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Impact of temperature and water activity on the

Cite this: DOI: 10.1039/d0fo01100b
aroma composition and flavor stability of pea
(Pisum sativum) protein isolates during storage
Hannah Mehle, Laurianne Paravisini and Devin G. Peterson *

Received 29th April 2020,
Accepted 3rd September 2020
DOI: 10.1039/d0fo01100b
rsc.li/food-function
Flavor stability of pea protein isolates (PPIs) during storage was investigated. Two commercial PPIs were
stored at three water activities (0.128–0.501) under refrigerated (7 °C) and accelerated (37 °C) tempera-
tures for 12 weeks. Eleven aroma compounds were monitored by gas chromatography-tandem mass
spectrometry (GC/MS/MS) and results revealed significant changes in the aroma concentrations among
the PPI samples during storage. In agreement with the chemical changes, significant differences in ortho-
nasal aroma profiles were demonstrated using a sensory difference-from-control test. The sample stored
under accelerated storage temperature (37 °C) and at the highest water activity showed the greatest
degree of aroma change. An aroma recombination sensory study indicated the generation of two specific
compounds, 1-octen-3-ol and nonanal, along with the degradation of 2–4-decadienal resulted in
sensory changes during storage indicating lipid oxidation was the main mechanism of flavor instability in
the PPI samples.

1. Introduction reduction in the total volatile compounds, whereas protein

Use of peas (Pisum sativum) as a plant-based protein source
has grown rapidly in recent years, and has an estimated global
market value of $176 million by 2025.1 The increased utiliz-
ation of plant protein by the food industry is supported by con-
sumer demand for high nutritional quality, inclusivity to many
diets (allergy-restricted, vegan, vegetarian, flexitarian), and low
environmental impact.2–5 While pea protein has many benefits
and applications, its use is still limited by negative flavor attri-
butes such as beany, green, earthy, hay-like, bitter, and
astringent,6,7 all of which are not typically desirable flavor
traits in the Western market.8 Among plant-based consumers,
approximately half reported flavor as the most important
aspect for eating plant-based protein alternatives.4 Strategies
that reduce the endogenous flavor attributes of pea proteins
would support increased utilization in food.
Inherent differences in pea cultivars, as well as environ-
mental factors associated with further processing and storage,
result in variation in the flavor profile of the pea protein iso-
lates (PPIs) that ultimately affect consumer acceptance.9,10
Multiple processing methods have been developed to process
peas into PPIs that include heat treatment and extraction
steps.6,11 Generally, heat treatment of whole peas results in a
Department of Food Science and Technology, The Ohio State University, 2015 Fyffe
Road, Columbus, Ohio 43210, USA. E-mail: peterson.892@osu.edu
extraction results in an increase in volatile compounds.12,13
The use of extraction pre-treatments such as soaking or
solvent extraction has been attempted to reduce bitterness and
total volatile aroma content.6,7 Murat et al.13 characterized
odor-active changes during isoelectric extraction of pea flour,
reporting five compounds were generated throughout proces-
sing due to lipid oxidation or thermal treatment. In this work,
although some lipid oxidation compounds were lost during
processing, others increased in concentration. In high-protein
snacks made with 60–90% pea flour, consumer liking was hin-
dered by pea and green flavor attributes for both omnivores
and flexitarians and the pea flour content was negatively corre-
lated with acceptance.14 Changes in odor-active compounds
and sensory properties of ultra high temperature (UHT) pea
protein beverages as impacted by processing and storage were
reported by Trikusuma et al.15 This study indicated raw PPI
beverages were mainly characterized with beany, potato, and
pasta notes, whereas after UHT heat processing and storage a
strong increase in the perceived intensity of sawdust, oxidized/
painty, and cardboard attributes was observed, while the
characteristic beany note of pea was nearly undetected. Several
compounds identified in the protein aroma profile were
inherent to pea; however, the majority of compounds were gen-
erated during UHT processing and storage from lipid oxidation
and the Maillard reaction.
Several strategies to specifically control general lipid oxi-
dation have been considered. Removal of lipids from protein

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ingredients by solvent or supercritical extraction has been
investigated and shown to reduce lipid oxidation volatile com-
pounds, however, this can negatively affect certain functional
properties or may not be industrially feasible7,13,16–18
Additional processing methods have been investigated includ-
ing alkaline extraction, solid-dispersion spray-drying, and
enzymatic treatment, which both improve functional pro-
perties and reduce off-flavor perception or volatile
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composition19–21 Others have suggested utilizing pea dextrin
to mask certain specific lipid oxidation compounds.22
Flavor stability of protein powders during storage is also
known to impact product performance in food applications.23
PPIs are low moisture (water activity aw < 0.6), shelf-stable
ingredients24 and are typically stored in warehouses for
extended periods of time under uncontrolled temperature and
humidity conditions. A better understanding of the role of
storage conditions on the off-flavor generation of pea protein
isolates and the specific compounds that impact flavor stabi-
lity would support the industry’s ability to develop high quality
pea protein ingredients. The overall goal of this work was to
investigate the molecular changes in the aroma profile of PPI
stored under different water activity levels and at different
temperatures on flavor stability. Changes in the aroma
composition were monitored by gas chromatography-tandem
mass spectrometry (GC/MS/MS) analysis and changes in the
orthonasal aroma perception were determined by sensory
analysis.
2. Materials and methods
2.1 Chemicals
Chemical grade sodium bromide anhydrous was purchased
from Sigma Aldrich (St. Louis, MO). Pure (>95%) commercial
standards of 4-heptanone, 1-pentanol, hexanal, methional,
2-pentyl furan, 2,5-dimethylpyrazine, 2-heptanone, heptanal,
octanoic acid, isovaleric acid, (E,E)-2,4-nonadienal, (E,E)-2,4-
decadienal, 4-hydroxybenzaldehyde, γ-nonalactone, 1-octen-3-
ol, (E)-2-octenal, 2-isobutyl-3-methoxypyrazine, p-vinylguaiacol,
4-methyl-5-thiazoleethanol, and maltol were purchased from
Sigma Aldrich. Nonanal was purchased from Alfa Aesar
(Haverhill, MA). Ethanol was purchased from Fisher Scientific
(Pittsburg, PA). The deuterated standard of 2-acetyl-1-pyrroline
([2H3]-2AP) was synthesized in-house.
2.2 Pea protein isolates (PPI)
Two commercial PPIs were acquired within 12 weeks from
their manufacturing date. The PPIs contained between
80–85% w/w protein and the remaining content included <8%
water, <8% fat, <5% fiber, and <6% ash by the supplier certifi-
cates of analysis. Prior to experiments, samples were stored at
−40 °C in glass amber bottles with a PFTE lined lid. For both
analytical and sensory analysis, samples were prepared at a 3%
w/w suspension in water.
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2.3 Water activity (aw) measurement
The water activities of the samples and salt solutions were
measured with a Novasina Water Activity Meter (Novasina AG,
Switzerland) calibrated from 0.11–0.75 with standard solu-
tions. Measurements were determined in triplicate.
2.4 Storage study
PPI samples were stored at three different water activities (aw)
of 0.1, 0.3, and 0.5 to simulate different storage environments.
The initial aw of the PPIs as is was about 0.3 (Table 1), which
corresponds to the middle of the range for low moisture
foods.24 For the lower aw sample, PPIs were lyophilized with a
Virtis Sentry 2.0 vacuum freezer at −60 °C and 100 mTorr for
15 min to achieve a low aw between 0.10–0.15. High aw
samples were placed under saturated salt solution of sodium
bromide to achieve targeted aw of ∼0.5 (Table 1). In brevity, a
saturated salt solution of sodium bromide was prepared and
layered into the bottom of glass desiccators. Three layers of
aluminum pans were stacked within the desiccators on top of
a perforated platform. Flat wooden sticks were used to stack
the aluminum pans and allow for airflow between each of the
layers. Fifty grams of each PPI were spread into each of the
aluminum pans. The desiccators were sealed with vacuum
grease and powders were stirred frequently (every 2–3 days)
until equilibrium. The desiccators were stored at ambient
temperature (23 ± 2 desiccators) for 35 days to achieve equili-
bration at aw of 0.5.
After equilibration, samples were then placed in amber
glass bottles fitted with a PFTE lid and stored for 12 weeks
under refrigerated (7 ± 2 °C) and accelerated (37 ± 2 °C) con-
ditions. Accelerated conditions were chosen to mimic storage
at ambient temperature (23 °C) for approximately 7 months.
The estimated accelerated time is based on a temperature
coefficient, Q10 equation.25 A Q10 of 2 is a typical value to esti-
mate reaction rates in food, particularly dehydrated foods
where the lowest expected Q10 is 1.5–2 for lipid oxidation.26,27
After storage, samples were immediately placed in amber glass
bottles with PFTE lid and stored at −40 °C in a freezer until
further analysis.
2.5 Quantification of volatile aroma compounds using
dynamic headspace-gas chromatography/mass spectrometry
(DHS-GC/MS)
Twenty-one volatile aroma compounds previously identified in
UHT PPI beverages by Trikusuma et al.15 were monitored in
the PPI samples over storage. Analyses were performed using
an Agilent 7890B GC (Agilent Technologies, Santa Clara, CA)
Table 1 Average water activity (aw) values (±standard deviation) for two
commercial pea protein isolates (PPIs) samples
Low aw Medium aw High aw
PPI A 0.128 ± 0.006 0.347 ± 0.003 0.501 ± 0.020
PPI B 0.120 ± 0.004 0.337 ± 0.001 0.493 ± 0.004
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equipped with a DB-Wax column (60 m × 0.25 mm ID ×
0.25 μm film thickness; Agilent Technologies) and coupled to
an Agilent 7010B triple quadrupole mass spectrometer (MS)
(Agilent Technologies). The GC/MS system was also equipped
with a multipurpose sampler (MPS; Gerstel, Mulheim an der
Ruhr, Germany) dynamic headspace (DHS) unit (Gerstel), pro-
grammable temperature vaporization (PTV) inlet (CIS4,
Gerstel), and a thermal desorption unit (TDU; Gerstel). PPIs
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test, anchored with no difference (0), very little difference
(1–2), little difference (3–4), medium difference (5–6), strong
difference (7–8), and extreme difference (9–10). Panelist then
used Compusense Cloud v7.2 software (Compusense Inc.,
Guelph, Ontario, Canada) to collect the data.
2.6.1. PPI samples. Aliquots (10 mL) of a 3% PPI (unaged
control, aged) suspension were placed in 60 mL amber bottles
fitted with PFTE lids for orthonasal aroma analysis and placed

were placed in suspension (3% w/w) in water and spiked with at room temperature for 30 min for equilibration.
an ethanolic internal standard solution of 4-heptanone 2.6.2. Sensory recombination samples. To evaluate the con-
(sample concentration of 50 µg L−1). Samples (2 mL) were tribution of aroma compounds to the overall sensory changes
placed into 20 mL glass vials for DHS volatile extraction. occurring during storage, recombination models were created
Samples were incubated and agitated for 10 min at 35 °C. by adding the compounds in water at their concentrations in
Following incubation, vials were purged with 200 mL of high control and aged samples in 10 mL aliquots in 60 mL amber
purity nitrogen gas at a flow rate of 50 mL min−1 to collect bottles with PTFE lids (Table 2). Based on the sensory DFC
volatile compounds on a Tenax adsorbent trap. The trap was results, the sample with the greatest sensory changes (PPI A at
subsequently dry purged for 4 min at a rate of 100 mL min−1 high aw and 37 °C) was selected as target for recombination.
and transferred to the TDU at 40 °C. The volatiles were ther- One model was prepared by adding all 11 compounds at their
mally desorbed from the trap at 250 °C for 3 min at a flow rate determined concentration prior to storage to mimic the
of 50 mL min−1. After desorption, the volatiles were cryofo- control sample (PPI A). Two models were prepared by changing
cused on the CIS maintained at −50 °C then ramped to 240 °C the concentration of the compounds that exhibited statistical
for 3 min and introduced into the column in splitless mode differences either from increasing or decreasing in concen-
for 0.5 min. The GC oven was programmed at 40 °C initially, tration during storage. Additionally, individual compound con-
ramped to 80 °C at 20 °C min−1, and then at 5 °C min−1 until tribution was evaluated by having panelists rate the size of the
240 °C, and was held for 1 minute at 240 °C. Helium carrier change induced by the change in concentration of each indi-
gas was set to a constant flow rate of 1.2 mL min−1. MS data vidual compound according to their levels in the aged
were collected in multiple reaction monitoring (MRM) mode samples.
adapted from Trikusuma et al.,15 obtained at 70 eV in electron 2.6.3 Data analysis. To evaluate change in volatile aroma
ionization (EI) mode with an acquisition rate of 50 scans per s. compounds, the effect of storage conditions on concentrations
Compounds were positively identified using mass spectra, was evaluated by one-way Analysis of Variance (ANOVA).
linear retention index (LRI) and injection of pure compound Whenever a significant effect of sample was observed, a Tukey
standards. LRI values were calculated with injection of post-hoc test was performed (α < 0.05) to determine differences
n-alkane from C6–C26 in the same condition as the samples. A between samples. For the sensory data, the effect of storage
standard solution of the compounds at a concentration corres- conditions on overall degree of difference was evaluated by
ponding to their odor threshold in water was analyzed as a two-way ANOVA (sample, panelist, and interaction). Whenever
reference. Compounds detected above odor threshold in at a significant effect of sample was observed, a Fisher LSD post-
least one storage condition were quantified using five-point
standard addition curves. Data were collected in triplicate.
Curves indicated good linearity (R2 > 0.95) and good Table 2 Aroma recombination model composition of unaged PPI
reproducibility. control and PPI aged for compounds significantly increasing or decreas-
ing during storage
2.6 Sensory analysis degree-of-difference
All sensory evaluations were conducted in compliance with the Unaged Aged PPI model of Aged PPI model of
PPI increasing decreasing
Institutional Review Board (#2019E0534, #2019E0743). A differ- model compounds compounds
ence-from-control (DFC) test was conducted according to the Compound (μg L−1) (μg L−1) (μg L−1)
methods outlined by Meilgaard et al. to determine if there was Hexanal 1188 1188 1188
a perceptible difference in orthonasal aroma between 2-Heptanone 35.6 144.7a 35.6
samples.28 Sixteen experienced sensory panelists (9 female, Heptanal 4.57 11.1a 4.57
2-Pentylfuran 71.5 159.1a 71.5
7 male, ages 24–45) were recruited participate. All sixteen Nonanal 15.6 26.5a 15.6
panelists participated in the initial evaluation of the control (E)-2-Octenal 4.47 4.47 4.47
and aged samples, 10 of which later evaluated the recombina- 1-Octen-3-ol 1.51 12.51a 1.51
2-Isobutyl-3- 0.026 0.026 0.026
tion samples. In each session, panelists were prompted to methoxypyrazine
smell an unaged, control sample and then were presented (E,E)-2,4-Nonadienal 1.15 1.15 0.53a
with four coded samples in a counterbalanced order, includ- (E,E)-2,4-Decadienal 13.3 13.3 3.0a
p-Vinylguaiacol 7.74 7.74 7.74
ing one blind control. Panelists used a visualized 10-point
a
paper scale to rate samples as they progressed through the Indicate compound concentration changed for aged models.

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hoc multiple comparison test was performed. The panelist*re-
plicate interaction was not significant for any of the samples
indicating panelist performance and panelist’s replicates were
therefore considered independent. All statistical analyses were
performed with SPSS® 25 (IBM Corporation, Armonk, NY).
3. Results and discussion
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3.1 Quantification of volatile aroma compounds of pea
protein isolates during storage
Three different aw and two temperatures were selected to simu-
late different storage environments for two PPI samples (A and
B). Twenty-one aroma compounds in PPI previously identified
by Trikusuma et al.15 were monitored during storage and the
eleven compounds detected above the odor thresholds are
quantitatively reported in Tables 3 and 4. Under refrigerated
conditions (7 °C), significant effect of aw during storage was
observed ( p-values < 0.05) for 8 and 7 compounds in PPI A and
B, respectively. Under accelerated conditions (37 °C), the effect
of aw was more pronounced with 10 and 9 compounds signifi-
cantly changing during storage in PPI A and B, respectively.
Inherent differences in aroma profiles between the two PPIs
were observed; for instance, 2-heptanone was unique to PPI B,
suggesting possible variation due to the crop and/or manufac-
turing conditions. 2-Heptanone is reportedly formed from
linoleic acid oxidation as a minor secondary oxidation
product.29 In this work, 2-heptanone, a brothy aroma com-
pound, was only detected at levels above odor threshold in PPI
A after 12 weeks of storage at 37 °C under medium and high
aw conditions. Crop cultivars are typically selected for a high
protein content, but there is also variation in the volatile com-
pound profile among cultivars.6,7,12,30 Additionally, many pro-
cessing techniques can be used for protein extraction and have
different effects on resulting flavor profiles.6,7
Review of the changes in aroma compound profiles during
storage indicated that autoxidation was a primary mechanism
of flavor instability with the low water activity storage con-
dition having the greatest impact. Heptanal, a lipid autoxida-
tion product of linoleic acid, showed significant increases in
concentration over storage at both temperatures and in both
PPIs. In PPI A, after 12 weeks heptanal concentration exhibited
the largest increase at low aw with 1.5-fold increase at 7 °C
storage. In PPI B, heptanal concentration did not significantly
change at 7 °C but when stored at 37 °C, showed the largest
change at low aw with about 2-fold increase. In general, com-
pound formation was favored by low water activity.
The linoleic acid degradation product 2-pentylfuran has
been reported to contribute to the beany aroma note of oxi-
dized soybean oil.31 In PPI A, significant increases in 2-pentyl-
furan, between 2 to 2.5-fold the initial concentrations, were
observed only at 37 °C across all aw levels and indicated the
effect of temperature on the formation of this compound. In
PPI B, however, a significant decrease in concentration was
observed in the high aw condition at low temperature but at
37 °C, significant increases were observed at low and medium
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aw. 2-Pentylfuran was present above odor threshold (approxi-
mately 10 to 30 times), thus suggesting contribution to the
fruity, green, and earthy notes of the protein aroma.
Nonanal is reported to derive from oleic acid and can be
impacted by differences between fatty acid profiles of the
protein brands.32,33 The longer chain aldehyde, nonanal,
showed an 8-fold increase in concentration in PPI A at the low
aw condition when stored at 7 °C, whereas no significant
changes were observed at medium and high aw conditions. In
the same protein sample, at 37 °C, nonanal concentration sig-
nificantly increased between 2 to 2.3-fold across all aw levels,
with a greater increase at the low aw condition (2.3-fold).
Nonanal in PPI B did not show any significant changes at 7 °C
storage temperature; it only significantly increased at low aw by
about 1.3-fold in the accelerated temperature condition.
Nonanal formation was thus the most impacted by the low aw
condition during storage and was further catalyzed at a higher
temperature as indicated by changes regardless of the aw level.
The involvement of nonanal in concomitant reaction pathways
can explain the lack of a linear trend during storage.
The progression of hexanal formation over storage was
different in the two PPIs; in PPI A, hexanal concentration sig-
nificantly increased at the medium aw at 7 °C, but did not sig-
nificantly change in PPI B under the same conditions. When
stored at 37 °C, the concentration in PPI A significantly
decreased in low and medium aw conditions but did not sig-
nificantly change at high aw. However, in PPI B, hexanal con-
centration significantly increased from the control in the low
aw condition but was not significantly different from the
control in the other conditions at 37 °C. Hexanal is known as a
ubiquitous oxidation marker of food quality34–37 and is formed
by oxidation of fatty acids with double bonds in the ω-6-posi-
tion, such as linoleic acid, and can also further autoxidize.38
In this study, hexanal was detected at levels 200 to 600 times
above its odor threshold, potentially contributing to green
bean aroma notes in PPIs or beany aroma characteristic in
combination with several other non-beany compounds, includ-
ing (E)-2-octenal, (E,E)-2,4-nonadienal, and (E,E)-2,4-
decadienal.39
(E)-2-Octenal has been reported as dusty, musty, and moldy
in pea protein15 and can contribute to a beany aroma in com-
bination with other compounds.39 In PPI A, (E)-2-octenal
changes were minimal, with a significant increase only in the
low aw in 7 °C storage. Whereas in PPI B, (E)-2-octenal showed
a significant decrease in concentration at low and high aw at
7 °C storage however when stored at 37 °C the concentration
significantly decreased at the medium aw. (E)-2-Octenal is
formed from the oxidation of linoleic acid or oxidation of (E,
E)-2,4-decadienal and can be both generated over storage and
act as reactive intermediate to form hexanal, heptanal, and
further reaction products,38,40,41
The unsaturated aldehydes with two double bonds, (E,E)-
2,4-nonadienal and (E,E)-2,4-decadienal, exhibited both sig-
nificant increases and decreases over storage. (E,E)-2,4-
Nonadienal in PPI A showed an increase in concentration at
low aw under refrigerated conditions only. At 37 °C, the level in
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Table 3 Concentrations of aroma compounds in 3% pea protein isolate A aqueous samples (PPI A) over 12-week storagea

Storage at 7 °C

LRI (DB-wax) Compound Control Low aw (0.128) Medium aw (0.347) High aw (0.501) Odor thresholdb (μg L−1) Storage effect p-valuec

1095 Hexanal 1188 ± 215ab 990± 56ab 1419 ± 76b 774 ± 55a 4.5 0.028
1195 2-Heptanone 35.6 ± 0.8a 42.8 ± 0.7b 46.4 ± 1.4c 47.3 ± 2.4c 140 <0.001
1197 Heptanal 4.57 ± 0.38a 6.99 ± 0.46c 5.69 ± 0.12b 5.85 ± 0.15b 3 0.001

This journal is © The Royal Society of Chemistry 2020

1238 2-Pentyl-furan 71.5 ± 8.1 74.1 ± 0.5 76.8 ± 2.0 74.3 ± 2.9 6 Ns
1405 Nonanal 15.6 ± 0.4a 125.5 ± 20.57b 23.03 ± 0.7a 24.64 ± 1.91a 1 <0.001
1444 (E)-2-Octenal 4.47 ± 1.53a 8.23 ± 0.36b 4.68 ± 0.08a 5.65 ± 0.47a 3 0.009
1451 1-Octen-3-ol 1.51 ± 0.13a 2.86 ± 0.13b 5.39 ± 0.21c 6.19 ± 0.11d 1 <0.001
1539 2-Isobutyl-3-methoxypyrazine 0.026 ± 0.001 0.036 ± 0.007 0.029 ± 0.002 0.029 ± 0.005 0.002 Ns
1717 (E,E)-2,4-Nonadienal 1.15 ± 0.09a 2.72 ± 0.46b 1.21 ± 0.04a 1.33 ± 0.13a 0.09 0.001
1814 (E,E)-2,4-Decadienal 13.32 ± 1.59a 22.74 ± 5.17b 11.32 ± 0.69a 10.34 ± 1.31a 0.07 0.003
2138 p-Vinylguaiacol 7.74 ± 0.66a 9.52 ± 0.18b 8.87 ± 0.21ab 7.89 ± 0.48a 3 0.015

Storage at 37 °C

LRI (DB-wax) Compound Control Low aw (0.128) Medium aw (0.347) High aw (0.501) Odor thresholdb (μg L−1) Storage effect p-valuec

1095 Hexanal 1188 ± 215c 942 ± 68ab 803 ± 77a 1142 ± 210c 4.5 0.009
1195 2-Heptanone 35.6 ± 0.8a 135.6 ± 4.8b 154.1 ± 14.2b 144.7 ± 6.2b 140 <0.001
1197 Heptanal 4.57 ± 0.38a 13.09 ± 0.64b 12.89 ± 1.33b 11.14 ± 0.75b 3 0.002
1238 2-Pentyl-furan 71.5 ± 8.1a 177.5 ± 6.7b 165.9 ± 18.9b 159.1 ± 6.9b 6 0.002
1405 Nonanal 15.6 ± 0.4a 36.4 ± 3.1c 29.2 ± 2.3bc 26.52 ± 1.6b 1 0.004
1444 (E)-2-Octenal 4.47 ± 1.53 7.30 ± 1.38 7.28 ± 0.96 7.90 ± 0.48 3 Ns
1451 1-Octen-3-ol 1.51 ± 0.13a 9.30 ± 0.52b 11.4 ± 1.0bc 12.55 ± 0.82c 1 <0.001
1539 2-Isobutyl-3-methoxypyrazine 0.026 ± 0.001a 0.038 ± 0.007b 0.033 ± 0.003ab 0.034 ± 0.004ab 0.002 0.048
1717 (E,E)-2,4-Nonadienal 1.15 ± 0.09b 1.12 ± 0.09b 0.70 ± 0.09a 0.53 ± 0.01a 0.09 <0.001
1814 (E,E)-2,4-Decadienal 13.32 ± 1.59c 8.46 ± 0.95b 4.08 ± 0.64a 2.67 ± 0.30a 0.07 0.006
2138 p-Vinylguaiacol 7.74 ± 0.66 10.08 ± 0.68 7.15 ± 0.60 7.71 ± 1.90 3 Ns
a
Mean ± standard deviation. b Odor threshold value in water; retrieved from Leffingwell &Associates. c In each row, values with different letters are significantly different according to Tukey’s
HSD (α = 0.05), ns: not significant.
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Table 4 Concentrations of aroma compounds in 3% pea protein isolate B aqueous samples (PPI B) over 12-week storagea

Storage at 7 °C

LRI (DB-wax) Compound Control Low aw (0.120) Medium aw (0.337) High aw (0.439) Odor thresholdb (μg L−1) Storage effect p-valuec

1095 Hexanal 1750 ± 193 1866 ± 51 1841 ± 116 1913 ± 78 4.5 Ns

1197 Heptanal 7.60 ± 0.24 7.43 ± 0.27 7.86 ± 0.15 7.83 ± 0.44 3 Ns
1238 2-Pentyl-furan 64.9 ± 2.7b 57.0 ± 1.2b 55.1 ± 3.6b 46.7 ± 1.2a 6 0.012
1405 Nonanal 14.7 ± 1.6a 15.6 ± 1.2b 13.0 ± 1.0a 11.0 ± 3.3a 1 Ns
1444 (E)-2-Octenal 8.15 ± 1.57b 2.18 ± 0.16a 7.16 ± 0.84ab 2.93 ± 0.65a 3 0.015
1451 1-Octen-3-ol 2.09 ± 0.15b 1.58 ± 0.15b 3.10 ± 0.50c 0.75 ± 0.15a 1 0.002
1539 2-Isobutyl-3-methoxypyrazine 0.038 ± 0.005 0.039 ± 0.002 0.038 ± 0.004 0.030 ± 0.007 0.002 Ns
1717 (E,E)-2,4-Nonadienal 3.65 ± 1.09b 2.12 ± 0.04ab 1.07 ± 0.20a 0.37 ± 0.03a 0.09 0.004
1814 (E,E)-2,4-Decadienal 60.69 ± 10.31b 42.69 ± 1.38b 15.65 ± 3.10a 4.01 ± 0.35a 0.07 <0.001
2138 p-Vinylguaiacol 22.61± 1.53b 14.90 ± 5.21b 5.52 ± 0.80a 3.42 ± 1.39a 3 0.003

Storage at 37 °C

LRI (DB-wax) Compound Control Low aw (0.128) Medium aw (0.347) High aw (0.501) Odor thresholdb (μg L−1) Storage effect p-valuec

1095 Hexanal 1750 ± 193a 2433 ± 230b 2214 ± 198ab 1587 ± 171a 4.5 0.022
1197 Heptanal 7.60 ± 0.24a 15.53 ± 0.98c 14.03 ± 0.26c 10.53 ± 0.23b 3 <0.001
1238 2-Pentyl-furan 64.9 ± 2.7a 113.1 ± 7.0b 99.1 ± 13.4b 93.2 ± 5.6a 6 0.009
1405 Nonanal 14.7 ± 1.6a 19.1 ± 0.3b 15.1 ± 0.8a 13.1 ± 0.8a 1 0.003
1444 (E)-2-Octenal 8.15 ± 1.57b 4.30 ± 0.38ab 1.14 ± 0.14a 7.23 ± 3.10ab 3 0.024
1451 1-Octen-3-ol 2.09 ± 0.15a 4.68 ± 0.69b 4.84 ± 0.79b 8.20 ± 1.10c 1 <0.001
1539 2-Isobutyl-3-methoxypyrazine 0.038 ± 0.005a 0.049 ± 0.002b 0.042 ± 0.003ab 0.038 ± 0.004ab 0.002 0.029
1717 (E,E)-2,4-Nonadienal 3.65 ± 1.09b 1.16 ± 0.05a 0.04 ± 0.03a 0.05 ± 0.03a 0.09 0.012
1814 (E,E)-2,4-Decadienal 60.69 ± 10.31b 15.55 ± 1.06a 5.86 ± 1.19a 2.60 ± 0.40a 0.07 <0.001
2138 p-Vinylguaiacol 22.61± 1.53c 6.57 ± 2.24b 3.48 ± 0.43ab 2.10 ± 0.48a 3 <0.001
a
Mean ± standard deviation. b Odor threshold value in water; retrieved from Leffingwell &Associates. c In each row, values with different letters are significantly different according to Tukey’s
HSD (α = 0.05), ns: not significant.
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PPI A did not significantly change at low aw, but significantly
decreases by about 2-fold in the medium and high aw con-
ditions. Similar changes were observed for (E,E)-2,4-nonadie-
nal in PPI B. All three aw levels resulted in the same significant
differences at 37 °C, with a significant decrease in concen-
tration up to 60-fold in the medium aw sample. (E,E)-2,4-
Nonadienal is known to exhibit floral, brothy, and waxy odor
notes, typically found in foods containing high linoleic acid
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content and subjected to high temperature processing con-
ditions. This compound has been also identified in other
spray-dried products, and losses could be a result of sub-
sequent reactivity of the double bonds to produce degradation
compounds.42 (E,E)-2,4-Decadienal showed similar changes in
both PPIs. More specifically, in PPI A at 7 °C, (E,E)-2,4-decadie-
nal concentration almost doubled under storage at low aw and
refrigerated conditions. Under accelerated conditions of 37 °C,
this compound significantly decreased at all three aw levels
with greatest decreases of 3 to 5-fold at medium and high aw,
respectively. In PPI B, the behavior of (E,E)-2,4-decadienal was
different at 7 °C; this compound significantly decreased at
medium and high aw. Whereas at 37 °C, changes similar to
PPI A were observed with large decreases suggesting it was
degrading to other products. Decreases of (E,E)-2,4-decadienal
could result in the formation of hexanal;38 however, this
increase was not observed as the level of hexanal were 25 to
100 times greater than the concentration of (E,E)-2,4-decadie-
nal in initial conditions. Another study using an isotope label-
ing experiment showed that (E,E)-2,4-decadienal can autoxi-
dize to form (E,E)-2,4-decanoic acid, heptanal, 2-octenal,
2-nonenal, glyoxal, and malonaldehyde in soybean oil.43
1-Octen-3-ol is a vinyl alcohol with a potent mushroom
aroma arising from linoleic acid oxidation in the presence of
enzymes.44 In the PPI samples, 1-octen-3-ol was present at con-
centrations 2 to 10 times greater than threshold. In PPI A,
1-octen-3-ol concentration exhibited significant increases
under all the aw conditions and at both 7 and 37 °C. Similarly,
in PPI B at 37 °C, 1-octen-3-ol concentration increased at all
aw; however, at 7 °C the concentration of 1-octen-3-ol signifi-
cantly increased at medium aw. The generation of 1-octen-3-ol
was generally favored at higher aw over lower aw suggesting
enzymatic pathways were involved in product formation.
Ullrich and Grosch reported 1-octen-3-ol to be one of the
main products of linoleic acid oxidation that increases over
storage.45 Hoffman44 observed in soybean oil, that linoleic
acid can degrade to form both (E)-2-octenal and 1-octen-3-ol,
but when pure linoleic acid was reacted only 1-octen-3-ol was
detected suggesting there may be other constituents that effect
formation and stabilization in the matrix, such as enzymatic
activity. Furthermore, 1-octen-3-ol can be further oxidized to
produce the more potent mushroom compound, 1-octen-
3-one,43 which was not quantified in this study.
Review of the changes in the lipid oxidation products of the
PPIs during storage in relation to aw were consistent with a
J-shaped relative reaction curve.46 As expected, higher tempera-
ture conditions resulted in more drastic changes during
storage, which is consistent with lipid oxidation in food.47
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During storage of peas the concentration of total volatile com-
pounds is higher at 37 °C than 4 °C.48
The potent pyrazine compound 2-isobutyl-3-methoxypyra-
zine (IBMP) with a green pepper aroma has been reported in
peas and other raw vegetables49,50 as well as in UHT processed
PPI.15 In the current study, IBMP concentration did not signifi-
cantly change over storage in PPI A nor in PPI B at 7 °C, but
did increase at elevated temperature for the low aw condition
in both samples.51 IBMP has a very low odor threshold
(0.002 μg L−1) and was >13-fold above the threshold value in
the PPI samples supporting its contribution to the aroma
attributes.
Levels of p-vinylguaiacol, a phenolic degradation compound
with a smoky, vanilla-like aroma changed slightly throughout
storage of PPI A when stored at low temperature and low aw.
However, in PPI B, significant decreases in concentration were
observed over storage at medium and high aw in the 7 °C con-
dition and in all the conditions at 37 °C. This compound is a
ferulic acid degradation product found in plant materials.52 It
was shown that amino acid L-cysteine can inhibit p-vinylguaia-
col formation in stored orange juice by up to 80%.53 Cysteine
is part of the amino acid profile of pea and may be preventing
ferulic acid degradation.54 A decrease in p-vinylguaiacol during
storage suggests further degradation of this compound. For
example, during beer storage, p-vinylguaiacol can be oxidized
to form vanillin or hydrated to form apocynol.55 Alternatively,
p-vinylguaiacol has been shown to interfere with the Maillard
reaction and may be decreasing due to formation of sugar-
phenol adducts.56
These results can be compared to several other storage
studies of pea and other legume products. In one study, pea
flour, concentrate, and starch were stored between 3.8–13.6%
moisture at 7, 21, and 30 °C for one year.57 Flavor changes as
compared to the control were correlated with higher moisture
levels and temperatures compared to the control. Sumner et al.
associated a decrease in linoleic acid content over storage that
suggested oxidation or polymerization reactions.57
Furthermore, in peanuts stored at 0.19 and 0.60 aw for 7 weeks
increased changes in the lipid oxidation volatiles were
observed at 0.19 aw than 0.6 aw.58 In another study, fava bean
flours and concentrates were stored under accelerated storage
conditions (38 °C, 65% relative humidity) and were compared
to a year-old sample from the previous crop year.59 These
authors’ results showed that free fatty acids increased over
storage, although the characteristic dried pea flavor did not
change. Heat treatment was also investigated, which resulted
in enzyme inactivation and a decrease in dried pea flavor;
however, additional objectionable off-flavors also developed.
3.2 Sensory evaluation and aroma recombination models
The overall perceived orthonasal change in the PPI samples
during storage is shown in Fig. 1 and 2. Significant changes in
aroma over storage ( p-value < 0.05) were observed for both
PPIs with the extent of changes depending on the PPI brand
and the storage temperature.
Food Funct.

Paper
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Fig. 1 Average difference from control (DFC) ratings (n = 32) for 3% aqueous pea protein isolates PPI A (i and ii) and PPI B (iii and iv) for unaged and
aged samples under modified aw conditions at 7 °C and 37 °C for 12 weeks. Within each temperature condition, different letters indicate significant
difference according to LSD post-hoc (α = 0.05).
Fig. 2 Average difference from control (DFC) ratings (n = 20) for
unaged PPI (blind control) and aged recombination models. Different
letters indicate significant difference according to LSD post-hoc (α =
0.05).
Under refrigerated conditions (versus higher temperature),
the PPIs were more stable, with a lower observed change in the
aroma profiles during storage. For PPI A, only high aw con-
Food Funct.
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ditions resulted in significant aroma changes for PPI A with a DFC
score of 3.7 corresponding to a ‘little’ difference according to the
sensory scale (Fig. 1i and ii); whereas for PPI B significant change
was observed at both the medium and high aw conditions (Fig. 1iii
and iv). No aroma changes were noted for the low aw condition for
PPI A and B. At 37 °C storage, all the samples for PPI A and B
samples were rated as significantly different from the blind
control. At this temperature, the aged PPI A resulted in the greatest
changes when stored at high aw condition with a DFC score of 6.8,
corresponding to a difference between ‘medium’ and ‘strong’ on
the scale. Furthermore, the low and medium aw samples were sig-
nificantly different from the blind control with ‘little’ to ‘medium’
difference DFC scores of 4.2 and 4.9, respectively, but were not sig-
nificantly different from each other. For PPI B, both the medium
and high aw samples resulted in the largest changes with DFC
scores in the ‘medium’ to ‘strong’ difference range of 5.9 and 6.3,
respectively. The low aw sample had a DFC score of 4.3.
The aged samples with significant difference scores indi-
cated that the chemical changes occurring during storage were
sufficient to alter the perceived aroma. Qualitative descriptions
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Food & Function Paper

of the samples were also collected to give an indication of the
nature of the chemical changes, such as intensity, aroma
characteristics, or a combination of the two. The comments
from this study revealed that both aroma intensity and quality
differences were detected compared to the control.
Aroma recombination models of the PPI A sample at high
aw condition for the unaged aged samples were subsequently
analyzed (shown in Fig. 2) to determine the sensory relevance
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(control) and two aged samples that reflected changes in the
compounds that significantly increased or decreased in con-
centration during storage (see Table 2). The two recombination
aged models of the increasing and decreasing compounds
were rated at 3.2 and 3.0, respectively, corresponding to ‘little’
difference from the blind control (Fig. 2). Thus, the reported
aroma compositional changes induced a perceptible difference
in orthonasal aroma.

of the noted quantitative changes in the aroma compounds To further define the specific role of the seven individual
during storage (Table 3 and Fig. 1). The recombination models compounds that significantly changed in concentration during
contained a mixture of all compounds for the unaged sample storage on the reported flavor change of the PPI samples

Fig. 3 Average difference from control (DFC) ratings (n = 20) for recombination models of unaged PPI (blind control), (i) aged PPI for individual
compounds increasing in concentration (unaged PPI recombination model + 11, 109.1, 87.6, 6.53, 10.9 µg L−1 of 1-octen-3-ol, 2-heptanone, 2-pen-
tylfuran, heptanal, and nonanal, respectively) and (ii) aged PPI recombination model for individual compounds decreasing in concentration (unaged
PPI recombination model − 0.62, 10.3 µg L−1 of (E,E)-2,4-nonadienal and (E,E)-2,4-decadienal, respectively); different letters indicate significant
difference according to LSD post-hoc (α = 0.05).

This journal is © The Royal Society of Chemistry 2020 Food Funct.


Paper
(Fig. 1 and 2), the unaged PPI recombination model (Table 2,
11 compounds) was compared to seven different aged recombi-
nation models representing the concentration for each individ-
ual compound (unaged PPI recombination model adjusted to
the concentration of each individual compound after storage)
and is shown in Fig. 3. Among the compounds that signifi-
cantly increased in concentration during storage (Fig. 3i),
1-octen-3-ol and, to a lesser extent, nonanal had a significant
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impact on the noted DFC score, whereas the remaining three
compounds did not exhibit significant changes from the
unaged PPI model mixture. While 1-octen-3-ol has a mush-
room odor, it has been reported to have synergy with other
compounds to contribute to a beany aroma and suggests how
it may be contributing to the aroma profile of PPIs.39 During
storage, 1-octen-3-ol concentration increased 8-fold in this
storage condition (high aw at 37 °C), which was the greatest
increase of all the compounds in this storage condition.
Nonanal showed a 2-fold increase in concentration for the
high aw and high temperature storage. Altogether these
changes result in different aroma composition ratios that have
been previously reported to alter the aroma properties.39
Review of the two compounds that significantly decreased
in concentration during storage (Fig. 3ii) indicated only (E,E)-
2,4-decadienal had a significant impact on the DFC sensory
score in the model mixture. This compound showed an 80%
decrease in concentration over storage whereas (E,E)-2,4-nona-
dienal showed 50% decrease (Table 3).
Although seven compounds were identified in the PPI A
sample that signficantly changed in concentration during
storge and were at levels above odor threshold, only three com-
pounds (1-octen-3-ol, nonanal, and 2,4-decadienal) had a
noticable impact on the odor properties of the samples. This
finding further supports the need to evaluate the flavor contri-
bution of individual compounds in an aroma mixture (as per-
ceived) rather than solely relying on reductionistic approaches,
such as odor threshold values.
4. Conclusions
Secondary oxidation products of linoleic and oleic acid were
reported to impact flavor stability during storage of PPIs.
Specifically the generation of 1-octen-3-ol, nonanal, and the
degradation of (E,E)-2,4-decadienal impacted sensory aroma
changes in PPIs and provide selective molecular targets for
flavor optimization strategies. Although hexanal is typically
an autoxidation marker, at the levels found in this study,
this compound did not have an impact on sensory changes
reported during storage nor was it predictive of flavor stabi-
lity. The generation of 1-octen-3-ol had the greatest contri-
bution to flavor instability that was generally favored at
higher water activity values during storage. This finding
suggests 1-octen-3-ol is formed, in part, by enzymatic path-
ways and further lipoxygenase inactivation is needed for
flavor improvement.
Food Funct.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors would like to acknowledge the financial support
provided the Flavor Research and Education Center at The
Ohio State University and its supporting members.
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