Chenopodium quinoa Wild)
Owing to its usability by the people having celiac disease, the rea-
son being that it does not contain gluten, quinoa stands out among the
cereal grains as one of the best sources of complete protein contain-
ing all of the nine crucial AAs. Quinoa is one of the best suppliers of
AA lysine. Klose et al. (2009), observed reduction or upgradation in
AAs might be witnessed for all protein fragments due to varying rates
of their metabolism. Lysine is considered as the limiting amino acid of
many cereal grains. Lorusso et al. (2017) recognized AA lysine as re-
stricting in products of quinoa. Alas, the scientific works did not seem
to encompass findings on protein quality in the observed pseudocereal
products. A more precise estimate of protein quality was provided by
the simultaneous consideration of urinary protein losses (as nitrogenous
end products) and discovered that urinary nitrogen losses were lower
in rats fed with Quinoa than in rats which were fed with casein. How-
ever, rats fed with quinoa showed slightly lower overall nitrogen balance
than in those which were fed with casein. After observing AA make-up
and findings on animals altogether, it may be concluded that the qual-
ity of proteins of quinoa matched up with casein. Mixing the grains of
quinoa with other cereals efficiently increases the protein quality of the
resultant product in this manner. But protein level in cereal-based di-
ets can be raised in limited amounts as the level of protein in quinoa
is only a bit upward of other cereals. The apparent retention factor in
% was estimated on a dry basis (U.S. Department of Agriculture, Na-
tional Agricultural Statistics Service 2007). The values were in between
94.9-95.4% for all AAs with cysteine having the lowest share, despite
A.M. Malik and A. Singh
the fact that higher levels were stated in the current work (56-73%).
The extent of degradation might be credited to the variety or cultivar
because of dissimilarities among the grains and also to the changes in
processing parameters utilized for the research. A big positive change in
%TR of almost all AAs was witnessed, excluding cysteine in amaranth
and glutamic acid in quinoa. Chemical score of uncooked quinoas was
assessed after enzymatic hydrolysis and the values came out as 72. A
drop in pH value (in proteins) prior to hydrolysis using blend of diges-
tive enzymes was used to measure in-vitro protein digestibility (%D)
and values came out as 82%, 78% and 91% for hydrated sample, un-
cooked and casein respectively. In terms of protein digestibility, black
quinoa protein isolate had a value of 95.71%, and yellow quinoa protein
isolate had a value of 95.80%. Elsohaimy et al. (2015) reported 78.37%
for the same material, but these values were higher than values reported
for other grains such as wheat and rice. For raw and washed quinoa, in
vitro digestibility rates were 77.7% and 83.3%, respectively, while an
extruded product extruded at 120-150°C provided a protein digestibil-
ity value of 86.1%. Different heat-treatments and pH levels were used
to analyze the in-vitro digestibility of quinoa protein isolates. Protein
aggregation/protein crosslinking could be the cause of the decrease in
digestibility with increasing extraction pH. The saponins in quinoa seeds
have the potential to negatively affect their digestion. Among the raw
and washed quinoa tested, they found digestibility rates of 83.3% for
the washed quinoa, which had saponins removed and 77.7% for the raw
one. Comparing the results with those from the black and yellow quinoa
protein isolate samples, the results were lower. Thus, leftover saponins
were not detrimental to the protein digestibility of the isolates (Sánchez-
Reséndiz et al., 2019). Appeal, endurance and digestibility of certain
foods containing quinoa-wheat food systems with sufficient reserves of
energy and proteins for the babies had been studied by López de Ro-
maña et al. (1978). Low nitrogen and fat absorption were shown by
kids supplied with oat and quinoa made foods. Evidence points towards
restriction on growth of rats resulting from saponins of Chenopodiaceae
family. The data received in these research findings point towards better
quality of proteins of quinoa grains and absence of deleterious effects
(due to saponins present) on the dietary value of protein. The digestibil-
ity of grains was lower if the elimination of saponins was not done from
quinoa. The IVPD in quinoa proteins were measured on the basis of drop
of pH of proteins prior to application of digestive enzymes that had been
utilized to hydrolyze the quinoa proteins. IVPD value for raw quinoa
was 78% which is good for raw grains. The PER values of the quinoa
seed proteins have been determined by using three distinct techniques
to evaluate the protein quality. PER values as determined and corrected
values for quinoa proteins had been a little better than for protein from
other sources (especially casein for reference). The PER values have been
reported as 3.8 for determined and 2.7 for corrected PER. This occurred
even though lysine concentration in quinoa was somewhat lower than
in casein (7.2g/100g protein). Both sources probably met the lysine re-
quirement adequately (National Research Council 1987) () of rats, but
both apparently failed to meet the need for methionine (primary re-
stricting AA in quinoa and casein). Animal derived foods show better
digestibility than grain-derived foods. Significant differences were ob-
served in protein digestibility between quinoa and casein. Fecal protein
loss is the basis for apparent protein digestibility. A more precise esti-
mate of protein quality was provided by the simultaneous consideration
of urinary protein losses (as nitrogenous end products) and revealed that
urinary nitrogen losses were lower in rats fed with quinoa than in rats
fed with casein. However, rats fed with quinoa showed slightly lower
overall nitrogen balance than in those which were fed with casein. PER
and NPR values were significantly different between the quinoa flour-
fed groups and groups fed with other cereal flours. Thus, quinoa flour
has an impact on weight gain due to its proteins being of high quality
with PER values greater than 2.0, in comparison with other diets hav-
ing medium quality proteins of PER values between 1.5 to 2.0. Quinoa’s
contribution as a source of high-quality protein and also complementing
for limiting amino acids for certain cereals could allow the quinoa flour
11
Food Chemistry Advances 1 (2022) 100001
or quinoa flour combined with other grains to improve protein quality
(Martinez et al., 2020).
4.4. Protein quality of Chia (Salvia hispanica L.)
Chia seeds are one of the best sources of proteins among the cereal
grains consisting of protein in the range of about 20-25%. They are also
considered high quality cereals when we talk about characteristics of the
proteins in them in terms of AAs composition and availability for people
of all age groups. Chia seed flour fulfills the AA needs of babies as 100%
fulfilling in regard to sulfur AAs, while the rest of the vital AAs are in the
range of 52-76%. The important AAs of flour varied between 66-126%
which was much better regarding coverage for adults. Wider ranges of
coverage of requirements were exhibited by the globulin fraction than
the flour obtained from the grains; twenty-seven to two hundred ten per-
cent in case of babies and thirty-four to two hundred eighty eight percent
in case of adults due to cereals being poor in lysine and degradation of
some of it during the separation. The share of important AAs reported
is near to 50% (46.5%), that is lot better regarding the values suggested
for soybeans (41.0%), also values for safflower (38.1%); an essential fac-
tor to keep in mind regarding chia protein quality (Sandoval-Oliveros &
Paredes-López, 2013). The important AA composition in seed proteins
of chia lies in between 41.8-42.8%. High amounts of glutamic acid, as-
partic acid and arginine as well as valine and leucine are present in the
proteins of chia seed. Comparable results were produced by the IVPD
of deoiled chia flour as were formerly suggested; these numeric data
of around 77.5%, comparing with values for Phaseolus vulgaris (77.5 %)
but better in comparison to grains such as corn, rice, sorghum and wheat
(66.6% and below). Globular proteins at 82.5% indicates slightly bet-
ter IVPD than deoiled flour at 78.9%, but a bit downward to values of
casein at 88.6% which was utilized as reference. Anti-nutritive factors
for chia have not been reported, excluding chances of detecting pro-
tease hindrance that could slow down the IVPD. Nutritional quality of
proteins is indicated by their digestibility and is related to spatial posi-
tioning, since the higher protein structures offer varying vulnerability
to degradation enzymes. 49.4% was the confirmed IVPD data for seg-
regates of chia protein after in-vitro protein digestibility values (%D)
of chia protein isolate were determined. Thermal treatment given to the
chia seeds as it enhances their digestibility, mainly the vegetal content is
impacted the most. Proteins are made available for attack by enzymes as
thermal treatment destroys the cell wall. Structural degradation of pro-
teins which follows heat treatments can lead to elevated digestibility of
proteins. An additional reason that is accountable for enhancing the di-
gestibility of proteins is the alkaline environment in the beginning of the
segregation method (pH 12, 90 minutes). The highest percentage of di-
gestibility was shown by the isolates obtained from chia during alkaline
treatment which led to disruption of higher structures of protein and
improvement in digestibility. Protein degradation enzymes are found to
be responsible for this in relation to peptide bonds. Glutamic acid was
one of the most important AA. Amongst all pseudocereals, chia seeds
contained the highest glutamic acid content. 37.87% came out as the
value of essential to total amino acids for chia seeds. Proteins with such
high values are considered as high-quality protein. Contrary to this, in
all the analyzed pseudocereals, methionine and cysteine values came as
lowest (< 2.9 %), in accordance with the study of Nowak et al. (2016).
Proteins of chia have high conversion efficiency to support increase in
weight, about 50 % with regards to casein, PER can be used as a tool
for this. Higher values of PER were witnessed earlier, but not with the
current study. Protein quality of sorghum were analyzed (Moraes et al.,
2012) and greater digestibility of proteins (avg 86%) and decreased PER
was recorded; suggesting better quality of AA profile of chia than other
grains, as the thing supports increase in weight. Different diets contain-
ing chia were fed to animals and they showcased equality of glucose
ranges in blood, compared with animals given casein. Such observa-
tions are due to chia flour containing soluble fiber fragments, which is
not the case for casein which contains cellulose. Viscosity of intestinal
A.M. Malik and A. Singh
lumen was increased by the soluble dietary fiber, thereby decreasing
enterocyte touch by glucose ultimately lowering their uptake (da Silva
et al., 2016).
4.5. Protein quality of album (Chenopodium album)
The presence of a high proportion of the major AAs in Album seeds
makes them one of the best sources of good quality proteins. Since the
existence of these AAs is significant enough, these grains can be sup-
plemented with major staple diet grains to complete the lacking pro-
tein quality characteristics of the diet. The tests required to assess the
quality parameters of AAs must be non-volatile so as they do not af-
fect the stability of the AAs present inside the album grains. Certain
dietary characteristics of album protein extracts were assessed as per
their AA composition, through the PER (Protein efficiency ratio) recog-
nized as the protein quality index. Based on the density of leucine and
proline (PER1), leucine and tyrosine (PER2), and methionine, leucine,
histidine and tyrosine (PER3), the protein efficiency ratio from literature
is well connected with the original PER values. To determine the pro-
tein quality, the techniques well documented in literature for estimating
PER are in-vivo techniques to quantify PER which are not enough for
most of the occasions because of the inflated body mass of animals un-
der observation which mostly do not show the protein absorption. Low
quality proteins have PER ranges under 1.5 while the PER ranges over
2 are superior quality proteins. As indicated by the current study, the
PER values of the API are over 2 and termed as superior quality pro-
teins. The PER values obtained are higher than the PER found in peanut,
L. sativus and cowpea. The results are similar to those documented by
Vioque et al. (2012) for Vicia protein extracts from Faba and allied iso-
lates. The PER is proportionate to the Leu amount; so minute differ-
ences in leucine content would impact PER. The EAAI for API was found
greater as compared to the majority of the grains. The outcomes of the
EAAI are corresponding to the outcomes by Abugoch et al. (2008), who
mentioned about EAAI of quinoa-grains to be matching with soybean
grains. The percent of protein which blends into the human body pro-
teins is quantified as biological value. Their value is between 70-100%
for good-quality protein food. The study shows the API BV at 72.82%
indicating a high BV. For the formulation of the protein efficient di-
ets, Chenopodium seeds can be used. The nutritional index is the basis
of the sample protein content and its essential amino acid index. The
higher protein contents result in higher dietary indices of the samples.
Protein derived diets prove to be of higher quality upon EAAI of 90 and
above. They are of great help if their EAAI values fall in the range of
70-80. They are termed as inadequate when the EAAI is below 70. The
EAAI for the API was above 70 and hence considered useful protein iso-
lates. Leucine and isoleucine participate in catalyzing the anabolic pro-
cedures in protein digestion and are therefore known as branched chain
amino acids (BCAA). They are useful in stopping proteins from degrada-
tion, promoting muscle growth after physical activity and are associated
with protein synthesis. Improvement of protein synthesis and retarda-
tion of muscle degeneration is due to the increase of the leucine content
of the protein. As when compared to normal diets, the foods consist-
ing of larger leucine to isoleucine proportions grant greater endurance.
For measuring cholesterolomic and atherogenic effects of proteins, ly-
sine to arginine proportions (LA ratio) is employed as an indicator of a
lower ratio is indicative of less lipidemic and atherogenic effects. API
had LA ratio of 0.67, lesser for soy protein extracts (0.71), whey pro-
tein extracts (5.38) which indicated good protein composition of Album
proteinaceous mass to promote cardiovascular health and can be used
for both infants and adults. In order to anticipate the protein quality in
terms of inherent potential of the dietary proteins to allow a proper form
of food crucial AAs, the AA score (AAS) could be utilized (FAO, 2013).
The protein quality present in a sample food is directly proportional to
the amino acids. The AAs existing in minute amounts are termed as re-
stricting AAs. Valine is the first limiting AA in case of newborn babies,
playschool kids (1-2 years) and grown-up men while isoleucine was the
12
Food Chemistry Advances 1 (2022) 100001
second limiting one. The Album protein isolate nutritional profile con-
firms the exceptional protein supplementing capability of album grains
(Mir et al., 2019).
5. Challenges related to different pseudo-cereal protein isolation
techniques
Out of all processes for protein isolation, the alkali method has sheer
efficiency in extracting proteins from the pseudocereals with one defi-
ciency of the presences of impurities besides the isolated protein. So,
there is a need to further improvisation and standardization of this
methodology to minimize the impurities. Another challenge is appli-
cation of enzymatic technique for protein isolation in certain pseudoce-
reals particularly album and wattleseed. The commercialization of the
isolation technique to obtain pure protein isolate is still a task to be
accomplished. The diversity of the biochemical composition of pseudo-
cereals harvested world over further make purification strategies a dif-
ficult task. Different proteins from chia and quinoa are high in quality
due to their eminent biological value (BV), net protein utilization (NPU),
protein efficiency ratio (PER), in-vitro protein digestibility (IVPD), etc.
however they remain underutilized due to dearth of knowledge among
the population.
6. Conclusion
The protein isolation methods were time consuming, energy inten-
sive and consume large quantities of chemicals. The yield and purity of
the protein isolates from pseudocereals extracted by the alkali method
was highest in comparison with other methods. The comparison of the
amino acid profiles of the different pseudocereals, showed that chia pro-
tein isolates had the highest amount of essential and Sulphur containing
amino-acids whereas album and wattleseed had lowest content. The val-
ues for aromatic amino acids were comparable for all the pseudocereals,
with chia faring better than the rest. The amino-acid scores of all the
pseudocereals were almost in the same range, with chia and album hav-
ing better scores than the other grains. The in-vitro digestibility values
of amaranth, buckwheat and quinoa were high and comparable whereas
chia had poor digestibility. The protein efficiency ratio of the amaranth
and chia was lower whereas that of buckwheat and quinoa were higher
indicating better utilization of buckwheat and quinoa proteins by the
body. As animal diseases have spread across the globe, healthier foods
have become more popular, and the demand for wholesome and eco-
nomic foods has increased. The environmental impact of animal-derived
proteins is greater, and they are more expensive than diets based on
plant proteins. To meet the consumer’s needs and provide nutritional
levels equal to animal proteins, we must develop new innovative food
products from plant proteins, like replacement of traditional meat and
dairy products with proteins from plant sources and this segment is wit-
nessing an enormous growth. Plant protein-based films, packaging ma-
terials and edible containers are also developed keeping in mind the
stress caused on the environment by the use of plastic-based packaging
and containers. Pseudo-cereal flours can lower the glycemic response to
the consumption of extruded breakfast cereal when they are combined
or replaced with the traditional extrusion flours. By fortifying and sup-
plementing pseudo cereals with cereal flour, nutritionally dense food
preparations can be made to meet or combat protein-energy deficiency.
Furthermore, the high protein content and balanced amino acid profile
of these neglected cereal grains may be a positive factor in masking the
limitations of cereals in terms of certain essential amino acids. Quinoa
and amaranth, for example, both contain amino acids similar to those
in casein and eggs respectively, besides they also contain bioactive pep-
tides that can improve human health and health processes after getting
absorbed in the active form. In addition, since pseudocereals do not con-
tain gluten, they are suitable for people suffering from celiac disease. It
can be concluded that pseudocereals-based protein isolates are relevant
A.M. Malik and A. Singh
to supplement the protein deficient diets in addition to enhancing the
life span of celiac disease population.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Amanda Manoj Malik reports a relationship with Sant Longowal In-
stitute of Engineering and Technology that includes employment.
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