Technologies for the production of protein isolates
from legumes
Tecnologías para la obtención de aislados proteicos a partir
de leguminosas
Miguel Enriquez
1
Klever Villarroel
2
Cristian Nunez
3
Franklin Villafuerte
4
Bonifaz Lilian
5
Abstract: The consumption of legume proteins has increased several
years ago due to the high cost of animal protein, which has motivated
the search for methods to obtain protein isolates, therefore, the objective
of the research was to analyze the different technologies proposed in
the literature to obtain protein isolates from legumes. For the
development of the research, the SALSA methodology modified by
Gunnarsdottir et al. (2020)(2020), which consists of 5 steps: search,
evaluation, snowball technique, synthesis and analysis. The most
widely used technology according to the literature for obtaining protein
isolates from legumes is alkaline extraction where, proteins are
solubilized at alkaline pH ranging between 8 and 11 to separate them
from the rest of non-soluble compounds, followed by isoelectric
precipitation by changing pH to ranges ranging between 3 and 5,
However, for the application of this technology it is important to
consider the composition of the raw material, since if it is rich in lipids,
the sample must be degreased in order to increase the yield of protein
extraction. Another technology used to obtain protein isolates consists
of extracting proteins by solubilization at alkaline pH followed by
ultrafiltration using membranes of different molecular weight exclusion
limits (10, 50 kDa). Obtaining protein isolates from legumes by the
above technologies depends on the process conditions (ratio of the raw
material-water solution, pH of protein precipitation or solubilization,
extraction temperature, membrane exclusion limits) and composition of
the raw material.
Keywords: Isoelectric point, precipitation, solubilization, protein,
ultrafiltration, ultrafiltration
Published
Instituto Tecnológico Superior Edwards
Deming. Quito – Ecuador
Periodicity
April - June
Dates of receipt
Received: January 09, 2023
Approved: April 02, 2023
http://centrosuragraria.com/index.php/revista
vol. 2. Num. 18. 2023.
pp. 68-98
Correspondence author
menriquez@uea.edu.ec
Creative Commons License
Creative Commons License, Attribution-
NonCommercial-ShareAlike 4.0
International.https://creativecommons.org/lice
nses/by-nc-sa/4.0/deed.es
1 Msc. Agroindustrial Engineering, Faculty of Earth Sciences, Universidad Estatal Amazónica, Puyo-Ecuador. menriquez@uea.edu.ec,
https://orcid.org/0000-0002-6113-450X
2 Msc. Agroindustrial Engineering, Faculty of Earth Sciences, Universidad Estatal Amazónica, Puyo-Ecuador. kvillarroel@uea.edu.ec,
https://orcid.org/0000-0002-8244-7097
3 Msc. Agroindustrial Engineering, Faculty of Earth Sciences, Universidad Estatal Amazónica, Puyo-Ecuador. cmunez@uea.edu.ec,
https://orcid.org/0000-0002-6120-984X
4 Msc. Agroindustrial Engineering, Faculty of Earth Sciences, Universidad Estatal Amazónica, Puyo-Ecuador. fvillafuerte@uea.edu.ec,
https://orcid.org/0000-0001-8322-6213
5 Msc. Independent Researcher, bonifazlilian@gmail.com, https://orcid.org/0000-0002-8937-9664
July - September vol. 1. Num. 18 - 2023
69
Resumen: El consumo de proteínas de leguminosas se ha incrementado
hace varios años debido al elevado costo de la proteína animal, lo que
ha motivado la búsqueda de métodos para la obtención aislados
proteicos, por ende, el objetivo de la investigación fue analizar las
diferentes tecnologías propuestas en la literatura para la obtención de
aislados proteicos a partir de leguminosas. Para el desarrollo de la
investigación se utilizó la metodología SALSA modificada por
Gunnarsdottir et al. (2020), misma que consta de 5 pasos que son:
búsqueda, evaluación, técnica de la bola de nieve, síntesis y análisis. La
tecnología más utilizada según la literatura para la obtención de aislados
proteicos a partir de leguminosas es la extracción alcalina donde, las
proteínas son solubilizadas a pH alcalino que oscila entre 8 y 11 para
separarlas del resto de compuestos no solubles, seguida de la
precipitación isoeléctrica mediante el cambio de pH a rangos que
oscilan entre 3 y 5, siendo el pH 4,5 más utilizado para la precipitación
de las proteínas, sin embargo, para la aplicación de esta tecnología es
importante considerar la composición de la materia prima, ya que si es
rica en lípidos se debe desengrasar la muestra con la finalidad de
incrementar el rendimiento de la extracción de proteínas. Otra
tecnología utilizada para la obtención de aislados proteicos consiste en
extraer las proteínas mediante la solubilización a pH alcalino seguida
de la ultrafiltración que utiliza membranas de diferentes límites de
exclusión de peso molecular (10, 50 kDa). La obtención de aislados
proteicos de leguminosas mediante las tecnologías mencionadas
depende de las condiciones de proceso (relación de la solución materia
prima-agua, pH de precipitación o solubilización de las proteínas,
temperatura de extracción, límites de exclusión de la membrana) y
composición de la materia prima.
Palabras clave: Punto isoeléctrico, precipitación, solubilización,
proteínas, ultrafiltración
Introduction
Legumes are considered one of the main sources of nutrients, especially
for low-income populations in developing countries. Among the most
consumed by humans are soybean (!"#$%&'()*), bean (+,)-'."/-0
1/"2)3%-), lentil (4'&-0 $/"%&)3%-), broad bean (5%$%)0 6)7)), chickpea
(8%$'3)3%'9%&/(), pea (+%-/(-)9%1/() and chocho (4/:%&/-0(/9)7%"%-),
which stand out for their high protein content ranging from 17 to 40 %
depending on the spice (Caro, 2015; Jaimes '90)"., 2012; Tello, 2018).
Olmedilla Alonso '90)";0(2010)Caro (2015) and Tello (2018) mention
that legumes have a higher protein content than cereals, easily
assimilated carbohydrates and, compared to meats, a low lipid content.
In addition, they are also characterized by having other nutrients such
as dietary fiber, minerals (iron, zinc, calcium), B complex vitamins and
in smaller amounts some bioactive compounds such as polyphenols,
alpha-galactosides, isoflavones.
Technologies for obtaining protein isolates from legumes
70
However, legumes have negative characteristics since the biological
value and bioavailability of nutrients is not as high as in that of animal
origin, due to the presence of toxic and anti-nutritional substances such
as phytates, trypsin inhibitors, hemagglutinins or lectins, saponins, -
alkaloids and cyanogenic glycosides. (Caro, 2015; Gallegos & Polo,
2013; González-Pérez & Arellano, 2009; Olmedilla Alonso '90 )";,
2010).
The most common ways to increase the nutritional value of legume
components, especially proteins, are cooking, fermentation and
germination, since these treatments reduce or eliminate anti-nutritional
substances, thermolabile toxins and oligosaccharides while maintaining
the protein and fiber content. Another way to take advantage of the
nutrients and especially the nutritional potential of legume proteins is
to isolate them from the rest of the antinutritional components found in
legumes, eliminating protease inhibitors and increasing protein
digestibility (Cantonal et al., 1995). (Cantonal '90)"., 1995; Olmedilla
Alonso '90 )";<0 2010).. The consumption of soy-based beverages has
increased due to the development of processing technologies that
improve their organoleptic properties based on their protein content,
which is essential for the human diet (Enriquez Estrella et al., 2022).
The consumption of legume proteins has increased several years ago
due to the high cost of animal protein, which has motivated the search
for methods to obtain protein concentrates and isolates by separating
the rest of the legume components (Cantonal et al., 1995). (Cantonal '90
)"., 1995).. Furthermore, considering that protein provides energy and
taking into account the origin of the term protein (from the Greek
"proteios" which means "primordial" or "first place")(Gonzalez-Torres
'90)"., 2007)means that it is the nutrient that provides the body with the
amino acids necessary for the creation, repair, development and
maintenance of cells and tissues during all stages of life, and many
proteins perform metabolic functions (act as enzymes, hormones,
antibodies). (González-Torres '90)";, 2007).In order to obtain protein
isolates as a source of this nutrient, it is necessary to obtain protein
isolates.
Obtaining protein isolates from legumes has advantages compared to
obtaining animal protein, since the production of animal protein uses a
large amount of water and feed, which increases deforestation and
greenhouse gas emissions, while obtaining protein from legumes
enriches the soil through nitrogen fixation, is economical, has a water
July - September vol. 1. Num. 18 - 2023
71
footprint, has a low greenhouse gas level and constitutes a sustainable
source of protein (González-Pérez & Arellano, 2009; Semba et al.
(González-Pérez & Arellano, 2009; Semba '90)"., 2021).
The importance of the consumption of protein isolates lies in the
nutritional contribution they provide, which is why they are used as
food for athletes, as ingredients in the supplementation of raw materials
low in protein, in the preparation of different types of foods such as
infant formulas, meat products, confectionery, desserts, emulsions and
beverages, since proteins provide improvements in the characteristics
of products due to the functional properties they possess. (Cantonal '90
)"., 1995; Fonseca, 2019; González-Pérez & Arellano, 2009; Jaimes '90
)";, 2012; Mercado '90al., 2015; Ulloa '90)"., 2012)..
By saying that proteins provide improvements in the characteristics of
products due to the functional properties they possess, we mean that
when using protein isolates in food production it is feasible to determine
the behavior of these during processing, storage or consumption, i.e.,
these properties and the way in which proteins act with other
components directly or indirectly affect their applications, quality and
acceptance of foods(Ulloa '90 )";, 2012).. Among the functional
properties of proteins are solubility, water-holding capacity, oil
absorption capacity, emulsifying capacity, foaming capacity and
gelling capacity (Cantonal '90)"., 1995; Fonseca, 2019; González-Pérez
& Arellano, 2009; Jaimes '90)";, 2012; Mercado '90)";, 2015; Ulloa '90
)"., 2012)..
The importance of the properties mentioned above vary with the type
of product in which the protein is intended to be used, for example,
protein isolates with proteins with high water or oil retention capacities
are used in meat and bakery or confectionery products, while proteins
with high emulsification capacities are suitable in the preparation of
salad dressings, sausages, bologna, confectionery and pastries (Ulloa '90
)";, 2012).. Additionally, obtaining protein isolates is the first step in
obtaining bioactive peptides that help human beings in the reduction of
diseases (Diaz, 2016).
A product may be considered as a protein isolate if it complies with the
Codex Alimentarius (2007)This is achieved by reducing or eliminating
the main non-protein constituents (water, lipids, carbohydrates,
vitamins, minerals, anti-nutritional and toxic substances).
For the elimination of non-protein compounds and in order to obtain
protein isolates, there are different technologies such as aqueous
extraction, saline extraction and ultrafiltration, the most widely used
being alkaline extraction/isoelectric precipitation. However, emerging
methods have also been developed with the aim of increasing extraction
Technologies for obtaining protein isolates from legumes
72
yields, increasing their nutritional properties and increasing their
technological applications. Among the emerging methods, enzyme-
assisted protein extraction, electrostatic protein separation, protein
extraction assisted by cell disruption techniques are mentioned
(Fonseca, 2019)
In obtaining protein isolates from legumes, the most commonly used
method is alkaline extraction and isoelectric precipitation of proteins,
where, proteins are solubilized at alkaline pH to separate them from the
rest of the non-soluble compounds, for subsequent precipitation by
changing pH (Fonseca, 2019; Mercado '90 )";, 2015; Vioque '90 )";,
2001).. Examples of this are: a) the protein isolate with a protein content
of 92 % protein on a dry basis obtained by Thompson (1977) by
solubilizing bean (+,)-'."/-0)/3'/-) proteins at pH 9 and 25 °C for 20
minutes followed by precipitation at pH 4; b) protein isolate from pea
(+%-/(0-)9%1/() with 90% protein obtained by (Sumner '90)"., 1981) by
solubilizing the proteins at pH 9 for 20 minutes followed by
precipitation at pH 4.5.
Another technology used in obtaining protein isolates from legumes
consists of alkaline extraction of proteins and subsequent filtration of
the protein solution through membranes with different molecular
weight exclusion limits, e.g. Des Marchais '90)"0.(2011) used a 50kDa
membrane to obtain a protein isolate with 96.1 % protein from peas.
Taking into account the above mentioned, the variations in the
parameters for obtaining protein isolates, the importance and usefulness
of protein isolates, the objective of this research was to analyze the
different technologies proposed in the literature for obtaining protein
isolates from leguminous plants.
Materials and methods
The research carried out was specifically a literature review and the
SALSA (Search, Appraisal, Synthesis, Analysis) methodology
modified by Gunnarsdottir '90)". (2020). The traditional SALSA method
for systematic reviews involves four steps: search, appraisal, synthesis
and analysis; however, Gunnarsdottir '90 )". added an additional step
known as the snowball technique as shown in Figure 1 and 2.
July - September vol. 1. Num. 18 - 2023
73
Figure 1.
!'&'3)"0=%)23)(0.609,'0>?4>?0('9,.=0)-0(.=%6%'=07#0!/&&)3-=.99%30'90
)";0@ABABC
The SALSA method consists of a comprehensive search process and
critical review that allows papers to be produced using the best of the
available information while minimizing the potential for bias
(Gunnarsdottir '90)"., 2020; Luengo '90)";, 2016).. The modified SALSA
method used for the elaboration of this research is described below and
can be visualized schematically in Figure 2.
The first step of the SALSA method consisted of searching for relevant
information on legume proteins and protein isolates in undergraduate
and graduate theses, scientific articles and books found in search
engines and databases such as Google Scholar, Web of Science,
Science, Direct, Scopus, Pubmed, Scielo. The information searched
was found in English, Spanish and Portuguese, and corresponds to the
years 2000 onwards, except for five documents from earlier dates.
The second step allowed further evaluation of whether the search results
met the criteria for inclusion (quantitative and/or qualitative research,
complete access to protein information, methods of obtaining protein
isolates from legumes) and exclusion (research outside the data
collection period, methods of obtaining protein from sources other than
legumes, opinion articles or blogs). This point allowed the evaluation
and classification of the literature used, and also served as a basis for
continuing with step three.
Search
(búsqueda)
Appraisal
(evaluación)
Synthesis
(síntesis)
Analysis
(análisis)
snowball
technique
(técnica de bola
de nieve)
Technologies for obtaining protein isolates from legumes
74
Figure 2.
D".E$,)390.609,'0(.=%6%'=0>?4>?0('9,.=0/-'=06.309,'0'")7.3)9%.&0.60
9,'0article.
Source: Gunnarsdottir '90 )". (2020) with modifications for the
elaboration of the work
The third step, the snowballing technique consists of using references
and citations of articles to identify more relevant literature, i.e., the
BúsquedaEvaluación
SíntesisAnálisis
Realizada en
buscadores y bases
de datos
Estudios de texto
completo según
criterios de
elegibilidad
Estudios incluidos en
síntesis cualitativa
Criterios de inclusión: investigaciones cuantitativas y/o
cualitativas, acceso completo sobre proteínas y aislados
proteicos de leguminosas.
Criterios de exclusión: investigaciones fuera
del período de recogida de datos, de proteínas de otras
fuentes. y artículos de opinión o blocs
Estudios incluidos en
análisis cualitativo y
cuantitativo
Filtros: año de publicación: de preferencia artículos de
2000 en adelante y 5 de años anteriores.
Área: alimentos
Tipos de documentos: artículos científicos, tesis, libros
Lenguaje: Ingles, español, portugués
Términos clave: aislados, leguminosas, proteínas,
Técnica de
bola de nieve
Elaboración o desarrollo
del documento
Permitió encontrar más
información bibliográfica
relevante sobre la obtención
de aislados proteicos
Análisis descriptivo por
categorías: Proteínas de
leguminosas, métodos
para obtener aislado
proteicos
July - September vol. 1. Num. 18 - 2023
75
review literatures found through the initial search served as the basis for
snowballing to find 15 additional bibliographic investigations.
The fourth stage consisted of the synthesis or elaboration of the
document based on the relevant information from the literature selected
under the criteria mentioned above. For this purpose, the publications
identified and evaluated in the previous stages were carefully read in
order to relate the relevant information in the written document.
Finally, the synthesized information was analyzed to fulfill the
objective of the research. For this purpose, unit operations, processes
and raw materials involved in obtaining protein isolates from legumes
were analyzed for the development of the results and discussion of the
current work.
3. Result
Legumes have a higher protein content than cereals (Olmedilla Alonso
et al., 2010). (Olmedilla Alonso '90 )";<02010).which makes them the
ideal raw material for obtaining protein isolates. Among the
technologies for obtaining protein isolates, two types of technology
stand out: a) obtaining protein isolates by alkaline extraction and
subsequent isoelectric precipitation and b) obtaining protein isolates by
alkaline extraction and subsequent filtration of the protein solution.
This technology consists of solubilizing the proteins at alkaline or basic
pH followed by isoelectric precipitation under acid pH conditions.
Tables 1, 2, 3, 4 and 5 show the aforementioned technology for
obtaining protein isolates.
The first step, prior to protein solubilization, is to reduce the particle
size of the grain (González-Pérez & Arellano, 2009). (Gonzalez-Perez
& Arellano, 2009)in order to increase the contact area or surface area
of the particles with the extracting medium or solution (solution at
alkaline pH), which would improve the yield of the extraction process
of the protein present in the food matrix. According to the research,
tables 1, 2, 3, 4 and 5 show that in order to obtain protein isolates, the
researchers start from flour, or in turn, the grains are reduced to flour.
Another point to consider before protein extraction is the lipid content
of the legumes, since high lipid contents interfere during the process.
Furthermore, this component can cause changes in the final product
because it can be associated with the proteins of the isolates, giving rise
to rancidity problems during processing and storage (Vioque et al.,
Technologies for obtaining protein isolates from legumes
76
2001). (Vioque '90)";<02001)Therefore, it is advisable to defat the raw
materials or to start protein extraction from defatted flour. This is
especially ratified in Table 2, due to the high lipid content of soybean,
which is 19.94% (Delgado-Andrade et al., 2001). (Delgado-Andrade '90
)";<02016)additionally in table 3, two authors perform defatting of beans
while one does not, due to the fact that the fat content of beans is
between 1.2 to 1.5 %. However, in Table 4, three out of four authors
defat the chocho flour because it has a lipid content of 9.74%, a content
that according to the results of the research affects the amount of protein
in the final product (55.58%) since it is lower than the other treatments.
In the technologies reviewed in tables 1 to 5, the pH for protein
extraction or solubilization ranges from 8 to 11, while the most
commonly used pH for protein precipitation is 4.5. This is justified by
the research carried out by Adebowale & Lawal (2003) and Sánchez-
Vioque '90)", (1999)(1999), who mention that the solubility profile of
the protein present in the protein concentrate or isolate is pH dependent
(Figure 3), i.e., the lowest protein solubility is found in the pH range of
4 to 5, however, while protein solubility increases as the pH increases,
which is why a pH close to 9 is used for the extraction of proteins from
the food matrix. (Vioque '90)"., 2001) some proteins, such as glutelins,
require pH equal to or higher than 11 to be extracted.
Figure 3. Effect of pH on protein solubility.
.
Source: (Adebowale & Lawal, 2003)
July - September vol. 1. Num. 18 - 2023
77
Table 1 presents the steps for obtaining protein isolates from the field
bean (+,)-'."/-0"/&)9/-04), for this, Tello (2018) and Tamayo (2018)
work at pH 8, Chel-Guerrero '90)";0 (2002) at pH 11 to solubilize or
extract the proteins from the food dye, while to precipitate the proteins
Tello (2018) and Tamayo (2018) work at pH 3, 4, 5 and Chel-Guerrero
'90)";0(2002) at pH 4.5. From these investigations Tamayo and Tello
obtain the highest amount of protein in the final product when working
at an isoelectric precipitation pH of 5, agreeing that the optimum pH for
protein precipitation is between 4 and 5.
Table 1. F'$,&.".2%'-06.309,'0:3.=/$9%.&0.60:3.9'%&0%-.")9'-063.(06%'"=0
7')&-0 @+,)-'."/-0 "/&)9/-0 4C0 7#0 )"G)"%&'0 '*93)$9%.&0 )&=0 %-.'"'$93%$0
:3'$%:%9)9%.&;0
Operations
Author
Tello (2018)
Tamayo
(2018)
Chel-Guerrero '90
)". (2002)
Suspension
5g of fava
bean flour in
50 ml of
distilled water
The flour was
suspended in
water in a 1:10
(w/v) ratio.
Flour/water was
mixed in a 1:6
w/v ratio.
Protein
solubilization
Adjust pH to 8
with NaOH 1
M with
agitation for
maintaining
the pH.
Adjusting pH 8
with NaOH 2
M for one hour
and constant
agitation
between 800-
900 rpm.
Adjusted to pH
11 with NaOH 1
N and soaked for
1 h.
Separation of
soluble
proteins
(liquid or
supernatant
phase)
By
centrifugation
for 30 minutes
at 4400 rpm
and room
temperature.
The
precipitate is
discarded and
the
supernatant is
continued with
the
supernatant.
By
centrifugation
at 4400 rpm for
30 min.
The precipitate
is discarded
and the
supernatant is
continued with
the
supernatant.
The suspension
is ground and
filtered to
separate the solid
fraction from the
liquid fraction
containing
protein and
starch. The
residual solids
are washed 5
times with
distilled water
and passed
Technologies for obtaining protein isolates from legumes
78
through a 150
mesh sieve; the
wash water is
then mixed with
the initial
supernatants. It is
allowed to settle
for 30 min to
recover the
starch and
separate the
solubilized
protein.
Precipitation
of proteins
from the
supernatant
pH adjustment
to 3, 4, 5 and 6
with 1M HCl
Adjustment of
pH to 3, 4, 5
and 6 with 2M
HCl. Shake for
5 minutes
The pH was
adjusted with 1 N
HCl to the
isoelectric point
4.5.
Rest
For 24 h
At 4° C for 24
h
Protein
separation
Remove
supernatant
and use the
precipitate.
Remove
supernatant
and use the
precipitate.
By
centrifugation at
1317 g for 12
min. Remove
supernatant and
use the
precipitate.
Drying/
Freeze-drying
At a pressure
of 0.2 Pa and a
temperature of
-57°C
At -50 °C and
0.2 Pa pressure
At -47 °C and 13
× 10
-3
mbar.
Storage
In sterile
flasks at -80
°C
In sterile flasks
at -80 °C
Extraction
yield
pH 3 8.64a ±
0.10%.
pH 4 18.56 ±
1.14 % pH 4
pH 3 19.55a ±
1.55 %.
pH 4 10.56b ±
0.83% 10.56b
± 0.83% pH 4
July - September vol. 1. Num. 18 - 2023
79
18.56 ± 1.14
%.
pH 5 12.86b ±
0.16%.
pH 6 32.58d ±
0.30%.
pH 5 19.56a ±
1.55%.
pH 6 27.59c ±
0.24%.
Quantification
of protein
content
Biuret's
method
pH 3 69.96 c ±
0.78% c ±
0.78% pH 3
69.96 c ±
0.78% pH 3
pH 4 62.39 b ±
0.35% pH 4
62.39 b ±
0.35% pH 4
62.39 b ±
0.35% pH 4
pH 5 71.33 c ±
0.92%.
pH 6 56.80 at
± 1.77%.
Kjeldahl
method.
pH 3 41.19 c ±
1.10% pH 3
41.19 c ±
1.10% pH 3
41.19 c ±
1.10% pH 3
pH 4 38.92 b ±
0.14% pH 4
38.92 b ±
0.14% pH 4
38.92 b ±
0.14% pH 4
pH 5 42.31 c ±
0.58% c ±
0.58% pH 5
42.31 c ±
0.58% pH 5
Biuret's
method
pH 3 52.96a ±
0.031% pH 3
52.96a ±
0.031% pH 3
52.96a ± 0.031
pH 4 57.85b ±
0.013% ±
0.013%.
pH 5 62.53c ±
0.018% ±
0.018%.
pH 6 56.74b ±
0.026% ±
0.026%.
71.13 ± 0.92%
Technologies for obtaining protein isolates from legumes
80
pH 6 34.36 at
± 0.44%.
a, b, c, d. Indicates that there are significant differences between
treatments.
Table 2. F'$,&.".2%'-0 6.30 .79)%&%&20 :3.9'%&0 %-.")9'-0 63.(0 -.#7')&0
@!"#$%&'0()*C0
Operations
Author
Avila (2011)
L'HOCINE et al.
(2006)
Puppo et al.
(1995)
Flour
production
Defatted
soybean meal
is milled
(particle size
45 µm).
Flour defatting
with hexane
Yes Part
defatted
soybean meal
Suspension
Flour/water
mixture in a 1:6
w/v ratio with
10 min
agitation.
Flour/water
mixture in a 1:15
w/v ratio at
55°C.
Flour/water
mixture in a
1:10 w/v ratio
with 10 min
agitation.
Protein
solubilization
At pH 9.5
adjusted with
NaOH 6N and
stirring for 30
min.
The pH was
adjusted and
maintained at
9.0 with NaOH
2 N with stirring
for 45 min at
55ºC. ºC
At pH 8
adjusted with
NaOH 2N and
stirring for 2 h
at room
temperature
Separation of
soluble
proteins
(liquid or
supernatant
phase)
By
centrifugation
at 12000 rpm.
The precipitate
is discarded
and the
supernatant is
continued with
the
supernatant.
The suspension
rests for 15 min
at room
temperature,
then centrifuged
at 4 °C for 30
min at 14300g.
The supernatant
is continued
with the
supernatant
By
centrifugation
at 13300g for
20 min at
15°C. Work
with the
supernatant
July - September vol. 1. Num. 18 - 2023
81
Precipitation
of proteins
from the
supernatant
pH adjustment
to 4.5 with HCl
6N
Adjust pH to 4.5
with 2 N HCl
and stirring for
45 min at 25 °C.
At pH 4.5
adjusted with
HCl 2 N
Rest
Protein
separation
By
centrifugation
at 12000 rpm.
Remove
supernatant
and use the
precipitate.
By
centrifugation at
2830 × g (4 °C)
for 15 min.
By
centrifugation
at 3300 for 20
min.
Washing
The precipitate
is washed twice
with water and
centrifuged each
time at 2830 × g
for 10 min.
Neutralization
With NaOH 2N
up to pH 7
Storage
Freezing
Drying/
Freeze-drying
At a pressure of
0.2 Pa and a
temperature of
-57°C
Freeze-dried
Lyophilized
Storage
AT 4°C
Extraction
yield
24.55%
Recovered
protein: 51%.
Quantification
of protein
content
85.45%
92.8 ± 0.08% on
dry weight basis
82.4 ± 0.4
Table 3. F'$,&.".2%'-06.30.79)%&%&20:3.9'%&0%-.")9'-063.(0+,)-'."/-0
1/"2)3%-0@7')&C0
Operations
Author
Rui '90)";(2011)
Ahmed '90
)".(2015)
Morales-de
León '90 )";0
(2007)
Technologies for obtaining protein isolates from legumes
82
Soaking
1 kg of sample
in 4 L of water
and kept at 4
°C for 12 h.
Subsequently,
the shells are
removed.
Particle size
reduction
By milling
With blender
3 kg of fresh
grain is mixed
with 30 L of
water and
ground.
Degreasing
With hexane (1:3,
w/v)
Suspension
Ratio of flour to
distilled water
(1:15)
The
suspension
obtained was
diluted ten
times (v/v)
Protein
solubilization
At pH: 9. With
agitation for 1 h at
55°C
With pH to 10
using NaOH
0.5 M. Mixing
for 1 h.
Adjustment of
pH to 8.0 with
0.2 M NaOH.
Extraction is
carried out at
35 ◦C by
stirring.
Separation of
the soluble
phase or
supernatant
(soluble
proteins)
Centrifugation at
11 000 × g at 20 °C
for 30 min.
Discard the
precipitate and
continue with the
supernatant.
Filtered
through a 75
μm mesh
sieve and
centrifuged at
3000 ×g for
30 min at 10
°C.
By
centrifugation
at 5000 × g for
15 min at 25
◦C. The
supernatant is
continued with
the supernatant
once filtered.
Rest
At 4 °C for about
15 h to sediment
the starch and then
July - September vol. 1. Num. 18 - 2023
83
centrifuged as
above.
Precipitation
of proteins
from the
supernatant
Adjusting pH 4.5
with HCl 1M
Adjusted to
pH 4.5 with
0.1 N HCl.
The pH is
adjusted to 4.3
with dilute
HCl.
Protein
separation
By centrifugation
at 11 000 × g at 20
°C for 30 min.
By
centrifugation
at 8000 ×g for
10 min at 5 °C
By
centrifugation
at 10000 × g
for 20 min
(repeat protein
solubilization).
Washing
Twice more with
Millipore water at
1:5 (w/w).
Wash with
distilled water
Neutralization
With 10% NaOH
solution (pH 6.5-
7). Then
centrifuged at
4000 rpm
for 5 min
Adjusting to
pH 7
Drying
Lyophilized
Freeze-drying
Freeze-drying
Extraction
yield
36.15% hard
bean
45.37% fresh
beans
Quantification
of protein
content
89,25 %
83,96 %
Range of
76.96-
83.96%.
71.9% hard
bean
75.6% fresh
beans
Table 4. F'$,&.".2%'-0 6.30 .79)%&%&20 :3.9'%&0 %-.")9'-0 63.(0 $,.$,.0
@4/:%&/-0(/9)7%"%-C0
Operations
Author
(Guerra &
Pozo, 2018)
(Aguinda,
2019)
(Acuña &
Caiza
Jimena,
2010)
(Urrutia
Gutiérrez,
2010).
Technologies for obtaining protein isolates from legumes
84
Flour
preparation
The
hydrated
grain is
peeled, dried
and ground.
Grain
milling
The chocho
beans are
ground
The grains are
soaked (1
day), cooked
(1h), washed
(5 days), dried
and ground.
Degreasing
By means of
an
extraction
system
continuous
(Soxhlet)
with hexane
With hexane
with
isolation for
18 h at TA
With 95%
ethanol in a
flour: solution
ratio of 1:3.
Three
extractions
during 5
hours each
Suspension
Dissolves in
water in a
1:10 (w/v)
ratio.
Flour to
distilled
water ratio
(1:7,
1:9,1:11)
At 43°C with
a flour:
solvent ratio
(1:20).
Protein
solubilizatio
n
At pH 6.8-
10. Adjusted
with 10 %
NaOH
At pH 8
adjusted
with NaOH
2 M. It is left
in agitation
for 1 h
pH (8.5, 9.5,
10.5)
adjusted
with NaOH
1N, stirred
for 30 min.
At pH 9.3, at
43°C
Separation
of soluble
proteins
(liquid or
supernatant
phase)
By
centrifugatio
n at 12000
rpm. The
precipitate is
discarded
and the
supernatant
is continued
with the
supernatant.
By
centrifugatio
n for 45 min
and 4400
rpm. The
precipitate is
discarded
and the
supernatant
is continued
with the
supernatant.
By
centrifugatio
n at 8000
rpm. The
precipitate is
discarded
and the
supernatant
is continued
with the
supernatant.
By
centrifugation
at 4000 rpm
for 20 min.
Precipitatio
n of proteins
from the
supernatant
Adjusting
pH 4.5 with
10 % HCl
solution
Adjust pH 3,
4, 5, 6 with
2M HCl,
shake for 5
minutes,
pH to 4.5
with H Cl
2N, stirred
for 15min.
At pH 4.5 and
stirring for 15
min.
July - September vol. 1. Num. 18 - 2023
85
then stir for
5 minutes.
Protein
separation
By
centrifugatio
n at 4000
rpm for 5
min.
In
refrigeration
(4 °C) for 24
hours. Then
remove the
supernatant
and obtain
the protein
precipitate.
By
centrifugatio
n at
12000rpm
Centrifugatio
n 4000 rpm
for 20 min.
Washing
2 times at a
rate of
of 1:5
(protein/water
). Elimination
of wash water
by
centrifugation
at 4000 rpm
for 15 min.
Table 4 @$.&9%&/'=C;0F'$,&.".2%'-06.30.79)%&%&20:3.9'%&0%-.")9'-063.(0
$,.$,.0@4/:%&/-0(/9)7%"%-C0
Neutralization
With 10%
NaOH
solution
(pH 6.5-7).
Then
centrifuge
d at 4000
rpm
for 5 min
Drying/
Freeze-drying
Drying for
24h in an
oven at 85
ºC.
Lyophilize
d at
pressure: <
0.2 atm;
temperature
< -55 °C)
and stored
at 4 °C
Freeze-
dried
Drying
Extraction
yield
pH3 18.5±
0.62%.
At pH
10.5 it
Technologies for obtaining protein isolates from legumes
86
pH4 28.6±
0.69%.
pH5 31.4±
0.32%.
pH6 24.7±
0.66%.
records
72.2%
protein
recover
y and
42.6%
yield by
weight.
Quantificatio
n of protein
content
67,25 ±
5,75%
pH3 76.8±
1.44%.
pH4 95.0±
3.17%.
pH5 96.9±
0.51%.
pH6 95.6±
2.82%.
55,58%
92,83±0,18
%
In addition to considering the solubilization pH of proteins, it is
important to take into account that the use of extreme pH (higher than
9 and lower than 4) negatively affects the characteristics of the protein
and can even cause its hydrolysis or denaturation, racemization of
amino acids and therefore loss of essential amino acids such as cysteine
and lysine, which would cause a reduction in protein digestibility, in
addition to producing the precipitation of non-protein components that
can affect the purity of the isolate (Callisaya & Alvarado, 2009;
González-Pérez & Arellano, 2009). (Callisaya & Alvarado, 2009;
González-Pérez & Arellano, 2009).. Therefore, for most vegetable
sources, the pH values for isolating proteins should be between 4 and 9
as this should not be harmful to the proteins (González-Pérez &
Arellano, 2009).
Additionally, according to what is observed in tables 1, 2, 3, 4, and 5,
the process to isolate proteins from legumes is based on the same
principle which is alkaline extraction and isoelectric precipitation,
however, there are small variations proposed by the authors, among
which is the use of centrifugation to separate the soluble part from the
solid part, while others do this process by natural precipitation, that is,
they wait for a time of 12 to 14 hours. Therefore, centrifugation is used
to make the process faster and more efficient.
July - September vol. 1. Num. 18 - 2023
87
Table 5. F'$,&.".2%'-0 6.30 .79)%&%&20 :3.9'%&0 %-.")9'-0 63.(0 .9,'30
"'2/(%&./-0:")&9-0
Operations
Author/legume
(Villafuerte et
al., 2019)/
chachafruit
(Erythrina
edulis Triana)
(Sanchez-
Vioque et al.,
1999)/
chickpea
(8%$'30
)3%'9%&/(C
(Atiencia
Pazmiño, 2021)/
Pea (Pisum
Sativum)
Flour
production
The grains
were cut and
dehydrated at
45 °C for
24 h, and then
grind them
Flour part
Suspension
The flour was
dispersed in
distilled water
(1:10 w/v).
20 g of flour
was
suspended in
200 ml of
(a) 0.2%
NaOH
solution pH 12
b) 0.25%
Na2SO3
solution at pH
10.5
10 g of flour in
100 mL of
distilled water
(ratio 1:10)
Protein
solubilization
At pH
adjusted to
11.0 with 0.1
M NaOH and
kept under
stirring for 1
hour.
At pH 12 or
10.5 with
stirring for 1h
At 8.0 with
Na(OH) 1N, and
stirred for 60
minutes by
means of a
magnetic stirrer.
Separation of
the soluble
phase or
supernatant
(soluble
proteins)
By
centrifugation
at 2,500 rpm
for 20
minutes. The
supernatant is
used
By
centrifugation
at 8000 g. Two
additional
extractions are
made
By centrifugation
for 20 minutes at
8000 rpm.
Insoluble
residues are
suspended with
distilled water at
a 1:5 ratio, then
pH is adjusted
Technologies for obtaining protein isolates from legumes
88
with 1N sodium
hydroxide to 10.0
by mechanical
shaking for 10
minutes and
centrifuged.
Precipitation
of proteins
from the
supernatant
Adjusting to
pH 4 and
stirring for 1
hour
At pH 4.3
At pH 4 with
agitation for 20
minutes
Protein
separation
By
centrifugation
at 2,500 rpm
for 30 minutes
By
centrifugation
at 8000 g
By centrifugation
at 12000 rpm
Washing
a) With
distilled water
adjusted to pH
4.3
b) with 100 ml
of distilled
water adjusted
to pH 4.3,
ethanol and
acetone.
Drying
Freeze-drying
a)
Lyophilization
b) Ambient
temperature
Extraction
yield
Final protein
recovered
from chickpea
flour was 65.9
and 62.1%.
11%
Quantification
of protein
content
96.01 % on
dry basis
80.9% at pH
12
87.1% at pH
10.5
79,14%
July - September vol. 1. Num. 18 - 2023
89
Most researchers use freeze-drying instead of conventional drying as
the operation to remove water from proteins. This is because freeze-
drying works at low temperatures, which causes sensory alterations
(color, shape, size, flavor, texture) and nutritional losses of the
dehydrated products to be significantly reduced (García-Mora et al.,
2019; Peña & Parra, 2015).. The yield in the processes of obtaining
proteins from raw material is less than 50% due to the fact that legumes
have a series of components such as lipids, carbohydrates, among
others, however, when talking about the percentage of protein recovery,
this does not exceed 80%. This is due to the fact that in a matrix there
are different types of proteins that have different pH solubilization
(close to neutrality for example) or precipitation, but it is preferred to
extract proteins at alkaline pH to favor the solubilization of proteins
denatured during the preparation of the concentrates. (Vioque et al.,
2001).
The technologies mentioned in Tables 1, 2 ,3 ,3 ,4 and 5 are applied to
obtain protein isolates from various types of legumes, e.g., Li '90)".
(2010) applied the technology of alkaline extraction and isoelectric
precipitation to obtain protein isolates from 16 varieties of mung bean
(5%2&)03)=%)9)). For this, they prepared five percent (w/v) solutions of
the mung bean flour suspension, adjusted to pH 9 at room temperature.
The solutions were mixed for 1 h and subsequently centrifuged for 15
min at 2000 - g. To obtain higher yields, the extraction and
centrifugation procedures were repeated once on the residue. The
extracts were combined and the pH adjusted to 4.5 to precipitate the
protein. Proteins were recovered by centrifugation at 2000 - g for 15
min followed by removal of the supernatant. This procedure allowed
them to obtain products with protein contents ranging from 69.22 to
74.84%.
Kaur & Singh, (2007) obtained protein isolates from chickpea (8%$'30
)3%'9%&/(04.) cultures by working under the same pH conditions as Li
'90)". (2010). The protein content of the product obtained ranged from
89.9% to 94.4%. Many researches mention obtaining protein isolates,
however, we must clarify that according to Codex Alimentarius (2007)
products can be classified according to protein content as follows:
a) Protein content greater than 50 and less than 65 percent: protein meal
b) Protein content greater than 65 and less than 90 percent: protein
concentrate
Technologies for obtaining protein isolates from legumes
90
c) Protein content greater than 90 percent: protein isolate
That is, according to the Codex Alimentarius, the use of the alkaline
extraction method and isoelectric precipitation of proteins allows
obtaining not only protein isolates but also concentrates, according to
the protein content reported in tables 1, 2, 3, 4 and 5.
Obtaining protein isolates by alkaline extraction and subsequent
filtration.
This technology consists of extracting the proteins by solubilization at
alkaline pH followed by filtration of the solution containing the proteins
using filters or membranes that allow non-protein substances to pass
through. The following are examples of the technology used to obtain
isolates:
Des Marchais '90 )", (2011) to obtain a protein isolate proceeded as
follows: they first mixed pea flour with water at room temperature in a
1:15 w/w ratio, then adjusted to pH 7.5 which allowed the proteins to
be extracted from the legume matrix. Following this, they discarded the
precipitate and proceeded with dissolution to ultrafiltration/diafiltration
(UF/DF) operation using 50 kDa hollow fiber membranes. The product
is lyophilized and after analysis they determined a protein content of
96.1±0.2% on a dry basis.
Boye '90 )"<0 (2010) conducted a comparative investigation of two
methods for obtaining protein isolates, these methods are ultrafiltration
(UF) and isoelectric precipitation (IEP). The first stage for the two
technologies is the same, i.e. protein solubilization was performed
under the following extraction conditions: pH 9.5 with solid/liquid ratio
1/15 at 35 °C for yellow pea, desi chickpea and kabuli and pH 9.0 with
solid/liquid ratio 1/10 at 25 °C for red and green lentils. Subsequently,
to obtain protein isolates by IEP the pH was adjusted to 4.5, while to
obtain isolates by UF/DF they used a 50 kDa membrane with
diafiltration (4X) at pH 6.0. The results obtained showed that the
technology using UF/DF allows obtaining products with higher protein
content as shown in Table 6.
July - September vol. 1. Num. 18 - 2023
91
+3.9'%&0 $.&9'&90 .60 %-.'"'$93%$)""#0 :3'$%:%9)9'=0 @HI+C0 )&=0
/"93)6%"93)9'=0@JDC0"'2/('0:3.9'%&0'*93)$9-. 0
Sample
Protein
IEP
UF
yellow pea
81.7 ± 0.3
83.9 ± 0.15
desi chickpea
73.6 ± 0.1
76.5 ± 0.05
kabuli chickpea
63.9 ± 1.3
68.5 ± 0.15
red lentils
78.2 ± 0.2
82.7 ± 0.20
green lentils
79.1 ± 0.3
88.6 ± 0.05
Source: Boye '90)"<0(2010)
The results by Boye '90)"<0(2010) confirm the studies carried out by
Fuhrmeister & Meuser (2003) as they found that rough pea concentrates
prepared by ultrafiltration had a higher protein content (70-80 %) than
concentrates obtained by isoelectric precipitation (68%). Chew '90
)",(2003) also obtained similar results when comparing the two
technologies since by IEP (working at solubilization pH 8-9 and
precipitation pH 4.5) they obtained a lupin protein concentrate with a
protein concentration of 671 g/kg, while by UF using a 10kD membrane
the protein content of the final product was higher (751 g/kg).
The bibliographic research carried out shows that the technology used
to obtain the highest protein content in the final product is UF/DF;
however, it also shows that the most widely used method for obtaining
protein isolate is isoelectric precipitation, information that is confirmed
by González-Pérez & Arellano, (2009)
4. Conclusions
According to bibliographic research, legumes have a higher protein
content than cereals, and therefore represent an important source for
obtaining protein isolates. However, in order to obtain protein isolates
from legumes, it is important to take into account the composition of
the raw material used, especially if it has a high lipid content, such as
soybean or chocho. This considering that it must be defatted to obtain
better results in the protein content of the final product,
The most commonly used technology for obtaining protein isolates
from legumes is alkaline extraction and isoelectric precipitation of
Technologies for obtaining protein isolates from legumes
92
proteins; however, to achieve the best protein extraction yield, the
particle size and lipids must first be reduced, and process conditions
such as raw material-water solution ratio, pH of precipitation or
solubilization of proteins and extraction temperature must be taken into
account.
The solubilization of the proteins should not be carried out at very high
pH, while the pH of the precipitation should not be very acid because it
can produce the denaturation of the proteins, therefore for the
solubilization it is advisable to use a pH of maximum 10 while for the
precipitation the recommended pH is 4.5.
References
Acuña, O., & Caiza Jimena (2010). Obtaining enzymatic hydrolysate
of chocho (Lupinus mutabilis) protein from wholemeal flour.
K'1%-9)0 +."%90L'$&%$), AM(1), 70-77.
https://revistapolitecnica.epn.edu.ec/ojs2/index.php/revista_polite
cnica2/article/view/274/pdf
Adebowale, K. O., & Lawal, O. S. (2003). Foaming, gelation and
electrophoretic characteristics of mucuna bean (Mucuna pruriens)
protein concentrates. D..=0 8,'(%-93#, NO(2), 237-246.
https://doi.org/10.1016/S0308-8146(03)00086-4.
Aguinda, A. (2019). P'9'3(%&)9%.&0 .60 9,'0 )&9%.*%=)&90 )&=0 )&9%Q
%&6")(()9.3#0)$9%1%9#0.60$,.$,.0@4/:%&/-0(/9)7%"%-0>E''9C06"./30
:3.9'%&0 %-.")9'-0 )&=0 9,'%30 2)-93.%&9'-9%&)"0 =%2'-9%7%"%9#0 %&0 1%93.0
[[Undergraduate thesis, Universidad Técnica de Ambato]].
https://repositorio.uta.edu.ec/bitstream/123456789/29418/1/BQ
180.pdf
Ahmed, I., Singh, D., Shanker, U., & Singh, B. (2015). Physico-
chemical and functional properties of native and hydrolyzed
kidney bean ( Phaseolus vulgaris L .) protein isolates. D..=0
K'-')3$,0 H&9'3&)9%.&)"0 R./3&)", ST(1), 11-18.
https://doi.org/10.1016/j.foodres.2014.08.027
Atiencia Pazmiño, A. M. (2021). U79)%&%&20:3.9'%&0$.&$'&93)9'063.(0
+%-/(0 -)9%1/(0 )-0 )&0 '&3%$,%&20 -/7-9)&$'0 6.30 7)G'3#0 :3.=/$9-0
[[Undergraduate thesis, University of Guayaquil]].
http://repositorio.ug.edu.ec/bitstream/redug/57537/1/BINGQ-
MAPA-21M01.pdf
July - September vol. 1. Num. 18 - 2023
93
Avila, C. (2011). P'9'3(%&)9%.&0 .60 :,#-%$.$,'(%$)"0 )&=0 6/&$9%.&)"0
:3.:'39%'-0.60-.#0:3.9'%&0%-.")9'0)&=0,#=3."#-)9'0)90:%".90-$)"'<06.30
)::"%$)9%.&0 %&0 6..=0 V[Undergraduate thesis, Escuela Politécnica
Nacional]]. https://bibdigital.epn.edu.ec/handle/15000/4300
Bonino, J., Cagnassi, L., Giudicci, A., Ibarra, J., March, P. O., Vidal,
A., Molina, M., Flores, A., López, D., Duran, N. M., Galante, M.,
Spelzini, D., & Boeris, V. (2016). Physicochemical
characterization of plant protein isolates. I&'32'%), WX(14).
https://repositorio.uca.edu.ar/bitstream/123456789/5739/1/caract
erizacion-aislados-proteinas-vegetales.pdf.
Boye, J. I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth,
E., & Rajamohamed, S. H. (2010). Comparison of the functional
properties of pea , chickpea and lentil protein concentrates
processed using ultrafiltration and isoelectric precipitation
techniques. D..=0 K'-')3$,0 H&9'3&)9%.&)", XO(2), 537-546.
https://doi.org/10.1016/j.foodres.2009.07.021.
https://doi.org/10.1016/j.foodres.2009.07.021
Callisaya Aroa, J. C. (2011). +3.9'%&0 %-.")9'-0 .60 ?&=')&0 23)%&-0
YZ/'&.:.=%)$')-Y0 Z/%&.)0 Y8,'&.:.=%/(0 Z/%&.)Y0 )&=0 8)&%,/)0
Y8,'&.:.=%/(0 +)""%=%$)/"'Y0 7#0 %-.'"'93%$0 :3'$%:%9)9%.&0
[[Undergraduate thesis, Universidad Mayor de San Andrés]].
https://repositorio.umsa.bo/handle/123456789/1391
Callisaya, C., & Alvarado, A. (2009). Protein isolates of high Andean
chenopodiaceae grains; quinoa "Chenopodium quinoa" - cañahua
"Chenopodium pallidicaule" by isoelectric precipitation. K'1%-9)0
[."%1%)&)0 ='0 Z/\(%$), AT(1).
http://www.scielo.org.bo/pdf/rbq/v26n1/v26n1a02.pdf
Cantonal, R., Fernández-Quintela, A., Martínez, J. A., & Macarulla, M.
T. (1995). Comparative study of the composition and nutritive
value of legume seeds and protein concentrates. ?3$,%1.-0
")9%&.)('3%$)&.-0 ='0 &/93%$%]&, X^(3), 242-248.
https://www.alanrevista.org/ediciones/1995/3/art-15/.
Caro, G. A. (2015). U79)%&%&20)&=0'1)"/)9%.&0.60)0:3.9'%&0%-.")9'063.(0
)0E%"=0"'2/('063.(09,'0-9)9'0.60_)"%-$.0@4/:%&/-0'*)"9)9/-0`/$$;C0
[University of Guadalajara].
http://repositorio.cucba.udg.mx:8080/xmlui/bitstream/handle/123
456789/3270/Caro_Ojeda_Gabriel_Alejandro.pdf?sequence=1&i
sAllowed=y
Chel-Guerrero, L., Pérez-Flores, V., Betancur-Ancona, D., & Dávila-
Ortiz, G. (2002). Functional properties of flours and protein
Technologies for obtaining protein isolates from legumes
94
isolates from Phaseolus lunatus and Canavalia ensiformis seeds.
_./3&)"0 .60 ?23%$/"9/3)"0 )&=0 D..=0 8,'(%-93#, ^B(3), 584-591.
https://doi.org/10.1021/JF010778J
Chew, P. G., Casey, A. J., & Johnson, S. K. (2003). Protein quality and
physico-functionality of Australian sweet lupin (Lupinus
angustifolius cv. Gungurru) protein concentrates prepared by
isoelectric precipitation or ultrafiltration. D..=08,'(%-93#, NO(4),
575-583. https://doi.org/10.1016/S0308-8146(03)00156-0
Codex Alimentarius (2007). 8'3')"-<0+/"-'-<04'2/('-0)&=05'2'9)7"'0
+3.9'%&0+3.=/$9-0(First Edition).
Delgado-Andrade, C., Olías, R., Jiménez-López, J. C., & Clemente, A.
(2016). Nutritional and beneficial aspects of legumes for human
health. ?37.3, WMA(779).
http://dx.doi.org/10.3989/arbor.2016.779n3003
Des Marchais, L.-P., Foisy, M., Mercier, S., Villeneuve, S., & Mondor,
M. (2011). Bread-making potential of pea protein isolate produced
by a novel ultrafiltration/diafiltration process. +3.$'=%)0 D..=0
>$%'&$', W(Icef 11), 1425-1430.
https://doi.org/10.1016/j.profoo.2011.09.211.
Díaz, N. (2016). P'1'".:('&90 .60 )0 :3.$'--0 6.30 .79)%&%&20 )0 :3.9'%&0
%-.")9'063.(0Z/%&.)0@8,'&.:.=%/(0Z/%&.)C06"./306.30%9-0:.9'&9%)"0
'1)"/)9%.&0 %&0 9,'0 [[Undergraduate thesis, Escuela Politécnica
Nacional]] %&=/-93#;0
,99:-abb7%7=%2%9)";epn.edu.ec/bitstream/15000/16837/1/CD-
7416.pdf
Duranti, M. (2006). Grain legume proteins and nutraceutical properties.
+,#9.9,'3):#, SS(2), 67-82.
https://doi.org/10.1016/j.fitote.2005.11.008
Enriquez Estrella, M., Kevin, M. V., & Uvidia Cavadiana, H. (2022).
Functional Foods The Consumer Trend Of The 21st Century.
K'$%'&), W(3), 10-19. https://doi.org/10.47187/reciena.v1i3.22
Enriquez Estrella, M., & Ojeda Caiza, G. (2020). Bromatological
evaluation of feed diets, with the inclusion of reject plantain meal.
K'1%-9)0 I>+?c8HId8H?0 H>>d0 WOMBQNWBO, WW(1), 12 - 18.
https://doi.org/10.51260/revista_espamciencia.v11i1.200
July - September vol. 1. Num. 18 - 2023
95
Fonseca, E. P. (2019). ce9.=.-0 ='0 '*93)fg.0 ='0 :3.9'\&)-0 '(0
"'2/(%&.-)-0 [Universidade Federal de Uberlândia].
https://www.sei.ufu.br/sei/controlador_externo.php?
Fuhrmeister, H., & Meuser, F. (2003). Impact of processing on
functional properties of protein products from wrinkled peas.
_./3&)"0 .60 D..=0 I&2%&''3%&2, ^T(2-3), 119-129.
https://doi.org/10.1016/S0260-8774(02)00241-8
Gallegos, R., & Polo, I. (2013). Proximal determination of the main
components of six legumes. %&6.?d?4hFH8>, W(1), 103-114.
https://dialnet.unirioja.es/servlet/articulo?codigo=8382681
García-Mora, P., Plaza-Ibarra, L. A., Loor, H., Alarcón-Olarte, J. X., &
Tapia-Bastidas, C. V. (2019). Design, construction and validation
of a freeze dryer for drying food-derived samples. WS9,04?88IH0
H&9'3&)9%.&)"0c/"9%Q8.&6'3'&$'06.30I&2%&''3%&2<0I=/$)9%.&<0)&=0
F'$,&.".2#a0 YH&=/-93#<0 H&&.1)9%.&<0 ?&=0 H&63)-93/$9/3'0 6.30
>/-9)%&)7"'08%9%'-0)&=08.((/&%9%'-Y.
González-Pérez, S., & Arellano, J. B. (2009). Vegetable protein
isolates. In i)&=7..G0 .60 i#=3.$."".%=-0 (pp. 383-419).
https://doi.org/10.1533/9781845695873.383.
González-Torres, L., Téllez-Valencia, A., Sampedro, J. G., & Nájera,
H. (2007). PROTEINS IN NUTRITION. K'1%-9)0-)"/=0:/7"%$)0#0
&/93%$%]&, N(2).
https://www.medigraphic.com/pdfs/revsalpubnut/spn-
2007/spn072g.pdf.
https://www.medigraphic.com/pdfs/revsalpubnut/spn-
2007/spn072g.pdf.
Guerra, D., & Pozo, P. (2018). Proximal analysis and amino acid profile
of protein isolate from Ecuadorian Andean chocho (fabaceae:
Lupinus mutabilis ). %&6.?d?4hFH8>0 T, T(1), 55-66.
https://dialnet.unirioja.es/servlet/articulo?codigo=7113389
Gunnarsdottir, I., Davidsdottir, B., Worrell, E., & Sigurgeirsdottir, S.
(2020). Review of indicators for sustainable energy development.
In K'&'E)7"'0)&=0>/-9)%&)7"'0I&'32#0K'1%'E-0(Vol. 133, pp. 1-
22). Elsevier Ltd. https://doi.org/10.1016/j.rser.2020.110294
Jaimes, J., Retrepo, D., & Acevedo, D. (2012). Functional properties of
legume protein isolates. K'8%F'H?, WW(2), 21-32.
https://books.google.es/books?hl=en&lr=&id=-
Z1IElz4U6sC&oi=fnd&pg=PA23&dq=protein+isolates+of+legu
mes+as+like+the+bean&ots=BudtG-ClM5&sig=EdXdSy-
Technologies for obtaining protein isolates from legumes
96
4wNay4ofviiHc_C9y3IA#v=onepage&q=protein+isolates+from
+legumes+such+as+beans&f=false
Kaur, M., & Singh, N. (2007). Food Chemistry Characterization of
protein isolates from different Indian chickpea ( Cicer arietinum L
.) cultivars. D..=0 8,'(%-93#, WBA, 366-374.
https://doi.org/10.1016/j.foodchem.2006.05.029.
L'Hocine, L., Boye, J. I., & Arcand, Y. (2006). Food Chemistry and
Toxicology Composition and Functional Properties of Soy Protein
Isolates Prepared Using Alternative Defatting and Extraction
Procedures. D..=08,'(%-93#0)&=0F.*%$.".2#, SW(3), C137-C145.
https://doi.org/https://doi.org/10.1111/j.1365-
2621.2006.tb15609.x.
https://doi.org/https://doi.org/10.1111/j.1365-
2621.2006.tb15609.x
Li, W., Shu, C., Yan, S., & Shen, Q. (2010). 8,)3)$9'3%-9%$-0.60-%*9''&0
(/&207')&0$/"9%1)3-0)&=09,'%30:3.9'%&0%-.")9'-. X^(6), 1205-1211.
https://doi.org/10.1111/j.1365-2621.2010.02259.x
Luengo, C., Paravic, T., & Valenzuela, S. (2016). Causes of
underreporting of occupational accidents and adverse events in
Chile. K'1%-9)0 +)&)('3%$)&)0 ='0 >)"/=0 +/7"%$)b+)&0 ?('3%$)&0
_./3&)"0 .60 +/7"%$0 i')"9,, OM(2), 86-92.
https://www.scielosp.org/pdf/rpsp/2016.v39n2/86-92
Mercado, J. L., Elías, C. C., & Pascual, G. J. (2015). Obtaining a protein
isolate from sacha inchi ( Plukenetia volubilis L.) cake and
evaluation of its techno-functional properties. ?&)"'-08%'&9\6%$.-,
ST(1), 160-167.
https://dialnet.unirioja.es/servlet/articulo?codigo=6171097.
Morales-de León, J. C., Vázquez-Mata, N., Torres, N., Gil-Zenteno, L.,
& Bressani, R. (2007). Preparation and Characterization of Protein
Isolate from Fresh and Hardened Beans ( Phaseolus vulgaris L .).
D..=0 8,'(%-93#0 )&=0 F.*%$.".2#, SA(2), 96-102.
https://doi.org/10.1111/j.1750-3841.2006.00244.x.
https://doi.org/10.1111/j.1750-3841.2006.00244.x
Olmedilla Alonso, B., Farré Rovir, R., Asensio Vegas, C., & Martín
Pedrosa, M. (2010). Role of legumes in current diets. ?$9%1%=)=0
P%'9'9%$), WX(2), 72-76. https://doi.org/10.1016/S1138-
0322(10)70014-6.
July - September vol. 1. Num. 18 - 2023
97
Peña, S., & Parra, H. (2015). P'-%2&0)&=0$.&-93/$9%.&0.60)063''j'0=3#'30
6.30 9,'0 =3#%&20 .60 )3.()9%$0 :")&9-0 [[Undergraduate thesis,
University of Santo Tomas]].
https://repository.usta.edu.co/bitstream/handle/11634/2796/Peñas
abastian2015.pdf
Puppo, M. C., Lupano, C. E., & Cristina, M. (1995). !'")9%.&0 .60
>.#7')&0+3.9'%&0H-.")9'-0%&0?$%=%$08.&=%9%.&-;0I66'$90.60:i0)&=0
+3.9'%&0 8.&$'&93)9%.&. XO(9), 2356-2361.
https://doi.org/https://doi.org/10.1021/jf00057a008
Rui, X., Boye, J. I., Ribereau, S., Simpson, B. K., & Prasher, S. O.
(2011). Comparative study of the composition and thermal
properties of protein isolates prepared from nine Phaseolus
vulgaris legume varieties. D..=0 K'-')3$,0 H&9'3&)9%.&)", XX(8),
2497-2504. https://doi.org/10.1016/j.foodres.2011.01.008.
Sánchez-Vioque, R., Clemente, A., Vioque, J., Bautista, J., & Millán,
F. (1999). Protein isolates from chickpea (Cicer arietinum L.):
Chemical composition, functional properties and protein
characterization. D..=0 8,'(%-93#, TX(2), 237-243.
https://doi.org/10.1016/S0308-8146(98)00133-2.
Semba, R. D., Ramsing, R., Rahman, N., Kraemer, K., & Bloem, M.
W. (2021). Legumes as a sustainable source of protein in human
diets. !".7)"0 D..=0 >'$/3%9#, AN, 1-19.
https://doi.org/10.1016/J.GFS.2021.100520.
Sumner, A. K., Nielsen, M. A., & Youngs, C. G. (1981). Production
and Evaluation of Pea Protein Isolate. _./3&)"0.60D..=0>$%'&$',
XT(2), 364-366. https://doi.org/10.1111/j.1365-
2621.1981.tb04862.x
Tamayo, J. (2018). I1)"/)9%.&0.60)&9%Q%&6")(()9.3#<0)&9%.*%=)&90)&=0
)&9%(%$3.7%)"0)$9%1%9%'-0.607)7#0"%()07')&0@+,)-'."/-0"/&)9/-04;C0
%-.")9'-0 )&=0 :3.9'%&0 ,#=3."#-)9'0 )&=0 9,'%30 %&0 1%93.0 =%2'-9%7%"%9#;0
[Universidad Técnica de Ambato].
https://repositorio.uta.edu.ec/bitstream/123456789/28384/3/BQ
163.pdf
Tello, A. G. (2018). >9/=#0.60 9,'0 )&9%.*%=)&90 )$9%1%9#0 .60 :)"")30 7')&0
@+,)-'."/-0"/&)9/-04;C06"./30:3.9'%&0%-.")9'-0)&=09,'%302)-93%$0)&=0
=/.='&)"0=%2'-9%7%"%9#0@%&01%93.C0[Universidad Técnica de Ambato].
https://repositorio.uta.edu.ec/bitstream/123456789/28380/3/BQ
160.pdf.
Thompson, L. U. (1977). Preparation and evaluation of mung bean
protein isolates. _./3&)"0 .60 6..=0 -$%'&$', XA, 202-206.
Technologies for obtaining protein isolates from legumes
98
https://doi.org/https://doi.org/10.1111/j.1365-
2621.1977.tb01252.x
Ulloa, J. A., Rosas-Ulloa, P., Carmen Ramírez-Ramírez, J., & Ulloa-
Rangel, B. E. (2012). Production of protein isolates from industrial
by-products. K'1%-9)0 D/'&9'0 &/'1)0 e:.$)0 ?k., X(11), 9-15.
http://dspace.uan.mx:8080/xmlui/bitstream/handle/123456789/89
9/Producción de aislados proteicos a partir de subproductos
industriales.pdf?sequence=1&isAllowed=y
Urrutia Gutiérrez, W. (2010). P'9'3(%&)9%.&0 .60 .:9%()"0 )"G)"%&'0
'*93)$9%.&0 :)3)('9'3-0 6.30 .79)%&%&20 :3.9'%&0 %-.")9'0 63.(0 9)3E%0
@4/:%&/-0 (/9)7%"%-C0 [[pregraduate thesis, Universidad Nacional
Micaela Bastidas de Apurímac]].
http://repositorio.unamba.edu.pe/bitstream/handle/UNAMBA/31
1/T_0140.pdf?sequence=1
Vaz Patto, M. C., Amarowicz, R., Aryee, A. N. A., Boye, J. I., Chung,
H. J., Martín-Cabrejas, M. A., & Domoney, C. (2015).
Achievements and Challenges in Improving the Nutritional
Quality of Food Legumes. 83%9%$)"0 K'1%'E-0 %&0 +")&90 >$%'&$'-,
OX(November 2014), 105-143.
https://doi.org/10.1080/07352689.2014.897907.
https://doi.org/10.1080/07352689.2014.897907
Villafuerte, F., Pérez, E., Mahfoud, A., Valero, Y., & Pérez Martínez,
A. (2019). Obtaining low-phenylalanine protein hydrolysates from
sweet whey and chachafruit (Erythrina edulis Triana). ?3$,%1.-0
4)9%&.)('3%$)&.-0 P'0 d/93%$%]&, TM(1), 25-33.
https://doi.org/10.37527/2019.69.1.004.
https://doi.org/10.37527/2019.69.1.004
Vioque, J., Sánchez-Vioque, R., Pedroche, J., Del Mar Yust, M., &
Millán, F. (2001). Obtaining and applications of protein
concentrates and isolates. !3)-)-0 #0 ?$'%9'-, ^A(2), 127-131.
https://doi.org/10.3989/GYA.2001.V52.I2.384.
