Received: 2 January 2017 | Revised: 31 March 2017 | Accepted: 7 April 2017

DOI: 10.1111/jfpp.13375

ORIGINAL ARTICLE

Composition, digestibility, and functional properties of yellow

pea as affected by processing

reau1 | Alberta N.A. Aryee1,2

Sabine Ribe | Siriane Tanvier1,3 | Jay Han4 |
Joyce I. Boye5

1
Food Research and Development Centre,
3600 Casavant Blvd. W. St. Hyacinthe,
Abstract
Que
bec, J2S 8E3, Canada The proximate composition, digestibility, and functional properties of micronized, pre-germinated,
2
Verschuren Centre for Sustainability in and untreated yellow pea flours were investigated. The three flours had comparable proximate
Energy and the Environment, Cape Breton composition. Foaming capacity and solubility at pH 7 were lower in the treated flours compared
University, 1250 Grand Lake Rd. Sydney,
with the untreated flours. Water holding capacity (WHC) and fat absorption capacity (FAC) were
Nova Scotia, B1P 6L2, Canada
both improved in the micronized flour and only FAC in the pre-germinated flour. The degree of
3
De
partement Ge
nie Biologique,
Spe
cialisation dans les industries hydrolysis of the flours pre-hydrolyzed with bromelain, trypsin, or papain ranged between 8.89
alimentaires et biologiques, Institut and 19.80%. Pre-hydrolysis resulted in partial reduction in the molecular weight (MW) of the pro-
Universitaire de Technologie Cre
teil-Vitry,
teins and extensive reduction after in vitro protein digestion. The hydrolysates had lower trypsin
Cre
teil Cedex, 94010, France
4
inhibitor and higher total phenol and phytic acid contents than the flours.
Alberta Agriculture and Rural Development,
Food Processing Development Centre, 6309 - Practical applications
45 Street, Leduc, AB T9E 7C5, Canada
The use of yellow pea and other pulses is hampered by their low protein digestibility due to the
5
Summerland Research and Development
presence of anti-nutritional factors and protein complexation with carbohydrates. Milder process-
Centre, 4200 Highway 97 South, PO Box
5000, Summerland, British Columbia, V0H 1Z0 ing techniques have become attractive alternative to overcome these attributes. The results from
Correspondence this study suggest that micronization, pre-germination, and/or pre-hydrolysis can be conveniently
Alberta N.A. Aryee, Verschuren Centre for used to modify the nutritional and functional properties and bioactive potential of yellow pea
Sustainability in Energy and the
flours. This could markedly influence value, diversify use, and competitiveness of yellow pea.
Environment, Cape Breton University, 1250
Grand Lake Rd. Sydney, Nova Scotia, B1P
6L2, Canada.
Email: alberta.aryee@mail.mcgill.ca
Funding information
Alberta Innovates and Agriculture and
Agri-Food Canada

1 | INTRODUCTION hypertension, weight management, and reduction in low density lipo-

protein cholesterol (Boye & Ma, 2012; Smith et al., 2012; Lambert
Yellow pea production in 2016 was estimated to increase to 4.8 Mt et al., 2016). Pulses are also valued for their functional properties such
making Canada one of the largest pea producers in the world (Wang, as solubility, water and fat absorption capacity, foaming, gelation, and
2016). Yellow pea is the most important among the varieties of pea emulsification (Ma et al., 2011).
produced in Canada. Like other pulses, yellow pea is a good and inex- Despite these known health benefits and functional proprieties,
pensive source of protein, starch, fiber, vitamins, and minerals, low in interest in pulses, and other plant-derived foods are often lower than
fat, gluten-free, and low allergenicity (Boye & Ma, 2012; Smith, Mol- animal-derived foods (Boye & Ma, 2012; Smith et al., 2012). These
lard, Luhovyy, & Anderson, 2012). Recent studies have linked the con- have been attributed in part to their less digestible protein and starch
sumption of pulses alone or as part of a mixed-macronutrient meal to fractions (Bellaio, Zamprogna Rosenfeld, Jacobs, Basu, & Kappeler,
health-benefiting properties such as reduced risk of certain cancers 2012) and the presence of anti-nutritional factors (ANF) such as phytic
(colon and cervical), diabetes, cardiovascular disease, celiac disease, acid, trypsin inhibitors (TI), and polyphenols. For instance, phytic acid
.......................................................................................................................................................................................
V
C 2017 Her Majesty the Queen in Right of Canada. Journal of Food Processing and Preservation V
C 2017 Wiley Periodicals, Inc.

Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

J Food Process Preserv. 2017;e13375. wileyonlinelibrary.com/journal/jfpp | 1 of 9

https://doi.org/10.1111/jfpp.13375

2 of 9 | RIBEREAU
has been shown to reduce mineral bioavailability, inhibit activity of sev-
eral enzymes such as trypsin and pepsin, decrease in vitro protein
digestibility (IVPD), and affect starch digestion and the glycemic index
(Deshpande, 2002; Lajolo, Genovese, Pryme, & Dale, 2004). TI impedes
trypsin proteolytic activity which can reduce amino acids availability
(Lajolo et al., 2004). Polyphenols can form cross-linked complexes with
proteins, making them unavailable during digestion as well as inhibit
digestive enzymes such as trypsin and amylase (Deshpande, 2002). On
the other hand, several studies have shown positive effects of some of
these ANF as bioactive components such as; antioxidant and cancer-
risk reducing effects (Lajolo et al., 2004). These properties of ANF can
be harnessed when formulating functional food products.
In light of the low nutritional quality and market value of the whole
seed, various treatments methods such as soaking, fermentation, ger-
mination, cooking, and hydrolysis have been applied to alter nutritional
content, reduce/remove ANF and improve digestibility. Seeds have
also been processed into flour or fractions (e.g., protein and starch as
concentrates and isolates) to enrich and/or serve as functional ingre-
dients in food products (Aluko et al., 2015; Bellaio et al., 2012; Boye &
Ma, 2012; Ma et al., 2011).
Pre-germination has been used in various studies to reduce phytic
acid and increase nutrient bioavailability (Duhan, Khetarpaul, & Bishnoi,
2002; Hsu, Leung, Finney, & Morad, 1980). Micronization is an emerg-
ing processing technique and has not been extensively reported in
pulse processing. It involves the exposure of the material to infra-red
(IR) electromagnetic energy. Micronization has been shown to inacti-
vate heat-labile factors such as protease inhibitors, promote starch
gelatinization and increase dry matter digestibility of yellow pea
(McNab & Wilson, 1974; Wray & Cenkowski, 2002). Enzymatic hydro-
lysis is another processing method that has been used to produce
hydrolysates with preferred or low molecular weight (LMW) peptides
and liberate biologically active peptides for specific functions and appli-
cations due to better absorption compared with intact proteins and
amino acids, reduced allergenicity and improved functional properties
(Aluko et al., 2015; Sujith & Hymavathi, 2011). However, extensive
hydrolysis may impart bitterness, an undesirable sensory characteristic
(Humiski & Aluko, 2007; Sujith & Hymavathi, 2011).
The effect of pre-hydrolysis using food-sourced enzymes on in
vitro digestibility (under simulated conditions), ANFs content, and func-
tional properties of untreated and processed yellow pea seeds remain
unexplored. The objectives of this present study were to determine the
effects of the micronization, pre-germination, and enzymatic hydrolysis
on the nutritional and functional properties of yellow pea flour.
2 | MATERIALS AND METHODS
Yellow pea flours (untreated, micronized, pre-germinated) were pro-
vided by the Canadian International Grain Institute (Winnipeg, Mani-
toba, Canada) from large No. 1 Canada yellow pea seeds of good
natural color (yellow). Whole yellow pea were dehulled, micronized, or
pre-germinated prior to milling. Whole yellow pea seeds were pre-
milled using a Jacobson 120-B lab scale hammer mill (Minneapolis,


ET AL.
MN) and milled using a roller mill (Uzwil, Switzerland Buhler ML-202
lab). The hulls were removed as the seeds passed through the first to
the third break roll. The collected hulls were pin milled at 22,000 rpm
using a lab-scale Hosokawa Alpine 100 UPZ pin mill (Summit, NJ). The
pin-milled hull fraction was blended with the roller-milled flour. This
procedure was used to produce untreated, pre-germinated, and
micronized whole pea flours. Untreated dehulled pea flour was pro-
duced by collecting the roller milled flour. Micronized yellow pea flour
was produced by tempering pea to the target moisture of 16%. After
tempering, the seeds were exposed to IR heat using a laboratory scale
micronizer (Micronizing Company, UK) at 110–115 8C and then milled
as described above. Pre-germinated yellow pea was produced by the
PARGEM process, which was developed by Buhler AG in Switzerland.
The process consists of soaking, partial germination and kilning of the
seeds. Whole yellow pea were partially germinated at short germina-
tion time/higher drying temperature and then milled as described.
Papain (5–10 units/mg solid), bromelain (3 units/mg protein), trypsin
(6,000 BAEE units/mg protein), pepsin (2,500 units/mg protein),
pancreatin (>100 U/mg), and D-Serine were purchased from Sigma-
Aldrich (St. Louis, MO). The enzymes used for hydrolysis were chosen
either for their commercial availability (trypsin), or vegetal origin (bro-
melain and papain) and their propensity to produce less bitter
hydrolysates.
2.1 | Proximate analysis
Total protein (N x 5.52) (International Dairy Federation, 2006), fat con-
tent, and ash at 600 8C were determined according to AOAC 992.15
(AOAC, 1995), American Association of Cereal Chemistry (AACC) 30-
25, and AACC 08-03, respectively.
2.2 | Functional properties
Solubility of the flours at pH 7 was determined following the method
of (Achouri, Boye, Yaylayan, & Yeboah, 2005). Foaming properties
(foam capacity and stability) were measured according to the method
of Karakashev, Ozdemir, Hampton, and Nguyen (2011) with some
modification. Briefly, 1% (w/v) flour suspensions in phosphate buffer
(pH 7.0) were stirred at 1,000 rpm for 25 times during which measure-
ments were taken every 10 s during the 15 min mixing time. Water
holding capacity (WHC) was determined following AACC 88-04. Fat
absorption capacity (FAC) was measured according the method of Lin
and Humberd (1974) using corn oil.
2.3 | Pre-hydrolysis
The flours were pre-hydrolyzed using the parameters in Table 1. Fifty
grams of each flour sample was suspended in 1 L phosphate buffer at
the appropriate pH (Table 1) and stirred in a water bath until the appro-
priate temperature (Table 1) was reached. To the flour suspensions
were each added the various enzymes (bromelain, papain, or trypsin).
After 5 min of incubation, the suspensions were heated at 100 8C for
10 min to inactivate the enzymes and then cooled rapidly to approxi-
mately 4 8C. The suspensions were frozen and freeze-dried for further


RIBEREAU ET AL. | 3 of 9

T A B LE 1 Pre-hydrolysis enzymes and conditions

Enzyme Source Temperature 8C Buffer (phosphate) pH Enzyme:substrate ratio E:S

Bromelain Pineapple stem 40 8.0 1:25

Papain Papaya latex 40 6.5 1:50

Trypsin Bovine pancreas 37 8.0 1:25

analysis. The degree of hydrolysis (DH) was determined according to
the method of Nielsen, Petersen, and Dambmann (2001) using
o-phthaldialdehyde (OPA) with D-serine as the standard. b is 0.970; a
is 0.342; and htot is 7.8 for legume proteins according to the method of
Adler-Nissen (1986).
was used for the flours (micronized, pre-germinated, untreated) and
pre-digested flours and 1 mL/min for the hydrolysates from the
IVPD studies. Elution was monitored at 280 and 254 nm.

2.8 | Statistical analysis

2.4 | Antinutritional factors and bioactive components
The data was subjected to analysis of variance (SAS® Version 2.0.3,
The phytic acid, TI, and total phenolic content of the three flours and
2008, SAS Institute Inc., Cary, NC). Triplicate measurements were
their hydrolysates were determined as follows. Phytic acid content was
taken and results are presented as mean. Differences were considered
determined according to the method of Latta and Eskin (1980) using
to be significant when the p values were <.05.
5% (v/v) HCl. TI content was according to the method of Liu and Mar-
kakis (1989) with some modifications (i.e., appropriate amount of the
samples were used to obtained 30–70% inhibition). Total phenolic con-
tent was determined according to the method of Velioglu, Mazza, Gao, 3 | RESULTS AND DISCUSSION
and Oomah (1998) using 4 mL 80% (v/v) methanol (containing 1%
HCl). The results were expressed as Gallic acid equivalents (GAE). 3.1 | Proximate composition
The protein, ash and fat content of the three flours ranged between
2.5 | IVPD 19.60–20.66%, 2.40–2.45%, and 1.27–1.50%, respectively (Table 2).
The proximate composition of the untreated flour was similar to those
The IVPD of the flours and pre-digested samples were determined
reported by Boye and Ma (2012) (even though the N factor used in is
according to the method of Singh and Jambunathan (1981). The pooled
study was 5.52). These results were also consistent with Sun, Watts,
supernatants were analyzed by size exclusion high performance liquid
Lukow, and Arntfield (2006) who reported no changes in the protein
chromatography (SE-HPLC).
and ash contents of micronized wheat. Other authors (Hsu et al., 1980;
Shah et al., 2011) on the other hand reported increases in protein con-
2.6 | Sodium dodecyl sulfate-polyacrylamide gel
tent after germination, contrary to El-Adawy, Rahma, El-Bedawey, and
electrophoresis
El-Beltagy (2003) who reported a decrease in protein content with ger-
Samples and LMW standards were suspended in Laemli buffer under mination and an increase in ash and fat contents. The partial germina-
nonreducing conditions (without b-mercaptoethanol), boiled and cen- tion used in this study could explain the nonsignificant changes in
trifuged for 5 min at 1,400g. Each sample was then loaded on 10–20% proximate composition of the pre-germinated flour. Der (2010)
Tris-HCl Criterion precast gels (Bio-Rad). reported an increase in fat content after micronizing lentil seeds, similar
to the results of this study.
2.7 | SE-HPLC
The supernatants obtained after centrifuging (5 min at 1,400g) T A B LE 2 Proximate composite of yellow pea flours (on dry weight
basis)
10 mg of the flours or pre-digested flours dispersed in 500 mL
100 mM phosphate buffer (pH 6.8) were analyzed by SE-HPLC. The Flour Ash (%) Protein (%) Fat (%)
samples obtained after the IVPD studies were re-suspended in Untreated 2.45 a
20.66 a
1.27a
1 mL water for SE-HPLC analysis. A Biosep-SEC-3000 analytic col-
Micronized 2.40a 19.60a 1.50a
umn (300 3 7.8 mm) (Phemomenex, Torrance) on an Agilent series
Pre-germinated 2.45a 20.48a 1.43a
1200 was used during both runs. Thyroglobulin bovine (670 kDa),
g-globulin (150 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and Standard Error 60.01 60.02 60.04

vitamin B12 (1.35 kDa) were the standards used. A mobile phase of Values in the same column with the same superscript are not signifi-
100 mM phosphate buffer (pH 6.8) at a flow rate of 0.8 mL/min cantly different (p > .05).

4 of 9 | RIBEREAU
3.2 | Functional properties
The WHC, FAC, and solubility at pH 7 of yellow pea flours were signifi-
cantly (p < .05) affected by the treatments applied. Both micronization
and pre-germination increased WHC by 7–16% relative to the
untreated flour. These treatments may have modified the structure of
the flours which enhanced greater imbibition, swelling, and retention of
water. Micronization may have resulted in some form of protein dena-
turation and starch damage and increased ability to entrap water.
FAC was, respectively, increased and decreased in the pre-
germinated and micronized flours relative to the untreated flour. Simi-
lar results were obtained by Der (2010) who reported an increase in
WHC and a decrease in solubility when lentil seeds were micronized,
while FAC remained unchanged. El-Adawy et al. (2003) also observed
improved WHC but a loss in FAC in pre-germinated mung bean, pea,
and lentil seeds. Binding of fat is dependent on the surface availability
of hydrophobic amino acids. The enhanced FAC in the pre-germinated
flour may be attributed to increased availability of unmasked nonpolar
residues.
Micronization and pre-germination also reduced protein solubil-
ity at pH 7 by more than 35% relative to the untreated flour. Pro-
tein solubility was dependent on the micronization temperature,
with higher temperature (120 8C) lowering solubility while solubility
is improved at lower temperature (<90 8C) (Niu, Classen, & Scott,
2003). Protein denaturation, protein–starch cross-linking, and
aggregation may be implicated in the reduced solubility of the
treated flours.
FIGURE 1 Foam capacity (a) and stability (b) of untreated,
micronized, and pre-germinated yellow pea flours


ET AL.
Foam capacity and stability were reported to be improved in
pre-germinated flours (El-Adawy et al., 2003). Such changes were
not observed in this study (Figure 1a,b). Foam capacity was not sig-
nificantly affected by treatment, however; Figure 1a shows that the
untreated flour formed 25% more foam than the treated flours. The
availability of relatively rich amount of proteins such as albumins in
the native/untreated flour may account for the better foaming
properties, while partial denaturation and aggregation of the pro-
teins in the processed flours may have resulted in its reduction. The
micronized flour formed the least stable foam while the pre-
germinated flour formed slightly stable foam compared with the
micronized flour (Figure 1b; Table 3).
3.3 | ANFs and bioactive components
The treatments applied (micronization and pre-germinated) as well
as the enzymes used (trypsin, bromelain, and papain) significantly
(p < .05) affected ANF and bioactive components (except phytic
acid) (Table 4). The treatments had no effect on phytic acid content,
but increased the amount of total phenolic compounds by 25–
65% and TI content by 60–200%, with the pre-germinated flours
giving the highest levels in both cases. Polyphenol and phytic acid
contents were higher in the hydrolyzed flours compared to the
unhydrolyzed flours, except for the pre-germinated flour hydro-
lyzed with bromelain which showed mostly similar phenol content
as the unhydrolyzed pre-germinated flour. In addition, the hydroly-
sates prepared with papain had higher levels of phytic acid than
hydrolysates prepared with trypsin or bromelain. These results
were different from those reported by Chitra, Vimala, Singh, and
Geervani (1995) and El-Adawy et al. (2003) who observed a
decrease in phytic acid content in germinated pea, and that of Chen
et al. (2013) who reported an increase in phytic acid content after
micronization. These disparities can be explained by the effects of
the shorter/partial germination used in this study. Long germination
times (>2 days) were reported to reduce phytic acid content
(Urbano et al., 2005). Total phenol content decreased in micronized
and sprouted flours (Chen et al., 2013; Nithya, Ramachandramurty,
& Krishnamoorthy, 2006) and increased in micronized buckwheat
(Batham, Sharma, Khan, & Govindaraj, 2013). Increase in total phe-
nolic content with thermal processed has been reported (Randhir,
Kwon, Lin, & Shetty, 2009). The increase in total phenol content in
T A B LE 3 Functional properties of yellow pea flours
Solubility at Fat Absorption Water Holding
Flour pH 7 (%) capacity (%) Capacity (%)
Untreated 69.45b 189.30a 8.74a
Micronized 30.69a 184.26a 9.34ab
Pre-germinated 44.25a 195.08a 10.20b
Standard Error 62.31 62.11 60.15
Values in the same column with the same superscript are significantly
different (p < .05).


RIBEREAU ET AL. | 5 of 9

T A B LE 4 Effects of treatments on the anti-nutritional factors content in yellow pea flours and hydrolysates

Sample Total Phenol (mg GAE/g sample) Phytic acid (mg/g sample) Trypsin Inhibitor (TUI/mg sample)

Untreated flour 0.732 a

10.810 a
0.0099e

Untreated flour 1 Bromelain 1.285cd 15.464b 0.0057bc

Untreated flour 1 Papain 1.529e 19.834cd 0.0069cd

Untreated flour 1 Trypsin 1.411de 14.288ab 0.0046ab

Micronized flour 0.925b 10.966a 0.0160f

Micronized flour 1 Bromelain 1.420de 16.717bc 0.0056abc

Micronized flour 1 Papain 1.362cd 21.031d 0.0041ab

Micronized flour 1 Trypsin 1.402de 14.057ab 0.0040ab

Pre-germinated flour 1.214c 10.197a 0.0287g

Pre-germinated flour 1 Bromelain 1.207c 13.499ab 0.0050abc

Pre-germinated flour 1 Papain 1.414de 22.215d 0.0077d

Pre-germinated flour 1 Trypsin 1.339cd 14.162ab 0.0037a

Standard Error 60.027 60.681 60.00032

Values in the same column with the same superscript are significantly different (p < .05).

the micronized samples may have resulted from the cleavage of
covalent bonds and the liberation of these compounds (Niwa &
Mixachi, 1986; Niwa, Kanoh, & Neigishi, 1988), resulting in their
better and/greater access for quantification. These results support
the possible reaction between the amino groups and phenol or
phosphorus groups with the resultant increase in the amount of
measurable phenol and phytic acid. This could also explain the
higher content of phenolic compounds in most of the hydrolysates
compared to their flours. The proteolytic enzymes may have easily
hydrolyzed phytic acid compounds, appreciably enhancing the liber-
ation of the compounds and increasing their content and plausibly
prevented the formation of protein-phytic acid complexes known
to be resistant to proteolytic degradation. These results suggest
that micronization, pre-germination or pre-hydrolysis may have
caused the breakdown of cellular structure and released quantifi-
able cellular materials such as phenolic compounds and phytic acid
and that hydrolysis can be used to improve the bioactive content of
yellow pea flours (Figure 2).
Contrary to previous reports by El-Adawy et al. (2003) and
Chen et al. (2013), micronization and pre-germination increased TI
content. This may be due to the TI artifacts (Anderson, 1986)
formed with micronization and pre-germination, significantly
increasing TI content to 0.0160 and 0.0287%, respectively, com-
pared with 0.0099% in the untreated flour. The hydrolysates
obtained from the untreated and pre-germinated flours, however,
showed reduced TI content, similar to a previous study (Khalil,
Mohamed, Taha, & Nordberg Karlsson, 2006). A similar effect was
observed in the micronized flour hydrolysates. The low pH during
hydrolysis may have reduced the once favorable association
between endogenous proteases and TI resulting in their reduction
and/loss. The hydrolytic enzymes used in this study may have
also hydrolyzed the TI in the flours. This also suggests that the

FIGURE 2 SDS-PAGE of pre-hydrolyzed untreated (a); micronized (b); and pre-germinated (c) yellow pea flours (F) using (B: bromelain, P:
Papain; and T: Trypsin). Low molecular weight polypeptides (LMPP)


6 of 9 | RIBEREAU ET AL.

T A B LE 5Effects of treatments on the degree of hydrolysis of respectively, 10, 12, and 18% in the bromelain, trypsin, and papain
yellow pea flours treated samples, independent of the substrate used (untreated, pre-
Sample DH (%) DH (%)* germinated, or micronized). The DH quantification method is based
on the reaction of trinitrobenzenesulfonic acid with primary amino
Untreated flour 4.43 a
4.43a
groups and determines the free N-terminal amino groups in the
Untreated flour 1 Bromelain 14.66c 10.23bc
hydrolysate. A possible explanation to the low DH value is the lim-
Untreated flour 1 Papain 21.92de 17.49d ited number of these amino acids produced by endo-peptidases;
Untreated flour 1 Trypsin 16.69c 12.26c papain, bromelain, and trypsin. The short hydrolysis time may also
account for the low DH values.
Micronized flour 4.07 a
4.07 a

Micronized flour 1 Bromelain 14.12c 10.00bc

3.5 | Sodium dodecyl sulfate-polyacrylamide gel
Micronized flour 1 Papain 22.97e 18.85d
electrophoresis electrophoregram
Micronized flour 1 Trypsin 16.48c 12.35c
The electrophoretic profile of the flours show bands ranging
Pre-germinated flour 11.00b 11.00bc
between 97 and <14.4 kDa (Figure 2a–c). Proteins with MW of
Pre-germinated flour 1 Bromelain 19.89d 8.89b >16 kDa represent 67–100% of the hydrolyzed soluble proteins.
Pre-germinated flour 1 Papain 30.80f 19.80d About 60% of the protein in the untreated and micronized flours

Pre-germinated flour 1 Trypsin 22.93e 11.93c

Standard Error 60.642 60.642
*DH of hydrolysate - DH of flour (except for DH of the flour). Values in
the same column with the same superscript are significantly different
(p < .05).
activities and quantities of bromelain, papain, and trypsin used
were not inhibited by TI. The heat terminating step included in
the hydrolysate production protocol may also partially account for
reduction in heat-labile TI content (Hsu, Leung, Morad, Finney, &
Leung, 1982).
Overall, these results suggest that micronization, pre-germination,
and/or pre-hydrolysis can be used to modify both the nutritional prop-
erties and bioactive potential of yellow pea flours through reduction in
TI content and increase in phenolic compounds and phytic acid con-
tents. Processing yellow pea flour by these methods could thus be
used to generate antioxidants.
3.4 | Degree of hydrolysis
Table 5 shows the DH of the various flours using the three different
enzymes. The DH values were significantly different (p < .05). The
untreated and micronized flours had the same initial DH value
whereas the DH of pre-germinated flour was almost threefolds
higher. The higher DH values of the pre-germinated flour may be
due to the ability of the inherent enzymes to converted proteins to
quantifiable amino acids during germination. The reduction in some
ANF content (Table 4) could also explain the increase in DH. The
efficiency of the various enzymes to hydrolyze the flours followed
the order: papain > trypsin > bromelain. The second column of
Table 5 provides the “normalized” DH values, that is, DH values due
to the added enzyme (and not the inherent enzymes) by subtracting
the initial DH value of the flour from DH value obtained using the
exogenous enzymes. Papain was a more efficient endo-peptidase
than bromelain or trypsin. After 5 min of hydrolysis, the DH were,
were >35 kDa and <10% were <16 kDa. The distribution of the
proteins in the pre-germinated flour were; 40% >30 kDa, 30%
between 30 and 16 kDa and 30% <16 kDa. This is consistent with
the report of Hsu et al. (1982) who also observed more small pro-
tein subunits after germination compared with the nongerminated
(control) yellow pea and with Sun et al. (2006) who reported no dif-
ferences in the electrophoretic profile of the control and micronized
wheat sample. Lipoxygenase (97.2 kDa), convicilin (77.9–72.4 kDa),
legumin (63.6 kDa; a: 40.8 kDa; b: 23.1–22.3 kDa), vicilin (47.3
kDa, 37–31.8 kDa, and 28.7 kDa) were observed in different pea
bean varieties (Barac et al., 2010). All these proteins were discerned
in the flours with or without treatment, except a-legumin which
was absent in all the flours and vicillin (47.3 kDa) which was absent
in the untreated flour. Fainter vicilin bands can be seen in the
hydrolysates obtain from the untreated flour. The proteins in the
unhydrolyzed (control) flours with >16 kDa MW were cleaved by
all the enzymes used during pre-hydrolysis into fragments smaller
than 14.4 kDa. The results suggest that pre-hydrolysis degraded the
soluble proteins into smaller fragments.
3.6 | SE-HPLC
The SE-HPLC chromatograms (Figure 3a–c) show a loss or a reduction
in proteins with MW larger than 17 kDa with pre-hydrolysis and this
confirms the loss of high MW proteins in the hydrolysates observed in
the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) analysis. Almost all the peaks in all the samples were resolved
within 15 min. All the samples (flours and hydrolysates) had a major
peak between 17 and 1.35 kDa.
Figure 4a–c show the chromatogram of the pepsin-pancreatin
digested (treated: pre-germinated, micronized pre-hydrolyzed) and
untreated (control) samples. All the peaks observed in the unhydro-
lyzed and pre-hydrolyzed samples (Figure 3a–c) were reduced to
<1.35 kDa after in vitro digestion (Figure 4a–c). Additionally, pepsin–
pancreatin digestion following pre-hydrolysis produced a much lower
MW peptides that were achieved by bromelain, papain, or trypsin


RIBEREAU ET AL. | 7 of 9

FIGURE 3 SE-HPLC of pre-hydrolyzed (h) (a) untreated yellow pea flour (YPF); (b) micronized yellow pea flour (mYPF); and (c) pre-
germinated yellow pea flours (pYPF) with B: bromelain (YPFhB/mYPFhB/pYPFhB), P: Papain (YPFhP/mYPFhP/pYPFhP), and T: Trypsin
(YPFhT/mYPFhT/pYPFhT) and standards (1: 670 kDa; 2: 150 kDa; 3: 44 kDa; 4:17 kDa; 5: 1.35 kDa)

FIGURE 4 SE-HPLC of pre-hydrolyzed (h) (with B: Bromelain, P: Papain, and T: Trypsin) and subsequent in vitro protein digestion condi-
tions with pepsin-pancreatin (*) of (a) untreated yellow pea flour (YPF); (b) micronized yellow pea flour (mYPF); and (c) pre-germinated yel-
low pea flours (pYPF) and standards (1: 670 kDa; 2: 150 kDa; 3: 44 kDa; 4:17 kDa; 5: 1.35 kDa)

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alone. The minimal reduction in MW may be indicative of limited
hydrolysis and availability of active sites in the pre-hydrolyzed samples
for pepsin and pancreatin digestion. Limited/short hydrolysis has been
used to reduce the development of bitterness (Humiski & Aluko, 2007;
Sujith & Hymavathi, 2011), but long enough to reduce the size of the
proteins.
4 | CONCLUSIONS
Processing of yellow pea by micronization, pre-germination, or pre-
hydrolysis had varied effect on the functional and nutritional properties
of the flours. Micronization and pre-germination had no significant
effect on the protein, ash and fat content but caused significant reduc-
tion in foaming capacity and protein solubility at pH 7. High amount of
both phytic acid and phenol compounds were accumulation with pre-
hydrolysis while TI content was greatly reduced. Papain was a more
efficient endo-peptidase than bromelain or trypsin for the pre-
hydrolysis of yellow pea flours. Digesting the untreated, micronized,
pre-germinated, or pre-hydrolyzed flours with pepsin and pancreatin
resulted in further degradation and disappearance of large MW protein
and the production of smaller peptide fragments. Further studies on
the effect of hydrolysis on the proximate composition, functional prop-
erties, and bitterness are warranted. Since both micronized and pre-
germinated flours have to be cooked before consumption, the effect of
cooking on the nutritional and functional properties is another interest-
ing angle to consider. This study provided further insight into proper-
ties of yellow pea and the adequacy and effectiveness of processing
treatments in modifying their properties and enhancing their utilization
as food ingredients.
ACKNOWLE DGMENTS
This project was funded by Agriculture and Agri-Food Canada and
Alberta Innovates. The authors report no conflict of interest.
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reau S, Aryee ANA, Tanvier S, Han
How to cite this article: Ribe
J, Boye JI. Composition, digestibility, and functional properties
of yellow pea as affected by processing. J Food Process Preserv.
2017;e13375. https://doi.org/10.1111/jfpp.13375