Food Research International 65 (2014) 193–198

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Changes in chlorophylls, chlorophyll degradation products and lutein in

pistachio kernels (Pistacia vera L.) during roasting
Gloria Pumilia a,1, Morgan J. Cichon b,1, Jessica L. Cooperstone b,1, Daniele Giuffrida a,
Giacomo Dugo a, Steven J. Schwartz b,⁎
a
Department of Environmental Science, Safety, Territory, of Food and Health (S.A.S.T.A.S.) University of Messina, Viale Ferdinando Stagno d'Alcontres, 31, 98166 S. Agata. Messina, Italy
b
Department of Food Science & Technology, The Ohio State University, 2015 Fyffe Ct., 110 Parker Food Science & Technology Building, Columbus, OH 43210, USA

a r t i c l e i n f o a b s t r a c t

Article history: Bright green pistachio kernels are desirable in the food industry and degradation of color is often associated with
Received 31 January 2014 a loss in quality. Chlorophylls (a and b) and lutein are the major pigments in raw pistachios. The heat treatment of
Received in revised form 6 May 2014 chlorophyll-containing foods results in color alteration, due to the conversion of chlorophylls (bright green color)
Accepted 29 May 2014
to pheophytins and pyropheophytins (yellow-brown olive color). In this study, changes in the concentration of
Available online 7 June 2014
chlorophylls, chlorophyll degradation products and lutein in pistachios were monitored during oven roasting.
Keywords:
An efficient, high performance liquid chromatography method with photodiode-array detection (HPLC-PDA)
Pistachio was developed to separate and quantitate these pigments in pistachios. Extractable chlorophylls a and b were
Pistacia vera L. higher in pistachio kernels roasted for 5 and 10 min than in the raw kernels. The most drastic losses were ob-
Chlorophylls served with pheophytins a and b, which both decreased by approximately 85% after 60 min of roasting.
Pheophytins Pyropheophytins a and b increased significantly during roasting and were 10–12 fold higher in the pistachios
Pyropheophytins roasted for 60 min than in the raw pistachios. Extractable lutein concentration increased by 37% after 5 min,
Pyrochlorophylls but did not change significantly with longer roasting times. Initial increases in chlorophylls a and b and lutein
Lutein
were likely due to enhanced extractability with roasting. Color measurements of the pistachios corresponded
chlorophyll degradation products
with changes in concentration of chlorophyll pigments observed at the later roasting times. Interestingly,
pyrochlorophylls a and b were detected in pistachios as confirmed by mass spectrometry. There are no prior re-
ports of these compounds being detected in pistachios. We hypothesize that the conditions of pistachio roasting
favor the formation of pyrochlorophylls. Color changes observed after oven roasting significantly affect quality
and value perceptions of pistachios.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction 1997). Additionally, roasting is an effective method to increase the safety

Pistachios (Pistachia vera L.) are a commonly consumed tree nut cul-
tivated in dry and hot areas, under saline conditions (Metheney, Reyes,
& Ferguson, 1997). Iran, the United States, Turkey, Syria, Italy and
Greece are the main producers of pistachios, and global production is
steadily increasing (FAO, 2013). Pistachio nuts are sold shelled or un-
shelled, raw or roasted, with or without salt, and the kernel can be
used in confections and pistachio nut paste to contribute flavor and
color (Gamlı & Hayoğlu, 2007). The majority of pistachios sold directly
to consumers are roasted; this process is responsible for the characteris-
tic aroma and taste of pistachios (Nicoli, Anese, Manzocco, & Lerici,
⁎ Corresponding author at: 2015 Fyffe Ct., 235 Parker Food Science and Technology
Building, Columbus, OH 43210, USA. Tel.: +1 614 292 2934.
E-mail address: schwartz.177@osu.edu (S.J. Schwartz).
1
These authors contributed equally to this manuscript.
of pistachios as it can potentially reduce aflatoxin content (Yazdanpanah,
Mohammadi, Abouhossain, & Cheraghali, 2005).
The popularity of pistachios is growing due in part to their proposed
health benefits. Unsaturated fats, carotenoids, vitamins and antioxi-
dants in pistachios increase the value of this nut to consumers (Kay,
Gebauer, West, & Kris-Etherton, 2010; Robbins, Shin, Shewfelt,
Eitenmiller, & Pegg, 2011). Pistachios have been shown to have a pos-
itive impact on plasma lipid profiles (Kocyigit, Koylu, & Keles, 2006) and
may decrease the risk for cardiovascular disease (Gebauer et al., 2008).
With consumer interest in pistachios increasing, continued research on
factors affecting quality is needed.
Color is an important parameter for the evaluation of pistachios with
chlorophylls (a and b) and lutein being the major pigments in raw
pistachios. Chlorophylls are heat labile and can degrade to form
pheophytins and pyropheophytins. Degradation products of chloro-
phylls have been reported in thermally processed green fruits and veg-
etables (Heaton & Marangoni, 1996; Schwartz & Elbe, 1983). The bright

http://dx.doi.org/10.1016/j.foodres.2014.05.047
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
194 G. Pumilia et al. / Food Research International 65 (2014) 193–198
green color of fresh vegetables is often dulled after thermal processing
as a result of degradation of chlorophylls a and b to their respective
pheophytins and pyropheophytins. Pheophytins are formed from the
displacement of the central magnesium atom from the chlorophyll
porphyrin ring with hydrogen. Previous work has reported pheophytins
in pistachios after 11 months of storage at 25 °C (Bellomo, Fallico, &
Muratore, 2009) indicating that pheophytins can also be formed
under conditions other than thermal processing. Pyropheophytins,
which are formed from pheophytins after loss of the carbomethoxy
group at the C10 position, can be produced as a result of more extreme
forms of heat treatment (Fig. 1) (Canjura, Schwartz, & Nunes, 1991;
Schwartz & Elbe, 1983). Chlorophylls a′ and b′ are the C10 epimers of
chlorophylls a and b, respectively, with an inversion of stereochemistry
between the carbomethoxy and hydrogen substituents (Schwartz, von
Elbe, & Giusti, 2008).
The predominant carotenoid found in pistachios is lutein, a xan-
thophyll with a characteristic yellow color (Giuffrida, Saitta, Torre,
Bombaci, & Dugo, 2006). Previous work shows that lutein appears
to be quite heat-resistant, in contrast to chlorophylls, which are suscepti-
ble to degradation after thermal processing (Khachik et al., 1992). In one
study, lutein was shown to be resistant to degradation after thermal pro-
cessing in four different green and red vegetables (Khachik et al., 1992).
Additionally, it has been shown that lutein dissolved in oil is more resis-
tant to thermal processing compared to some hydrocarbon carotenoids
(Henry, Catignani, & Schwartz, 1998). Previous literature has also demon-
strated a perceived increase of lutein after thermal processing, which can
be attributed to a loss of soluble solids into the canning medium (Weckel,
Santos, Hernan, Laferriere, & Gabelman, 1962), inactivation of enzymes
able to oxidize the carotenoids and denaturation of carotenoid–protein
complexes facilitating extractability (Kirk & Tilnet-Basset, 1978).
This study characterizes the effect of roasting on pistachio pigments:
changes in chlorophylls, chlorophyll degradation products and lutein
Fig. 1. Chlorophyll and its derivatives.
were monitored using HPLC and confirmed using mass spectrometry.
Colorimetric data was collected to investigate the effects of roasting
on quantitative color parameters that also influence pistachio quality.
2. Materials and methods
2.1. Chemicals
All solvents were purchased from Fisher Scientific (Pittsburgh,
PA, USA). N-N-dimethyl-formamide (DMF), hexane, methyl tert-
butyl ether, methanol and water were HPLC grade and diethyl ether
was ACS grade. Sodium sulfate was purchased from Fisher Scientific
(Pittsburgh, PA, USA) and ammonium acetate was purchased from J.T.
Baker (Phillipsburg, NJ, USA). For mass spectrometry, Optima grade
methanol and water were used.
Chlorophylls a and b, and lutein standards were purchased from
Sigma Aldrich (St. Louis, MO, USA). Pheophytin a and b standards
were prepared by treating chlorophyll a and b standards, respectively,
in diethyl ether with 1 M HCl under nitrogen gas for five minutes
(Hynninen & Ellfolk, 1973). Standard curves were generated for
quantitation of chlorophylls a and b, pheophytins a and b, and lutein.
Chlorophylls a′ and b′ and pheophytins a′ and b′ were quantified
using the standard curves for chlorophylls a and b and pheophytins
a and b, respectively; and pyropheophytins a and b were also quantified
using relative slopes based on a ratio of molar extinction coefficient to
pheophytins a and b respectively. Pyrochlorophylls were detected, but
not quantified.
2.2. Pistachio roasting
Raw, unshelled commercial pistachio nuts from California were pur-
chased from Nuts.com (Cranford, NJ, USA). Pistachios were heated in a
 G. Pumilia et al. / Food Research International 65 (2014) 193–198
convection oven at 138 °C. Samples were removed from the oven after
5, 10, 30, and 60 min and frozen immediately at −80 °C. Chlorophylls,
chlorophyll degradation products and carotenoids were extracted
from samples at each time point in triplicate and were analyzed via
HPLC-PDA.
2.2.1. Pigment extraction
The samples were extracted by liquid-phase distribution between
DMF and hexane, according to a method previously described with
minor modifications (Giuffrida et al., 2006; Minguez-Mosquera,
Gandul-Rojas, & Gallardo-Guerrero, 1992). Briefly, approximately
50 g of pistachios were ground in order to sample 6 g for extraction
with 5 mL of acetone, repeated 5 times, for a total of 25 mL of extract.
Mild sonication was used during extraction with acetone to increase
contact between the sample and the solvent (Downes, Hrstich, &
Vincent, 1993). The acetone extracts were pooled and dried under
nitrogen. The extract was then dissolved in 6 mL of DMF, and treated
with 12 mL portions of hexane and centrifuged at 2000 ×g. The top
layer was then removed and extraction with hexane was repeated
two additional times. Chlorophylls, chlorophyll degradation products
and xanthophylls were retained in the DMF phase while the hexane
phase contained lipids and carotenes. The hexane phase was saponified
and subsequently analyzed by HPLC-PDA for carotenes. Beta-carotene
was detected at low levels, as previously reported (Giuffrida et al.,
2006), but was not quantified.
The DMF extract was treated with 6 mL of a 2% aqueous Na2SO4
(w/v) solution in bath of ice, followed by 3 successive 4 mL portions
of 1:1 hexane:diethyl ether. The hexane:diethyl ether phases were
centrifuged each time. The aqueous phase was discarded eliminating
polyphenols and other water-soluble compounds. The organic phase
was reconstituted in a known volume, aliquoted, and evaporated to
dryness under nitrogen. The dried residue was dissolved in an appropri-
ate volume of 1:1 methanol:MTBE, filtered through a 0.22 μm nylon
filter, and analyzed by HPLC.
2.3. Pigment analysis by HPLC and mass spectrometry
Chlorophylls, chlorophyll degradation products and xanthophylls
extracted from the pistachio samples were analyzed using a model
2695 HPLC with a model 996 PDA (Waters Corp., Milford, MA). For
HPLC separation, an YMC C30 analytical column (250 mm × 4.6 mm
i.d., 3 μm) (Waters Corp.) was used at 30 °C. The mobile phase consisted
of the following: A: 90% MeOH, 3% MTBE, 5% H2O, 2% of a 2% (w/v) so-
lution of aqueous ammonium acetate and B: 88% MTBE, 10% MeOH,
2% of a 2% (w/v) solution of aqueous ammonium acetate. A gradient
was employed flowing at 1.3 mL/min to separate the pigments of inter-
est as shown in Table 1. The same model HPLC-PDA was also interfaced
with a QToF Premier quadrupole time-of-flight mass spectrometer
(Waters Corp.) using atmospheric pressure chemical ionization (APcI)
in positive ion mode to confirm the identities of pyrochlorophylls and
Table 1
Gradient schematic for separation of pigments in pistachios by HPLC-PDA.
Time (min) Solvent A
0 95
5 60
10 50
12 48
14 45
16 40
18 40
20 20
22 20
24 5 95
27 5 95
27.01 95
30 95
195
pyropheophytins. The same HPLC method was used for chromatograph-
ic separation, with the following MS parameters: 7 μA corona current,
8 eV collision energy, 3 V cone voltage, 110 °C source temperature,
and 450 °C probe temperature.
Chromatograms were integrated for quantitation at the following
wavelengths: 661 nm (chlorophyll a/a′), 642 nm (chlorophyll b/b′),
667 nm (pheophytin a/a′ and pyropheophytin a), 655 nm (pheophytin
b/b′ and pyropheophytin b) and 444 nm (lutein). For those pigments
without authentic external standards, identities were confirmed by
spectral characteristics and accurate mass.
2.4. Color analysis
Color readings were taken on the raw and roasted, ground pistachios
using a Hunter ColorQuest XE (Hunter Labs, Reston, VA). All samples
were read in duplicate with a D65 illuminant and a 10° observer
angle. Color measurements were expressed using the CIELAB color
system.
2.5. Data analysis
Data were analyzed for differences between means using one-way
analysis of variance (ANOVA) and Tukey's post-hoc test, with statistical
significance when P b 0.05, using SPSS version 19 (IBM, Armonk, NY).
3. Results and discussion
Chlorophylls (and their epimers), pheophytins (and their epimers),
pyropheophytins and lutein were measured in raw and roasted pista-
chios. Although the epimers have the same spectra as their parent,
they can be differentiated by their elution order. On a C30 column,
chlorophylls a′ and b′ eluted immediately after their corresponding a
and b peaks; whereas pheophytins a′ and b′ eluted immediately before
their corresponding a and b peaks (Fig. 2). The epimers are always pres-
ent in lower concentration compared to the parent chlorophyll. Fig. 2
shows HPLC chromatograms of raw pistachios, and pistachios roasted
for 5, 10, 30 and 60 min. Table 2 shows the concentrations of the
pigments in the pistachios at the different roasting times. Extractable
chlorophylls a and b were higher in the pistachio kernels roasted for 5
and 10 min than in the raw kernels. The increase in concentration ob-
served after mild roasting could be a result of the breakdown of chloro-
phyll–protein complexes, which could improve the extractability of the
pigments from the nuts (Paulsen & Schmid, 2002; Yamane, Kashino,
Koike, & Satoh, 1997). Concentrations of extractable pheophytins a
and b were also higher in pistachio kernels roasted for 5 min compared
to raw pistachios, but after 60 min of roasting concentrations decreased
by approximately 85%. The presence of pheophytins a and b in raw pis-
tachios is likely due to the natural chlorophyll degradation observed at
room temperature after long term storage (Bellomo et al., 2009).
Pyropheophytin peaks showed a considerable increase in the chro-
matogram of pistachios roasted for 30 min with the highest value in pis-
tachios roasted for 60 min (reaching levels 10–12 times higher than in
the raw pistachios). Pyropheophytins are formed from pheophytins
during thermal processing as a result of the loss of the carbomethoxy
Solvent B
group at C10 (Schwartz & Elbe, 1983; Schwartz, Woo, & Von Elbe,
5 1981). Extractable lutein concentration increased by 37% after 5 min,
40
50
but did not change significantly after that; this observation is also likely
52 a result of increased carotenoid extractability from the nuts. Durmaz
55 and Gökmen report a significant decrease in lutein in oil derived from
60 pistachios roasted for 20 min, but at a higher roasting temperature
60
(180 °C) (Durmaz & Gökmen, 2011). However, other research shows
80
80 lutein in food products to be relatively resistant to heat treatments
(Khachik et al., 1992), as observed in this study.
Interestingly, two compounds were identified for the first time in
5 pistachio kernels: pyrochlorophylls a and b. These compounds were
5
confirmed by their spectral characteristics, retention time and mass
196 G. Pumilia et al. / Food Research International 65 (2014) 193–198

Fig. 2. The HPLC chromatograms (combined 665 and 650 nm) of the pistachio extracts from raw and 5, 10, 30 and 60 min of roasting. Peak identification: 1, chlorophyll b; 2, chlorophyll b′;
3, chlorophyll a; 4, chlorophyll a′; 5, pheophytin b′; 6, pheophytin b; 7, pheophytin a′; 8, pheophytin a; 9, pyrochlorophyll b; 10, pyrochlorophyll a; 11, pyropheophytin b; 12,
pyropheophytin a.

spectrometry (Figs. 3 and 4). Concentrations of pyrochlorophylls a and b the loss of a carbomethoxy group (Fig. 1). As pyrochlorophylls have
were highest in pistachios roasted for 60 min. Pyrochlorophylls are less the same absorption spectra to their parent chlorophylls, accurate
common chlorophyll degradation products than pheophytins and mass was used to differentiate between these compounds. We hypoth-
pyropheophytins. They can be formed directly from chlorophylls from esize that the high roasting temperatures and low moisture content of

Table 2
Changes in pistachio pigments from raw during roasting at 138 °C for 5, 10, 30 or 60 min. Different letters in the same row denote statistically significant differences (P b 0.05).

Pigment Quantity of Pigment (mg/kg wet weight)

Raw 5 min 10 min 30 min 60 min

Chlorophyll a 3.63 ± 0.57a 7.64 ± 1.25b 7.75 ± 1.39b 4.10 ± 0.46a 2.00 ± 0.47a
Chlorophyll a′ 0.80 ± 0.20a 1.92 ± 0.21bc 2.49 ± 0.29c 1.65 ± 0.27b 0.89 ± 0.12
Chlorophyll b 1.81 ± 0.38a 3.29 ± 0.27b 3.28 ± 0.61b 1.47 ± 0.10a 0.46 ± 0.09c
Chlorophyll b′ 0.68 ± 0.09a 1.40 ± 0.03b 1.37 ± 0.09b 0.64 ± 0.03a 0.22 ± 0.05a
Pheophytin a 6.29 ± 1.56a 7.16 ± 0.88a 5.93 ± 0.84ab 3.58 ± 0.78b 0.90 ± 0.29c
Pheophytin a′ 1.12 ± 0.21a 1.28 ± 0.11a 1.13 ± 0.11a 0.71 ± 0.14b 0.21 ± 0.07c
Pheophytin b 0.81 ± 0.07a 1.00 ± 0.06b 0.93 ± 0.07ab 0.39 ± 0.0303c 0.11 ± 0.10d
Pheophytin b′ 0.15 ± 0.02a 0.23 ± 0.03b 0.20 ± 0.05ab 0.05 ± 0.01c 0.01 ± 0.03c
Phyropheophytin a 0.54 ± 0.03a 0.70 ± 0.03a 1.05 ± 0.09a 4.59 ± 0.81b 6.70 ± 0.22b
Phyropheophytin b 0.13 ± 0.13a 0.22 ± 0.04a 0.34 ± 0.08a 0.93 ± 0.03b 1.37 ± 0.21b
Lutein 8.12 ± 0.71a 10.83 ± 0.77b 10.83 ± 1.54b 10.62 ± 0.60b 11.14 ± 0.25b
 G. Pumilia et al. / Food Research International 65 (2014) 193–198 197

Fig. 3. Extracted ion chromatograms for pyrochlorophyll a (tentative) and pyropheophytin a Fig. 4. Extracted ion chromatograms for pyrochlorophyll b (tentative) and pyropheophytin b
in the pistachios roasted for 60 min. Mass chromatograms (top two) are aligned with the in the pistachios roasted for 60 min. Mass chromatograms (top two) are aligned with the
HPLC chromatogram (bottom) at 665 nm. HPLC chromatogram (bottom) at 650 nm.

the pistachio may be responsible for the conversion of chlorophylls to pistachios significantly decreased during roasting, which indicates less
pyrochlorophylls. The formation of pyrochlorophylls has been previous- yellow color. These observed color changes are considered to be less de-
ly observed in spinach leaves during microwave heat treatment (Teng & sirable. Similar trends in L*-, a*- and b*-values were reported for oils
Chen, 1999), which supports our hypothesis. However, additional work from pistachios roasted at 180 °C for 5, 10, 20, 30 and 40 min
is needed to test the specific effect of low moisture, and high tempera- (Durmaz & Gökmen, 2011).
ture conditions on the formation of these compounds. Color is one of the most important quality attributes evaluated by
The pigment extraction used in this paper was faster than the most consumers, producers and distributors. Chlorophylls a and b are the pre-
common method used to extract chlorophylls and xanthophylls dominant pigments responsible for the green color of pistachios. Pig-
(Minguez-Mosquera et al., 1992) due to smaller sample sizes versus ment concentrations are influenced by a number of factors, including
the method presented in the literature. Previous pigment extractions geographic origin, degree of ripeness, storage conditions and processing
from pistachios have used sample sizes of 37 g where this study used methods. The concentrations and ratios of these pigments can be quan-
a smaller sample size (6 g) for a faster sample preparation requiring tified and used to determine the geographic origin of a sample (Bellomo
less solvent (Giuffrida et al., 2006). Extraction efficiencies of the two & Fallico, 2007). For quality and import purposes, region of origin may
methods were evaluated and found to be comparable. Additionally, a affect the value of pistachios, thus authentication of geographic origin
more efficient HPLC method was developed for the separation and anal- is valuable in preventing adulteration or mislabeling of pistachios.
ysis of these pigments in pistachios. This improved method has a shorter
run time while retaining good peak separation (Giuffrida et al., 2006). Table 3
Ammonium acetate was added to the mobile phase for better peak Color analysis of raw and roasted pistachios in duplicate. Different letters in the same
resolution, shape and on column recovery among the analytes (Hart & column denote statistically significant differences, as determined by ANOVA (P b 0.05)
and Tukey's post-hoc test for multiple comparisons.
Scott, 1995).
Colorimetric measurements of the pistachio samples after different Roasting time L* a* b* C* h* dE*
roasting times correspond with changes in concentration of chlorophyll Raw 60.7 a
1.3 a
26.9 ab
26.9 ab
87.3 a
66.4a
pigments determined by HPLC-PDA (Table 3). The L*-value of the pista- 5 min 60.8a 0.5b 27.1a 27.1b 88.9b 66.8a
chios decreased with roasting time, indicating a darkening of the nuts. 10 min 58.4b 2.5c 26.1a 26.2a 84.6c 64.0b
Additionally, the a*-value significantly increased during roasting, 30 min 53.8c 5.6d 21.0c 21.1c 75.0d 58.0c
60 min 50.2d 5.1d 17.2d 18.3d 70.9e 53.4d
which indicates less green in the sample, while the b*-value of the
198 G. Pumilia et al. / Food Research International 65 (2014) 193–198
Additionally, roasting pistachios can affect other nut parameters by
inducing the Maillard reaction, producing aroma compounds and
influencing lipid oxidation.
It has been shown that nut roasting decreases reducing sugars, as
these sugars likely take part in Maillard browning reactions (Kashani
& Valadon, 1984) and can lead to the formation of important aroma
compounds (Aceña, Vera, Guasch, Busto, & Mestres, 2010). Pistachio
nut oils produced from roasted nuts were found to have lower peroxide
and TBARS values after 30 days of storage compared to unroasted nut
oils (Durmaz & Gökmen, 2011). This suggests that roasting may inacti-
vate enzymes responsible for nut rancidity. All of these characteristics
play an important role in pistachio nut quality and acceptability.
4. Conclusions
Interesting changes in concentrations of chlorophylls, pheophytins,
pyropheophytins as well as pyrochlorophylls were observed during con-
vection oven roasting. After extended roasting times pyropheophytins a
and b are the predominant chlorophyll compounds. The formation of
pyrochlorophylls a and b was observed in oven roasted pistachios and
their presence was confirmed by mass spectrometry. We consider the
formation of these compounds to be a unique finding from this research.
We hypothesize that the conditions of pistachio roasting favor the forma-
tion of pyrochlorophylls. Colorimetric data of the oven roasted samples
corresponded with changes in pigment concentration; as chlorophylls
degraded to pheophytins and other degradations products, the color of
the pistachio changed from bright green to dull, olive green. Future
work may investigate ways to inhibit chlorophyll degradation and mini-
mize color changes in pistachios during roasting. This color change is sig-
nificant in perception of quality and value for consumers and producers
of roasted pistachio products.
Acknowledgments
We would like to acknowledge Thomas C. Haufe for his editorial as-
sistance in the preparation of this manuscript.
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