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Enzymatic Production, Bioactivity, and Bitterness of Chickpea ( Cicer

arietinum ) Peptides

Article  in  Comprehensive Reviews in Food Science and Food Safety · October 2019

DOI: 10.1111/1541-4337.12504

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Enzymatic Production, Bioactivity, and Bitterness
of Chickpea (Cicer arietinum) Peptides
Luis M. Real Hernandez and Elvira Gonzalez de Mejia

Abstract: Chickpeas are inexpensive, protein rich (approximately 20% dry mass) pulses available worldwide whose
consumption has been correlated with positive health outcomes. Dietary peptides are important molecules derived from
dietary proteins, but a comprehensive analysis of the peptides that can be produced from chickpea proteins is missing in the
literature. This review provides information from the past 20 years on the enzymatic production of peptides from chickpea
proteins, the reported bioactivities of chickpea protein hydrolysates and peptides, and the potential bitterness of chickpea
peptides in food products. Chickpea peptides have been enzymatically produced with pepsin, trypsin, chymotrypsin,
alcalase, flavorzyme, and papain either alone or in combination, but the sequences of many of the peptides in chickpea
protein hydrolysates remain unknown. In addition, a theoretical hydrolysis of chickpea legumin by stem bromelain
and ficin was performed by the authors to highlight the potential use of these enzymes to produce bioactive chickpea
peptides. Antioxidant activity, hypocholesterolemic, and angiotensin 1-converting enzyme inhibition are the most studied
bioactivities of chickpea protein hydrolysates and peptides, but anticarcinogenic, antimicrobial, and anti-inflammatory
effects have also been reported for chickpea protein hydrolysates and peptides. Chickpea bioactive peptides are not
currently commercialized, but their bitterness could be a major impediment to their incorporation in food products. Use
of flavorzyme in the production of chickpea protein hydrolysates has been proposed to decrease their bitterness. Future
research should focus on the optimization of chickpea bioactive peptide enzymatic production, studying the bioactivity
of chickpea peptides in humans, and systematically analyzing chickpea peptide bitterness.
Keywords: bioactive peptides, bitter peptides, enzymatic hydrolysis, garbanzo, pulses

Introduction potential bioactive ingredients in the future (Siegner, 2018; Wyers,

Pulse consumption has been associated with positive health
outcomes across multiple populations (Viguiliouk, Blanco Mejia,
Kendall, & Sievenpiper, 2017). Consumption of chickpeas, which
are commonly available pulses worldwide, has been proposed to
be beneficial in the managing of multiple chronic diseases such as
cardiovascular diseases and obesity (Gupta et al., 2017; Padhi &
Ramdath, 2017). Although the exact mechanisms of how com-
pounds present in chickpea, also known as garbanzo, can help al-
leviate symptoms associated with various chronic illnesses are still
relatively unknown, bioactive peptides produced from chickpea
proteins could play a role (Roy, Boye, & Simpson, 2010; Sánchez-
Chino et al., 2018). Bioactive peptides are amino acid polymers
produced from the hydrolysis of proteins that can exert benefi-
cial health effects through interactions with biological molecules
(Erdmann, Cheung, & Schröder, 2008). Bioactive chickpea pep-
tides are not currently added as ingredients to food products, but
given the rise in popularity of foods containing chickpea ingredi-
ents in recent years, chickpea peptides may be sought off after as
CRF3-2019-0103 Submitted 4/27/2019, Accepted 9/4/2019. Authors are with protein hydrolysates and peptides; third, the potential bitterness of
Dept. of Food Science and Human Nutrition, Univ. of Illinois at Urbana–Champaign, the protein hydrolysates and peptides. Overall, chickpea bioactive
Urbana, IL, 61801, U.S.A. Direct inquiries to author Gonzalez de Mejia
(E-mail: edemejia@illinois.edu).
2017).
The objective of this review was to descriptively analyze
published literature in English from 1999 to 2019 regarding the
potential for bioactive chickpea peptides to be added as bioactive
ingredients to foods. This review also includes an update,
performed by authors, on the theoretical hydrolysis of chickpea
legumin by ficin and stem bromelain. Google Scholar and Scopus
databases were used to search for articles using the main keywords:
chickpea peptides, chickpea bioactive peptides, chickpea protein
hydrolysates, chickpea protein bitterness, and chickpea peptide
bitterness. Protein hydrolysates were understood to be a mixture
of peptides derived from the enzymatic hydrolysis of a protein
sample. The timespan of the published literature analyzed is
extensive due to the lack of a comprehensive review on this topic
and due to the sparse number of articles published from 1999 to
2019 regarding bioactive chickpea peptides. This review contains
three major sections that highlight key topics associated with
commercializing chickpea bioactive peptides. First, the knowledge
on the enzymatic production methods of peptides from chickpea
proteins; second, the proposed biological activity of chickpea
peptides have the potential to be beneficial in the management of
chronic diseases, but more studies are needed to identify specific


C 2019 Institute of Food Technologists®

doi: 10.1111/1541-4337.12504 Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1913
Properties of chickpea peptides . . .

peptides with potent biological activity and how to most effec- minerals such as potassium (9.94 to 12.64 mg/g), phosphorus (3.94
tively produce them so that they can be used as food ingredients. to 4.52 mg/g), and calcium (0.82 to 2.72 mg/g). Differences in
protein content of chickpeas between Kabuli and Desi varieties are
Chickpea production not entirely clear, with variations observed in the literature based
Humans have been consuming chickpeas for at least the past more on specific genotypes instead of the two variants (Jukanti,
10,000 years as indicated by the presence of chickpea grains in Gaur, Gowda, & Chibbar, 2012). A Kabuli chickpea genotype
archaeological sites in northern Syria (Tanno & Willcox, 2006; from Tunisia was found to have more whole grain protein content
Willcox, Fornite, & Herveux, 2008). It has been hypothesized than a Desi genotype from the same area, but Kabuli (Bumper)
that chickpeas were selected for domestication and inclusion as and Desi (Amethyst & 9105–33N) genotypes grown in Australia
one of the founder crops of the Near East due to their high did not have a significant difference in whole grain protein con-
nutritional content and storage capabilities (Kerem, Lev-Yadun, tent (Ghribi, Maklouf, Blecker, Attia, & Besbes, 2015; Wood,
Gopher, Weinberg, & Abbo, 2007). In 2017, India, Australia, Knights, Campbell, & Choct, 2014). Based on the percentages of
and Myanmar were the top chickpea producing countries, with each essential amino acid found in a gram of chickpea protein,
9,075,000, 2,004,000, and 526,772 metric tons of chickpea, re- chickpea protein contains adequate amounts of all essential amino
spectively (FAOSTAT, 2019). India, Bangladesh, and Pakistan were acids for adults, but can be deficient in meeting leucine, pheny-
the top importers of chickpeas in 2016, whereas Australia, Russia, lalanine, threonine, and tryptophan requirements in children as
and Canada were the top exporters of chickpeas that same year recommended by the Food and Agriculture Organization of the
(FAOSTAT, 2019). Production of chickpeas in the United States United Nations (Xu et al., 2016). The nutritional composition
has significantly increased during the past 2 years (Parr, Bond, of many chickpea food products as reported in the USDA Food
& Minor, 2018). In the United States, Montana, Washington, Composition Databases is given in Table 2.
and Idaho are currently the largest producers of chickpeas (U.S. While Table 2 tabulates the nutritional profile of a variety of
Department of Agriculture, 2018). The United States imports dry and wet chickpea containing food products, it also displays the
chickpeas predominantly from Mexico and Canada, but it mostly versatility of the use of chickpeas in different food products. Cur-
exports chickpeas to Spain (Flexport Inc., 2018). Table 1 lists rent chickpea containing food products have different nutritional
chickpea processors in the United States currently recognized by profiles, and they are all not based on the nutritional profile of dry
the U.S. Dry Pea & Lentil Council. The majority of U.S. chickpea chickpeas. Hummus, for example, can have double or more the
processors in Table 1 are currently located in North Dakota and amount of fat (7 to 29 g of fat per 100 g of product) than boiled
Washington State. Whole and milled chickpeas are the main prod- chickpeas (3 to 4 g of fat per 100 g) alone (Table 2). The protein
ucts offered by U.S. chickpea processors, but Anchor Ingredients content of chickpeas is highly valued, but not all chickpea con-
also offers split chickpeas, which are popular in Southeast Asian taining food products are protein rich. Chickpea miso from South
cuisine (Table 1). Currently, chickpea protein isolates and concen- River Miso Co. Inc. has 0 g of protein per 100 g of wet product,
trates are not widely produced by major ingredient companies, but even though it is chickpea based (Table 2). Chickpea crumbs from
startups CHiCK.P in Israel and Nutriati Inc. in the United States Watusee Foods and linguine chickpea pasta from Banza LLC are
have started to offer these ingredients at a commercial scale. dry chickpea containing food products with a high protein content
(25 g protein/100 g of product). Mediterranean chickpea burgers
Botany and general composition from the Kellogg’s Co. USA have a wet based protein content
The modern, domesticated chickpea species Cicer arietinum is (16 g protein/100 g of product) similar to that found in boiled
a descendant of the species Cicer reticulatum (Abbo, Shtienberg, chickpeas with salt (Table 2).
Lichtenzveig, Lev-Yadun, & Gopher, 2003). Cicer arietinum is the
only currently domesticated species of the genus Cicer, and it has Chickpeas as ingredients in food products
not been found to grow in the wild without human intervention Chickpea ingredients are not currently recognized as major food
(Sharma, Upadhyaya, Roorkiwal, Varshney, & Gowda, 2013). The allergens by the Food Allergen Labeling and Consumer Protection
two varieties of chickpea currently produced are Desi and Kabuli. Act in the United States nor by Regulation 1169/2011 of The
Kabuli chickpeas have a thin seed coat that composes around European Parliament and the Council of the European Union,
5% of their mass and are light-brown to beige in color across making them an attractive replacement to soy ingredients that
genotypes, whereas Desi chickpeas have a thicker seed coat that require allergenicity labeling in the United States and Europe.
composes around 14% of their mass and can vary from tan to black Canned and dried chickpeas are the most basic chickpea food
in coloration across genotypes (Wood, Knights, & Choct, 2011). products available worldwide (Figure 1). Chickpeas can be split
A chickpea genotype refers to a specific strain of chickpeas with in half and then dried to form chana dal, which is a common
a specific genome. Desi chickpeas are characteristically smaller, form of dried chickpeas used in the cuisines of Southern Asia.
browner, and more angular in shape than Kabuli chickpeas. Dried chickpeas can be milled to a flour, which can be added to
Dried chickpeas are primarily composed of starch (30% to 56% baked goods to increase their protein content (Benkadri, Salvador,
w/w) and protein (19% to 27% w/w), in which globulins make Zidoune, & Sanz, 2018; Santos, Fratelli, Muniz, & Capriles, 2018).
up the largest protein fraction (Hall, Hillen, & Robinson, 2017). Chickpea flour can be pressure-cooked in an extruder to make
Globulins make up 53% to 60% of proteins in dry chickpeas, and pastas and puffed snacks (Specialty Food Association, 2017).
albumins (8% to 12%), glutelins (18% to 24%), and prolamins Cooked chickpeas, such as those that are canned, can be mashed
(3% to 7%) are also present (Hall et al., 2017). Legumin, vicilin, and blended with other ingredients to make hummus. Hummus
and convicilin are the most abundant globulin proteins in dried is a popular food that can be part of a balanced diet due to its
chickpeas (Rachwa-Rosiak, Nebesny, & Budryn, 2015). The gen- nutritional composition (Wallace, Murray, & Zelman, 2016). Al-
eral protein and amino acid composition of chickpeas has been though hummus is traditionally savory and salty, there are now
reviewed by Rachwa-Rosiak et al. (2015). Dry chickpeas also sweet versions of this food (Specialty Food Association, 2017). In
have a small percentage of oil (2% to 7% w/w), and they contain recent years, the often discarded water found in canned chickpeas,

1914 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019 
C 2019 Institute of Food Technologists®
Properties of chickpea peptides . . .
Table 1–Current chickpea processors in the United States.
Processor U.S. location
AGT Foods Bismarck, ND
Anchor Ingredients Fargo, ND
Bespoke Group LLC Irving, TX
Columbia Grain Intl. Portland, OR
George F. Brocke & Sons, Inc. Kendrick, ID
Great Northern Ag Plaza. ND
Hinrichs Trading Co. Pullman, WA
Inland Empire Milling Co. St. John, WA
Palouse Pulse Farmington, WA
PNW Farmers Cooperative Genesse, ID
Southwest Processors Exchange Tonopah, AZ
Spokane Seed Co. Spokane, WA
Stone Mill, LLC Richardton, ND
Viterra, USA LLC Ray, ND
Data adapted from U.S. Dry Pea & Lentil Council (https://www.usapulses.org/processors).
termed aquafaba, has gained popularity as a plant-based emulsifier
in emulsions such as mayonnaise because it can be used in ve-
gan food products (Mustafa, He, Shim, & Reaney, 2018). Canned
chickpeas can also be roasted to make different snack products. Al-
though cooked chickpeas are not commonly fermented, chickpea-
based miso is available commercially. Fermenting chickpeas could
enhance their protein digestibility (Chandra-Hioe, Wong, &
Arcot, 2016).
Patents regarding the production of chickpea peptides
Several patents have been recently published in the past
2 years regarding the production of protein hydrolysates or
peptides from chickpeas (Table 3). Patents published from
2017 to now were searched using the Google Patents database
(https://patents.google.com) using the keywords: chickpea, gar-
banzo, peptide, protease, and hydrolysate. The keyword “protease”
was used in every search to eliminate search entries that did not
reference the production of chickpea protein hydrolysates and
peptides. Only patents exclusively focused on chickpea protein
hydrolysates or peptides were analyzed. Patents detailing pharma-
ceutical or food mixtures containing chickpea ingredients or the
potential for a patented peptide to be found in a chickpea protein
were not tabulated.
The majority of recent patents regarding chickpea protein hy-
drolysates and peptides were filed in China. There are several
patents from China listing chickpea components as part of medic-
inal and herbal supplement formulations, but the role and presence
of chickpea ingredients in these products are not clearly detailed.
Antioxidant activity is the main biological activity claimed for
chickpea protein hydrolysates and peptides in recent patents filed
in China. Although patent CN107594249A does not mention the
use of commonly approved proteases used in food manufactur-
ing, it claims that activation of endogenous proteases in chickpeas
via germination is a more efficient way of producing peptides
from chickpea proteins. Patent CN106432445A mentions the N-
terminal sequence of a polypeptide, but none of the patents an-
alyzed were exclusively focused on patenting peptide sequences
found from the hydrolysis of chickpea proteins.
Enzymatic Production of Peptides from Chickpea
Proteins
Peptides are produced from the hydrolysis of proteins, which
can be achieved through enzymatic or acid–base methods (Álvarez,
Rendueles, & Dı́az, 2013; Daliri et al., 2017). In the production
of peptides from food proteins, enzymatic methods are preferred

C 2019 Institute of Food Technologists®
Chickpea ingredients offered Website
Flour http://www.agtfoods.com
Whole, split, and flour http://anchoringredients.com
Whole http://www.globalagco.com
Whole http://www.columbiagrain.com
Whole https://gfbrocke.com
Whole http://www.greatnorthernag.com
Whole http://www.hinrichstrading.com
Whole http://www.iemc.com
Whole and flour https://www.palousebrand.com
Whole http://www.pnw.coop
Whole and flour https://www.swprocessors.com
Whole http://www.spokaneseed.com
Whole http://www.stonemill.net
Whole http://www.viterra.com
due to their specificity, but enzymatic hydrolysis can be costlier
than acid–base hydrolysis.
Enzymatic hydrolysis of chickpea proteins can be performed on
chickpea flour, chickpea protein concentrate, or protein isolates.
Chickpea flour can contain lipid and carbohydrate molecules that
can interfere with the cleavage of chickpea proteins. Lipids can be
removed from the flour by defatting it with organic solvents, and
carbohydrates can be separated by purifying the protein using acid–
base precipitation methods (Arcan & Yemenicioǧlu, 2010). The
purification of proteins from chickpea flour leads to the produc-
tion of chickpea protein concentrates and protein isolates. Protein
isolates have a higher protein concentration than protein con-
centrates. Chickpea protein concentrates and protein isolates are
prefered over chickpea flour when performing protein hydrolysis
because chickpea flour contains a higher concentration of lipids
and carbohydrates that can decrease the rate of protein hydrolysis.
More research is needed regarding the optimization of chickpea
peptide production in the presence of different concentrations of
lipids or carbohydrates in the chickpea starting material.
Globally, the Codex Alimentarius provides general guidelines on
the use of enzymes as processing aids, whereas in the United States,
volume 3 chapter 1 of Title 21 in the Code of Federal Regula-
tions specifies approved enzymes for use in food products (Codex
Alimentarius Comission, 2010; U.S. Food and Drug Administra-
tion, 2018). Chickpea peptides have been reported to have been
produced by the enzymes alcalase, α-chymotrypsin, flavorzyme,
papain, pepsin, and trypsin either alone or in combination during
the past 20 years (Barbana & Boye, 2010; del Mar Yust et al.,
2012; Girón-Calle, Alaiz, & Vioque, 2010; Kou, Gao, Zhang,
Wang, & Wang, 2013; Milán-Noris, Gutiérrez-Uribe, Santacruz,
Serna-Saldı́var, & Martı́nez-Villaluenga, 2018; Pedroche et al.,
2002; Torres-Fuentes, Alaiz, & Vioque, 2011; Yust et al., 2003).
Unfortunately, the amino acid composition of chickpea protein
hydrolysates is sometimes given without any sequencing of the
peptides present in the protein hydrolysate (Yust et al., 2003).
Although knowing the amino acid composition of a protein
hydrolysate or peptide can be valuable in determining their
nutritional qualities, it is not very useful in understanding their
bioactivity. Table 4 shows reported peptide sequences from
the enzymatic hydrolysis of chickpea proteins. The majority of
sequenced chickpea peptides are those produced from chickpea
legumin, which is not surprising because legumin is the most
abundant storage protein found in chickpeas (Chang, Alli, Molina,
Konishi, & Boye, 2012). Sequenced peptides produced through
enzymatic hydrolysis have been obtained through simulated
gastrointestinal digestion with pepsin followed by pancreatin or by
Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1915
 Table 2–Nutritional composition of chickpeas and chickpea containing food products.

Dietary Total
USDA nutrient Mass Energy Protein Total lipid Carbohydrates fiber sugars Calcium Iron Sodium
Product database ID Manufacturer basis (kcal/100 g) (g/100 g) (g/100 g) (g/100 g) (g/100 g) (g/100 g) (mg/100 g) (mg/100 g) (mg/100 g)
Barbeque-flavored 45169836 Biena LLC Dry 429 18 11 64 21 4 71 5 857
roasted chickpeas
Chickpea crumbs 45269145 Watusee Foods Dry 400 25 5 65 20 5 0 7 50
Chickpea flour 16157 – Dry 387 22 7 58 11 11 45 5 64
Chickpea puffs 45333683 Green Park Snacks Inc. Dry 465 14 19 58 12 7 47 4 581
Chile lemon–flavored 45048197 Golden Packaging Co. Dry 393 14 11 53 4 0 143 5 1071
roasted chickpeas
Chili-flavored roasted 45221551 MTY San Miguel Dry 406 18 18 4 4 4 212 6 1058
chickpeas Distributors LLC
Dry chickpea grains 45036987 La Preferida Inc. Dry 244 18 4 64 31 2 133 4 56
Dry chickpea grains 45276035 The Kroger Co. Dry 314 20 6 60 17 11 114 5 29
Properties of chickpea peptides . . .

Dry chickpea grains 45228085 Target Stores Dry 244 18 4 64 31 2 133 4 56

Hummus chips 45009760 Dishaka LLC Dry 464 7 18 68 4 4 71 3 1071
Linguine chickpea pasta 45331365 Banza LLC Dry 333 25 6 56 14 9 70 9 105
Linguini chickpea pasta 45187209 RP’s Pasta Co. Dry 271 12 4 51 5 4 24 2 647
Penne chickpea pasta 45067858 Banza LLC Dry 386 23 6 58 12 11 35 9 114
Ranch-flavored roasted 45164648 American Importing Co. Dry 467 17 20 57 13 10 67 5 433
chickpeas Inc.
Raw mature chickpeas 16056 – Dry 378 20 6 63 12 1 57 4 24
Roasted chickpeas 45287927 Watusee Foods Dry 429 21 11 61 18 7 0 6 500
Salted roasted chickpeas 45170096 Pulse Foods LLC Dry 393 18 7 61 18 11 107 6 411
Salted roasted chickpeas 45170464 American Halal Co. Inc. Dry 433 20 13 60 17 3 67 5 767
Seasoned dry roasted 45162002 Emerald Kitchen LLC Dry 368 20 8 54 18 9 179 6 346
chickpeas
Boiled mature chickpeas 16357 – Wet 269 15 4 45 13 8 80 5 399
with salt

1916 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
Boiled mature chickpeas 16057 – Wet 164 9 3 27 8 8 49 3 7
without salt
Canned chickpeas 45043347 Wal-Mart Stores Inc. Wet 85 5 2 14 5 1 31 1 254
Canned chickpeas 45059114 Food Town Stores Inc. Wet 85 5 2 14 5 1 31 1 238
Canned chickpeas 45065027 Mercado Latino Inc. Wet 96 5 1 18 5 1 16 1 352
Canned chickpeas 45108296 Goya Foods Inc. Wet 82 5 2 16 6 1 33 4 295
Canned chickpeas 45114067 Meijer Inc. Wet 85 5 2 14 5 1 31 1 254
Canned chickpeas 45114581 Iberia Foods Corp. Wet 81 4 1 13 3 1 30 1 148
Canned chickpeas 45244342 Bush Brothers & Co. Wet 81 5 2 15 4 0 31 1 362
Canned chickpeas 45282529 Giant Eagle Inc. Wet 85 5 1 15 5 0 31 1 269
Canned chickpeas 45289948 Conservas La Costena Wet 100 5 2 15 5 2 31 1 138
S.A.
Canned chickpeas 45314787 The Kroger Co. Wet 92 5 2 15 5 2 31 1 100
Canned chickpeas 45343386 Seneca Foods Corp. Wet 96 5 1 18 5 1 16 1 240
Canned chickpeas 45346862 Raley’s Wet 96 5 1 18 5 1 16 1 176


Canned chickpeas 45079817 Whole Foods Market Inc. Wet 85 5 2 13 4 1 31 1 8
Canned mature chickpeas 16358 – Wet 139 7 3 23 6 4 45 1 246
Chan masala (cooked 45054284 Kohinoor Foods Ltd Wet 99 6 3 12 2 0 50 2 275
seasoned chickpeas)
Chickpea miso 45084424 South River Miso Co. Inc. Wet 167 0 0 33 – 0 – – 2833
Hummus dip 45122989 ZB Importing Inc. Wet 140 7 7 13 3 3 67 1 233
Hummus dip 45212676 Wild Oats Marketing LLC Wet 286 4 29 7 4 0 0 0 500
Mediterranean chickpea 45127967 Kellogg Co. US Wet – 16 7 19 9 1 97 2 374
burgers
Pumpkin spice-flavored 45180504 Sweetsop LLC Wet 350 14 6 57 19 – 107 3 864
hummus
Data adapted from USDA Food Composition Databases (https://ndb.nal.usda.gov/ndb/)
Only nutrient data reported for most chickpea-based food products in the database are shown.

C 2019 Institute of Food Technologists®

Properties of chickpea peptides . . .

Figure 1–Representative food products derived from chickpea. Product type is labeled in black, whereas main processing operations are labeled in
blue and italicized. All images were obtained from the respective manufacturers or through free-licensing.

vitro digestive models that use the same enzymes found in the
hydrolysis due to alcalase (Table 4). There is currently no distinct
pattern to the sequenced chickpea peptides produced by pepsin human digestive tract to study protein digestion. In simulated
followed by pancreatin digestion across studies, with sequenced in vitro digestion models, a pepsin solution at pH 2 is used to
peptides reported by Milán-Noris et al. (2018) mainly having a simulate gastric proteolysis. After pepsin hydrolysis, a solution of
terminal lysine residue, whereas those reported by Torres-Fuentes pancreatin, an enzymatic mix of enzymes that would be excreted
et al. (2015) mainly have a terminal leucine residue. Only small from the pancreas to the small intestine, at pH 7 is used to simu-
peptides composed of five amino acids or less have been sequenced late intestinal proteolysis (Minekus et al., 2014). The proteolytic
from those produced by alcalase hydrolysis of chickpea proteins enzymes in pancreatin are mainly trypsin and α-chymotrypsin.
(Table 4). Endogenous chickpea peptides that did not require Protein hydrolysis involving the use of pepsin, chymotrypsin, and
external enzymatic hydrolysis for their production have also been trypsin is usually performed at 37 °C to mimic average body
sequenced, such as Arietin, Cicerin, and Ciceranin (Table 4). temperature.
Pepsin (MEROPS Database ID: A01.071) is an aspartic acid en-
Proteases used in simulated gastrointestinal digestion doprotease that uses dual aspartic acids in its active site to catalyze
alone and in combination (pepsin, trypsin, and peptide bond hydrolysis (Fujinaga, Chernaia, Mosimann, James,
& Tarasova, 1995). Pepsin has a high preference for leucine to be
chymotrypsin)
present at or around the cleave site of a substrate protein or peptide
Studying digestive proteolysis in humans can be costly and time
(Rawlings et al., 2018). Although it is common practice to perform
consuming. Hence, there is a preference for using simulated in

Table 3–Recent patents regarding the production of peptides from chickpea proteins.

Country of Protein hydrolysate/ Starting material

Patenta Date published origin peptide bioactivity claim Enzymes mentioned hydrolyzed
CN106432445A February 22, 2017 China Antimicrobial and No enzyme mentioned Defatted chickpea flour
anti-carcinogenic
CN106947798A July 14, 2017 China None given Alkaline protease, flavorzyme, Precipitated chickpea
and protease complex proteins
CN106957833A July 18, 2017 China Suggests possible Alkaline protease, bromelain, Mashed chickpeas
thrombolytic activity neutral protease, papain,
pancreatin, and pepsin
CN107383159A November 24, 2017 China Antioxidant and decrease Alkaline protease, bromelain, Chickpea flour
physical fatigue neutral protease, papain,
pepsin, and trypsin
CN107385002A November 24, 2017 China Suggests possible Neutral protease Chickpea milk
antioxidant and
hypoglycemic activity
CN107594249A January 19, 2018 China Antioxidant Endogenous proteases Whole chickpeas
CN107805653A March 16, 2018 China Antioxidant Bromelain, flavorzyme, and Precipitated chickpea
pepsin proteins
CN107988301A May 4, 2018 China Antimicrobial and immune Alkaline protease, neutral Chickpea protein extract
system enhancer protease, papain, and
trypsin
TN2017000185A1 October 19, 2018 Tunisia Antioxidant Alcalase Chickpea protein isolate
CN104789629B October 26, 2018 China None given Alkaline protease, flavorzyme, Precipitated chickpea
and neutral protease proteins
a Patents were translated to English automatically by Google Patents (https://patents.google.com) if they were written in another language.


C 2019 Institute of Food Technologists® Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1917
 Table 4–Chickpea peptide sequences known to be produced through enzymatic hydrolysis.

Parent molecule
Parent protein or peptide MW (kDa)a UniProtKB IDb Peptide sequencec Enzyme scheme used Reference
U-box domain-containing protein 75 to 92 A0A1S2XF51∗ or A0A1S3E7A3∗ QERHQ Pepsin followed by pancreatin Torres-Fuentes et al., 2015
33-like isoforms X1 & X2
NADPH-quinone oxidoreductase 84 B5LMS9 ACF Alcalase Shi, Hou, Liu, et al., 2019
subunit 5
Globulin-1 S Allele 76 A0A1S2YZ56∗ SREEETTEWEEEVAK Pepsin followed by pancreatin Milán-Noris et al., 2018
Vicilin-Like 69 A0A1S2Y087∗ GRRGSEESEEGDAIVK, GSEESEEGDAIVK,
RGSEESEEGDAIVK, & SRNPIYSNKFGK
IIPAGHPV & LPQHIDAD Torres-Fuentes et al., 2015
Provicilin-Like 65 A0A1S2XYZ0∗ SPIYSNKFGK Milán-Noris et al., 2018
LPQHTDAD Torres-Fuentes et al., 2015
Legumin J-Like 60 A0A1S2XVG1∗ FSGNRGPLVQPR Milán-Noris et al., 2018
Properties of chickpea peptides . . .

ALEPDHR, LTETWNPNHPEL, PNHPEL, Torres-Fuentes et al., 2015

TETWNPNHPE, & TETWNPNHPEL
Legumin A-Like isoforms X1 &X2 52 to 59 A0A1S2XSB9∗ or A0A1S3E0V2∗ HIVDKLQGRDEDEEK Milán-Noris et al., 2018
AEHGSLH, SAEHGSL, & SAEHGSLH Torres-Fuentes et al., 2015
Legumin 56 Q9SMJ4 LHQNIGSSSSPDIYNPQAGR Milán-Noris et al., 2018
No enzymes used Hao et al., 2009
LHQNIGSSSSPDIYNPQAGRIK, Pepsin followed by pancreatin Milán-Noris et al., 2018
NEDEEKGAIVKVK, & SEGGLIETWNPSNK
FGSLH, FVPH, HKNAM, HQNIGSS, Torres-Fuentes et al., 2015
LAGNHEQE, RQPH, SSSSPDI, VPHYN,
YALKG, & YLAGNHEQE
Legumin-Like 56 A0A1S2XTK6∗ GGLSIITPPEKEPR, GGLSIITPPEKEPRQK, & Milán-Noris et al., 2018
VKGGLSIITPPEKEPR

1918 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
Sucrose-binding 54 A0A1S2XVJ8∗ IFKISKEDVHGLAPK
Protein-like
Adenosylhomocysteinase 53 A0A1S2YCQ2∗ IVGVSEETTTGVK
Vicilin 51 A0A1S2XQ88∗ QQSQETDVIVK
NADPH-quinone oxidoreductase 46 B5LMS0 QILP Alcalase Shi, Hou, Liu, et al., 2019
subunit H
RNA-binding protein 24-Like 26 A0A1S2XWV3∗ or A0A1S3E357∗ QYPIY
isoforms X1 & X2
P24 oleosin isoform 21 A0A1S2XJM3∗ DVGQKTKEVGQDIQAK Pepsin followed by pancreatin Milán-Noris et al., 2018
A-Like
Arietin 2 P83988 GVGYKVVVTTTAAADDDDVV No enzymes used Ye, Ng, & Rao, 2002
Cicerin 2 P83987 ARCENFADSYRQPPISSSQT


Ciceranin 2 P83986 VKSTGRADDDLAVKTKYLPP Chu, Liu, & Ng, 2003
Multiple possible parent proteins DHG & VGDI Alcalase Ghribi, Sila, et al., 2015
EQIEELSKNAK, EQIEELSKNAK, Pepsin followed by pancreatin Milán-Noris et al., 2018
ISREQIEELSKNAK, & VLLEEQEQKPK
AHH, EGEEEE, HGDE, KNPQLQD, LLPH, LPH, Torres-Fuentes et al., 2015
LPHFN, LPHFNS, PRP, RDSHQ, & RNENEQ
EEM, EIGV, EL, FNENN, MFPF, MNQ, NPM, Alcalase Shi, Hou, Liu, et al., 2019
NSPV, PVEP, PYIE, QTIVM, RTYSC, TMPD,
TSIAY, VEF, VEPI, VFVRN, VKF, & WEIM
a Molecular weights are based on specific protein sequences specified in UniProt Database (https://www.uniprot.org).
b Identifications with an asterisk (∗ ) have been submitted, but have not been manually reviewed in the UniProt Database. Only peptide sequences that could be found in a chickpea protein’s sequence in the UniProt Database were tabulated. Preference was given to reviewed protein
sequences.
c Peptide sequences were obtained from peptides isolated through fractionation.

C 2019 Institute of Food Technologists®

Properties of chickpea peptides . . .
digestion of a protein sample with pepsin before pancreatin, there
are studies that have digested chickpea proteins with pepsin alone.
Chickpea protein hydrolysates produced by pepsin alone have dis-
played antioxidant and angiotensin-I-converting enzyme (ACE)
inhibitory properties (Arcan & Yemenicioǧlu, 2010; Boschin,
Scigliuolo, Resta, & Arnoldi, 2014; Sánchez-Chino et al., 2018).
Antioxidant potential of chickpea protein hydrolysates produced
by pepsin alone found that the nondigested chickpea protein con-
centrate had better iron chelating activity, and prevention of olive
oil oxidation, than the pepsin complete protein hydrolysate made
from the same material (Arcan & Yemenicioǧlu, 2010). Pepsin was
found to digest <1% of unheated chickpea vicilin-like globulin
proteins after 2 hr, indicating that protein denaturation is probably
necessary for maximal chickpea peptide production using pepsin
(Tavano & Neves, 2008).
Trypsin is a serine endoprotease found in three isoforms (1, 2,
and mesotrypsin) in human pancreatic fluid (Chen, Radisky, &
Férec, 2013). Trypsin 1, referred to as “cationic” trypsin, is the
most abundant isoform. Trypsin has high selectivity for cleaving
the carboxylic acid side of arginine and lysine amino acids (Olsen,
Ong, & Mann, 2004). Although no studies were found regarding
the hydrolysis of chickpea protein isolate or concentrate by trypsin
alone, one study hydrolyzed chickpea vicilin-like globulin proteins
with only trypsin (Tavano & Neves, 2008). This study found
that trypsin could hydrolyze 7% of unheated chickpea vicilin-like
protein after 2 hr, but more interestingly, it found that chickpea
albumin had high trypsin inhibitory activity compared to chickpea
globulins.
Chymotrypsin is another serine endopeptidase found in hu-
man pancreatic fluid, which cleaves peptide bonds formed by
large hydrophobic amino acids instead of cationic ones (Hedstrom,
Perona, & Rutter, 1994). Two main isoforms of chymotrypsin (A
or B) are present in human pancreatic fluid. Depending on how
the proenzyme chymotrypsinogen A is activated, it will form ei-
ther α- or γ - chymotrypsin, in which both forms have the same
primary structure, but different tertiary structures and specificities
(Gráf, Szilágyi, & Venekei, 2013). Similar to trypsin, there have
been no studies in the past 20 years that have tested the bioac-
tivity of chickpea protein hydrolysates produced by chymotrypsin
hydrolysis alone.
It is more common to find literature that has performed chick-
pea protein hydrolysis using pepsin–pancreatin-simulated gastroin-
testinal digestion than finding literature that has hydrolyzed chick-
pea proteins with only one protease involved in digestion (Ribeiro
et al., 2017; Wang et al., 2010). Although analyzing peptides
formed through pepsin–pancreatin digestion might provide valu-
able information about the types of peptides that a protein sample
might naturally produce upon digestion, it may not provide infor-
mation about novel peptides that would not naturally be produced
by the body. Chickpea protein hydrolysates and peptides formed
through simulated gastrointestinal digestion have mainly shown
antioxidant, ACE inhibitory, anti-inflammatory, and anticarcino-
genic bioactivities (Barbana & Boye, 2010; Jamdar, Deshpande, &
Marathe, 2017; Milán-Noris et al., 2018; Sánchez-Chino et al.,
2018). Although chickpea proteins might be able to naturally pro-
duce health promoting peptides during digestion, chickpea storage
proteins have been found to resist gastrointestinal digestion, so the
quantity of peptides produced from these proteins might be lower
in reality than what has been measured in in vitro systems (Wang
et al., 2010). Furthermore, the production of chickpea peptides
using simulated gastrointestinal digestion has only been reported
to have been performed on isolated systems that do not consider

C 2019 Institute of Food Technologists® Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1919
other food components, such as lipids and carbohydrates, that
might be actually be present during digestion.
Microbial proteases (alcalase and flavorzyme)
Both alcalase and flavorzyme are commercially available prote-
olytic enzymes derived from microorganisms commonly used in
the production of food ingredients. At a commercial scale, pro-
teases derived from microorganisms are preferred to those derived
from plants and animals because they require less resources to
be obtained (Jisha et al., 2013). Alcalase and flavorzyme were
the only proteases of microbial origin to have been reported
in the literature of the past 20 years to have been used in the
production of chickpea protein hydrolysates and peptides. Al-
calase, a variant of the enzyme subtilisin, is a serine endopep-
tidase commonly obtained from Bacillus licheniformis (MEROPS
Database ID S08.001, https://www.ebi.ac.uk/merops/cgi-bin/
pepsum?id=S08.001;type=P). Although alcalase is considered to
have a broad specificity, it prefers to cleave substrates whose amino
acid at the P1 position is either leucine or alanine and whose P1
position is either alanine or serine (Rawlings et al., 2018). Ref-
erence to positions P4 to P4 refers to the eight amino acids of
a protein or peptide substrate present at or around the active site
of a protease. Cleave can occur between the amino acids present
in sites P1 and P1 of the substrate molecule. The three amino
acids on sites P4 to P2 are sequentially connected to the amino
acid present at site P1 , and the three amino acids in sites P4 to
P2 are sequentially connected to the amino acid at site P1. This
nomenclature allows for easy specification of the amino acids that
can occupy the active site of a protease for a protein or peptide to
be cleaved without having to detail the structure of the active site.
Alcalase has a high specificity for L-amino acids, making it useful
in stereospecific reactions (Chen, Hsiao, Chiou, Wu, & Wang,
1992).
Alcalase has been widely used in the production of chickpea
protein hydrolysates and peptides. Chickpea protein hydrolysates
formed by alcalase alone have been shown to have potential an-
tioxidant, ACE inhibitory, and hypolipidemic bioactives (Medina-
Godoy et al., 2012; Shi, Hou, Liu, Guo, & He, 2019; Zhang, Jiang,
Miao, Mu, & Li, 2012). Alcalase was used to hydrolyze chickpea
protein isolate to produce the peptide NRYHE, whose antioxidant
activity has been well studied in both chemical and cellular mod-
els (Guo, Zhang, Jiang, Miao, & Mu, 2014; Zhang, Li, Miao, &
Jiang, 2011). However, the sequence of the peptide NRYHE is not
currently found in any of the chickpea proteins submitted to the
UniProt Database (https://www.uniprot.org/), but it is found in
the sequence of two enzymes from the chickpea fungus Didymella
rabiei (UniProt IDs: A0A163BK66 and A0A162 × 5F2). Hydrol-
ysis of chickpea proteins by alcalase has been shown to increase
their solubility (Yust, Pedroche, Millán-Linares, Alcaide-Hidalgo,
& Millán, 2010; Ghribi, Maklouf Gafsi, 2015).
Flavorzyme is actually a mixture of enzymes obtained from As-
pergillus oryzae (MEROPS Database ID S10.016, https://www.ebi.
ac.uk/merops/cgi-bin/pepsum?id=S10.016;type=P). A proce-
dure to separate the individual enzymes found in flavorzyme has
been published, and it has been found that flavorzyme contains
two aminopeptidases, two dipeptidyl peptidases, and three en-
dopeptidases (Merz et al., 2015). Given the range of proteolytic
enzymes present in flavorzyme, predicting the theoretical pep-
tide sequences that can be derived from the hydrolysis of a pro-
tein by flavorzyme is not very feasible. Flavorzyme has not been
reported to have been used alone to hydrolyze chickpea pro-
tein, but it has been commonly been used to further hydrolyze
Properties of chickpea peptides . . .
chickpea protein hydrolysates produced by alcalase (Kou et al.,
2013; Xue et al., 2012). Flavorzyme is not commonly used
alone to hydrolyze proteins because it is mainly composed of
amino- and di-peptidases that can more rapidly hydrolyze pro-
tein that has undergone some hydrolysis by endopeptidases (Merz
et al., 2015). The peptide RQSHFANAQP was discovered from
the sequential hydrolysis of chickpea albumin by alcalase fol-
lowed by flavorzyme, but its sequence is actually found in provi-
cilin (Q304D4) and vicilin-like (A0A1S2XQR4, A0A1S3E1A0,
and A0A1S2XQ88) chickpea proteins in the UniProt Database
(Kou et al., 2013). The antioxidant, hypolipidemic, and anti-
inflammatory potential of the peptide RQSHFANAQP have all
been tested in animal models (Xue et al., 2012; Xue, Hou,
et al., 2018; Xue, Wang, Wen, Yu, & Kou, 2018). Flavorzyme
has been reported to reduce the bitterness of chickpea protein hy-
drolysates, and this point is discussed further in a later section (Kou
et al., 2013).
Cystine proteases from tropical fruits (papain, ficin, and
stem bromelain)
Some plant-based proteases are approved to be used in the pro-
duction of food grade protein hydrolysates and peptide ingredi-
ents. Although many plant-derived proteases could be used in the
production of peptides, only proteases derived from tropical fruits
that have cysteine amino acid residues in their active sites were
found to have been used to hydrolyze chickpea proteins. Papain
(MEROPS ID: C01.001) is a commercially available cysteine en-
doprotease derived from papaya (Cstorer & Ménard, 1994). Papain
has a relatively broad specificity, but it has a high preference for
arginine to be in the P1 position, leucine to be in the P2 position,
and glutamine to be in the P1 position of the substrate protein or
peptide (Dı́az & Martinez, 2013; Rawlings et al., 2018). Only two
studies were found to have hydrolyzed chickpea proteins with pa-
pain (Barbana & Boye, 2010; Medina-Godoy et al., 2012). Papain
hydrolysis was reported in both studies to have been performed
at 40 °C and at a pH of 6.5. Only the antioxidant capacity and
ACE inhibitory activity of papain-derived chickpea protein hy-
drolysates have been analyzed. No chickpea peptides derived from
papain hydrolysis have been sequenced, but this should be a focus
of future studies to know if papain is capable of producing novel
chickpea peptides that other food-grade enzymes might not be
able to provide.
Theoretical hydrolysis of chickpea legumin. Papain is not the
only plant-based cysteine protease that can be used in the produc-
tion of food ingredients. Pineapples and figs also contain cysteine
proteases that can be used in the production of food-grade pro-
tein hydrolysates and peptides (Arshad et al., 2014; U.S. Food and
Drug Administration, 2018). Bromelain is a common term for
the cysteine endoproteases found in the different parts of pineap-
ple (Ananas comosus) plants. The optimum pH and temperature of
bromelain depends on the substrate it is hydrolyzing, but generally,
an optimum pH range of 6.5 to 7.7 and an optimum tempera-
ture range of 55 to 59 °C should be used when hydrolyzing most
proteins with bromelain (Corzo, Waliszewski, & Welti-Chanes,
2012). Only one article to our knowledge has reported the hy-
drolysis of chickpea proteins with bromelain, but only sequenced
some small peptides in the protein hydrolysate with five or less
amino acids (Hindi-Tamelikecht, Dauphin, Hamon, Grangaud,
& Pradeau, 1997). Bromelain was mentioned as a possible en-
zyme used in patents CN106957833A and CN107383159A, but
whether it was actually used in the processes mentioned in the
patents is unknown (Table 3). It is also unknown if patents
1920 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
mentioning papain as a possible enzyme of use actually utilized
papain. Bromelain was mentioned to be part of an enzyme mix-
tures used to obtain bioactive peptides from chickpea flour in
patent CN107805653A, but the exact role of bromelain in the
enzyme mixture was not mentioned.
To update on the possible peptides that can be produced from
the enzymatic hydrolysis of chickpea proteins with bromelain,
a theoretical hydrolysis of chickpea legumin, the most abundant
protein in chickpeas, was manually performed using the pepti-
dase specificity of stem bromelain (Chang, Alli, Molina, Konishi,
& Boye, 2012). The peptidase specificity of stem bromelain was
obtained from the MEROPS database (MEROPS ID: C01.005),
but only information about known substrate amino acid sequences
from the P3 to P3 positions were used (Rawlings et al., 2018).
In essence, cuts to the chickpea legumin sequence (GenBank ID:
CAB60140.1) were made between the P1 and P1 sites if a possible
six amino acid sequence corresponding to P3 to P3 was found
in the legumin sequence that matched the enzyme specificity. For
the specificity of stem bromelain, possible amino acids for the P3
site were G, P, A, V, L, F, S, T, K, R, and H; for the P2 site were
G, P, V, L, F, Y, S, T, N, R, and H; and for the P1 site were G,
A, V, L, F, Y, S, T, N, Q, and R. Possible amino acids for the P1
site were G, V, L, I, F, Y, S, T, N, Q, D, E, K, and H; for the
P2 site were P, A, V, L, Y, S, T, N, Q, D, E, K, and H; and for the
P3 site were G, P, V, L, F, S, T, C, N, Q, D, E, R, and H.
A total of 69 possible peptide sequences were found with 10 or
less amino acids could be formed from the hydrolysis of chickpea
legumin by stem bromelain (Table 5). Stem bromelain has a broad
cleavage specificity; therefore, it is expected to produce peptides
of various molecular weights (192.07 to 1,284.63 Da), isoelectric
points (3.11 to 12.50), net charges (–1 to +3), and hydropho-
bicities (–1.33 to 17.17) as shown in Table 5. Stem bromelain is
expected to release the peptide HQNIGSS from chickpea legu-
min, which is a peptide that has also been reported to be released
from chickpea legumin by pepsin followed by pancreatin diges-
tion (Table 4 and 5). ACE and DPP-IV inhibitory fragments were
the main bioactive fragments found in the peptides expected to be
produced from the hydrolysis of chickpea legumin by stem brome-
lain (Table 5). The presence of a bioactive fragment in a peptide
indicates that there is the potential for the peptide to be bioactive.
Many of the peptides present in Table 5 have a bitter fragment that
contains at least one leucine residue, but as with bioactivity, the
presence of a bitter fragment in a peptide is not an indication that
the peptide will indeed be bitter. Overall, stem bromelain could
be used to produce a complex mixture of potentially bioactive
peptides from chickpea legumin, but it is not recommended for
the production of a few specific peptides given its broad cleavage
specificity.
Ficin (MEROPS ID: C01.006) is a cystine endoprotease present
in the latex of figs that resembles papain (Devaraj, Kumar, &
Prakash, 2008). Although approved for use in foods, no studies
were found in the past 20 years that have used ficin to produce
peptides from chickpea proteins (U.S. Food and Drug Administra-
tion, 2018). Methods have been developed to purify and extract
different ficin isoforms, and it is generally proposed that a 6 to 7
pH range and 50 to 60 °C temperature range are optimal for max-
imum ficin activity (Baeyens-Volant, Matagne, El Mahyaoui, Wat-
tiez, & Azarkan, 2015; Devaraj et al., 2008; Gagaoua et al., 2014).
Although different ficin isoforms have slightly varying specificity,
ficin favors a hydrophobic amino acid at the P2 position and argi-
nine at the P1 position of the substrate protein or peptide (Azarkan
et al., 2011; Rawlings et al., 2018).

C 2019 Institute of Food Technologists®
 
Table 5–Peptide sequences with 10 amino acids or less obtained from the theoretical hydrolysis of chickpea legumin by stem bromelain.

Theoretical peptide Isoelectric Net Hydrophobicity Potential biological activities Bitter fragments in Umami fragments in
sequencea MW (Da) point charge (Kcal/mol) (responsible sequence fragments)b peptide sequencec peptide sequencec
SS 192.07 5.38 0 8.82 None found None None
TS 206.09 5.30 0 8.61 DPP-IV inhibitor (TS)
LS 218.13 5.50 0 7.11 None found
VT 218.13 5.52 0 7.69 DPP-IV inhibitor (VT)
TL 232.14 5.36 0 6.90 DPP-IV inhibitor (TL)
LL 244.18 5.58 0 5.40 Glucose uptake stimulator (LL); DPP-IV inhibitor (LL) LL
YA 252.11 5.46 0 7.69 ACE inhibitor (YA); renin inhibitor (YA); None
DPP-IV inhibitor (YA)

C 2019 Institute of Food Technologists®

SSS 279.11 5.38 0 9.28 Vasoactive substance release stimulator (SSS)
Properties of chickpea peptides . . .

FQ 293.14 5.38 0 6.96 ACE inhibitor (FQ); DPP-IV inhibitor (FQ)

YQ 309.13 5.37 0 7.96 DPP-IV inhibitor (YQ)
FY 328.14 5.38 0 5.48 ACE inhibitor (FY) FY
TSF 353.16 5.32 0 6.90 ACE inhibitor (SF); renin inhibitor (SF); None
DPP-IV inhibitor (SF, TS)
SSSS 366.14 5.38 0 9.74 Vasoactive substance release stimulator (SSS)
LAGN 373.20 5.63 0 9.15 ACE Inhibitor (LA, AG); LA
Ubiquitin-mediated proteolysis activator (LA);
DPP-IV inhibitor (LA, AG)
HSR 398.20 10.73 1 12.50 DPP-IV Inhibitor (HS) None
QPNS 444.20 5.37 0 10.12 DPP-IV inhibitor (QP, PN)
LSRA 445.26 11.18 1 9.42 ACE inhibition (RA);
Ubiquitin-mediated proteolysis activator (RA);
DPP-IV inhibitor (RA)
IKTV 459.30 10.14 1 9.37 DPP-IV inhibitor (KT, TV)
FHPA 470.23 7.95 0 9.16 ACE inhibitor (HP); DPP-IV inhibitor (PA, HP) PA
DSHQ 485.19 4.98 −1 15.10 DPP-IV inhibitor (SH) None
IAVSL 501.32 5.70 0 6.03 ACE Inhibitor (IA, AV); DPP-IV inhibitor (IA, SL, AV, VS)
QTFL 507.27 5.44 0 5.96 ACE inhibitor (TF); DPP-IV inhibitor (FL, QT, TF) FL
IDTSS 521.23 3.11 −1 11.59 DPP-IV inhibitor (TS) ID
HSRQ 526.26 10.80 1 13.27 DPP-IV inhibitor (HS) None
MAKLLA 645.39 10.21 1 8.53 ACE inhibitor (LA, KL); glucose uptake stimulator (LL); LL, LA
Ubiquitin-mediated proteolysis activator (LA);
DPP-IV inhibitor (MA, LA, LL)
LNCKGN 647.31 8.51 1 12.28 ACE inhibitor (KG, LN); DPP-IV inhibitor (KG, LN) KG KG
IKTVTS 647.38 9.88 1 10.08 DPP-IV inhibitor (KT, TS, TV, VT) None None
TLQPNS 658.33 5.30 0 9.12 ACE inhibitor (LQP, LQ);
DPP-IV inhibitor (QP, LQP, PN, TL)
LSRATL 659.40 11.11 1 8.42 ACE inhibitor (RA);
Ubiquitin-mediated proteolysis activator (RA);
DPP-IV inhibitor (RA, AT, TL)
IDTSSF 668.30 3.11 −1 9.88 ACE inhibitor (SF); renin inhibitor (SF); ID
DPP-IV inhibitor (SF, TS)
FHPAVT 670.34 7.57 0 8.95 ACE inhibitor (HP, AV); PA
DPP-IV inhibitor (PA, HP, AV, VT)
(Continued)

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1921
 Table 5–Continued.

Theoretical peptide Isoelectric Net Hydrophobicity Potential biological activities Bitter fragments in Umami fragments in
sequencea MW (Da) point charge (Kcal/mol) (responsible sequence fragments)b peptide sequencec peptide sequencec
QPNSLL 670.36 5.44 0 7.62 Glucose uptake stimulator (LL); LL
DPP-IV inhibitor (LL, QP, SL, PN)
FYLAGN 683.33 5.27 0 6.73 ACE inhibitor (YL, FY, LA, AG); anxiolytic (YL); LA, FY
Ubiquitin-mediated proteolysis activator (LA);
DPP-IV inhibitor (LA, AG, YL)
LLQTFL 733.44 5.58 0 3.46 ACE inhibitor (LQ, TF); FL, LL
Vasoactive substance release stimulator (LLL);
Glucose uptake stimulator (LL);
DPP-IV inhibitor (LL, FL, QT, TF)
HQNIGSS 741.34 7.63 0 12.80 ACE inhibitor (IG, GS); DPP-IV inhibitor (QN) IG
GTCFALR 766.38 8.65 1 8.63 ACE inhibitor (GT, CF, FAL, LR); renin inhibitor (LR); None
DPP-IV inhibitor (FA, AL)
Properties of chickpea peptides . . .

SDRFSY 773.33 6.49 0 11.85 ACE inhibitor (RF, SY); DPP-IV inhibitor (DR, SY) RF
IKTVTSF 794.45 9.93 1 8.37 ACE inhibitor (SF); renin inhibitor (SF); None
DPP-IV inhibitor (KT, SF, TS, TV, VT)
IDTSSFQ 796.36 3.11 −1 10.65 ACE inhibitor (SF, FQ); renin inhibitor (SF); ID
DPP-IV inhibitor (FQ, SF, TS)
HQNIGSSS 828.37 7.63 0 13.26 ACE inhibitor (IG, GS); IG
Vasoactive substance release stimulator (SSS);
DPP-IV inhibitor (QN)
TAPFHFL 831.43 7.89 0 6.45 ACE Inhibition (TAP, AP); FL, PF
DPP-IV inhibitor (AP, TA, FL, HF, PF)
MAKLLALS 845.50 9.88 1 7.74 ACE inhibitor (LA, KL); glucose uptake stimulator (LL); LL, LA
Ubiquitin-mediated proteolysis activator (LA);
DPP-IV inhibitor (MA, LA, LL, AL)

1922 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
DLQALRF 861.47 6.56 0 10.41 ACE inhibitor (RF, LQ, LR); renin inhibitor (LR); RF, DL
DPP-IV inhibitor (AL, QA)
YALNCKGN 881.41 8.48 1 12.07 ACE inhibitor (YA, KG, LN); renin inhibitor (YA); KG KG
DPP-IV inhibitor (AL, KG, LN, YA)
TLQPNSLL 884.50 5.36 0 6.62 ACE inhibitor (LQP, LQ); glucose uptake stimulator (LL); LL None
DPP-IV inhibitor (LL, QP, SL, LQP, PN, TL)
KQFACAGVA 893.44 9.04 1 11.93 ACE inhibitor (AG, GV); renin inhibitor (QF); GV, VA
DPP-IV inhibitor (VA, FA, AG, GV, QF)
LKGRARLL 925.62 12.50 3 12.22 ACE inhibitor (RL, RA, GR, KG, AR); GR, LL, KG KG
Glucose uptake stimulator (LL); antioxidant (LK);
Ubiquitin-mediated proteolysis activator (RA);
DPP-IV inhibitor (LL, RA, KG, RL)


KLSAEFGSL 950.51 6.81 0 12.69 ACE inhibitor (FG, GS, KL); cAMP-DPDE inhibitor (EF); FG, EF, SAEFG AE
Renin inhibitor (EF); DPP-IV inhibitor (SL, AE)
HEQEFLR 957.47 5.23 −1 17.11 ACE inhibitor (LR); cAMP-DPDE inhibitor (EF); FL, EF None
Renin inhibitor (LR, EF); DPP-IV inhibitor (FL, HE, QE)
IETWNPSN 959.43 3.20 −1 10.87 ACE inhibitor (IE); antioxidant (TW); None ET
DPP-IV inhibitor (NP, WN, ET, PS, TW)
DLQALRFL 974.55 6.74 0 9.16 ACE inhibitor (RF, LQ, LR); renin inhibitor (LR); RF, FL, DL None
DPP-IV inhibitor (FL, AL, QA)
LSFCFLLF 988.51 5.20 0 −0.54 ACE inhibitor (LF, FCF, SF, CF, LLF); FL, LF, LL, FLL
Glucose uptake stimulator (LL); antioxidative (FC);
Renin inhibitor (SF); DPP-IV inhibitor (LL, FL, SF)
(Continued)

C 2019 Institute of Food Technologists®

 Table 5–Continued.


Theoretical peptide Isoelectric Net Hydrophobicity Potential biological activities Bitter fragments in Umami fragments in
sequencea MW (Da) point charge (Kcal/mol) (responsible sequence fragments)b peptide sequencec peptide sequencec
IAVSLIDTSS 1,004.54 3.11 −1 9.72 ACE inhibitor (IA, AV); glucose uptake stimulator (LI); LI, ID
DPP-IV inhibitor (IA, SL, AV, LI, TS, VS)
SSTAPFHFL 1,005.49 7.89 0 7.37 ACE inhibition (TAP, AP, ST); FL, PF
DPP-IV inhibitor (AP, TA, FL, HF, PF)
FDLQALRF 1,008.54 6.56 0 8.70 ACE inhibitor (RF, LQ, LR); renin inhibitor (LR); RF, DL
DPP-IV inhibitor (AL, QA)
LKLSAEFGSL 1,063.59 6.81 0 11.44 ACE inhibitor (LKL, FG, GS, KL); antioxidant (LK); FG, EF, SAEFG AE
cAMP-DPDE inhibitor (EF); renin inhibitor (EF);
DPP-IV inhibitor (SL, AE);
LSRATLQPNS 1,085.58 10.85 1 10.64 ACE inhibitor (LQP, RA, LQ); None None

C 2019 Institute of Food Technologists®

Ubiquitin-mediated proteolysis activator (RA);
Properties of chickpea peptides . . .

DPP-IV inhibitor (RA, QP, LQP, AT, PN, TL)

NLNANSILYA 1,091.56 5.40 0 7.58 ACE inhibitor (LY, YA, LN, IL); glucose uptake stimulator IL
(IL); antioxidant (LY); renin inhibitor (YA);
DPP-IV inhibitor (IL, LN, NA, NL, SI, YA)
SSSTAPFHFL 1,092.52 7.89 0 7.83 ACE inhibitor (TAP, AP, ST); FL, PF
Vasoactive substance release stimulator (SSS);
DPP-IV inhibitor (AP, TA, FL, HF, PF)
HEQEFLRY 1,120.53 5.23 −1 16.40 ACE inhibitor (RY, LRY, LR); cAMP-DPDE inhibitor (EF); FL, EF
Renin inhibitor (LR, EF); DPP-IV inhibitor (FL, HE, QE)
FDLQALRFL 1,121.62 6.74 0 7.45 ACE inhibitor (RF, LQ, LR); renin inhibitor (LR); RF, FL, DL
DPP-IV inhibitor (FL, AL, QA)
NQLDQMPRR 1,156.58 10.58 1 15.77 ACE inhibition (PR, RR); RR, PR, LD
DPP-IV inhibitor (MP, DQ, NQ, QL, RR)
QPNSLLQTFL 1,159.62 5.44 0 5.68 ACE inhibition (LQ, TF); glucose uptake stimulator (LL); FL, LL
DPP-IV inhibitor (LL, QP, FL, SL, PN, QT, TF)
LKGRARLLYA 1,159.72 11.46 3 12.01 ACE inhibitor (RL, LY, RA, YA, GR, KG, AR); GR, LL, KG KG
Glucose uptake stimulator (LL); immunostimulant (LLY);
antioxidant (LY, LK); renin inhibitor (YA);
Ubiquitin-mediated proteolysis activator (RA);
DPP-IV inhibitor (LL, RA, KG, RL, YA)
LSLSFCFLLF 1,188.62 5.20 0 −1.33 ACE inhibitor (LF, FCF, SF, CF, LLF); FL, LF, LL, FLL None
Glucose uptake stimulator (LL);
Antioxidative (FC); renin inhibitor (SF);
DPP-IV Inhibitor (LL, FL, SL, SF)
TSFDLQALRF 1,196.62 6.56 0 9.41 ACE inhibitor (RF, SF, LQ, LR); renin inhibitor (LR, SF); RF, DL
DPP-IV inhibitor (AL, QA, SF, TS)
HKNAMFVPHY 1,242.60 9.55 1 13.30 ACE inhibitor (MF, HY, VP, PH, HK, FVP); FV
antioxidant (PHY, FVPH);
DPP-IV inhibitor (VP, HY, MF, NA, PH)
HEQEFLRYQ 1,248.59 5.23 −1 17.17 ACE inhibitor (RY, LRY, RYQ, LR); antioxidant (RYQ); FL, EF
cAMP-DPDE inhibitor (EF); renin inhibitor (LR, EF);
DPP-IV inhibitor (FL, HE, QE, YQ)
QNQLDQMPRR 1,284.63 10.58 1 16.54 ACE inhibitor (PR, RR); RR, PR, LD
DPP-IV inhibitor (MP, DQ, NQ, QL, QN, RR)
Molecular weight, isoelectric point, net charge, and hydrophobicity were all determined using PepDraw (https://www.pepdraw.com).
a Amino acids are coded according to their one-letter abbreviation: A, alanine; C, cystine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine;
V, valine; W, tryptophan; Y, tyrosine.
b Potential bioactivities of found peptide sequences were accessed using the bioactive peptide library of the BIOPEP database (http://www.uwm.edu.pl/biochemia/index.php/en/biopep).
c Bitter and umami peptide sequence fragments were accessed using the sensory peptides and amino acids library of the BIOPEP database. Bitter and umami sequence fragments that are implicated in a peptide’s potential biological activity are bolded, with potentially bioactive bitter
fragments being underlined and potentially bioactive umami fragments being italicized.

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1923
Properties of chickpea peptides . . .

Table 6–Peptide sequences with 10 amino acids or less obtained from the theoretical hydrolysis of chickpea legumin by ficin.

Theoretical peptide Isoelectric Hydrophobicity Potential biological activities Bitter fragments in Umami fragments in
sequencea MW (Da) pointb (Kcal/mol) (responsible sequence fragments)c peptide sequenced peptide sequenced
FL 278.16 5.46 4.94 DPP-IV inhibitor (FL) FL None
KLSAEFGS 837.42 6.60 13.94 ACE inhibitor (FG, GS, KL); FG, EF, SAEFG AE
cAMP-DPDE inhibitor (EF);
Renin inhibitor (EF);
DPP-IV inhibitor (AE)
FLKLSAEFGS 1097.57 6.60 10.98 ACE inhibitor (LKL, FG, GS, KL); FG, FL, EF, SAEFG
antioxidant (LK);
cAMP-DPDE inhibitor (EF);
Renin Inhibitor (EF);
DPP-IV inhibitor (FL, AE)
Molecular weight, isoelectric point, and hydrophobicity were all determined using PepDraw (https://www.pepdraw.com).
a Amino acids are coded according to their one-letter abbreviation: A, alanine; E, glutamic acid; F, phenylalanine; G, glycine; K, lysine; L, leucine; S, serine.
b All peptides had a net charge of zero.
c Potential bioactivities of found peptide sequences were accessed using the bioactive peptide library of the BIOPEP database (http://www.uwm.edu.pl/biochemia/index.php/en/biopep).
d Bitter and umami peptide sequence fragments were accessed using the sensory peptides and amino acids library of the BIOPEP database. Bitter and umami sequence fragments that are implicated in a peptide’s
potential biological activity are bolded, with potentially bioactive bitter fragments being underlined and potentially bioactive umami fragments being italicized.

Similar to stem bromelain, a manual theoretical hydrolysis of rically measuring chemical conjugates formed from the reaction
chickpea legumin was performed using the specificity of ficin of a reporter molecule and an amine group of a protein or pep-
to update on possible dietary chickpea peptides that could be tide (Rutherfurd, 2010). In food products, the functionality of a
produced using this enzyme. The enzyme specificity of ficin was hydrolyzed protein ingredient, such as its emulsification and foam-
obtained from the MEROPS database, and only specificity data ing capacity, can be heavily influenced by its degree of hydrolysis
from the P3 to P3 sites was used (Rawlings et al., 2018). For the (Ghribi, Maklouf Gafsi, et al., 2015; Mune Mune, 2015). Un-
specificity of ficin, possible amino acids for the P3 site were G, der similar experimental conditions, chickpea protein concentrate
A, V, L, I, F, T, R, and H; for the P2 site were G, V, L, F, T, and had a higher degree of hydrolysis when hydrolyzed by alcalase than
K; and for the P1 site were G, L, F, Y, S, K, R, and H. Possible papain or pancreatin (Medina-Godoy et al., 2012). The highest
amino acids for the P1 site were L, F, Y, S, T, K, and H; for the degree of hydrolysis of chickpea proteins was observed for the
P2 site were V, L, Y, T, N, K, and H; and for the P3 site were sequential hydrolysis of chickpea proteins by alcalase followed by
P, A, V, L, S, T, E, and K. Only three possible peptide sequences flavorzyme (Barbana & Boye, 2010; Kou et al., 2013; Megı́as et al.,
were found with 10 or less amino acids that could be produced 2007; Pedroche et al., 2002; Yust et al., 2010). Hydrolysis with
from the hydrolysis of chickpea legumin by ficin (Table 6). Unlike two or more enzymes is preferential in obtaining a higher degree
stem bromelain, ficin has a narrow cleavage specificity. This can of hydrolysis, as observed for the hydrolysis of chickpea proteins by
be helpful in the production of specific peptides from chickpea pepsin followed by pancreatin (Girón-Calle et al., 2010; Torres-
proteins. Two of the peptides, FL and FLKLSAEFGS, found to be Fuentes et al., 2011).
potentially produced by ficin hydrolysis of chickpea legumin were A higher degree of hydrolysis is expected to produce peptides
not found during the theoretical hydrolysis of chickpea legumin by with shorter sequences, as the sequence of a protein would be
stem bromelain (Table 5 and 6). Ficin is expected to produce small cut into smaller and smaller fragments as it is further hydrolyzed.
peptides with a narrow isoelectric point range (5.46 to 6.60) and The peptide RQSHFANAQP, whose bioactivity will be discussed
peptides with DPP-IV inhibitory activity (Table 6). Interestingly, in the following sections, was isolated from a chickpea protein
the cleavage of the fragment FL from the amino terminus of hydrolysate with a degree of hydrolysis of 54.73% (Kou et al.,
the peptide FLKLSAEFGS, to become KLSAEFGS, causes the 2013). The degree of hydrolysis of a protein hydrolysate does not
peptide to lose an antioxidant fragment (LK) from its sequence always correlate with the bioactivity of the protein hydrolysate
(Table 6). Because ficin has a narrow cleave specificity, it can be (Pedroche et al., 2002). This might be due to bioactivity being
potentially combined with other proteases that have a different, but more dependent on the sequences present in a protein hydrolysate
also narrow cleavage specificity to produce other unique peptides rather than the degree of hydrolysis or the molecular weight of the
from chickpea proteins. peptides present in the protein hydrolysate. Degree of hydrolysis
measurements are more important in food product development
where, for example, high degrees of hydrolysis can start to degrade
Degrees of hydrolysis
the foaming capacity and stability of slightly hydrolyzed protein
As proteins are hydrolyzed, they convert into peptides, but this
(del Mar Yust et al., 2012). More research is needed on the scale
process is time dependent as different proteolytic enzymes have
up of chickpea protein hydrolysate production for commercial
different rates of hydrolysis for different substrates. When a pro-
applications.
tein is enzymatically hydrolyzed, the degree of hydrolysis is often
calculated to measure the extent of peptide production as a factor
of the time a specific enzyme, at a specific concentration, is allowed Biological Activity of Chickpea Peptides
to cleave a particular protein. Formally, the degree of hydrolysis of Compounds are termed bioactive if they are found to exert pos-
a protein is the ratio of the number of cleaved peptide bonds to the itive health effects upon their consumption (Daliri et al., 2017).
total number of peptide bonds in the protein (Nielsen, Petersen, Food bioactive compounds are not necessarily nutrients, and they
& Dambmann, 2001). do not have to come from the same chemical class or food source.
Degree of hydrolysis is often measured by either monitoring the One class of dietary bioactive compounds are bioactive peptides,
amount of acid or base that must be added to a solution to keep which can be produced endogenously as a result of normal pro-
a constant pH as a protein is hydrolyzed or by spectrophotomet- tein digestion or they can be produced exogenously during the

1924 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019 
C 2019 Institute of Food Technologists®
Properties of chickpea peptides . . .
production of food ingredients and products (Toldrá, Reig,
Aristoy, & Mora, 2018). Bioactive peptides exert their bioactivity
by modulating the expression or activity of cellular components.
Bioactive peptides can be beneficial in the management of symp-
toms associated with various chronic diseases, such as cardiovas-
cular diseases and obesity (Cicero, Fogacci, & Colletti, 2017; Li,
Liu, He, & Wu, 2018).
Although chickpea bioactive peptides are not currently added as
specific ingredients to food products, there are reports of their po-
tential bioactivities in the literature. Chickpea peptides have been
reported to have antioxidant, antihypertensive, anti-inflammatory,
anticarcinogenic, antifungal, hypoglycemic, anti-obesity, and
hypocholesterolemic bioactivities (Barbana & Boye, 2010; del
Mar Yust et al., 2012; Milán-Noris et al., 2018; Torres-Fuentes
et al., 2015; Xue et al., 2015). The main bioactivities studied
for chickpea peptides are their potential antioxidant, cholesterol
lowering, and ACE inhibitory properties. Future studies should
focus on testing the bioactivity of chickpea protein hydrolysates
and peptides in human subjects.
Although the bioactivity of chickpea protein hydrolysates is
mainly due to the peptides present in the protein hydrolysate, it
is possible that the presence of other bioactive substances, such
as phenolic compounds, could have an influence on the bioac-
tivity of the protein hydrolysate. Phenolic compounds could be
present in chickpea protein hydrolysates if chickpea flour, protein
concentrate, or protein isolate was hydrolyzed to produce the pro-
tein hydrolysates. Defatting chickpea flour before starting protein
extraction is one method that eliminates most of the phenolic
compounds present in the flour and subsequent extracts (Arcan
& Yemenicioǧlu, 2010). Phenolic compounds are not, though,
completely responsible for the bioactivity of protein hydrolysates
because artificially synthesized chickpea peptides, which do not
contain phenolic compounds, have been shown to be bioactive
(Shi, Hou, Guo, & He, 2019; Xue, Hou, et al., 2018).
Antioxidant capacity
Antioxidants are compounds that can reduce or stop radical-
based reduction–oxidation reactions from happening by quench-
ing or transforming reactive radical species and chelating metals
ions, such as iron ions that can participate in Fenton reactions
that produce reactive oxygen species (Yang et al., 2018). Although
oxidative stress is not always a negative biological phenomenon,
such as during cancer therapy, it can be harmful and lead to ox-
idative damage if it is not controlled (Pizzino et al., 2017). Food
scientists have studied antioxidants for a long time, not only due
to their health promoting properties upon ingestion, but also be-
cause they can stop the deterioration of food products caused by
reactive radical species (Cömert & Gökmen, 2018). Antioxidants
in foods are not only small organic compounds, dietary peptides
can also participate in reactions that would classify them as antiox-
idants (Lorenzo et al., 2018). Chickpea protein hydrolysates and
peptides shown to have potential antioxidant activity are reported
in Table 7.
The antioxidant potential of chickpea protein hydrolysates and
peptides is often reported due to the ease and commonality of the
chemical assays used to measure antioxidant potential. However,
chemical antioxidant assays are not always applicable to humans as
many biological processes are ignored by those assays. Although
chemical assays have predominantly been used to measure the
antioxidant potential of chickpea peptides, three studies have ana-
lyzed the antioxidant activity of chickpea protein hydrolysates and
peptides in cells (Table 7). Torres-Fuentes et al. (2015) tested the

C 2019 Institute of Food Technologists® Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1925
antioxidant activity in Caco-2 cells of a chickpea protein isolate
hydrolysate produced by pepsin followed by pancreatin hydrolysis,
but the results were given in an uncommon unit to compare them
across studies. Jamdar et al. (2017) hydrolyzed different chick-
pea flours to test in cells the antioxidant activity of the resulting
hydrolysates, but found that nonhydrolyzed flour from nontreated
chickpeas actually had the highest antioxidant activity out of all the
protein hydrolysates tested. Because these authors used chickpea
flour as the starting material for enzymatic hydrolysis, it is very
likely that compounds other than the peptides produced could
have influenced their results (Table 7).
Guo et al. (2014) analyzed the antioxidant activity of a single
purified chickpea peptide (NRYHE) in cells. They found that a
concentration of 0.5 mg/mL of the peptide NRYHE in cell media
produced positive antioxidant effects in cancer cells by increasing
the activity and expression of endogenous antioxidant enzymes in
the cells (Table 7). The peptide NRYHE can be produced from by
alcalase hydrolysis of chickpea protein isolate (Zhang et al., 2011).
The antioxidant activity of peptide NRYHE was also tested using
chemical assays, but relevance of those assays is lower compared to
the studies performed in cells (Zhang et al., 2011). Although not
tested in cells, the chickpea peptide RQSHFANAQP at a con-
centration of 20 mg per kilogram of body weight (BW) per day
was found to increase the activity of antioxidant enzyme superox-
ide dismutase in the blood of mice (Xue, Hou, et al., 2018). The
chickpea peptide RQSHFANAQP was isolated from a protein hy-
drolysate produced from the hydrolysis of chickpea albumin isolate
by alcalase followed by flavorzyme (Table 7). Crude chickpea albu-
min hydrolysate produced by alcalase followed by flavorzyme was
also found to increase the activity of blood superoxide dismutase in
mice. These results for crude protein hydrolysate might have been
due to the presence of the peptide RQSHFANAQP (Xue, Hou,
et al., 2018). To date, only the chickpea peptides NRYHE and
RQSHFANAQP have well supported evidence to be potentially
antioxidant in humans.
When it comes to antioxidant activity in food products, the pep-
tide NRYHE was shown to inhibit the oxidation of unsaturated
fatty acids in solution (Zhang et al., 2011). That indicates that the
peptide NRYHE could have antioxidant effects in both foods and
humans. A protein hydrolysate produced from chickpea protein
concentrate by pepsin was the only chickpea protein hydrolysate
to have its antioxidant activity tested in an actual food system
(Table 7). However, nonhydrolyzed chickpea protein concentrate
was better at inhibiting oxidation in olive oil than the protein hy-
drolysate produced through pepsin hydrolysis mentioned before
(Arcan & Yemenicioǧlu, 2010). More studies are needed on the
antioxidant activity of chickpea protein hydrolysates and peptides
in actual food products.
ACE inhibition
ACE (MEROPS Database ID: XM02.001) is a carboxypepti-
dase that requires zinc ions to hydrolyze several physiologically
important peptides such as angiotensin I (Wu et al., 2018). An-
giotensin I is a decapeptide that can be hydrolyzed by ACE
to angiotensin II, so that angiotensin II can bind to cellular
receptors that signal vasoconstriction, thereby increasing blood
pressure. Inhibitors of ACE can help reduce the incidence of
hypertension, although there is controversy whether it is best
to inhibit ACE directly or the receptors that bind angiotensin
II and signal vasoconstriction (Messerli, Bangalore, Bavishi, &
Rimoldi, 2018). Commercial kits to test the ACE inhibitory
potential of different samples are widely available, allowing for
 Table 7–Antioxidant capacity of chickpea protein hydrolysates and peptides.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
In vitro
Reactive oxygen species Flour from nonsoaked, Pepsin then trypsin 37 120 then 2 then 6.8 2,250 U/mg then Nondigested flour Approximately 35% SA/mg Jamdar et al.,
SA in MCF-7 breast noncooked, and and chymotrypsin 120 94 U/mg 2017
cancer cells nongerminated (trypsin)
chickpeas and 52 U/mg
(chymotrypsin)
Flour from chickpeas Approximately 25% SA/mg
soaked in water for 16 hr
Flour from chickpeas Nondigested flour from Approximately 15% SA/mg
Properties of chickpea peptides . . .

germinated for 24, 48, chickpeas germinated

or 72 hr for 24 hr
Flour from pressure cooked Nondigested flour Approximately 25% SA/mg
chickpeas
Caco-2 cellular PI Pepsin then 37 180 then 2.5 then 1:20 w/w 478 to 788 Da fraction Approximately 55 cellular Torres-Fuentes
antioxidant activity pancreatin 80 7.5 containing peptide antioxidant activity units et al., 2015
FVPH at 0.3 mg/mL
Catalase activity of Purified peptide NRYHE No enzyme used 0.5 mg/mL 106.65 ± 3.26% compared Guo et al., 2014
Caco-2 cell lysate to 100% negative control
Glutathione reductase No enzyme used 112.8 ± 2.94% compared
activity of Caco-2 cell to 100% negative control
lysate
Glutathione peroxidase No enzyme used Approximately 160%
activity of Caco-2 cell compared to 100%
lysate negative control

1926 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
Expression of Nrf2 mRNA No enzyme used 4-Fold increase in
in Caco-2 cells expression compared to
negative control
Expression of HO-1 mRNA No enzyme used 4-Fold increase in
in Caco-2 cells expression compared to
negative control
Expression of γ -GCS No enzyme used 4-Fold increase in
mRNA in Caco-2 cells expression compared to
negative control
Expression of NQO1 No enzyme used 0.25 or 0.5 mg/mL 4-Fold increase in
mRNA in Caco-2 cells expression compared to
negative control
Catalase activity of HT-29 No enzyme used 0.5 mg/mL 106.00 ± 3.56% compared
cell lysate to 100% negative control
Glutathione reductase No enzyme used 108.9 ± 2.09% compared


activity of HT-29 cell to 100% negative control
lysate
Glutathione peroxidase No enzyme used Approximately 160%
activity of HT-29 cell compared to 100%
lysate negative control
Expression of Nrf2 mRNA No enzyme used 2.08-Fold increase in
in HT-29 cells expression compared to
positive control
Expression of HO-1 mRNA No enzyme used 4-Fold increase in
in HT-29 cells expression compared to
negative control
(Continued)

C 2019 Institute of Food Technologists®

 
Table 7–Continued.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
Expression of γ -GCS No enzyme used 5-Fold increase in
mRNA in HT-29 cells expression compared to
positive control
Expression of NQO1 No enzyme used 4-Fold increase in
mRNA in HT-29 cells expression compared to
negative control
Chemical
ABTS radical SA PC Pepsin Unknown – 2 1:10 Protein hydrolysate 19.5 ± 0.3% inhibition of Sánchez-Chino

C 2019 Institute of Food Technologists®

from 90 min ABTS radical at unknown et al., 2018
hydrolysis concentration
Properties of chickpea peptides . . .

Pancreatin 7 1:10 18.9 ± 0.2% inhibition of

ABTS radical at unknown
concentration
Pepsin then 2 then 7 1:10 then 1:10 24.8 ± 1.0% inhibition of
pancreatin ABTS radical at unknown
concentration
Flour from nonsoaked, Pepsin then trypsin 37 120 then 2 then 6.8 2,250 U/mg then Digested flour Approximately 140 µM Jamdar et al.,
noncooked, and and chymotrypsin 120 94 U/mg trolox equivalents/mg 2017
nongerminated (trypsin) and
chickpeas 52 U/mg
(chymotrypsin)
Flour from chickpeas Approximately 150 µM
soaked in water for 16 hr trolox equivalents/mg
Flour from chickpeas Digested flour from Approximately 160 µM
germinated for 24, 48, chickpeas germinated trolox equivalents/mg
or 72 hr for 48 hr
Flour from pressure cooked Digested flour Approximately 140 µM
chickpeas trolox equivalents/mg
Purified or synthesized No enzyme used Purified peptide IC50 = 2.31 µmol/mL Xue et al., 2015
peptide RQSHFANAQP
PI from raw or boiled Pepsin then 37 120 then 2.5 then 1:100 w/w then <7 kDa fraction from PI IC50 = 42.39 ± 2.12 Karaś et al.,
(100 °C for 60 min) pancreatin 120 7.0 2:5 v/v of boiled chickpeas µg/mL 2015
chickpeas
Albumin isolate Alcalase then 50 60 then 8 then 7 0.3 U/g then Fraction containing IC50 = 0.97 mmol/L Kou et al., 2013
flavorzyme 120 50 U/g peptide
RQSHFANAQP
Hardened chickpea PC, Alcalase 50 120 9 1:25 w/w 1 mg hardened albumin Approximately 53 mM Medina-Godoy
globulin concentrate, or concentrate or TEAC et al., 2012
albumin concentrate globulin concentrate
(Kabuli) hydrolysate
Papain 40 6.5 1 mg hardened albumin Approximately 58 mM
concentrate TEAC
hydrolysate
Pancreatin 39 8 Approximately 75 mM
TEAC
Fresh chickpea PC, globulin Alcalase 50 9 1 mg fresh chickpea PC Approximately 95 mM
concentrate, or albumin hydrolysate TEAC
concentrate (Kabuli)
Papain 40 6.5 Approximately 69 mM
TEAC
Pancreatin 39 8 Approximately 47 mM
TEAC
(Continued)

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1927
 Table 7–Continued.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
PC Pepsin 37 1440 2.5 17:500 w/w Hydrolyzed PC 277 ± 10 µmol trolox Arcan &
equivalents/g Yemenicioǧlu,
2010
DPPH radical SA Purified or synthesized No enzyme used Purified peptide IC50 = 3.15 µmol/mL Xue et al., 2015
peptide RQSHFANAQP
PI from raw or boiled Pepsin then 37 2 then 2 2.5 then 1:100 w/w then 2:5 3.5 to 7 kDa fraction IC50 = from 20.94 ± 3.36 Karaś et al.,
(100 °C for 60 min) pancreatin 7.0 v/v from PI of raw µg/mL 2015
chickpeas chickpeas
PI Pepsin then 37 180 then 2.5 then 1:20 w/w 363 to 836 Da and 363 Approximately 95% DPPH Torres-Fuentes
Properties of chickpea peptides . . .

pancreatin 180 7.5 to 713 Da fractions radical SA et al., 2015

containing the
peptide FVPH
PC Alcalase 50 210 8 1:1 U/mg Fraction containing 67.32% DPPH radical SA Ghribi, Sila,
peptide VGDI at et al., 2015
200 µg/mL
Albumin isolate Alcalase then 50 60 then 8 then 7 0.3 U/g then Fraction containing 57.0 ± 3.5% DPPH radical Kou et al., 2013
flavorzyme 120 50 U/g peptide SA
RQSHFANAQP at
1 mg/mL
PI Alcalase then 50 – 8 then 7 4.6:50 v/w then 1 hr alcalase then Approximately 78% DPPH del Mar Yust
flavorzyme 3.7:100 v/w 120 min flavorzyme radical SA et al., 2012
fraction at 10 mg/mL
Hardened chickpea PC, Alcalase 50 120 9 1:25 w/w 1 mg hardened albumin Approximately 45 mM Medina-Godoy
globulin concentrate, or concentrate TEAC et al., 2012

1928 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
albumin concentrate hydrolysate
(Kabuli)
Papain 40 120 6.5 1:25 w/w Approximately 34 mM
TEAC
Pancreatin 39 120 8 1:25 w/w 1 mg hardened chickpea Approximately 42 mM
PC hydrolysate TEAC
Fresh chickpea PC, globulin Alcalase 50 120 9 1:25 w/w 1 mg fresh chickpea PC Approximately 43 mM
concentrate, or albumin hydrolysate TEAC
concentrate (Kabuli)
Papain 40 120 6.5 1:25 w/w 1 mg fresh albumin Approximately 47 mM
concentrate TEAC
hydrolysate
Pancreatin 39 120 8 1:25 w/w 1 mg fresh albumin Approximately 44 mM
concentrate or TEAC
globulin concentrate


hydrolysate
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 45.33% DPPH radical SA Zhang et al.,
at 1.0 mg/mL 2011
PI Alcalase 50 40 8 1:50 v/w Low molecular weight 85.8 ± 1.3% DPPH radical Li, Jiang, Zhang,
fraction at SA Mu, & Liu,
1.0 mg/mL 2008
Fe2+ chelation Flour from nonsoaked, Pepsin then trypsin 37 120 then 2 then 6.8 2,250 U/mg then Digested flour Approximately 250 µM Jamdar et al.,
noncooked, and and chymotrypsin 120 94 U/mg EDTA equivalents/mg 2017
nongerminated (trypsin) and
chickpeas 52 U/mg
(chymotrypsin)
(Continued)

C 2019 Institute of Food Technologists®

 
Table 7–Continued.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
Flour from chickpeas Nondigested flour Approximately 140 µM
soaked in water for 16 hr EDTA equivalents/mg
Flour from chickpeas Nondigested flour from Approximately 290 µM
germinated for 24, 48, chickpeas germinated EDTA equivalents/mg
or 72 hr for 48 hr
Flour from pressure cooked Digested flour Approximately 200 µM

C 2019 Institute of Food Technologists®

chickpeas EDTA equivalents/mg
PI from raw or boiled Pepsin then 37 2 then 2 2.5 then 1:100 w/w then <3.5 kDa fraction from IC50 = 81.60 ± 1.08 Karaś et al.,
Properties of chickpea peptides . . .

(100 °C for 60 min) pancreatin 7.0 2:5 v/v PI of raw chickpeas µg/mL 2015
chickpeas
PC Alcalase 50 3.5 8 1:1 U/mg Whole hydrolysate at 50.45% Fe2+ chelating Ghribi, Sila,
5 mg/mL capability et al., 2015
PI Pepsin then 37 180 then 2.5 then 1:20 w/w then 363 to 628 Da fraction Approximately 75% Fe2+ Torres-Fuentes,
pancreatin 180 7.5 1:20 w/w at 0.1 mg/mL chelating capability Alaiz, &
Vioque, 2012
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 63.08% Fe2+ chelating Zhang et al.,
at 50 µg/mL capability 2011
PC Pepsin 37 1440 2.5 17:500 w/w Nonhydrolyzed PC 156 µmol equivalents of Arcan &
Na2 EDTA/g Yemenicioǧlu,
2010
Reduction of Fe3+ Purified or synthesized No enzyme used Synthesized peptide at Same reduction caused by Xue et al., 2015
peptide RQSHFANAQP 10.0 µmol/mL 0.5 µmol/mL of ascorbic
acid
PI from raw or boiled Pepsin then 37 120 then 2.5 then 1:100 w/w then 2:5 <3.5 kDa fraction from A700 = 0.362 ± 0.005 per Karaś et al.,
(100 °C for 60 min) pancreatin 120 7.0 v/v PI of boiled chickpeas µg/mL 2015
chickpeas
PI Pepsin then 37 180 then 2.5 then 1:20 w/w 363 to 628 Da fraction A700 = approximately 0.55 Torres-Fuentes
pancreatin 180 7.5 containing peptide at 25 µg/mL et al., 2015
FVPH
PC Alcalase 50 210 8 1:1 U/mg Fraction containing A700 = 0.857 at 1 mg/mL Ghribi, Sila,
peptides VGDI and et al., 2015
DHG
Albumin isolate Alcalase then 50 60 then 8 then 7 0.3 U/g then Fraction containing A700 = 0.32 ± 0.06 Kou et al., 2013
flavorzyme 120 50 U/g peptide
RQSHFANAQP at
1 mg/mL
PI Alcalase 50 Under Hydrostatic 1:20 w/v 200 MPa of hydrostatic A700 = 0.406 ± 0.006 Zhang et al.,
Pressure pressure for 20 min 2012
PI Alcalase then 50 – 8 then 7 4.6:50 v/w then 1 hr alcalase then 1.5 hr A700 = approximately 0.32 del Mar Yust
flavorzyme 3.7:100 v/w flavorzyme fraction at 10 mg/mL et al., 2012
PI Alcalase 50 40 8 1:50 v/w Low molecular weight A700 = approximately 0.30 Li et al., 2008
fraction at 2.5 mg/mL
Ferric ion reducing Flour from nonsoaked, Pepsin then trypsin 37 120 then 2 then 6.8 2,250 U/mg then Digested flour Approximately 50 µM Fe2+ Jamdar et al.,
antioxidant power noncooked, and and chymotrypsin 120 94 U/mg produced/mg 2017
nongerminated (trypsin) and
chickpeas 52 U/mg
(chymotrypsin)
(Continued)

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1929
 Table 7–Continued.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
Flour from chickpeas Approximately 40 µM Fe2+
soaked in water for 16 hr produced/mg
Flour from chickpeas Digested flour from
Properties of chickpea peptides . . .

germinated for 24, 48, chickpeas germinated

or 72 hr for 24 hr
Flour from pressure cooked Digested flour
chickpeas
Total antioxidant capacity Flour from nonsoaked, Pepsin then trypsin 37 120 then 2 then 6.8 2,250 U/mg then Digested flour 15 µmol α-tocopherol/mg Jamdar et al.,
(reduction of Mo6+ ) noncooked, and and chymotrypsin 120 94 U/mg 2017
nongerminated (trypsin) and
chickpeas 52 U/mg
(chymotrypsin)
Flour from chickpeas 12 µmol α-tocopherol/mg
soaked in water for 16 hr
Flour from chickpeas Digested flour from 14 µmol α-tocopherol/mg
germinated for 24, 48, chickpeas germinated
or 72 hr for 24 or 48 hr
Flour from pressure cooked Digested flour 12 µmol α-tocopherol/mg

1930 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
chickpeas
Inhibition of β-carotene PC Alcalase 50 210 8 1:1 U/mg Whole hydrolysate 66.92% reduction of Ghribi, Sila,
bleaching at 5 mg/mL bleaching compared to et al., 2015
control
PI (Kabuli) Pepsin then 37 180 then 2.5 then 1:20 w/w then 363 to 836 Da fraction 85.2% reduction of Torres-Fuentes,
pancreatin 180 7.5 1:20 w/w containing the bleaching compared to Alaiz, &
peptide FVPH control at 0.12 mg/mL Vioque, 2014
PI Alcalase then 50 – 8 then 7 4.6:50 v/w then 30 min alcalase fraction Approximately 50% del Mar Yust
flavorzyme 3.7:100 v/w at 10 mg/mL reduction of bleaching et al., 2012
compared to control
PI Alcalase then 50 – 8 then 7 0.3 U/g then Hydrolysate formed by 4.1 Times less oxidation Megı́as et al.,
flavorzyme 100 U/g alcalase for 10 min at compared to histidine 2007
0.1 µg/mL
Hydroxyl radical SA Purified or synthesized No enzyme used Purified peptide IC50 = Xue et al., 2015
peptide RQSHFANAQP 2.03 µmol/mL


Albumin isolate Alcalase then 50 60 then 8 then 7 0.3 U/g then Fraction containing IC50 = approximately Kou et al., 2013
flavorzyme 120 50 U/g peptide 0.2 mg/mL
RQSHFANAQP
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 60.09% hydroxyl radical SA Zhang et al.,
at 1.5 mg/mL 2011
(Continued)

C 2019 Institute of Food Technologists®

 
Table 7–Continued.

Enzymatic hydrolysis conditions

Chickpea-based
starting material Temperature Time Most potent Greatest effect
Assay (variety if known) Identity (°C) (min) pH E:S ratio sample/treatment observeda Reference
PI Alcalase 50 40 8 1:50 v/w
Low molecular weight 81.39 ± 1.21% hydroxyl Li et al., 2008
fraction at radical SA

C 2019 Institute of Food Technologists®

1.5 mg/mL
Properties of chickpea peptides . . .

Superoxide radical SA High hydrostatic pressure Alcalase 50 – 8 1:20 w/v 400 MPa PI 45.54% superoxide radical Zhang et al.,
treated PI pretreatment and SA 2012
hydrolysis for
approximately
35 min
PI Under Hydrostatic 200 MPa of hydrostatic 66.26 ± 1.67% superoxide
Pressure pressure for 20 min radical SA
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 79.81% superoxide radical Zhang et al.,
at 2.0 mg/mL SA 2011
PI Alcalase 50 40 8 1:50 v/w Low molecular weight 69.15 ± 1.23% superoxide Li et al., 2008
fraction at radical SA
2.0 mg/mL
Cu2+ chelation PI from raw or boiled Pepsin then 37 2 to 2 2.5 to 7.0 1:100 w/w to 3.5 to 7 kDa fraction IC50 = 119.62 ± 5.98 Karaś et al.,
(100 °C for 60 min) pancreatin 2:5 v/v from PI of boiled µg/mL 2015
chickpeas chickpeas
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 76.92% Cu2+ chelating Zhang et al.,
at 50 µg/mL capability 2011
PI Pepsin then 37 180 then 2.5 then 1:20 w/w then 836 to 1,806 Da Approximately 97% Cu2+ Torres-Fuentes
pancreatin 180 7.5 1:20 w/w fraction at chelating capability et al., 2011
0.24 mg/mL
Inhibition of unsaturated PI (Kabuli) Pepsin then 37 180 then 2.5 then 1:20 w/w then 1:20 836 to 1,806 Da and 100% inhibition of fatty Torres-Fuentes
fatty acid oxidation pancreatin 180 7.5 w/w 478 to 788 Da acid peroxidation at et al., 2014
fractions 0.03 mg/mL
PI Alcalase 50 40 8 1:50 v/w Purified peptide NRYHE 88.81% inhibition of Zhang et al.,
at 50 µg/mL linoleic acid peroxidation 2011
after 8 days
PI Alcalase 50 40 8 1:50 v/w Low molecular weight 81.13% inhibition of Li et al., 2008
fraction at linoleic acid peroxidation
0.4 mg/mL
Inhibition of LDL PI (Kabuli) Pepsin then 37 180 then 2.5 then 1:20 w/w then 363 to 713 Da fraction 100% inhibition of LDL Torres-Fuentes
oxidation pancreatin 180 7.5 1:20 w/w containing the oxidation at 0.03 mg/mL et al., 2014
peptide FVPH
Inhibition of olive oil PC Pepsin 37 1440 2.5 17:500 w/w Nonhydrolyzed PC Delayed oxidation for Arcan &
oxidation 25 days Yemenicioǧlu,
2010
a For assaying the reduction of Fe3+ , samples were incubated with ferric chloride solution for approximately 10 min and then the absorbance of the solution was measured at 700 nm. A higher absorbance value indicates a higher reducing power, which is a favorable antioxidant parameter.

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1931
 Table 8–Angiotensin I-converting enzyme (ACE) inhibitory activity of chickpea protein hydrolysates and peptides.

Enzymatic hydrolysis conditions

Chickpea-based starting Temperature Most potent
material (variety if known) Identity (°C) Time (min) pH E:S Ratio sample/treatment ACE IC50 Reference
Peptide extract from No enzyme used Complete extract 0.152 ± 0.003 mg/mL Mamilla & Mishra,
nongerminated chickpeas 2017
Peptide extract from No Enzyme Used Extract from chickpeas 0.047 ± 0.001 mg/mL
chickpeas germinated at germinated at 40 °C
30 or 40 °C for 5 days
Flour from nonsoaked, Pepsin then trypsin & 37 120 then 120 2 then 6.8 2,250 U/mg then Digested flour Decreased to 0.7 ± 0.1 Jamdar et al.,
noncooked, and chymotrypsin 94 U/mg (trypsin) mg/mL after digestion 2017
Properties of chickpea peptides . . .

nongerminated chickpeas & 52 U/mg

(chymotrypsin)
Flour from chickpeas soaked Decreased to 0.6 mg/mL
in water for 16 hr after digestion
Flour from chickpeas Digested flour from Decreased to 0.6 ± 0.1
germinated for 24, 48, or chickpeas germinated mg/mL after digestion
72 hr for 72 hr
Flour from pressure cooked Digested flour Decreased to 0.6 mg/mL
chickpeas after digestion
PI Pepsin 37 1080 2 1:100 w/w Complete hydrolysate 673 ± 15.3 µg/mL Boschin et al.,
2014
Hardened chickpea PC, Alcalase 50 120 9 1:25 w/w Hardened albumin 0.033 µg/mL Medina-Godoy
globulin concentrate, or concentrate et al., 2012

1932 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
albumin concentrate hydrolysate
(Kabuli)
Papain 40 120 6.5 1:25 w/w Hardened chickpea PC 0.010 µg/mL
Pancreatin 39 120 8 1:25 w/w Hardened globulin 0.116 µg/mL
concentrate
hydrolysate
Fresh chickpea PC, globulin Alcalase 50 120 9 1:25 w/w Fresh globulin 0.307 µg/mL
concentrate, or albumin concentrate
concentrate (Kabuli) hydrolysate
Papain 40 120 6.5 1:25 w/w 0.016 µg/mL
Pancreatin 39 120 8 1:25 w/w 0.585 µg/mL
PC (Kabuli or Desi) Alcalase then 50 60 then 90 7 then 8 1:8 w/w then 1:10 PC from Desi chickpeas 228 ± 3 µg/mL Barbana & Boye,
flavorzyme w/w 2010


Papain 40 240 6.5 1:25 w/w 180 ± 1 µg/mL
Pepsin then trypsin & 37 120 then 150 2 then 6.5 1:250 w/w then 140 ± 1 µg/mL
chymotrypsin 1:250 w/w
Legumin isolate (Kabuli) Alcalase 50 30 7 0.3 U/g Peptide containing M, D, 0.011 mg/mL Yust et al., 2003
F, L, and I amino acids
PI (Kabuli) Alcalase then 50 – 7 then 8 0.3 U/g then 50 U/g Hydrolysate produced 0.19 mg/mL Pedroche et al.,
flavorzyme from alcalase for 30 2002
min

C 2019 Institute of Food Technologists®

Properties of chickpea peptides . . .
the testing of the ACE inhibitory potential. Multiple peptides
from dietary sources, including legumes, have shown ACE in-
hibitory potential (Balgir, Kaur, & Sharma, 2016). The ACE 50%
inhibitory concentrations of various chickpea protein hydrolysates
and peptides reported in the literature are shown in Table 8. Be-
cause the assay used to assess ACE inhibition is well standard-
ized, the results of this assay across studies can be compared us-
ing the 50% inhibitory concentrations of the samples analyzed
(Table 8). The most potent chickpea sample to inhibit ACE
(IC50 = 0.010 µg/mL), has been a protein hydrolysate from
hardened chickpea protein concentrate produced through papain-
mediated hydrolysis (Medina-Godoy et al., 2012). Furthermore,
these researchers found that chickpea protein hydrolysates pro-
duced by papain alone had the most potent ACE inhibitory ac-
tivity. The least potent samples to inhibit ACE were protein hy-
drolysates produced directly from chickpea flour, but the ACE
inhibitory activity of single chickpea peptides has not been de-
termined (Table 8). There is a need for studies on the effect of
chickpea protein hydrolysates or peptides on blood pressure in
animal models to conclude if the ACE inhibitory data currently
reported in the literature is biologically relevant.
Regulation of blood serum and liver cholesterol
Animals require cholesterol to maintain the integrity of their
cellular membranes and to synthesize vitamin D, hormones, and
bile acids, but excessive amount of blood serum cholesterol is a risk
factor for the development of cardiovascular diseases (Cerqueira
et al., 2016). Cholesterol is synthesized in the liver, and it becomes
incorporated into low-density lipoproteins (LDL) so that it can be
distributed to tissues throughout the body. Reduction of blood
serum LDL cholesterol is currently the main clinical strategy to
prevent or manage the progression of cardiovascular diseases, and
this can be achieved through dietary changes and the use of statins,
drugs that inhibit cholesterol synthesis by inhibiting 3-hydroxy-
3-methyl glutaryl coenzyme A (HMG-CoA) reductase (Wadhera,
Steen, Khan, Giugliano, & Foody, 2016). Cholesterol is transferred
to the liver from peripheral tissues by high-density lipoproteins
(HDL). While the elevation of blood serum HDL levels have
historically been seen as a positive health outcome, this might only
apply to individuals without cardiovascular disease (März et al.,
2017; Tietge, 2019). Consumption of legumes, such as chickpeas,
has been associated with a lower incidence of cardiovascular disease
(Afshin, Micha, Khatibzadeh, & Mozaffarian, 2014; Marventano
et al., 2016). The most recent studies on the biological activity
of chickpea protein hydrolysates and peptides have focused on the
improvement to blood serum and liver cholesterol (Table 9).
Six studies using animal models have been conducted to assess
the hypolipidemic effects of chickpea protein hydrolysates and
peptides (Table 9). The two most recent animal studies of this
category assessed the hypolipidemic effects of a chickpea protein
hydrolysate formed through alcalase hydrolysis on male and female
rats. In male rats, a 100 mg per kilogram of BW per day dose of
a chickpea protein hydrolysate produced by alcalase was the most
effective dose in reducing overall cholesterol concentrations in the
body of male rats eating high-fat (HFD) and nonhigh-fat diets (Shi,
Hou, Guo, et al., 2019). In female rats, a 200 mg per kilogram of
BW per day dose of a chickpea protein hydrolysate formed through
alcalase was the most effective dose in improving markers associated
with hyperlipidemia (Shi, Hou, Liu, et al., 2019). The chickpea
protein hydrolysate formed through alcalase showed to decrease
cholesterol in rats by inhibiting enzymes that promote cholesterol
synthesis in cells and increasing the fecal excretion of cholesterol

C 2019 Institute of Food Technologists® Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1933
(Table 9). The peptide VFVRN was identified in the chickpea
protein hydrolysate produced through alcalase mentioned, but its
hypolipidemic effects have only been tested in cells (Shi, Hou,
Guo, et al., 2019; Shi, Hou, Liu, et al., 2019). VFVRN was
shown to decrease the expression and activity of HMG-CoA, a
key enzyme in cholesterol synthesis (Table 10).
Two other studies performed on male mice fed a HFD fo-
cused on the antioxidant potential of the chickpea peptide RQSH-
FANAQP mentioned in the previous section (Xue, Hou, et al.,
2018; Xue, Wang, et al., 2018). Those studies also found a
decrease in lipid content in the bodies of mice; this effect
was due to an increase in the expression of proteins involved
in cholesterol metabolism and removal (Table 9). A dose of
20 mg per kilogram of BW per day of the peptide RQSH-
FANAQP was the most effective in regulating the expression
of proteins associated with cholesterol homeostasis (Xue, Wang,
et al., 2018). A mixture of the peptides VFVRN, SDHPF, AMHPF,
and VNASAL was not effective in significantly decreasing choles-
terol in cancer cells, so only the chickpea peptides VFVRN
and RQSHFANAQP have well supported evidence to be po-
tentially helpful in helping to regulate cholesterol levels in humans
(Table 9 and 10). It should be noted that the animal studies done
on the peptides VFVRN and RQSHFANAQP were performed
by manually inserting the peptides into the bodies of the animals,
which could lead to different results if the peptides would have
been consumed in the diet and had to travel the entirety of the
gastrointestinal track.
Serum cholesterol levels in mice with cancer have been found to
be comparable to those of mice without cancer as a result of a 10 to
30 mg per kilogram of BW per day treatment of a chickpea protein
concentrate hydrolysate formed through the sequential hydrolysis
by pepsin followed by pancreatin (Sánchez-Chino et al., 2018).
That same study found that mice with cancer fed a HFD had a
significant decrease in blood cholesterol levels when treated with
a chickpea protein concentrate hydrolysate at a dose of 30 mg
per kilogram of BW per day. No peptide fraction or peptides
were identified as responsible for the observed effects (Table 9).
Similarly, a chickpea albumin hydrolysate (100 to 150 mg per
kilogram of BW per day) formed from the sequential hydrolysis of
alcalase followed by flavorzyme decreased blood cholesterol levels
in mice fed a HFD (Xue et al., 2012). The mechanisms for the
reduction of cholesterol in mice fed a HFD and chickpea protein
hydrolysates or peptides are still not well resolved, but it might be
due to the inhibition of cholesterol absorption by chickpea protein
hydrolysates (del Mar Yust et al., 2012).
Other reported bioactivities
Antioxidant, hypolipidemic, and ACE inhibitory activities are
not the only bioactives that chickpea peptides have been reported
to have. Chickpea protein hydrolysates and peptides have been
reported to have anticarcinogenic, antifungal, anti-inflammatory,
antidiabetic, and skin health promoting properties. Potential bioac-
tives aside from antioxidant capacity, hypolipidemic effects, and
ACE inhibitory potential reported for chickpea protein hy-
drolysates and peptides are shown in Table 9 and 10.
There are two animal studies that have accessed the anticar-
cinogenic effects of chickpea protein hydrolysates (Table 9). The
chickpea protein concentrate hydrolysate formed from the se-
quential hydrolysis of pepsin followed by pancreatin that was re-
ported to regulate cholesterol homeostasis in mice did not sig-
nificantly decrease the number of aberrant crypts in mice with
cancer (Sánchez-Chino et al., 2018). On the other hand, a
 Table 9–Significant biological effects observed in animals administered chickpea protein hydrolysates and peptides.
Animal model
Target condition analyzed Chickpea-based treatment Assay
Hyperlipidemia Male Wistar rats fed a Chickpea protein isolate Total serum cholesterol
HFD hydrolysate formed by
alcalase
Total serum triglycerides
Serum LDL cholesterol
Serum HDL cholesterol
Serum LDL/HDL ratio
Hepatic LDL cholesterol
Hepatic HDL cholesterol
Hepatic apolipoprotein B expression
Total fecal cholesterol
Hepatic HMG-CoA reductase activity
Hepatic fatty acid synthase activity
Hepatic sterol regulatory
element-binding protein-1c
protein expression
Hepatic peroxisome
proliferator-activated receptor γ
protein expression
Hepatic LDL receptor protein
expression
Male Wistar rats fed a Serum LDL cholesterol
normocaloric diet
Hepatic HDL cholesterol
1934 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
Total fecal cholesterol
Hepatic fatty acid synthase activity
Ovariectomized Chickpea protein isolate Adipose tissue weight after 6 weeks
female SD rats hydrolysate formed by
alcalase
Total serum cholesterol
Total serum triglycerides
Serum LDL cholesterol
Serum HDL cholesterol
Serum LDL/HDL ratio
Total hepatic cholesterol
Total hepatic triglycerides
Total hepatic bile acids

Total fecal cholesterol
Total fecal triglycerides
Total fecal bile acids
Hepatic HMG-CoA reductase activity
Hepatic fatty acid synthase activity
Hepatic estrogen receptor α protein
expression
C 2019 Institute of Food Technologists®
Most potent dose Greatest effect observed Reference
100 mg/kg BW 23.62% reduction compared to NTC Shi, Hou, Guo,
et al., 2019
50 mg/kg BW 43.44% reduction compared to NTC
100 mg/kg BW 29.82% reduction compared to NTC
200 mg/kg BW 22.11% increase compared to NTC
100 mg/kg BW 51.83% reduction compared to NTC
200 mg/kg BW 61.76% reduction compared to NTC
100 mg/kg BW 60.71% reduction compared to NTC
44.44% reduction compared to NTC
50 mg/kg BW 74.60% increase compared to NTC
Properties of chickpea peptides . . .
200 mg/kg BW Approximately 1.6-fold decrease in
activity compared to NTC
50 mg/kg BW Approximately 2-fold decrease in
activity compared to NTC
200 mg/kg BW Similar expression as found in Male
Wistar rats fed a normocaloric diet
Approximately 2-fold increase in
expression compared to NTC
100 mg/kg BW Approximately 2-fold increase in
expression compared to NTC
100 mg/kg BW 53.13% reduction compared to NTC
45.71% reduction compared to NTC
17.14% increase compared to NTC
Approximately 1.4-fold decrease in
activity compared to NTC
50, 100, or Approximately 1.6-fold decrease in Shi, Hou, Liu, et al.,
200 mg/kg BW adipose tissue weight compared to 2019
NTC
100 mg/kg BW 16.67% reduction compared to NTC
200 mg/kg BW 28.57% reduction compared to NTC
50 or 200 mg/kg BW 21.62% reduction compared to NTC
200 mg/kg BW 31.37% increase compared to NTC
100 or 200 mg/kg Approximately 2.5-fold decrease
BW compared to NTC
50 mg/kg BW 19.71% reduction compared to NTC
100 mg/kg BW 20.87% reduction compared to NTC
50, 100, or Similar concentration to that of
200 mg/kg BW non-ovariectomized female SD rats
200 mg/kg BW Similar concentration to that of
non-ovariectomized female SD rats
100 mg/kg BW 32.17% increase compared to NTC
200 mg/kg BW 25.25% increase compared to NTC
50, 100, or Similar activity as found in
200 mg/kg BW non-ovariectomized female SD rats
50, 100, or Similar activity as found in
200 mg/kg BW non-ovariectomized female SD rats
200 mg/kg BW Approximately 10-fold increase in
expression compared to NTC
(Continued)
 
Table 9–Continued.

Animal model
Target condition analyzed Chickpea-based treatment Assay Most potent dose Greatest effect observed Reference
Hepatic estrogen receptor β protein Approximately 1.7-fold increase in
expression expression compared to NTC
Hepatic sterol regulatory 100 mg/kg BW Similar expression as found in
element-binding protein-1c non-ovariectomized female SD rats
expression
Hepatic peroxisome 200 mg/kg BW Similar expression as found in
proliferator-activated receptor γ non-ovariectomized female SD rats
protein expression

C 2019 Institute of Food Technologists®

Hepatic liver X receptor α protein Approximately 2.7-fold increase in
Properties of chickpea peptides . . .

expression expression compared to NTC

Male KM mice fed a Synthesized peptide Hepatic expression of acetyl-CoA 20 mg/kg BW/day 1.97-fold decrease in expression Xue, Wang, et al.,
HFD RQSHFANAQP carboxylase mRNA compared to NTC 2018
Hepatic expression of fatty acid 40 mg/kg BW/day Approximately 1.8-fold decrease in
synthase mRNA expression compared to NTC
Hepatic expression of peroxisome 20 mg/kg BW/day 1.4-Fold increase in expression
proliferator-activated receptor α compared to NTC
mRNA
Hepatic expression of fatty acid 1.4-Fold increase in expression
binding protein 1 mRNA compared to NTC
Hepatic expression of adipose 1.5-Fold increase in expression
triglyceride lipase mRNA compared to NTC
Hepatic expression of sterol 1.62-Fold decrease in expression
regulatory element-binding compared to NTC
protein 2 mRNA
Hepatic expression of liver X receptor 1.47-fold increase in expression
α mRNA compared to NTC
Hepatic expression of HMG-CoA 1.55-Fold decrease in expression
reductase mRNA compared to NTC
Hepatic expression of LDL receptor 1.65-Fold decrease in expression
mRNA compared to NTC
Hepatic expression of proprotein 1.65-Fold decrease in expression
convertase subtilisin/kexin 9 compared to NTC
mRNA
Hepatic expression of 1.43-Fold increase in expression
cholesterol-7α-hydroxylase mRNA compared to NTC
Hepatic expression of ATP binding 1.43-Fold increase in expression
cassette subfamily A member 1 compared to NTC
mRNA
Hepatic expression of scavenger 1.56-Fold increase in expression
receptor class B member 1 mRNA compared to NTC
Hepatic protein expression of ATP 1.82-Fold increase in expression
binding cassette subfamily A compared to NTC
member 1
Hepatic protein expression of 1.85-Fold increase in expression
cholesterol-7α-hydroxylase compared to NTC
Hepatic protein expression of sterol 1.5-Fold decrease in expression
regulatory element- binding compared to NTC
protein 2
(Continued)

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1935
 Table 9–Continued.

Animal model
Target condition analyzed Chickpea-based treatment Assay Most potent dose Greatest effect observed Reference
Hepatic protein expression of 2.8-Fold decrease in expression
proprotein convertase compared to NTC
subtilisin/kexin 9
Hepatic protein expression of LDL Approximately 1.3-fold increase in
receptor expression compared to NTC
Male KM mice fed a Synthesized peptide Total serum cholesterol 10 mg/kg BW/day Decrease to approximately 4 mmol/L Xue, Hou, et al.,
HFD RQSHFANAQP from approximately 6 mmol/L for 2018
Properties of chickpea peptides . . .

Total serum triglycerides
Total hepatic triglycerides
Hepatic lipase activity
Hepatic lipoprotein lipase activity
Fecal lipid content
Liver index
NTC
Decrease to approximately 1.5
mmol/L from approximately 2
mmol/L for NTC
40 mg/kg BW/day 66.06% reduction compared to NTC
20 mg/kg BW/day Increase to approximately 3 U/mg
from approximately 1.5 U/mg for
NTC
40 mg/kg BW/day Increase to approximately 2.5 U/mg
from approximately 1.5 U/mg for
NTC
20 mg/kg BW/day Increase to approximately 2 g from
approximately 1 g for NTC after 4
weeks
Decreased to 3.8 ± 0.4 g/100 g BW

1936 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
from 4.1 ± 0.3 g/100 g BW for
NTC
Kidney index 40 mg/kg BW/day Decreased to 1.2 ± 0.1 g/100 g BW
from 1.4 ± 0.1 g/100 g BW for
NTC
Epididymal fat index Decreased to 2.5 ± 0.6 g/100 g BW
from 1.8 ± 0.4 g/100 g BW for
NTC
Male ICR mice with Chickpea protein Total serum cholesterol 10 mg/kg BW Similar cholesterol concentration Sánchez-Chino
AOMICC fed a concentrate hydrolysate compared to mice without cancer et al., 2018
normocaloric diet formed by sequential
hydrolysis with pepsin
followed by pancreatin
Serum LDL cholesterol 20 mg/kg BW Similar LDL concentration compared
to mice without cancer


Serum LDL/HDL ratio 10 mg/kg BW Similar LDL/HDL ratio compared to
mice without cancer
Male ICR mice with 30 mg/kg BW Decrease to 6.5 ± 0.8 from 11.7 ±
AOMICC fed a 0.52 for NTC
hypercaloric diet
Male ICR mice fed a Total serum cholesterol 30 mg/kg BW Decrease to 82.5 ± 6.0 mM from
hypercaloric diet 105.5 ± 11.1 mM for NTC
(Continued)

C 2019 Institute of Food Technologists®

 
Table 9–Continued.

Animal model
Target condition analyzed Chickpea-based treatment Assay Most potent dose Greatest effect observed Reference
Serum LDL cholesterol Decrease to 73.1 ± 7.1 mM from
97.4 ± 11.1 mM for NTC
Serum LDL/HDL ratio Decrease to 6.2 ± 0.6 from 13.3 ±
2.1 for NTC
Male KM mice fed a Chickpea albumin isolate Total serum cholesterol 100 or 150 mg/kg Decreased to approximately Xue et al., 2012
HFD hydrolysate formed by BW 2.5 mmol/L from 4.6 ± 0.8
sequential hydrolysis mmol/L for NTC
with alcalase followed by

C 2019 Institute of Food Technologists®

flavorzyme
Total serum triglycerides 150 mg/kg BW Decreased to 2.2 ± 0.3 mmol/L from
Properties of chickpea peptides . . .

3.4 ± 0.7 mmol/L for NTC

Serum LDL cholesterol Decreased to 2.1 ± 0.5 mmol/L from
4.1 ± 0.6 mmol/L for NTC
Weight gain 50 mg/kg BW 3.4 ± 1.0 g weight gain compared to
7.5 ± 2.2 g weight gain for NTC
Cancer Male ICR mice with Chickpea protein Number of aberrant crypts 30 mg/kg BW Similar number of aberrant crypts Sánchez-Chino
AOMICC fed a concentrate hydrolysate compared to mice without cancer et al., 2018
normocaloric diet formed by sequential
hydrolysis with pepsin
followed by pancreatin
Male ICR mice with
AOMICC fed a
hypercaloric diet
Male KM mice Chickpea albumin isolate Tumor volume 100 mg/kg BW Decreased to 1,172 ± 345 mm3 from Xue et al., 2012
transplanted with hydrolysate formed by 2,344 ± 281 mm3 for NTC
H22 liver cancer sequential hydrolysis
cells with alcalase followed by
flavorzyme
Spleen index Increase to 11.5 ± 1.0 mg/g from
6.4 ± 0.5 mg/g for NTC
Proliferation of spleen lymphocytes 200 mg/kg BW Approximately 2-fold increase in
proliferation compared to NTC
Tumor inhibition rate 100 mg/kg BW 53.37% tumor inhibition rate
compared to NTC
Oxidative Stress Male KM mice fed a Synthesized peptide Serum superoxide dismutase activity 20 mg/kg BW/day Increase to approximately 400 U/mL Xue, Hou, et al.,
HFD RQSHFANAQP from approximately 300 U/mL for 2018
NTC
Hepatic MDA concentration Decreased to 0.1 U/mg from
0.5 U/mg for NTC
Male KM mice Chickpea albumin isolate Serum superoxide dismutase activity 200 mg/kg BW Increased to 352 ± 21 U/mg protein Xue et al., 2012
transplanted with hydrolysate formed by from 295 ± 22 U/mg protein for
H22 liver cancer sequential hydrolysis NTC
cells with alcalase followed by
flavorzyme
Serum MDA concentration Decreased to 2.55 ± 0.28 nmol
MDA/mg protein from 3.29 ±
0.17 nmol MDA/mg protein for
NTC
Inflammation Male KM mice fed a Synthesized peptide Hepatic expression of tumor necrosis 20 mg/kg BW/day 1.6-Fold decrease in expression Xue, Wang, et al.,
HFD RQSHFANAQP factor α mRNA compared to NTC 2018

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1937
 Table 10–Other bioactivities of chickpea protein hydrolysates and peptides reported in the literature.

Enzymatic hydrolysis conditions

Chickpea-based Most potent
Target starting material Temperature sample/ Greatest effect
Condition Assay (variety if known) Identity (°C) Time (min) pH E:S ratio treatment observed Reference
In vitro
Hyperlipidemia Cholesterol concentration Peptides VFVRN, No enzyme used VFVRN at 1 mM Similar concentration Shi, Hou, Guo,
in HepG2 liver cancer SDHPF, AMHPF, and for 24 hr as NTC et al., 2019
cells VNASAL
Sterol regulatory Peptide VFVRN No enzyme used Similar expression as
element-binding NTC
protein-1c expression in
HepG2 liver cancer cells
Properties of chickpea peptides . . .

Sterol regulatory No enzyme used Similar expression as

element-binding NTC
protein-2 expression in
HepG2 liver cancer cells
Liver X receptor α protein No enzyme used Similar expression as
expression in HepG2 NTC
liver cancer cells
HMG-CoA reductase No enzyme used Similar expression as
protein expression in NTC
HepG2 liver cancer cells
Inhibition of porcine No enzyme used VFVRN at 0.4 mM 64.38% inhibition
hepatic HMG-CoA compared to NTC
reductase activity
Cholesterol concentration PI Alcalase 55 80 8 1:50 w/v <1 kDa fraction at Approximately

1938 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
in HepG2 liver cancer 3 mg/mL 1.4-fold reduction
cells in concentration
compared to NTC
Peptide VFVRN No Enzyme Used VFVRN at 1 mM Approximately Shi, Hou, Liu,
1.8-fold reduction et al., 2019
in concentration
compared to NTC
Cholesterol uptake in PI Alcalase 55 80 8 1:50 w/v <1 kDa and Approximately
HepG2 liver cancer cells <5 kDa 1.4-fold increase in
fractions at 3 uptake compared to
mg/mL NTC
HMG-CoA reductase Peptide VFVRN No enzyme used VFVRN at 1 mM Approximately 2-fold
protein expression in reduction in
HepG2 liver cancer cells expression


compared to NTC
Cancer MCF-7 breast cancer cell Peptide RQSHFANAQP Pepsin then 37 120 then 120 1 to 7 9:100 w/w Peptide fragment EC50 = 2.672 ± Xue et al., 2016
viability trypsin then SH for 72 hr 0.559 mmol/L
19:100
w/w
MDA-MB-231 breast Purified or synthesized No enzyme used Synthesized EC50 = 1.50 µmol/mL Xue et al., 2015
cancer cell viability peptide RQSHFANAQP
RQSHFANAQP
MCF-7 breast cancer cell No enzyme used EC50 = 2.38 µmol/mL
viability

(Continued)

C 2019 Institute of Food Technologists®

 
Table 10–Continued.

Enzymatic hydrolysis conditions

Chickpea-based Most potent
Target starting material Temperature sample/ Greatest effect
Condition Assay (variety if known) Identity (°C) Time (min) pH E:S ratio treatment observed Reference
Protease-inhibitor rich No enzyme used No significant effect observed at 25, 50, Magee, Owusu-
PC 100, 200, or 400 mg/mL Apenten,
McCann, Gill,
& Rowland,
2012
MDA-MB-231 breast No enzyme used PC at 200 µg/mL Approximately 15%
cancer cell viability for 48 hr decrease in cell

C 2019 Institute of Food Technologists®

viability compared
to NTC
Properties of chickpea peptides . . .

LNCaP prostate cancer No enzyme used Approximately 35%

cell viability decrease in cell
viability compared
to NTC
PC-3 prostate cancer cell No enzyme used PC at 400 µg/mL 36.3 ± 2.4% decrease
viability for 48 hr in cell viability
compared to NTC
THP-1 cell viability PI (Kabuli) Pepsin then 37 – 2 then 7.5 1:20 w/w Pepsin for 72 min 78% decrease in cell Girón-Calle
pancre- then 1:20 followed by viability compared et al., 2010
atin w/w pancreatin for to NTC
approximately
100 min sample
at 10% for
96 hr
Skin diseases Filaggrin mRNA Brine extract of No enzyme used No significant effect observed at 1 mg/mL Rizzello et al.,
expression in Caco-2 chickpea flour dough 2015
colon cancer cells
Brine extract of No enzyme used Extract at 1 Approximately
fermented chickpea mg/mL for 8 hr 10-fold increase in
flour dough mRNA expression
compared to NTC
Involucrin mRNA Brine extract of No enzyme used No significant effect observed at 1 mg/mL
expression in Caco-2 chickpea flour dough
colon cancer cells
Brine extract of No enzyme used Extract at 1 Approximately 5-fold
fermented chickpea mg/mL for 8 hr increase in mRNA
flour dough expression
compared to NTC
BJ ATCC CRL2522 human PI from raw or boiled Pepsin then 37 2 then 2 2.5 then 7.0 1:100 w/w 3.5-7 kDa fraction 40% increase in cell Karaś et al.,
fibroblast cell viability (100 °C for 60 min) pancre- then 2:5 from boiled viability 2015
chickpeas atin v/v chickpeas at
25 µg/mL
Inflammation Inhibition of nitric oxide PC from chickpeas Pepsin then 37 60 then 120 2 then 6.5 2250 U/mL Most hydrophobic IC50 = 97.31 ± 19.13 Milán-Noris
production by germinated for 5 days pancre- then 1980 peptide fraction µg/mL et al., 2018
RAW264.7 or boiled for 30 min atin U/mL from PC of
macrophages (Desi) germinated
chickpeas

(Continued)

Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1939
 Table 10–Continued.

Enzymatic hydrolysis conditions

Chickpea-based Most potent
Target starting material Temperature sample/ Greatest effect
Condition Assay (variety if known) Identity (°C) Time (min) pH E:S ratio treatment observed Reference
PC from chickpeas IC50 = 90.70 ± 20.21
germinated for 5 days µg/mL
or boiled for 30 min
(Kabuli)
Microbiological
Fungal infection Inhibition of Whole green chickpea No enzyme used Cicerarin peptide IC50 = 15.3 µM Chu et al., 2003
Properties of chickpea peptides . . .

Mycosphaerella flour VKSTGRADDD-

arachidicola growth LAVKTKYLPP
Inhibition of Physalospora No enzyme used IC50 = 8.2 µM
piricola growth
Inhibition of Botrytis No enzyme used IC50 = 20.6 µM
cinerea growth
Inhibition of Botrytis Whole chickpea flour No enzyme used Arietin IC50 = 2 µM Ye et al., 2002
cinerea growth GVGYKVVVTT-
TAAADDDDVV
Biochemical
Diabetes Human α-amylase Whole chickpea flour No enzyme used Peptide 73.6 ± 0.5% Hao et al., 2009
inhibition LHQNIGSSSSP- inhibition at
DIYNPQAGR unknown
concentration

1940 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019
Fungal infection Inhibition of human Whole green chickpea No Enzyme Cicerarin peptide IC50 = 87.5 µM Chu et al., 2003
immunodeficiency virus flour Used VKSTGRADDD-
reverse-transcriptase LAVKTKYLPP
activity
Fungal infection Inhibition of rabbit Whole chickpea flour No enzyme used Cicerin IC50 = 4.5 µM Ye et al., 2002
reticulocyte lysate ARCENFADSY-
translation activity RQPPISSSQT
Chemical
Hyperlipidemia Reduction of cholesterol PI Alcalase 50 – 8 then 7 4.6:50 v/w Hydrolysate 50% reduction at del Mar Yust
micellar solubility then fla- then produced by 1 2.5 mg/mL et al., 2012
vorzyme 3.7:100 hr alcalase then
v/w 30, 90, or
120 min


flavorzyme
In silico
Hyperlipidemia Binding to cholesteryl Peptide RQSHFANAQP No enzyme used – −76.5574 kcal/mol Xue, Hou, et al.,
ester transfer protein complex binding 2018
energy
Cancer Binding energy with SH, HF, and ANAQ No enzyme used Peptide fragment −7.52 kcal/mol Xue et al., 2016
p53-R273H mutant fragments from ANAQ binding energy
peptide
RQSHFANAQP

C 2019 Institute of Food Technologists®

Properties of chickpea peptides . . .
chickpea albumin hydrolysate (100 mg per kilogram of BW per
day) formed from the sequential hydrolysis of alcalase followed by
flavorzyme did have a positive effect by inhibiting and decreasing
the number of tumors in mice (Xue et al., 2012). It is still unclear
if chickpea protein hydrolysates and peptides will potentially have
anticarcinogenic effects in humans. Chickpea protein hydrolysates
have been found to decrease the viability of cancerous cells, but
the efficacy of these results is questionable until they are vali-
dated in animal models (Table 10). The peptide RQSHFANAQP,
mentioned previously as being antioxidant and having hypolipi-
demic effects, was also found to decrease the viability of cancer
cells (Table 10). Interestingly, the peptide fragment ANAQ of the
peptide RQSHFANAQP was found to favorably interact in silico
with the structure of the protein p53, which is heavily involved
in cancer signaling (Xue et al., 2016). More research is needed on
the anticarcinogenic effects of chickpea protein hydrolysates and
peptides.
The peptide RQSHFANAQP is also implicated to have anti-
inflammatory effects as it significantly decreased the expression of
tumor necrosis factor-α in mice that received 20 mg of the peptide
per kilogram of BW per day (Xue, Wang, et al., 2018). Hydropho-
bic peptides from a protein hydrolysate produced from germinated
chickpeas were found to inhibit the production of nitric oxide, an
inflammatory signaling molecule, by macrophages, but the pep-
tides were not sequenced (Milán-Noris et al., 2018). Antimicrobial
peptides extracted from chickpeas have also been described, but
there have not been recent studies on these peptides (Table 10).
Figure 2 displays the chemical structures of these peptides and the
structures of peptides that have demonstrated well characterized
bioactivity discussed in the previous sections. There are some re-
ports that chickpea protein hydrolysates and peptides might be
beneficial to skin heath, but these studies are not very well sup-
ported (Table 10). The peptide LHQNIGSSSSPDIYNPQAGR
has been reported to significantly inhibit human α-amylase, but
this finding is also not very well supported (Hao et al., 2009).
More research is needed on the anti-inflammatory and antidia-
betic potential of chickpea protein hydrolysates and peptides.
Figure 2–Chemical structures of bioactive peptides with potent potential biological activity. Chemical structures were obtained using the PepDraw
tool interface (https://www.pepdraw.com).

C 2019 Institute of Food Technologists®
Bitterness of Chickpea Bioactive Peptides
Bitterness is defined as a disagreeable, acrid, and pungent taste
that opposes sweetness. It is hypothesized that humans developed
the ability to sense bitter compounds as a defense mechanism,
because many toxicants induce a bitter taste in humans (Aliani &
Eskin, 2017). Bitterness has traditionally been analyzed using hu-
man sensory panels; however, with advancements in bioinformat-
ics, the bitterness of small organic compounds is now more com-
monly predicted using structure-based computational methods.
These methods predict the bitterness of a compound depending
on how similar its structure is to that of known bitter compounds
(Bahia, Nissim, & Niv, 2018). Structure-based methods to predict
bitterness are currently focused on small organic compounds and
not peptides. The chemical interactions bitter compounds have
with bitter taste receptors are currently unknown because the
atomic structures of bitter taste receptors are currently unresolved
(Di Pizio, Shoshan-Galeczki, Hayes, & Niv, 2018).
A major issue in incorporating bioactive peptide ingredients
into commercialized food products is that many peptide solutions
can have a bitter taste, making them unpopular with consumers
(Li-Chan, 2015). Current methods of reducing the bitterness of
peptides in food products include extensively hydrolyzing peptide
ingredients, filtering out known bitter peptides using membrane
filtration or chromatographic separations, and masking the bitter-
ness of peptide ingredients by adding compounds that can module
or overpower their bitter taste (Chakrabarti, Guha, & Majumder,
2018). Extensive hydrolysis of peptide ingredients to reduce their
bitterness is undesirable as it can eliminate their bioactivity and
their technological functionality, but having to fractionate them
or adding more ingredients to a food product to mask their bit-
terness can increase the price of the food product.
Reported bitterness of chickpea protein hydrolysates
Studies on the bitterness of chickpeas are sparse. Chickpeas
contain bitter compounds, such as saponins, but the perception
of bitter taste from the consumption of chickpeas appears to be
genotype dependent (Jukanti et al., 2012; Mukhopadhyay et al.,
Vol. 18, 2019 r Comprehensive Reviews in Food Science and Food Safety 1941
Properties of chickpea peptides . . .

2018). Only one study was found to directly analyze the bitterness
of chickpea protein hydrolysates (Kou et al., 2013). This study
employed a trained sensory panel that evaluated the bitterness of
various chickpea albumin hydrolysates produced by the enzymes
alcalase and flavorzyme in comparison to varying concentrations
of quinoline, a known bitter compound. As found before for peas,
hydrolysis with flavorzyme produced a less bitter hydrolysate than
the protein hydrolysate produced by alcalase alone (Humiski &
Aluko, 2007). More studies are necessary on the topic of chick-
pea protein hydrolysate and peptide bitterness in order for these
ingredients to be commercialized, because bitterness can deter
consumers from buying certain food products.
antihypertensive, anticarcinogenic, antifungal, anti-inflammatory,
antidiabetic, and skin health promoting bioactivities. Although the
peptides VFVRN, NRYHE, and RQSHFANAQP have already
been isolated and characterized from chickpea protein hydrolysates
and have shown potential biological activity, human studies are still
necessary to determine the biological activity of these peptides in
humans. Much is still unknown about the bitterness of chickpea
protein hydrolysates and peptides, and more testing of the bit-
terness of these potential ingredients will be necessary for them
to be commercialized. Overall, chickpea protein hydrolysates and
peptides show great potential as bioactive ingredients that can be
feasibly produced and added to food products.

Potential bitterness of chickpea peptides derived from the Acknowledgment

hydrolysis of chickpea legumin by stem bromelain and ficin Research was supported by the USDA-NIFA-HATCH project
Table 5 and 6 show potential peptides with 10 amino acids or 1014457
less in their sequence that can be potentially produced from the
hydrolysis of chickpea legumin by stem bromelain and ficin, re-
spectively. Using the sensory peptides and amino acids library of
Author Contributions
L.M.R.H. searched and analyzed the literature and wrote the
the BIOPEP database, bitter and umami fragments in the theoret-
initial manuscript. E.G.d.M. conceived the idea, suggested the
ical peptides were determined. Although the presence of a bitter
in silico study to deliver original information, provided scientific
peptide fragment in a peptide will not innately make the pep-
guidance through the process, and edited the manuscript. All au-
tide bitter, it can be an indication of potential bitterness (Li-Chan,
thors critically revised the manuscript and gave final approval for
2015). Not all of the peptide sequences reported from the theoret-
its submission.
ical hydrolysis of chickpea legumin by stem bromelain were found
to have bitter fragments. However, all of the peptide sequences
reported from the same procedure using ficin were found to have Conflicts of Interest
bitter fragments. Interestingly, the peptide sequences GTCFALR, Authors declare no conflict of interest.
IETWNPSN, IKTVTSF, and LSRATLQPNS found from the
theoretical hydrolysis by stem bromelain were not found to have Nomenclature
bitter fragments, even though other sequences of similar size were A Alanine
found to contain bitter fragments. A caveat to predicting potential AOMICC Azoxymethane-induced colon cancer
peptide bitterness by searching for bitter fragments is that large ABTS 2,2 -Azino-bis(3-ethylbenzothiazoline-6-
peptides have a larger probability of having bitter fragments, even sulfonic acid)
though there is a point where the peptide would be too large ACE Angiotensin-I-converting enzyme
to easily interact with bitter taste receptors to actually be bitter BW Body weight
(Aluko, 2017). There is currently no standard way of predicting C Cystine
peptide bitterness, and future research should focus on describing cAMP-DPDE Cyclic adenosine monophosphate-dependent
accurate and feasible methods for predicting peptide bitterness. phosphodiesterase
Umami tasting fragments found in the peptides reported were D Aspartic acid
also documented because umami peptides have been found to DPP-IV Dipeptidyl peptidase-IV
suppress the perception of bitter compounds (Kim, Son, Kim, DPPH 2,2-Diphenyl-1-picrylhydrazyl
Misaka, & Rhyu, 2015). As with bitterness, the presence of an E Glutamic acid
umami fragment in a peptide only indicates the potential of the E:S Enzyme:substrate
peptide to have an umami taste. Only the umami fragments AE, F Phenylalanine
ET, and KG were found in the theoretical peptides reported. The G Glycine
umami fragment KG was found to be both bitter and umami γ -GCS γ -Glutamylcysteine synthetase
tasting, making it unknown whether its presence in a peptide H Histidine
will potentially make it bitter. The umami fragment ET was only HDL High-density lipoprotein
found in the peptide IETWNPSN from the theoretical hydrolysis HFD High-fat diet
of chickpea legumin by stem bromelain (Table 5). HMG-CoA 3-Hydroxy-3-methyl glutaryl coenzyme A
HO-1 Heme oxygenase-1
Conclusions I Isoleucine
Bioactive peptides are compounds that can help manage disease K Lysine
symptoms and promote health. These peptides can be produced KM Kunming
through enzymatic hydrolysis of chickpea proteins with a va- L Leucine
riety of food-approved enzymes such as alcalase, chymotrypsin, LDL Low-density lipoprotein
flavorzyme, papain, pancreatin, pepsin, and trypsin. Although not M Methionine
reported to have been used in the literature, the enzymes ficin and MDA Malondialdehyde
stem bromelain can potentially be used to produce bioactive pep- MW Molecular weight
tides from chickpea proteins. Chickpea protein hydrolysates and N Asparagine
peptides have been shown to have antioxidant, hypolipidemic, NQO1 NADPH quinone oxidoreductase 1

1942 Comprehensive Reviews in Food Science and Food Safety r Vol. 18, 2019 
C 2019 Institute of Food Technologists®
Properties of chickpea peptides . . .
Nrf2 Nuclear transcription factor E2-related factor 2
NTC No treatment control
P Proline
PC Protein concentrate
PI Protein isolate
Q Glutamine
R Arginine
S Serine
SA Scavenging activity
SD Sprague Dawley
T Threonine
TEAC Trolox equivalent antioxidant capacity
V Valine
W Tryptophan
Y Tyrosine
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