577

Environment and Ecology 40 (2A) : 577—583, April—June 2022

ISSN 0970-0420

Use of Sprouted Legumes as Sources of Antioxidants

in Functional Dairy Product Development
Serlene Tomar, Annada Das,
Narendra Singh Tomar

Received 17 December 2021, Accepted 5 March 2022, Published on 12 April 2022

ABSTRACT This review provides a summary of the antioxidant

potential of sprouted legumes as assessed by various
The potential health benefits of phytochemicals and methods and thereby providing their suitability as
their ability to be incorporated into dairy foods as potential candidates in the development of functional
nutraceuticals has received considerable interest in foods of livestock origin.
food industry. Phenolic compounds are added to dairy
products to improve both their technological func- Keywords Sprouted legumes, Antioxidants, Dairy
tionality and nutritional value. Sprouted legumes are products, Functional food, Phytochemicals.
considered to be power houses of nutrients and hold
various bioactivities, which could have many health
benefits, improve its nutritional value and prevent
oxidation in number of food matrices. Extraction of
phytochemicals from these sprouts and incorporation
into food based products can be suitable method to
INTRODUCTION
improve the functionality of the food and increase
the shelf life of fat rich foods due to the potent free
Free radicals are generated through normal reactions
radical quenching ability of these phytochemicals.
in human body. These physiologically produced rad-
icals can exert diverse functions like signaling roles
and providing defense against infections. However,
an excessive amount of reactive radicals can results
in cellular damage, which in turn, initiates several
Serlene Tomar diseases including atherosclerosis, arthritis, diabetes
Assistant Professor, Department of Livestock Products Technology, and cancer (Phaniendra et al. 2015). Under normal
College of Veterinary Science and AH, Rewa, MP, India conditions, antioxidant defense systems can remove
Annada Das* reactive species through enzymatic (like superoxide
PhD Scholar, Department of Livestock Products Technology, dismutase and glutathione peroxidase) and non-en-
College of Veterinary Science, WBUFAS, Kolkata 700037, India zymatic antioxidants (like antioxidant vitamins, trace
elements, coenzymes and cofactors). However, in
Narendra Singh Tomar
Veterinary Assistant Surgeon, Veterinary Hospital, Khajua, Rewa, certain circumstances, the endogenous defense system
MP, India fails to protect the body against reactive radicals on
Email: [email protected] its own resulting in oxidative stress, a condition in
*Corresponding author which the generation of highly reactive molecules
578

such as reactive oxygen species and reactive nitro- fected by conditions such as planting, environmental
gen species exceed their elimination and/or when and genetic factors such as cultivar, cultivation year,
their elimination is inadequate (Liu et al. 2008). In cultivation location and temperature with storage
humans, oxidative stress usually plays the role of a time and germination period having marked effects
promoter rather than an initiator of chronic diseases, on the phenolic compounds and is oflavones (Randhir
which has fostered research on plant sources and et al. 2004). Selection of the right legume varieties
the screening of raw materials for identifying new combined with a suitable germination process could
antioxidants. The antioxidant property is mainly im- provide good sources of bioactive compounds and
parted to their phenol-derived structure. Antioxidants significantly enhance the antioxidant properties of
“sacrifice themselves” by giving up a hydrogen atom, legumes is critical if legumes are to become a com-
then rearrange to a stable conformation. Plant-based petitive source of phytonutrients and incorporation in
foods, such as fruits, vegetables, whole grains and products as desirable functional ingredients or natural
legumes, which contain significant amounts of bio- antioxidants in new food formulations.
active phytochemicals, may provide desirable health
benefits beyond basic nutrition to reduce the risk of Sprouted legumes
developing chronic diseases (Liu 2004).
Germination causes important changes in the bio-
MATERIALS AND METHODS chemical, nutritional and sensory characteristics of
seeds (Smith et al. 2018). The reserve materials of
Legume - power house of bioactive compounds the seeds are degraded and used partly for respiration
and partly for synthesis of new cell constituents of the
Legumes believed to be rich sources of bioactive com- developing embryo during germination. In addition
pounds are recently been studied for their antioxidant of being a good source of basic nutrients, they also
properties. Consumption of legumes is also correlated contain important health promoting phytochemi-
to number of positive health benefits such as hypo- cals compared with their mature counterparts with
cholesterolemic, antiatherogenic, anticarcinogenic disease preventive and health promoting properties
and hypoglycemic properties, reduction in obesity, (Fernandez-Orozco et al. 2006). Sprouting mobilizes
reduction of coronary heart diseases, which has been polymerized forms, such as concentrated starch and
noted and confirmed (Angeles et al. 2021). These protein, into carbohydrates and free amino acids,
health benefits are known to be associated with dietary respectively (Randhir et al. 2004) and have substan-
fiber and phytochemicals like polyphenols which are tial nutritional benefits for the human body because
considered to be natural antioxidants, representing an of their high concentration of nutrients which can be
important group of bioactive compounds to be used used readily by the body. As a consequence of germi-
in functional foods and other applications. Wu et al. nation, some anti-nutritional factors such as trypsin
(2004) categorized a wide array of foods in terms of inhibitors, phytic acid and saponins, decrease or even
antioxidant capacity and concluded that most legume disappear during germination while some compounds
seeds ranked in the group with the highest activity with antioxidant effects such as phenolics, vitamins
(>2000 µmol Trolox equiv. /serving). Legumes, being and a variety of other bioactive compounds that act
excellent sources of bioactive compounds can be im- as reducing agents, free radical terminators, metal
portant sources of ingredients for uses in functional chelators and singlet oxygen quenchers increase
foods and other applications. Legumes are also rich (Doblado et al. 2007) increase during germination
in phenolic compounds ranging from 1.1 to 68 mg/g process.They are confirmed to possess diverse bio-
of dry weight (Vaz Patto et al. 2015). Phenolic acids activities, which could have many health benefits
and flavonoids are the most abundant phenolics in such as reduce the risks of cancer, heart disease and
legumes and they generally occur as free and bound diabetes, inhibition of plasma platelet aggregation, cy-
forms, the latter representing up to 50% of the total clooxygenase (COX) activity and histamine release,
phenolic content (Yeo and Shahidi 2015). The content in vitro antibacterial, antiviral, anti-inflammatory
of bioactive compounds of legumes is generally af- and anti-allergenic activities (Oak et al. 2005) and

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 579

prevent oxidation in foods and also act as protective lentil seeds and their sprouts against DPPH radical
factors against oxidative damage in the human body and observed that germination process modified the
(Kikuzaki et al. 2002). antiradical efficacy against DPPH of the legume seeds
but differently for each. Four to six day old germs
Effect of germination on phytochemical profile showed higher antiradical capacity than the raw seeds
and antioxidant activity of legumes and it was most remarkable after 4 days.

Legume germination has also been suggested as a Effect of different extraction solvents on antioxi-
powerful strategy to increase total antioxidant activity dant capacity of different legume extracts
(Fernandz-Orozco et al. 2006). During germination
the hydrolytic enzymes modify the endosperm and Solvent extraction is most frequently used technique
may liberate some of the bound components that play for isolation of plant antioxidant compounds. The
a role in antioxidant activity (Doblado et al. 2007). selection of solvent is supported by their different
Xue et al. (2016) reported that optimum germination chemical characteristics. Recovery of antioxidant
time for sprouts was 3-5 days when total bioactive compounds from plant materials is typically accom-
compound content and antioxidant activities both plished through different extraction techniques taking
reached their peak values. Phenolics and flavanoid into account their chemistry and even distribution
contributed the highest towards the antioxidant in the plant matrix. Soluble phenolics are present in
activity of mung, soyabean and black gram sprouts. higher concentrations in the outer tissues of grains
Xue et al. (2016) observed that vitamin C content than the inner tissues (Antolovich et al. 2000). An-
increased in a time-dependent pattern and reached tioxidant capacity of different plant extracts is de-
a peak on Day 6, amounting to 1.97, 1.94 and 1.93 pendent on the solvent nature, temperature, time, pH,
mg/g FW for mungbean, soybean and black bean, presence of oxygen, oxidative agents and enzymes.
respectively. The vitamin C contents increased in Polar solvents have been frequently employed for the
a time-dependent manner and reached peak on day recovery of polyphenol from a plant matrix. The most
8 at the concentration of 285±25.7 mg/100 g DW, suitable of these solvents are (hot or cold) aqueous
almost 24 times higher than the initial concentration mixtures containing ethanol, methanol, acetone and
in mungbean seeds (Guo et al. 2012). Doblado et ethyl acetate (Peschel et al. 2006). Methanol and
al. (2007) reported an increase in vitamin C content ethanol have been extensively used to extract anti-
in sprouted cowpeas and the content reached upto oxidant compounds from legumes. Studies have also
23.3 and 25.2 mg/100 g DM after 4 and 6 days of demonstrated the efficacy of ethyl acetate to extract
germination, respectively while no vitamin C was polyphenolic compounds from Vigna sinensis (Emy-
detected in raw seeds. Sood and Malhotra (2002) nur Shafekh et al. 2012). Polarity of the solvent is an
observed that vitamin C content of chickpea increased important factor in optimizing the antioxidant activity
between 4-7 times during germination while Plaza et of extracts. In general, the amount of total extractable
al. (2003) observed 54%, 218% and 919% increase compounds decreased with decreasing polarity of the
respectively in wheat, soybean and alfalfa after 96 solvent in the order of water, ethyl acetate, methanol
h at 28°C in the dark. Pajak et al. (2014) observed and hexane (Mariod et al. 2009).
more than 10-fold increase of antioxidant activity Hydrogen bonding may induce dramatic changes
against ABTS and DPPH radicals in mungbean after in the H atom donor activities of phenolic antioxi-
5-day germination and reported that this increment dants. Xu and Chang (2007) suggested 50% acetone
to be related with changes in the content of antioxi- was the best solvent for extraction of phenolics from
dants, such as vitamins and polyphenols while Guo peas, chickpea and yellow soybean, whereas acidic
et al. (2012) reported that the antioxidant activity of 70% acetone was the best for lentil, black soybean and
mungbean sprouts were six times higher than in the common beans while Nithiyanantham et al. (2012)
seeds as measured by the hydrophilic peroxyl radical found that 80% methanol served to be a better solvent
scavenging capacity. López-Amorós et al. (2006) for extraction of phenolics and tannins in chickpea
studied the antiradical efficacy of pea, bean and while 70% acetone, for green peas.

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580

RESULTS AND DISCUSSION Effect of germination on various antioxidant

assays
Effect of germination on total phenolic content
DPPH radical scavenging activity
Phenolic compounds occur as secondary metabo-
lites in all plants (Lin et al. 2016). They embrace Model of scavenging free radicals is widely used to
a considerable range of substances possessing an evaluate the antioxidant properties in relatively short
aromatic ring bearing one or more hydroxysubstitu- period, as compared to other methods. DPPH assay is
ents, although a more precise definition is based on widely used for assessment of free radical scavenging
metabolic origin as those substances derived from the potential due to its stability and simplicity, involving
shikimate pathway and phenyl propanoid metabolism only the direct reaction between the radical and an
(Ryan and Robards 1998). Antioxidant properties antioxidant (Kedare and Singh 2011). DPPH free
of polyphenols arise from their high reactivity as radical scavenging activity by antioxidants is due to
hydrogen or electron donors which can stabilize their hydrogen donating ability; the more the num-
and delocalize the unpaired electron and from their ber of hydroxyl groups, the higher the possibility of
potential to chelate metal ions (termination of Fenton free radical scavenging ability. Marathe et al. (2011)
reaction) (Rice-Evans et al. 1996). classified cowpea (brown, red), common beans, pea-
nut, horse gram and fenugreek as legumes with high
antioxidant activity while pigeonpea, green gram,
Sreeramulu et al. (2009) reported that pulses and chickpea (small brown), cowpea (white), common
legumes had a TPC that ranged from 62.35 to 418.34 bean (maroon), black gram, lentils and soyabean ex-
mg/100 g while Marathe et al. (2011) reported a TPC hibited moderate DPPH activities. Chickpea (cream,
in the range of 0.325- 6.378 mg/g GAE in various green and big brown), moth bean, butter bean, pea
commonly consumed legumes with the highest and common bean (white) had lowest antioxidant
concentration being recorded in cowpea (red and activities. DPPH radical scavenging activity ranged
brown). Karthiga and Jaganathan (2013) investigated from 0.26–1.07 trolox equ mg/g among different
commonly used pulses namely, bengal gram, black pulses as observed by Sreeramulu et al. (2009). They
gram, green gram, red gram and rajmah to conclude reported highest activity in P. vulgaris followed by
that TPC ranged from 345.2 to 54.43 mg GAE/100 green pea, green gram, red gram, bengal gram and
g. Fouad and Rehab (2015) observed a significant black gram. Dark seed colored cowpea extracts had
increase in the TPC (mg/100 g DW) of lentil seeds higher antioxidant activities as compared to light
during germination which increased from 1341.13 colored seeds however the activity was significantly
in raw lentil seeds to 1630.20 during germination. lower than that of standard antioxidants (Siddhuraja
Pajak et al. (2014) reported that TPC (mg/100 g and Becker 2007). Xu and Chang (2007) suggested
DM) ranged from 0.72 in mung seeds to 9.95 in 70% methanol to be the best solvent for study of
mung sprouts where gallic and ferulic acids were DPPH activity from yellow pea and chickpea while
the predominant compounds. Huang et al. (2014) 80% acetone to be a better solvent for lentil, black
reported 330% increase in TPC in 4 day germinated soyabean, yellow soyabean and red kidney beans.
soyabean as compared to their seeds. Lin and Lai Nithiyanantham et al. (2012) observed significant dif-
(2006) reported that germination enhanced phenolics ferences between antioxidant activities of methanolic
content in soybean with the highest content in 4-day (IC50 of 12.17g extract/g DPPH) and acetonic (3.72 g)
germinated bean. Gharachorloo et al. (2013) observed raw chickpea extracts thereby reflecting the fact that
that germinated seeds of lentil, chickpea, white bean acetone extracts had a greater antioxidant potential
and common vetch had higher TPC than their seed while in case of raw peas higher DPPH (IC50 13.67 g
counterparts irrespective of the solvent used. Pasko et extract/g DPPH) scavenging activity was observed in
al. (2009) also reported higher TPC in sprouts com- methanolic extracts. Higher DPPH inhibition activ-
pared to seeds, suggesting that synthesis of phenolic ities were registered in ethyl acetate extracts of raw
antioxidants may occur during germination. and sprouted green beansas compared to methanol,

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 581

n-hexane and n-butanol extracts (Kim et al. 2012). associated with the presence of reductones and the an-
Gharchaloo et al. (2013) reported that acetone extracts tioxidant action of reductones is based on the breaking
of chickpea sprouts functioned more efficiently at of free-radical chain by donating a hydrogen atom.
delaying oxidation in tallow as compared with hexane The reducing power of a compound serves as a signifi-
and methanolic extracts, which might be due to the cant indicator of its potential antioxidant activity (Das
differences in solvents polarity and consequently the et al. 2012). Reducing power explains some aspect
type of the extracted compounds. Ethanolic extracts of antioxidant activity and can be directly related to
of raw and sprouted lentil seed exhibited 40.76 to the amount of phenolic compounds. Djordjevic et
62.19% inhibition of DPPH free radical (Fouad and al. (2011) reported significantly greater FRAP for
Rehab 2015). The antioxidant capacity of raw cow- mungbeanethanolic extract (24.98 nmol mg-1 extract)
peas (27.4 µmol TEAC/g DM) increased by 58% and when compared with soybean ethanolic extract (8.34
67% after 4 and 6 days of germination respectively nmolmg-1extract). Kumar et al. (2010) observed
(Doblado et al. 2007). that FRAP value of yellow soybeans ranged from 11
to 28 FE µmol g-1, while of black soybeans and red
ABTS radical scavenging activity kidney beans ranged from 12.7-99.3 and 28.5-92.2 FE
µmol g-1 respectively. Anwar et al. (2007) observed a
ABTS, generated from oxidation of 2, 2-azino-bis (3 linear correlation between TPC and reducing power.
ethylbenzothiazoline-6-sulphonic acid) by potassium Siddhuraju and Becker (2007) observed that raw seed
persulfate, is presented as an excellent tool for deter- extracts of cowpea showed higher FRAP antioxidant
mining the antioxidant activity of hydrogen-donating activity, DPPH and ABTS activity besides high
antioxidants (scavengers of lipid peroxyl radicals). correlation between total phenolics and antioxidant
Marathe et al. (2011) grouped cowpea (red and activity as compared to processed cowpea extracts.
brown), soyabean, common beans (red, brown), horse
gram and fenugreek under legumes showing high Application in dairy products
ABTS activity (>12 µmol TEAC/g) and high phenolic
content while common bean (white), lablab bean, pea Currently, the potential health benefits of the phy-
(black), pigeonpea, green gram, chickpea, cowpea tochemicals and their ability to be incorporated into
(white), common bean, lentil and black gram showed dairy foods as nutraceuticals has received consid-
moderate activity (6-12 µmol TEAC/g) against ABTS erable interest in food industry. There has been an
free radical. Legumes with low phenolic content increase in the addition of phenolic compounds to
such as lablab bean (white, cream), chickpea (cream, dairy products, thus improving their technological
green and big brown), butter bean and pea (white functionality and nutritional value (O’Connell and
and green) showed lowest antioxidant activity (<6 Fox 2001). Attempts have been made to produce
µmol TEAC/g). Acetone extracts of raw chickpea fermented dairy foods supplemented with poly-
exhibited better ability to quench free radicals by phenolic compounds from grape wine (Howard et
the ABTS assay as compared to their methanolic al. 2000), grape pomace (Tseng and Zhao 2013),
counterparts (Nithiyanantham et al. 2012) while the aronia juice (Nguyen and Hwang 2016), Hibiscus
same authors reported that methanolic extracts of raw extract (Iwalokun and Shittu 2007), tea infusions
pea exhibited higher ABTS (6155 Troequ (mmol/g (Najgebauer- Lejko et al. 2011), apple polyphenols
extract) scavenging activity. Siddhuraju and Becker (Sun-Waterhouse et al. 2012), polyphenolic extract
(2007) reported that raw cowpea seed samples with of strawberry (Singh et al. 2013), sour cherry pulp
the highest TPC have more ability to quench free (Sengul et al. 2012), strawberry extract (Singh et
radicals (ABTS+). Villalobos et al. (2016) reported al. 2013) and ethanolic extract of pomegranate peel
60 % inhibition of the ABTS radical in defatted (Sandhya et al. 2018). Various legume extracts have
soyabean flour. been suitably used to prepare milk shakes containing
legume extract-bovine milk blends in the ratio 50:50.
Reducing power assay
These milk blends were found to be sensorially ac-
The reducing properties of antioxidants are generally ceptable to consumers (Preeti and Sheel 2015). These

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582

products are considered functional dairy products Fernandez-Orozco R, Piskula MK, Zielinski H, Kozlowska H, Frias
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