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PII: S0308-8146(18)30058-X
DOI: https://doi.org/10.1016/j.foodchem.2018.01.049
Reference: FOCH 22238
Please cite this article as: Xu, M., Jin, Z., Peckrul, A., Chen, B., Pulse Seed Germination Improves Antioxidative
Activity of Phenolic Compounds in Stripped Soybean Oil-in-Water Emulsions, Food Chemistry (2018), doi: https://
doi.org/10.1016/j.foodchem.2018.01.049
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Pulse Seed Germination Improves Antioxidative Activity of
Emulsions
Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA
*
To whom correspondence should be addressed. Tel. (701) 231-9450, e-mail:
bingcan.chen@ndsu.edu
1
ABSTRACT
compounds extracted from germinated pulse seed including chickpeas, lentils and
yellow peas. Phenolic compounds were extracted at different germination time and
emulsions. The results suggested that germination time is critical for phenolic
emulsion system with the order of chickpea>yellow pea> lentil. On the basis of these
2
1. Introduction
Pulse grains are a dry edible variety of beans, peas, and lentils that have been
consumed as staple foods in many regions throughout the world for centuries. Pulses
are rich in plant based proteins, carbohydrates, dietary fibers, and minerals (Vaz Patto
et al., 2015). In addition to nutritional properties, a great deal of attention have been
devoted to the phenolic compounds in pulses due to their health benefits for the
Fernandez-Orozco et al., 2009; Shahidi & Yeo, 2016). However, the application of
food is related to high total phenolic content (TPC). Unlike fruits which have been
(GAE)/100 g), most of pulse crops can only be categorized as having a low to
medium TPC (~100 mg GAE/ 100 g) (Vasco, Ruales, & Kamal-Eldin, 2008). In
and varieties. Padhi et al reported TPC of 14 commercial Canadian pulse cultivars and
found green lentil had the highest TPC being about 6 times higher than in pea and
great interest to effectively boost the TPC and antioxidative activity of pulses as
functional ingredients.
3
Germination is an efficient and economical means for improving antioxidant contents
wheat and oat (Mäkinen & Arendt, 2015). Phenolic compounds can be classified as
free, soluble bound and insoluble bound phenolics; free phenolics mainly exist as
phenolic acids and flavonoids; soluble bound phenolics are recognized as esterified
phenolics; insoluble bound phenolics are typically involved in the cell wall combined
with cellulose, lignin and arabinoglycan (Cheynier, Comte, Davies, Lattanzio, &
transformed. For instance, both soluble and insoluble phenolics were reported to
and yellow pea (Yeo & Shahidi, 2015). However, the decrease of soluble phenolics in
lentil were also reported after germination for 2 days (López-Amorós, Hernández, &
Estrella, 2006), while Yeo and Shahidi (Yeo & Shahidi, 2015) reported that both
during germination are complicated, which making it difficult to estimate the most
effective extract antioxidant composition over the course of germination using in vitro
assay.
In this study, oil-in-water emulsions prepared using stripped soybean oil (free of
yellow pea (Pisum sativum L.) and lentil (Lens culinaris Merr.) at varying stage of
4
germination process. The objectives of this study were therefore to i) assess the
impact of germination time on TPC; ii) investigate the impact of germination time on
the antioxidative activity in vitro and in the emulsion system; iii) make comparison of
germination time.
Chickpea (Cicer aretinium L.), lentil (Lens culinaris Merr.) and yellow pea (Pisum
sativum L.) were gifted from JM Grain (Garrison, ND, USA), Viterra Inc (Grand
Forks, ND, USA), and AGT Food and Ingredients (Minot, ND, USA). Commercial
ferrozine were purchased from Acros Organics (Morris Plains, NJ, USA); 2, 2’-azobis
thiocyanate, hexanal, hexanol, gallic acid, Folin Ciocalteu’s phenol reagent were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were
seeds were soaked in 2000 mL of 0.07% sodium hypochlorite solution for 30 min.
The seeds were then washed with distilled water until reaching neutral pH and
subsequently soaked in 2000 mL of distilled water for 5.5 h with shaking every 30
min. The hydrated seeds were stored on and covered by wet laboratory paper in
germination trays, which were in contact with circulating moist air to maintain the
relative humidity and the moisture of seeds at 99% and 50%, respectively. Seeds were
germinated at 25 °C in the dark for 6 days. The sprouted seeds at 2, 4, or 6 days into
the germination process were collected, freeze-dried, and ground in a IKA M20 mill
(Staufen, Germany) fitted with a 0.5 mm sieve. The ground flour was placed in plastic
Twelve grams of ground sample was defatted with 60 mL of n-hexane by shaking for
1 h under nitrogen, and then centrifuged at 2,000×g for 5 min and supernatants
collected. The defatting was repeated and the two defatted meal samples were
combined. The defatted meal was mixed with 60 mL of acetone/H2O (7/3, v/v),
purged with N2, and then shaken for 1 h. After centrifugation of the resulting slurry
for 5 min at 2,000×g, the supernatant was collected. The extraction was repeated and
6
supernatants were combined. The crude phenolic solution was then prepared by
evaporating the acetone from the supernatants at 40 °C with rotary evaporation (RE
111 Buchi, Flawil, Switzerland), and the pH of the solution was adjusted to 2~3. Free
phenolic compounds were extracted from the crude solution by partitioning 3 times
with ethyl ether/ethyl acetate (1/1, v/v). The supernatants were combined and the
solvent removed with rotary evaporation. Remaining solvent traces were removed
with nitrogen purge. The dried samples were redissolved using 15 mL methanol, the
solution transferred into storage vials and the methanol removed by purging with
nitrogen. The vials were stored at 4 °C. Soluble bound phenolic compounds were
collected by evaporating any remaining organic solvent from the subnatant of the
ethyl ether/ethyl acetate solution. The remaining extract was transferred to vials using
The TPC of each extract was determined using the method described by Alshikh and
Shahidi (Alshikh, de Camargo, & Shahidi, 2015) with modification. The mixture of
deionized water (3 mL), the phenolic extract (150 µL), and Folin-Ciocalteu’s reagent
solution (250 µL) were vortexed and incubated for 5 min at room temperature, followed
by the addition of 20% sodium carbonate (750 µL) to each tube. The solution were
stored in dark at ambient temperature for 1 h. The absorbance was measured at 765 nm
(VWR 6300 Double Beam UV-Vis Spectrophotometer, VWR, Palo Alto, CA, USA)
using distilled water as a blank. TPC was expressed as gallic acid equivalents (mg of
7
GAE/g dry pulse) using the calibration curve of gallic acid.
radical scavenging capacity (DPPH) of extracts from the pulse and their two fractions
were monitored as described by Yu (Yu, 2008) using a Fluostar Optima plate reader
(BMG Labtech, Chicago, IL, USA). The final ORAC and DPPH values were
calculated as Trolox equivalents (TE) per mL extract (µmol TE/mL) using a standard
The ability of phenolics extracted from germinated pulses to bind iron was determined
using a modified method of Chen et al (Chen, Mcclements, & Decker, 2010). The
iron-binding capacity was expressed as % iron bound per mL of extracts, which was
total × 100%
Stripped soybean oil (SSO) was prepared according to the method of Chen et al (Chen,
Mcclements, & Decker, 2014). In short, silicic acid (100 g) was washed three times
8
sequentially with 22.5 g of silicic acid, followed by 5.625 g of activated charcoal and
then another 22.5 g of silicic acid by the dry method. Thirty grams of soybean oil was
flask oil was covered with aluminum foil. The solvent in the stripped oil solution was
removed with a vacuum rotary evaporator (RE 111 Buchi, Flawil, Switzerland) at
37 °C (Buchi 461 water bath, Flawil, Switzerland), and the remaining trace solvent
removed by flushing with nitrogen for at least one hour. Fifteen grams of colorless
SSO were collected, mixed, and kept at -80 °C for subsequent studies.
0.2 wt% Tween 20 and 0.01 M phosphate buffer using a high-speed blender
Processor, Microfluidics, Newton, MA) for one pass at a pressure of 5,000 psi and
two passes at a pressure of 10,000 psi. The emulsions were kept on ice over the whole
procedure to minimize oxidation. After homogenization, 0.04 wt% sodium azide was
Pulse extract was added into freshly prepared SSO-in-water emulsion at the
aliquot of mixture was removed and placed into a 20 mL vial sealed with an
9
aluminum cap. These vials were stored in an incubator at 25 °C and covered with
The emulsion particle size was directly measured using a laser light scattering
Lipid hydroperoxides were determined using a method adapted from Chen and
Hexanal and hexanol were determined directly from the sealed vial. Analyses were
performed with Agilent Technologies 7890B GC system and 5977A mass detector
equipped with a PAL RSI 120 autosampler. Subsamples (0.5 mL) in 20 mL glass vials
capped with aluminum caps with PTFE/silicone septa were incubated at 55 °C for 15
Supelco, Bellefonte, USA) was inserted into the vial for 2 min to absorb volatiles and
then transferred to the injector port (250 °C) for 3 min. The injection port was
operated in split mode, and the split ratio was set at 5:1. Volatiles were separated on a
10
ZB-Wax column (60 m × 0.25 mm i.d. × 0.25 µm film thickness). Oven temperature
program set as: Initial temp 40 °C, hold 4 min, to 70 °C at 10 °C/min, hold 6 min,
then from 70 to 250 °C at 100 °C/min, hold 3 min. Helium carrier gas was used at a
flow rate of 1 mL/min. Carry over and peaks originating from the fiber was regularly
assessed by running blank samples. After each analysis fiber was immediately
The MS was operated in electron impact (EI) ionization mode at 70 eV. Ion source
temperature was 230 °C. Selected ion monitoring (SIM) was used for the detection of
hexanal (m/z: 72 and 82) and hexanol (m/z: 55, 56, and 69). The dwell time was set to
100 ms. Identification of hexanal and hexanol were carried out by comparing GC
and hexanol were determined in duplicate as relative area using the calibration curve.
measurements. The data were statistically analyzed using statistical software, SAS
version 9.4 (SAS institute Inc. Cary, NC). One-way analysis of variance (ANOVA)
was conducted, and significant difference was defined at p < 0.05 by Tukey’s test.
Total phenolic content of soluble bound phenolic compounds and free phenolic
11
compounds were determined with Folin-Ciocalteu’s assay, and results were shown in
Fig. 1. Total soluble phenolic compounds (free + soluble bound phenolics) of the
ungerminated chickpea, lentil and yellow pea were 1.04, 8.43, and 1.17 mg/g dry
pulse, respectively, which was close to the value summarized by Amarowicz and Pegg
(Amarowicz & Pegg, 2008), with chickpea, lentil, and yellow pea being in the range
Figure 1 inserted
After germination, the content of total phenolics have changed in both soluble bound
and free phenolics. For instance, germination time was directly proportional to TPC in
chickpea and yellow pea, while it was inversely proportional to TPC in lentil. In
chickpea (Fig. 1A) and yellow pea (Fig. 1C), the original TPC of soluble bound
phenolics (0.81 mg GAE/g dry chickpea and 0.85 mg GAE/g dry yellow pea) and free
phenolics (0.23 mg GAE/g dry chickpea and 0.33 mg GAE/g dry yellow pea) were
lower. After 6 days germination, TPC values of both soluble bound (1.40 mg GAE/g
dry chickpea and 1.95 mg GAE/g dry yellow pea) and free phenolics (0.96 mg GAE/g
dry chickpea and 0.75 mg GAE/g dry yellow pea) increased significantly (P<0.5).
This trend was the same as the germination effect on antioxidant concentration
al., 2006), both of whom observed the significant increase in TPC of chickpea and
yellow pea after germination. On the contrary, when it came to lentil (Fig. 1B), TPC
decreased from 8.43 to 3.07 mg/g dry pulse, which was mainly caused by the decrease
12
of soluble bound phenolic compounds from 7.68 to 2.59 mg/g dry pulse. The TPC
change of free phenolic compounds was relatively small, from 0.377 to 0.748 mg/g
dry pulse, and the ungerminated lentil had the biggest TPC value. It was also observed
lentil (IC50 ~4.9-5.1 ) exhibits greater antioxidant activity than the germinated form
(IC50 ~5.9-10.9).
The various pulse crop seeds had different germination attributes. It can be assumed
that, in chickpea and yellow pea, the synthesis rate of soluble phenolic compound was
larger than the consumption rate, whereas the opposite trend was observed in lentil.
This trend was also reported by some researchers (Bartolomé et al., 1997;
López-Amorós et al., 2006; Tarzi et al., 2012) that chickpea and yellow pea had an
In addition, it was worth noting that soluble phenolic compounds in lentil were
initially so high that even after 6 days of decreasing, TPC value was still greater than
that in chickpea and yellow pea. This was reasonable because TPC value of
ungerminated lentil was reported to be 6-8 times larger than chickpea and yellow pea
(Amarowicz & Pegg, 2008; Vaz Patto et al., 2015; Xu & Chang, 2007).
extracts
13
It was obvious that the content of phenolic compounds had changed during
germination, the question was that if the composition of phenolic compounds had also
changed during germination and how would it impact antioxidative activity? To sort
this out, all the extracts of pulses were diluted into the same concentration of TPC, i.e.,
187 µg GAE/mL extracts and antioxidative activity of each extract was assessed using
in vitro assays, including free radical scavenging ability and metal chelating ability.
Free radical scavenging capacity of natural antioxidant can be classified as two major
mechanisms: hydrogen atom transfer (HAT) and electron transfer (ET) (Shahidi &
antioxidants to inactivate free radicals generated in the system, thus suspending the
with the transfer of electron from antioxidants to the free radicals (Galano et al.,
2016).
The ORAC assay is an HAT-based radical scavenging method, which measures the
inhibition ability against the peroxyl radical-induced oxidation. As the peroxyl radical
is the predominant initiating radical found in food system, ORAC values are
radicals generated by AAPH can damage the fluorescent probe. Phenolic compounds
extracted from pulses could react with the peroxyl radical by donating the hydrogen
atom to retard the damage of fluorescent probe (Shahidi & Zhong, 2015).
Table 1 inserted
14
With the data shown in Table 1, free phenolic compounds in chickpea and yellow pea
had an increasing trend of ORAC during germination, from 2.85 and 2.18 µmol
TE/mL extraction to 5.82 and 4.60 µmol TE/mL extraction, respectively. With the
lentil, the ORAC of free phenolic compounds maintained steady within 4 days
germination (p > 0.05) and increased to 3.51 µmol TE/mL extraction after 6 days. As
to the ORAC of soluble bound phenolic compounds, germination had little influence
on chickpea in the first four days. However, we observed a 2.5-fold increase after 6
days germination. Conversely, a decline of ORAC over the course of germination was
exhibited from soluble bound phenolic compounds of both lentil and yellow pea.
Brunswick (Rock & Brunswick, 2005) argued that HAT had also happened as a
marginal pathway. DPPH is a stable radical due to the resonance stabilization and
compounds extracted from pulse based on ET, which also called reducing power
(Shahidi & Zhong, 2015). DPPH radical scavenging assay results were shown in
Table 1. Each of pulse seeds displayed unique pattern for DPPH. Much the same as
ORAC value, free phenolic compounds in chickpea had an increasing trend of DPPH
radical scavenging ability during germination, from 11.9 initially to 80.3 µmol TE/L
extraction after 6 days germination. The DPPH of free phenolics in yellow pea was
independent of germination. However, the DPPH radical scavenging ability of the free
phenolic compounds in lentil had a moderate decrease from 104.8 to 82.1 µmol TE/L
extraction.
15
When it came to the DPPH radical scavenging ability of soluble bound phenolic
compounds, the soluble bound phenolic compounds extracted from lentil had the
influence on lentil. The DPPH radical scavenging ability had an increase in the
chickpea during germination being from 45.7 to 75.7 µmol TE/L extraction.
in yellow pea since continuous decrement was observed, with DPPH dropped from
Conclusions drawn from radical scavenging ability assays, both ORAC and DPPH,
indicated that radical scavenging ability of both extracts from chickpea and free form
from yellow pea increased during germination. Khattak and coworkers (Khattak, Zeb,
Nizakat, Khalil, & Khattak, 2007) reported that the methanol extracted phenolics
indicated that the composition of phenolic compounds had changed over the course of
faster in chickpea and yellow pea than that of soluble bound phenolic compounds,
which can infer that the newly synthesized species of free phenolic compounds have
higher in vitro antioxidative activity than that of soluble bound phenolic compounds.
during germination, which contrasted with chickpea and yellow pea. The sharp
Bartolomé and coworkers (Bartolomé et al., 1997) may result in the decrease of
16
antioxidative activity of lentil.
The antioxidative activity of extracts from pulses are closely related to the
Previous studies suggested that the major phenolic compound in chickpea (e.g. ferulic
acid, caffeic acid, p-coumaric acid, chlorogenic acid, vanillic acid, protocatechuic acid,
syringic acid, sinapic acid), lentil (e.g. catechins, trans p-coumaric acid, quercetin
hydroxybenzoic acid), and yellow pea (e.g. kaempferol, ferulic acid, sinapic acid,
quercetin, vanillic acid, p-coumaric acid, caffeic acid) were responsible for the
antioxidative activity of pulse (Vaz Patto et al., 2015). Further research is needed to
elucidate the relationship between phenolic composition and free radical scavenging
Metal ions are critical factors for lipid oxidation since they can actively react with
2011). In addition, metal ions can also promote the decomposition of lipid
McClements, & Decker, 2012). The antioxidative properties of a chelator can often be
dependent on the synergistic effect of functional groups such as –OH, –SH, –COOH,
–PO3H2, C=O, –NR2, –S– and –O– (Fennema, 1996). As to the free phenolic
compounds, the chelating ability of antioxidants are most probably formatted by the
extract was added into 5 mM Fe2+ solution to determine their iron binding capacity.
Free phenolic compounds from ungerminated chickpea had the highest iron binding
capacity and the values decreased during first 4 days germination (Table 1). The iron
binding capacity of lentil free phenolic compounds reached maximum after 2 days
germination, then gradually decreased over the course of germination. Yellow pea
showed similar pattern as chickpea and ungerminated one had the highest iron binding
capacity. This result indicated that the structure of free phenolic compounds in
It would be expected that soluble bound phenolics will carry certain iron binding
bound with. For instance, some research have demonstrated that certain anionic
polysaccharides such as xanthan gum, pectin and sodium alginate could potential act
as antioxidants in oil-in-water emulsions due to its higher iron binding capacity (Qiu,
Zhao, Decker, & McClements, 2015; Salvia-Trujillo, Decker, & McClements, 2016).
antioxidative activity in food system and strong iron binding capacity was one of the
major mechanisms (Tong, Sasaki, McClements, & Decker, 2000). It was unclear why
all the soluble bound phenolic compounds extracted from pulses had little to no iron
binding capacity. One possible reason was that large molecule of polysaccharides or
protein trapped the phenolic compounds and formatted steric hindrance between
metals and phenolics when conjugated with phenolic compounds (Fennema, 1996;
18
Mozuraityte, Kristinova, Rustad, & Storr∅, 2016).
oil-in-water emulsions
In vitro assays proved that the phenolic compositions of pulse extracts were variable
in vitro assays mainly focused on specific or part of the antioxidative activity rather
than oxidative stability of real foods. The antioxidative activity assessed by in vitro
time.
The initial average droplet size (d32) of SSO emulsified by Tween 20 in phosphate
buffer solution (pH 7.0) was 0.446 µm. After adding 200 µg GAE/g oil phenolic
compounds, the average droplet size ranged from 0.446-0.602 µm during the storage
emulsion during storage. Both droplet analysis and observed results indicated no
hexanol.
pea. Free phenolic compounds extracted from both 4 and 6 days germinated chickpea
suppressed the formation of lipid hydroperoxides and retarded lag phase for 3 days,
while chickpea free phenolic compounds germinated for 0 and 2 days showed 2 days
lag phase same as control (Fig. 2). Among all the extracts of free phenolic compounds,
emulsions incorporated with yellow pea extracts with 6 days germination showed the
longest lag phase of 4 days (data not shown). Surprisingly, the lag phase of emulsion
in the presence of free phenolic compounds extracted from lentil remained the same
These results showed that antioxidative activity of free phenolic compounds increased
with the germination time in chickpea and yellow pea. As proved by in vitro assay, the
antioxidative activity of free phenolic compound of chickpea and yellow pea had
increased during germination. This can account for the enhancement of antioxidative
activity of these extracts in emulsion system. The relationship between in vitro assay
and emulsion system hold in germinated lentil in that both TPC and DPPH had
decreased during germination, probably explaining why the lentil free phenolic
3.4. Antioxidative activity of soluble bound phenolic extracts from germinated pulses
in oil-in-water emulsions
much more complicated than that of free phenolic compounds. Soluble bound
20
phenolic compounds from chickpea and yellow pea showed more sustainable
antioxidative activity in emulsions than the free forms. The soluble bound phenolic
compounds extracted from both ungerminated chickpea (Fig. 4A) and yellow pea
(data not shown) generated 10 days lag phase as indicated by the formation of lipid
germination time is proportional to the antioxidative activity. The longest lag phase of
14 days was observed when soluble bound phenolic compounds extracted from
chickpea with 6 days germination was presented in emulsion. However, this was not
the case for both lentil and yellow pea. The germination time has no impact on the
extracted from yellow pea as evidenced by lipid hydroperoxides (data not shown).
Hexanal was widely used as secondary oxidation markers for high linoleic oil systems
oxidation study. It was true in emulsion system when free phenolic compounds were
presented (Fig. 2-3). Unexpectedly, hexanal did not increase significantly during
storage in the presence of soluble bound phenolic compounds, ever though the lipid
volatile marker to differentiate the lag phase of emulsions with different soluble
bound phenolic compounds (Fig. 4B & 5B). The TIC scan of emulsion oxidation
21
volatiles indicated the remarkable production of hexanol during storage (graphical
abstract). In this situation, the formation of hexanol in emulsions was also recorded
along with hexanal during storage. As one can see in Fig. 4C & 5C, the formation of
antioxidative activity as aroma threshold of hexanol is 4000 µg/L (Siebert et al., 2005)
which is much higher than that of hexanal, 75 µg/L (Buttery, Guadagni, & Ling, 1973).
However, it was unclear if there were any constituents owning strong reducing power
Unlike free phenolic compounds which might also be oxidized by oxygen and
transition metals rather than retarding lipid oxidation (Zhou & Elias, 2012), soluble
bound phenolic compounds extracted from chickpea and yellow pea exerted stronger
them would be transported to the cell wall and part of them would become soluble
& Yeo, 2016). Some of constituents conjugating with phenolic compounds were
compounds (Xie, Hu, Wang, & Zeng, 2014). Our iron binding capacity study
suggested that the greater antioxidative activity exerted by soluble bound phenolic
22
compounds was not because of its metal chelating ability. It was more likely that the
Markovic, & Amic, 2013). Moreover, steric hindrance, caused by large molecular of
conjugated biopolymers, may happen to the soluble bound phenolic compounds which
phenolic compounds from lentil had little antioxidative activity, similar as its free
form, to prevent emulsion oxidation. It may be caused by the TPC of lentil which was
2-8 times greater than that of chickpea and yellow pea, so as the dilution ratio to the
final concentration (i.e., 187 µg GAE/mL), which may dilute synergistic effect upon
antioxidative activity.
emulsion system cannot be correlated with in vitro assays. The antioxidative activity
of soluble bound phenolic compounds described by ORAC and DPPH was improved
by germination much less than that of free phenolic compounds in chickpea and
yellow pea (Table 1), whereas soluble bound phenolic compounds of chickpea (Fig. 4)
with 6 days germination and ungerminated yellow pea (data not shown) had
and in real food system has been reported (Alamed, Chaiyasit, McClements, &
Decker, 2009). This again suggests that in vitro assays have limited value in
23
Alternatively, the results from in vitro assay could be applied to unravel the
4. Conclusion
assay. Phenolic compounds extracts from germinated chickpea and yellow pea
germinated chickpea and yellow pea and can be exploited as novel natural
interesting phenomenon was observed that the soluble bound phenolic compound
extracted from pulses had the ability to generate more hexanol than hexanal.
Acknowledgment
This work is supported by the USDA National Institute of Food and Agriculture,
24
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Figure captions
Figure 1 Total phenolic content of chickpea (A), lentil (B), and yellow pea (C) over the course of germination. F, B, S, C, L and Y denotes free
phenolics, soluble bound phenolics, total soluble phenolics (free phenolics + soluble bound phenolics), chickpea, lentil and yellow pea
respectively. For example, CF stands for free phenolics in chickpea. Data points represent means ± standard deviations. Different letters around
Figure 2 Formation of lipid hydroperoxides (A) and hexanal (B) in SSO-in-water emulsion system, without (control) and with addition of 200
μg GAE/g oil free phenolic compounds extracted from chickpea. Data points represent means ± standard deviations.
Figure 3 Formation of lipid hydroperoxides (A) and hexanal (B) in SSO-in-water emulsion system, without (control) and with addition of 200
μg GAE/g oil free phenolic compounds extracted from lentil. Data points represent means ± standard deviations.
Figure 4 Formation of lipid hydroperoxides (A), hexanal (B), and hexanol (C) in SSO-in-water emulsion system, without (control) and with
addition of 200 μg GAE/g oil bound phenolic compounds extracted from chickpea. Data points represent means ± standard deviations.
Figure 5 Formation of lipid hydroperoxides (A), hexanal (B), and hexanol (C) in SSO-in-water emulsion system, without (control) and with
addition of 200 μg GAE/g oil bound phenolic compounds extracted from lentil. Data points represent means ± standard deviations.
Figure 1
2.5 10 3.0
CF A LF B YF dS C
dS 9 aS
CB cS LB YB
Total phenolic content (mg/g dry pulse)
Control Control
A 300 B
20 ungerminated ungerminated
germinated 2 days germinated 2 days
germinated 4 days 250 germinated 4 days
Lipid hydroperoxide (mmol/L)
Hexanal (μmol/L)
200
10 150
100
5
50
0
0
1 2 3 4 5 6 1 2 3 4 5
Incubation time (days) Incubation time (days)
Figure 3
Control Control
A 300 B
20 ungerminated ungerminated
germinated 2 days germinated 2 days
germinated 4 days 250 germinated 4 days
Lipid hydroperoxide (mmol/L)
Hexanal (μmol/L)
200
10 150
100
5
50
0
0
1 2 3 4 5 6 1 2 3 4 5
Incubation time (days) Incubation time (days)
Figure 4
400 120
25 Control Control Control
A B C
ungerminated ungerminated ungerminated
germinated 2 days germinated 2 days 100 germinated 2 days
20 germinated 4 days 300 germinated 4 days germinated 4 days
Lipid hydroperoxide (mmol/L)
Hexanol (μmol/L)
15
200 60
10
40
100
5
20
0
0
0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Incubation time (days) Incubation time (days) Incubation time (days)
Figure 5
400 120
Control Control Control
A B C
25 ungerminated ungerminated ungerminated
germinated 2 days germinated 2 days 100 germinated 2 days
germinated 4 days 300 germinated 4 days germinated 4 days
Lipid hydroperoxide (mmol/L)
Hexanal (μmol/L)
Hexanol (μmol/L)
15 200 60
10
40
100
5 20
0
0 0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Incubation time (days) Incubation time (days) Incubation time (days)
Graphic Abstract
Table 1 Antioxidative activity of ungerminated and germinated pulse estimated by in
vitro assaya
DPPHb ORACc Iron binding capacity
Germination
(µmol TE/L (µmol TE/mL (µmol Fe2+/mL
(days)
extracts) extracts) extracts)
------Free phenolics------
0 11.9±5.6 a 2.85±0.15 a 0.91±0.04 d
Chickpe 2 62.3±1.4 b 2.96±0.48 a 0.33±0.00 b
a 4 77.6±1.2 c 4.96±0.84 b 0.10±0.01 a
6 80.3±1.6 c 5.82±0.57 b 0.49±0.02 c
0 104.8±0.7 c 2.14±0.12 a 0.25±0.01 a
2 101.4±1.1 c 2.10±0.09 a 0.71±0.02 d
Lentil
4 87.0±2.1 b 2.28±0.25 a 0.66±0.00 c
6 82.1±2.3 a 3.51±0.26 b 0.53±0.01 b
0 66.3±3.1 a 2.18±0.03 a 0.84±0.01 c
Yellow 2 62.0±2.4 a 1.73±0.13 a 0.63±0.01 b
pea 4 65.6±1.8 a 3.27±0.20 b 0.77±0.01 bc
6 61.5±1.4 a 4.60±0.79 c 0.22±0.11 a
------Soluble bound phenolics------
0 45.7±3.6 a 0.61±0.04 a Ndd
Chickpe 2 64.9±6.4 bc 0.91±0.13 a Nd
a 4 61.1±5.1 b 0.80±0.24 a Nd
6 75.7±3.5 c 1.51±0.30 b Nd
0 122.4±0.5 a 2.49±0.40 c Nd
2 122.4±0.3 a 1.69±0.24 b Nd
Lentil
4 119.3±3.7 a 0.86±0.02 a Nd
6 117.6±0.0 a 0.89±0.17 a Nd
0 101.4±5.6 c 0.65±0.02 d Nd
Yellow 2 63.8±1.3 b 0.44±0.02 c Nd
pea 4 39.8±1.6 a 0.14±0.05 a Nd
6 45.5±4.9 a 0.19±0.10 b Nd
a
Data points represent mean (n=3) ± standard deviation. Different letters indicate statistically
significant differences intraspecies (p<0.05).
b
DPPH; 2,2-diphenyl-1-picryhydrazyl radical scavenging capacity.
c
ORAC; oxygen radical absorbance capacity.
d
Nd; not detected.
30
Highlights
• TPC in chickpea and yellow pea increases over the course of germination
• Lentil has highest TPC and germination has negative impact on its TPC
• Free phenolics have strong iron binding capacity than soluble bond phenolics
• Soluble bond phenolics from chickpea can effectively prevent emulsion oxidation
31