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agronomy
Article
Changes in Capsiate Content in Four Chili Pepper
Genotypes (Capsicum spp.) at Different
Ripening Stages
Mercedes Vázquez-Espinosa 1, Oreto Fayos 2, Ana V. González-de-Peredo 1,
Estrella Espada-Bellido 1, Marta Ferreiro-González 1, Miguel Palma 1,
Ana Garcés-Claver 2and Gerardo F. Barbero 1, *
1Department of Analytical Chemistry, Faculty of Sciences, University of Cadiz, Agrifood Campus of
International Excellence (ceiA3), IVAGRO, 11510 Puerto Real, Cadiz, Spain;
mercedes.vazquez@uca.es (M.V.-E.); ana.velascogope@uca.es (A.V.G.-d.-P.); estrella.espada@uca.es (E.E.-B.);
marta.ferreiro@uca.es (M.F.-G.); miguel.palma@uca.es (M.P.)
2
Centro de Investigaci
ó
n y Tecnolog
í
a Agroalimentaria de Arag
ó
n, Instituto Agroalimentario de Arag
ó
n-IA2,
CITA-Universidad de Zaragoza, Avda. Montañana 930, 50059 Zaragoza, Spain; ofayos@cita-aragon.es (O.F.);
agarces@cita-aragon.es (A.G.-C.)
*Correspondence: gerardo.fernandez@uca.es; Tel.: +34-956-016355; Fax: +34-956-016460
Received: 18 July 2020; Accepted: 3 September 2020; Published: 5 September 2020
Abstract:
Interest in the consumption of the fruits of pepper (Capsicum spp.) is not only due
to its organoleptic characteristics, but also due to its bioactive compounds content, which are
reported to provide essential benefits to human health. However, the amount and diversity of
these compounds in each fruit specimen depend on its genotype and on a number of environmental
factors. This work describes the quantitative ultra-high-performance liquid chromatography coupled
to photodiode-array (UHPLC-PDA) analysis of the capsinoids content in four varieties of pepper
(‘Habanero’, ‘Habanero Roxo’, ‘Bode’, and ‘Malagueta’) grown until different development stages in a
greenhouse under controlled conditions. In all the varieties analyzed, capsiate was the only capsinoid
found. The accumulation of capsiate, in all the pepper varieties, started from the 10th to the 20th
day post-anthesis (dpa), and increased during the first days (between the 20th and the 27th dpa).
From that moment a drastic reduction took place until the end of the ripening stage, except for ‘Bode’
peppers, where the capsiate content increased from the first harvest point on the 20th dpa up to the
76th dpa. The capsiate accumulation patterns over the development of the fruit has been related to the
capsaicionoids accumulation patterns in the same samples of the four varieties of pepper. According
to our results, the content evolution of both families of compounds will vary depending on each
fruit’s genotype, as well as on environmental conditions. No clear trends have been established and,
therefore, an in-depth analysis under controlled conditions should be carried out.
Keywords:
‘Bode’ pepper; capsiate content; Capsicum spp.; capsinoids; fruit ripening; ‘Habanero’
pepper; ‘Habanero Roxo’ pepper; ‘Malagueta’ pepper; UHPLC
1. Introduction
Pepper belongs to the genus Capsicum and the Solanaceae family, original from tropical areas in
America. From the 35 described, only five species have been domesticated: Capsicum chinense Jacq.,
C. frutescens L., C. annuum L., C. baccatum L., and C. pubescens Ruiz & Pav., with significant economic and
social impact worldwide [
1
]. Capsicum fruits vary in size (thick or thin), shape (round, elongated, etc.),
color (green, purple, chocolate, yellow, orange, or red, depending on pepper variety and maturation
stage.), flavor, and pungency (from the non-pungent varieties to the hottest species) [
2
]. Due to the
Agronomy 2020,10, 1337; doi:10.3390/agronomy10091337 www.mdpi.com/journal/agronomy
Agronomy 2020,10, 1337 2 of 14
vast quantity and the diverse varieties consumed, pepper is among the most valued and commonly
cultivated produce because of their color, flavor, and taste sensory attributes. The food industry is
the principal user of pepper fruits. It is often used as a coloring and flavoring agent in sauce, soup,
processed meat, snacks, candies, soft drinks, and alcoholic beverages [
3
]. In addition to their sensory
features, oleoresin is extracted from pepper fruits and used as an ingredient in numerous commercial
products such as insect repellent or even self-defense sprays; and peppers can be also employed in
medicinal applications, since they are an important source of the kind of bioactive compounds that
provide health benefits to consumers [
4
]. Among such bioactive compounds two families should
be noted: capsaicinoids and capsinoids, exclusive to the genus Capsicum and responsible for pepper
pungency [
5
]. Both are widely known for their pharmacological properties, such as anti-inflammatory,
anti-carcinogenic, neurological, antimicrobial, and antioxidant. They also contribute to weight loss
treatments, relieve pain, and provide gastrointestinal and cardiovascular benefits when ingested
regularly [6–10].
Capsaicinoid and capsinoid biosynthesis takes place in the placenta between the 10th and the
20th days post anthesis (dpa), but they can also be detected in some of the fruit tissues, such as seeds
or pericarp, due to the fact that they are eventually excreted [
11
]. Capsaicinoids, and also probably
capsinoids, are ultimately produced by capsaicin synthase through the condensation of an aromatic
moiety, derived from vanillin, with fatty acid branched-chains of 9–11 carbon atoms [
12
]. In addition,
their fundamental chemical structures are rather similar, with the exception of their central bond.
Thus, while capsinoids have an ester group, capsaicinoids have an amide group. This difference in
their structure seems to be responsible for the lower pungency of capsinoids, roughly determined
as 1000 times lower compared to that of capsaicinoids. Therefore, the employment of capsinoids is
particularly attractive as a food additive or medicinal product, since they do not present the side effects
of capsaicinoids such as irritation or burning sensations [13,14].
Capsinoids were first reported by Yazawa et al., in a non-pungent pepper cultivar CH-19 Sweet [
15
].
To date, three capsinoids, capsiate, dihydrocapsiate, and nordihydrocapsiate, have been described
in pepper fruits, capsiate being the major one. Later on, these compounds were detected in other
varieties of non-pungent and low pungent peppers, as well as in hot and super-hot pepper cultivars,
although in considerably lower concentrations than capsaicinoids. In pungent peppers, vanillin is
converted to both vanillylamine and vanillyl alcohol, which in turns gives place to the production of
capsaicinoids and capsinoids, respectively [
16
]. The putative aminotransferase (p-AMT) gene encodes
the aminotransferase enzyme (p-AMT) that catalyzes the formation of vanillylamine from vanillin in
the capsaicinoid biosynthetic pathway. Different mutations in the p-AMT gene have been described
as always leading to a loss-of-function with the subsequent increment in the production of vanillyl
alcohol. Consequently, pepper genotypes carrying a pamt allele are non or low-pungent due to the
high production of capsinoids over that of capsaicinoids [
17
,
18
]. Therefore p-AMT allele could be
considered as a useful gene to control the content of capsaicinoids and capsinoids in pepper breeding
programs [19,20].
The content of capsaicinoid and capsinoid compounds in peppers can be affected by different
factors, including water availability (there is a significant reduction in fruit yield when a reduced
amount of water is applied during the periods of vegetative growth, flowering, and fruiting) [
21
],
light (it regulates morphological characteristics and acts as a source of energy for the primary
metabolism and photosynthetic processes) [
22
], temperature, climatic conditions, genotype, cultivation
techniques, mineral supply, growing conditions, and maturity stage (during the fruit ripening stage,
several biochemical, physiological, and structural changes take place, and those changes govern the
characteristics of the final fruit) [
23
,
24
]. Sampling and storage conditions need to be closely controlled
to produce high-quality plant material for its characterization and further use [25].
The present study has focused on four of the main pepper varieties consumed in Brazil: ‘Habanero’,
‘Habanero Roxo’, ‘Bode’, and ‘Malagueta’. ‘Habanero’ peppers are from the C. chinense family [
26
].
This intensely aromatic fruit is claimed to be one of the hottest varieties in the world, with pungency
Agronomy 2020,10, 1337 3 of 14
values between 100.000 and 300.000 Scoville Heat Units (SHUs). This chili pepper is dark green
changing to orange, orange-red, red, or even chocolate (‘Habanero Roxo’) when fully ripe. Pod size
normally varies from 2.9 to 6.0 cm in length, 2.5 to 4.6 cm in width, and 7 to 12 g in weight, and it
is mainly used in sauces, chutneys, and marinades for seafood or pickles [
27
,
28
]. Its unique aroma,
pungency and color are its most attractive properties and a quality reference for consumers. Brazil is
considered a center of diversity for some Capsicum species (domesticated and wild) [
29
]. However,
‘Habanero’ pepper holds an enormous social and commercial relevance in other American countries,
such as Mexico. The main production zones of ‘Habanero’ chili pepper in M
é
xico are located in the
states of Yucatan, Campeche, and Quintana Roo [
3
]. There is a current great interest in exporting
this crop as a whole dehydrated product to the USA and Europe, where it is becoming an important
source of extractable oleoresin. Pepper fruits and its derivatives are also commercialized worldwide as
condiments, additives, and as the lachrymatory agent in pepper sprays; as well as a fungicidal and
cytotoxic agent [30].
The ‘Bode’ pepper variety also belongs to the C. chinense family and is native to Recife. It is
widely cultivated throughout the northern and northwestern regions in Brazil [
31
]. Its fruits, of an
intermediate pungency (15.000–30.000 SHU), are round and small, and their coloration varies between
yellow, orange, and red when fully mature. This variety is highly valued in the kitchen for its smoky
and fruity flavor and for its aroma. It is mostly consumed as pickles [32].
‘Malagueta’ pepper is a variety of the C. frutescens species; mostly cultivated and consumed in
Brazil, and particularly in the states of Minas Gerais, S
ã
o Paulo, Bahia, and Goi
á
s. It is widely used in
the production of sauces and also in preserves, jams, and pastes [
33
]. Its color changes rapidly from
green (unripe fruit) into red (ripe fruit) and, in some cases, it may present a light red color intermediate
stage. As for the size of the fruit, it varies from 1 to 3 cm long and 0.4 to 0.5 cm wide, and they are
conical with very thin walls [34].
Capsaicinoid accumulation patterns for the four species above mentioned have been previously
studied [
35
,
36
]. However, no assessment of the capsinoid accumulation patterns over the different fruit
development stages, as well as a description of the correlation between capsinoid and capsaicinoid
contents throughout those fruit development stages have been reported. For the purposes of this study,
ultrasound-assisted extraction techniques will be used. Thus, high frequency ultrasonic waves, capable
of causing cavitation due to the expansion and contraction cycles that the material goes through, will
be applied. Such expansion and contraction cycles disrupt the cell walls in the vegetable matrix to
favor the penetration of a solvent and, in turn, the mass transfer, which results in increasing extraction
rates and yields [37].
This paper intends to cast some light on two aspects that have been scarcely studied in relation
to pepper cultivation: capsinoids accumulation at the different ripening stages of pepper fruits; and
the potential correlation between capsinoid and capsaicinoid accumulation patterns in the varieties
studied over their fruit development. The conclusions that may be reached with regards to these two
aspects should help pepper breeders to determine the optimum harvesting moment that allow them to
obtain the maximum added value from their crops.
2. Materials and Methods
2.1. Plant Material
Chili pepper seeds of the var. ‘Habanero’ (C. chinense), ‘Habanero Roxo’ (C. chinense), ‘Bode’
(C. chinense), and ‘Malagueta’ (C. frutescens) were supplied by the Vegetable Germplasm Bank in
Zaragoza at the CITA of Arag
ó
n (Zaragoza, Spain). The seeds of the four varieties were germinated
in Petri dishes, and then 10 plants per genotype were grown in a random distribution inside an
acclimatized greenhouse in 17 cm diameter black plastic pots (one plant per pot), filled with a substrate
mixture formed by peat, sand, and clay-loam soil as well as Humin Substrat (Klasman-Deilmann,
Geeste, Germany) (1:1:1:1, v/v). Two grams of a slow-release fertilizer (Osmocote 16N-4P-9K, Scotts,
Agronomy 2020,10, 1337 4 of 14
Tarragona, Spain) were used as a topdressing for each pot. The plants were also watered daily by
a drip irrigation system to maintain their optimum humidity levels for growth. Temperature levels
were controlled of the whole process with values between 12–24
◦
C in the spring and 20–28
◦
C in
the summer.
The flowers were labeled at the onset of their anthesis, so as to allow the fruit stage of development
to be determined and hence each pepper’s age at the time of harvesting. The peppers were
harvested during the last week of September, since the plants stopped producing new peppers
(around 6-month-old plants). A total pepper weight varying between 232 and 346 g was harvested
from all the plants at different stages of development in order to avoid particular effects from individual
pepper plants. The maturation stages of the peppers at the time of harvest varied between immature
green and senescent. Once the samples were harvested, all the fruits from all the plants of each variety
were grouped together according to their dpa. The stem and seeds of the peppers were discarded
before their analysis, while their pericarp and placenta were ground together in an Ultra-Turrax blender
(IKA, Staufen, Germany) to produce a fully homogeneous sample that was then frozen at
−
20
◦
C
until analysis.
2.2. Chemicals and Reagents
The analytical standards of the two major capsinoids, capsiate (CTE) (4-hydroxy-3-methoxybenzyl
(E)-8-methyl-6-nonenoate) and dihydrocapsiate (DHCTE) (4-hydroxy-3-methoxybenzyl 8-methylnonanoate),
were synthesized in the Department of Organic Chemistry at the University of Cadiz by Barbero et al. [
38
].
All of the samples were prepared in a mixture of HPLC grade methanol and ethyl acetate (99.9%) from
Panreac Qu
í
mica S.L.U. (Castellar del Vall
é
s, Barcelona, Spain), and Milli-Q water provided by a deionization
system (Millipore, Bedford, MA, USA). For the chromatographic separation, HPLC grade acetonitrile
(99.99%) from Panreac Qu
í
mica S.L.U. (Castellar del Vall
é
s, Barcelona, Spain), glacial acetic acid (99%) from
Merck (Darmstadt, Germany), and Milli-Q water were employed.
2.3. Fresh Pepper Extraction Procedure
The capsinoid extraction process from peppers at the different maturation stages was performed
following a method previously developed by our research team [
39
]. The ultrasounds were applied by
means of a Sonoplus probe (BANDELIN ELECTRONIC, Heinrichstra
β
e, Berl
í
n, Germany) coupled to
a 7 L refrigerated circulator for temperature control (PolyScience, Niles, IL, USA). The sample was
immersed into a temperature-insulated double-walled vessel. Approximately 0.5 g of chili pepper
from each different ripening stages were placed in a 50 mL plastic holder, followed by the addition of
15 mL of extraction solvent (which was composed by 42% methanol +58% ethyl acetate). The sample
was sonicated for 2 min at 5.5
◦
C, under 80% of the maximum allowed power (70 W) and applying duty
cycles of 0.5 s. The extraction process was carried out in duplicate for each group of homogeneous
samples. The average of the two values obtained would be considered as the final results. The extracts
were centrifuged twice for 5 min at 7500 rpm (orbital radius 9.5 cm) and the supernatants were
transferred to a 25 mL volumetric flask, which was made up to the mark with the same extraction
solvent. The samples were filtered using a 0.22
µ
m nylon syringe filter (Membrane Solution, Dallas,
TX, USA) and analyzed by means of a ultra-high-performance liquid chromatography coupled to
photodiode-array (UHPLC-PDA) to confirm the presence of capsinoids.
2.4. UHPLC-Q-ToF-MS Identification of Capsinoids
In order to identify the capsinoids present in the pepper samples, a UHPLC system (Waters
Corporation, Milford, MA, USA) with a 2.1
×
100 mm, 1.7
µ
m particle size rp-C18 analytical column
(Acquity UPLC BEH C-18, Waters, MA, USA) was used. The UHPLC system was coupled to a
quadrupole time-of-flight mass spectrometer (Q-ToF-MS) equipped with an electrospray ionization
source (ESI) interface (Xevo G2 QToF, Waters Corporation, Milford, MA, USA) operating in positive ion
mode. For the control of the equipment, its integration, and the subsequent data analysis, Masslynx
Agronomy 2020,10, 1337 5 of 14
software version 4.1 was employed. The UHPLC variables, as well as the operating conditions of
the mass spectrometer were performed according to the method described by V
á
zquez-Espinosa et
al. [
40
]. Spectra were acquired in the full-scan mode (m/z=100–600). The molecular ions [M +H]
+
and
[M +Na]
+
monitored for their identification were: CTE (m/z307 and m/z329), and DCHTE (m/z309
and m/z330), respectively. However, CTE was the only capsinoid detected and, therefore, quantified in
the different varieties of pepper analyzed. Its chemical structure is shown in Figure 1. Furthermore,
the mass spectrum showing the characteristic fragments that allow their identification can be found in
supplementary material (Figure S1).
Agronomy 2020, 10, x 5 of 14
Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy
Masslynx software version 4.1 was employed. The UHPLC variables, as well as the operating
conditions of the mass spectrometer were performed according to the method described by
Vázquez-Espinosa et al. [40]. Spectra were acquired in the full-scan mode (m/z = 100–600). The
molecular ions [M + H]+ and [M + Na]+ monitored for their identification were: CTE (m/z 307 and m/z
329), and DCHTE (m/z 309 and m/z 330), respectively. However, CTE was the only capsinoid
detected and, therefore, quantified in the different varieties of pepper analyzed. Its chemical
structure is shown in Figure 1. Furthermore, the mass spectrum showing the characteristic
fragments that allow their identification can be found in supplementary material (Figure S1).
Figure 1. Chemical structure of capsiate (CTE).
2.5. UHPLC-PDA Analysis of Capsinoids
After identifying the only capsinoid present in these pepper samples (CTE), the extracts were
subjected to UHPLC-PDA using an Acquity Ultra Performance LC Class system (Waters
Corporation, Milford, MA, USA) equipped with an autosampler operated at 15 °C, a Quaternary
Pump System, and a Photodiode Array Detector (PDA) set to a wavelength of 280 nm for the
detection and subsequent quantification of the compound present in the different pepper varieties.
A Waters ACQUITY UPLC BEH rp-C18 100 x 2.1 mm column with 1.7 µ m particle size, maintained
at 50 °C, was used as the analytical column. Empower 3 software (Waters Corp., Milford, MA, USA)
was used for the data treatment and equipment control. The UHPLC equipment variables were the
same as in the method previously described by Vazquez-Espinosa et al. [40]. CTE, the major
capsinoid, was the only one found in all the four pepper varieties that were analyzed. A calibration
curve (y = 1682.50x + 164.74) was used for CTE quantification. Its regression equation and correlation
coefficients (R2 = 0.9997), limit of detection (LOD = 3.60 ng g−1 of fresh weight (FW)) and
quantification (LOQ = 12.00 ng g−1 of FW) were all determined. The quantitative data were obtained
based on the integration of the UHPLC peak areas corresponding to three injections of the CTE
analytical standard. A chromatogram of each variety obtained by UHPLC-PDA (280 nm), at the time
of maximum capsiate concentration for each variety, has been included in supplementary material
(Figure S2).
2.6. Statistical Analysis
A one-way analysis of variance (ANOVA), followed by a Tukey’s test, were performed to
determine any significant differences (p-value < 0.05) in CTE contents depending on ripening stage.
The results were expressed as the mean ± standard deviation (SD) for duplicate analysis. All of the
data obtained from the analyses were dealt with by means of Statgraphic Centurion Version XVII
(Statgraphics Technologies, Inc., The Plains, VA, USA).
3. Results and Discussion
3.1. Evolution of the Total Capsinoids Content
As mentioned above, capsinoids have excellent pharmacological effects on human health. In
addition, they are considerably less spicy than capsaicinoids, which makes them more attractive and
favorable for a regular daily intake, so that they provide all their benefits without the pungency
side-effects. This is what makes any study on the correlation between fruit development stage and
Figure 1. Chemical structure of capsiate (CTE).
2.5. UHPLC-PDA Analysis of Capsinoids
After identifying the only capsinoid present in these pepper samples (CTE), the extracts were
subjected to UHPLC-PDA using an Acquity Ultra Performance LC Class system (Waters Corporation,
Milford, MA, USA) equipped with an autosampler operated at 15
◦
C, a Quaternary Pump System, and
a Photodiode Array Detector (PDA) set to a wavelength of 280 nm for the detection and subsequent
quantification of the compound present in the different pepper varieties. A Waters ACQUITY UPLC
BEH rp-C18 100 x 2.1 mm column with 1.7
µ
m particle size, maintained at 50
◦
C, was used as the
analytical column. Empower 3 software (Waters Corp., Milford, MA, USA) was used for the data
treatment and equipment control. The UHPLC equipment variables were the same as in the method
previously described by Vazquez-Espinosa et al. [
40
]. CTE, the major capsinoid, was the only one
found in all the four pepper varieties that were analyzed. A calibration curve (y =1682.50x +164.74)
was used for CTE quantification. Its regression equation and correlation coefficients (R
2
=0.9997),
limit of detection (LOD =3.60 ng g
−1
of fresh weight (FW)) and quantification (LOQ =12.00 ng g
−1
of
FW) were all determined. The quantitative data were obtained based on the integration of the UHPLC
peak areas corresponding to three injections of the CTE analytical standard. A chromatogram of each
variety obtained by UHPLC-PDA (280 nm), at the time of maximum capsiate concentration for each
variety, has been included in supplementary material (Figure S2).
2.6. Statistical Analysis
A one-way analysis of variance (ANOVA), followed by a Tukey’s test, were performed to determine
any significant differences (p-value <0.05) in CTE contents depending on ripening stage. The results
were expressed as the mean
±
standard deviation (SD) for duplicate analysis. All of the data obtained
from the analyses were dealt with by means of Statgraphic Centurion Version XVII (Statgraphics
Technologies, Inc., The Plains, VA, USA).
3. Results and Discussion
3.1. Evolution of the Total Capsinoids Content
As mentioned above, capsinoids have excellent pharmacological effects on human health.
In addition, they are considerably less spicy than capsaicinoids, which makes them more attractive
and favorable for a regular daily intake, so that they provide all their benefits without the pungency
side-effects. This is what makes any study on the correlation between fruit development stage
Agronomy 2020,10, 1337 6 of 14
and capsinoids content so interesting, so that harvesting can take place when highest capsinoid
concentration is to be expected.
The number of reports that can be found in the literature on the evolution of capsinoids content in
the different varieties of peppers is very low. Fayos et al., analyzed capsinoids content in the varieties
‘Chiltep
í
n’, ‘Tampiqueño 74
0
, and ‘Bhut Jolokia’, but only at four specific moments over the fruit ripening
period, specifically on the 10th, 20th, 40th, and 60th dpa [
41
]. According to their study, similar trends
could be observed in the three genotypes, with the accumulation of capsinoids beginning between the
10th and the 20th dpa and then increasing up to their maximum concentration on the 40th dpa (with
values reaching 276.39
µ
g g
−1
of FW, 69.31
µ
g g
−1
of FW, and 122.62
µ
g g
−1
of FW, respectively). Finally,
the content gradually decreased over the last stages of development [
41
]. Jang et al., also studied the
evolution of these compounds at four different moments during the ripening process of four different
varieties of pepper that ranged between spicy and slightly spicy ones. Similarly, they registered the
largest content of capsinoids at the intermediate stages of the fruit development, i.e., between the 30th
and the 40th dpa (with values around 603.66
µ
g g
−1
of DW for ‘Habanero’ and around 300–400
µ
g g
−1
for the other varieties) [
42
]. Finally, Jarret et al. carried out the same study on the variety C. annuum
‘509-45-1
0
. Immature green, mature green, turning, and mature red stages were considered in that
occasion. As expected, capsinoid concentrations in the fruits increased rapidly on the 10th dpa and
reached their maximum value over the mature green stage (at 1013
µ
g g
−1
of DW), followed by a fall in
capsinoid concentration levels [43].
The aim of the present work was to complete a much more detailed monitoring process by
increasing the number of time lapses analyzed throughout the ripening of the fruit, so that a deeper
knowledge of and more detailed information about pepper’s beneficial compounds was attained,
since previous studies had not reached such a thorough understanding of the process for any of the
pepper varieties of interest. The ultimate objective is to precisely determine the moment of largest
level of capsinoid concentration, and therefore, their optimal harvesting time. ‘Habanero’, ‘Habanero
Roxo’, ‘Bode’, and ‘Malagueta’ are the pepper varieties selected for the study, and their content has been
controlled at 10 different fruit developing stages (specifically on the 13th, 20th, 27th, 34th, 41st, 48th,
55th, 62nd, 69th, and 76th dpa). The visual appearance after the harvesting of the peppers analyzed at
each one of the developing stages is shown in Figure 2. The samples were crushed before carrying out
the extraction in order to increase the contact surface and facilitate the penetration of the solvent to
favor a larger recovery [44].
Capsiate, the major capsinoid, was the olnly one to be found in all of the pepper varieties analyzed.
The evolution with regards to capsiate content (
µ
g g
−1
of FW) over the ripening process of the fruits is
represented in Figure 3.
Similar behavior was observed in the ‘Habanero’, ‘Habanero Roxo’, and ‘Malagueta’ peppers, where
the maximum CTE content was registered on the 27th dpa (with 137.84
µ
g g
−1
of FW, 398.28
µ
g g
−1
of FW, and 431.10
µ
g g
−1
of FW, respectively). After that, ‘Habanero Roxo’ and ‘Malagueta’ presented
drastic reductions in CTE content between the 27th and 48th days, corresponding to 77.66% and 83.96%
of their maximum registered concentration, respectively. After the 48th day, their CTE concentration
remained practically stable until the end of the ripening process. In the case of ‘Habanero’, after reaching
its maximum level of CTE content, there was a substantial reduction (88.47% decrease) over this last
period until the day 76th. All of these results are in agreement with those obtained by Jarret et al.,
for C. annuum ‘509-45-1
0
pepper, as above explained. This evolution has also been reported by other
researchers in relation to other Capsicum spp. such as ‘Chiltep
í
n’, ‘Tampiqueño 74
0
or ‘Bhut Jolokia’,
although in their case, they reached their maximum concentration a few days later; this difference
could be attributed to growing conditions or genotype reasons [41].
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Agronomy 2020, 10, x 6 of 14
Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy
capsinoids content so interesting, so that harvesting can take place when highest capsinoid
concentration is to be expected.
The number of reports that can be found in the literature on the evolution of capsinoids content
in the different varieties of peppers is very low. Fayos et al. analyzed capsinoids content in the
varieties ‘Chiltepín’, ‘Tampiqueño 74′, and ‘Bhut Jolokia’, but only at four specific moments over the
fruit ripening period, specifically on the 10th, 20th, 40th, and 60th dpa [41]. According to their study,
similar trends could be observed in the three genotypes, with the accumulation of capsinoids
beginning between the 10th and the 20th dpa and then increasing up to their maximum
concentration on the 40th dpa (with values reaching 276.39 µg g−1 of FW, 69.31 µ g g−1 of FW, and
122.62 µg g−1 of FW, respectively). Finally, the content gradually decreased over the last stages of
development [41]. Jang et al. also studied the evolution of these compounds at four different
moments during the ripening process of four different varieties of pepper that ranged between spicy
and slightly spicy ones. Similarly, they registered the largest content of capsinoids at the
intermediate stages of the fruit development, i.e., between the 30th and the 40th dpa (with values
around 603.66 µ g g−1 of DW for ‘Habanero’ and around 300–400 µ g g−1 for the other varieties) [42].
Finally, Jarret et al. carried out the same study on the variety C. annuum ‘509-45-1′. Immature green,
mature green, turning, and mature red stages were considered in that occasion. As expected,
capsinoid concentrations in the fruits increased rapidly on the 10th dpa and reached their maximum
value over the mature green stage (at 1013 µ g g−1 of DW), followed by a fall in capsinoid
concentration levels [43].
The aim of the present work was to complete a much more detailed monitoring process by
increasing the number of time lapses analyzed throughout the ripening of the fruit, so that a deeper
knowledge of and more detailed information about pepper’s beneficial compounds was attained,
since previous studies had not reached such a thorough understanding of the process for any of the
pepper varieties of interest. The ultimate objective is to precisely determine the moment of largest
level of capsinoid concentration, and therefore, their optimal harvesting time. ‘Habanero’,
‘Habanero Roxo’, ‘Bode’, and ‘Malagueta’ are the pepper varieties selected for the study, and their
content has been controlled at 10 different fruit developing stages (specifically on the 13th, 20th,
27th, 34th, 41st, 48th, 55th, 62nd, 69th, and 76th dpa). The visual appearance after the harvesting of
the peppers analyzed at each one of the developing stages is shown in Figure 2. The samples were
crushed before carrying out the extraction in order to increase the contact surface and facilitate the
penetration of the solvent to favor a larger recovery [44].
Figure 2. Pepper fruits assayed for their capsiate accumulation patterns during development and
maturation. Fruits of ‘Habanero’ (A), ‘Habanero Roxo’ (B), ‘Bode’ (C), and ‘Malagueta (D) at 13, 20,
27, 34, 41, 48, 55, 62, 69, and 76 dpa from left-to-right.
Figure 2.
Pepper fruits assayed for their capsiate accumulation patterns during development and
maturation. Fruits of ‘Habanero’ (
A
), ‘Habanero Roxo’ (
B
), ‘Bode’ (
C
), and ‘Malagueta (
D
) at 13, 20, 27, 34,
41, 48, 55, 62, 69, and 76 dpa from left-to-right.
Agronomy 2020, 10, x 7 of 14
Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy
Capsiate, the major capsinoid, was the olnly one to be found in all of the pepper varieties
analyzed. The evolution with regards to capsiate content (µ g g−1 of FW) over the ripening process of
the fruits is represented in Figure 3.
Figure 3. Evolution of total capsiate content (µg g−1 of FW) during the development of pepper fruits
(n = 2). According to the Tuckey’s test, results with a p-value less than 0.05 were considered to be
statistically different. Taking this into account, the use of different letters in this figure indicates that
there is a significant difference between results depending on Tuckey’s test. In turn, the letters of
each color refer to their respective variety, that would be from left-to-right, blue for ‘Habanero’,
orange for ‘Habanero Roxo’, grey for ‘Bode’, and yellow for ‘Malagueta’.
Similar behavior was observed in the ‘Habanero’, ‘Habanero Roxo’, and ‘Malagueta’ peppers,
where the maximum CTE content was registered on the 27th dpa (with 137.84 µ g g−1 of FW, 398.28
µg g−1 of FW, and 431.10 µ g g−1 of FW, respectively). After that, ‘Habanero Roxo’ and ‘Malagueta’
presented drastic reductions in CTE content between the 27th and 48th days, corresponding to
77.66% and 83.96% of their maximum registered concentration, respectively. After the 48th day, their
CTE concentration remained practically stable until the end of the ripening process. In the case of
‘Habanero’, after reaching its maximum level of CTE content, there was a substantial reduction
(88.47% decrease) over this last period until the day 76th. All of these results are in agreement with
those obtained by Jarret et al. for C. annuum ‘509-45-1′ pepper, as above explained. This evolution has
also been reported by other researchers in relation to other Capsicum spp. such as ‘Chiltepín’,
‘Tampiqueño 74′ or ‘Bhut Jolokia’, although in their case, they reached their maximum concentration
a few days later; this difference could be attributed to growing conditions or genotype reasons [41].
Generally, the short number of studies that have been conducted on the accumulation of
capsinoids in Capsicum fruits have shown that the concentration of these compounds increase during
the early stages of the fruit development, and this trend goes on during the first stages of the
ripening process until a maximum value is reached, usually between the 20th and 40th dpa [41–43].
After that time, there is an inversion of the trend and a marked reduction in capsinoids content is
observed. Such reduction in capsinoids content over the last stages of the fruit development, could
be associated with a reduction in the biosynthesis of capsinoids inside the pepper according to the
specific cultivation conditions in the greenhouse [45] or, alternatively, to the effect of the peroxidases
that can be found in peppers. Hot pepper peroxidases, especially peroxidase isoenzyme 6, oxidizes
the phenolic precursors involved in capsaicin biosynthesis. Basic peroxidase isoenzyme 6 is located
in the placental epidermal cells and in the vacuoles, where capsaicinoids are synthesized, and their
capacity to degrade these compounds is due to the strong capsaicin-oxidizing activity of this
Figure 3.
Evolution of total capsiate content (
µ
g g
−1
of FW) during the development of pepper fruits
(n=2). According to the Tuckey’s test, results with a p-value less than 0.05 were considered to be
statistically different. Taking this into account, the use of different letters in this figure indicates that
there is a significant difference between results depending on Tuckey’s test. In turn, the letters of each
color refer to their respective variety, that would be from left-to-right, blue for ‘Habanero’, orange for
‘Habanero Roxo’, grey for ‘Bode’, and yellow for ‘Malagueta’.
Generally, the short number of studies that have been conducted on the accumulation of capsinoids
in Capsicum fruits have shown that the concentration of these compounds increase during the early
stages of the fruit development, and this trend goes on during the first stages of the ripening process
until a maximum value is reached, usually between the 20th and 40th dpa [
41
–
43
]. After that time,
there is an inversion of the trend and a marked reduction in capsinoids content is observed. Such
reduction in capsinoids content over the last stages of the fruit development, could be associated with
Agronomy 2020,10, 1337 8 of 14
a reduction in the biosynthesis of capsinoids inside the pepper according to the specific cultivation
conditions in the greenhouse [
45
] or, alternatively, to the effect of the peroxidases that can be found in
peppers. Hot pepper peroxidases, especially peroxidase isoenzyme 6, oxidizes the phenolic precursors
involved in capsaicin biosynthesis. Basic peroxidase isoenzyme 6 is located in the placental epidermal
cells and in the vacuoles, where capsaicinoids are synthesized, and their capacity to degrade these
compounds is due to the strong capsaicin-oxidizing activity of this isoenzyme [
46
,
47
]. Cell walls
and vacuoles are also the places where capsiate is accumulated. Based on this fact, Lema et al., have
suggested that the same chili peroxidases that oxidize capsaicinoids vanillyl residues were also capable
of oxidizing the vanillyl residues from capsinoids. The use of different inhibitors allowed to confirm
that this peroxidase actually has the capacity to perform such oxidation. These results strongly support
the assumption that the basic peroxidases that can be found in C. annuum could be responsible for the
oxidation of their own CTE content [48].
Conversely, it can be seen from Figure 3that, in a global context, the total capsiate content in ‘Bode’
peppers raised from the first point of harvest on the 13th dpa until the 76th dpa, at which point there
was a concentration of 329.46
µ
g g
−1
of FW, which corresponds to 350% increment compared to the
initial content. And this was so, despite two perceptible CTE content falls that were registered during
the maturation period: specifically, a decrease of 46.15% between the 27th dpa and the 34th dpa, and a
slight reduction by just 1.63% between the 55th and the 62nd dpa. These falls in CTE content could
be attributed to the action of the peroxidases in the peppers and to the subsequent reduction in the
synthesis of their capsinoid content. In contrast to what is generally reported, a significant increase in
the content of CTE was observed over the first ripening days, specifically from the first point of harvest
on the 13th dpa (91.96
µ
g g
−1
of FW) up to the 27th dpa, where it reached a concentration of 217.03
µ
g
g
-1
of FW. This was followed by an increment in CTE content between the 34th and 55th dpa. The final
increase in the total content of CTE that took place in the last stage of the maturation process could be
due to the loss of water suffered by overripen peppers. Nevertheless, these facts will not be considered
for the object of this study, since such overripe peppers are not suitable for commercialization due to
inadequate organoleptic attributes.
Most of the studies conducted on the different pepper varieties have reported that capsinoid
content decreased rapidly as the fruits matured and changed color. This substantial reduction in
capsinoid content as pepper ripens supports the need to perform sampling of the fruit at the appropriate
development stages. In addition to all the factors that affect capsinoid content and that been already
mentioned, including the decreased gene expression or peroxidase action, the instability of these
compounds in different solvents should also be taken into account. Capsinoids are esters of fatty acid
and vanillyl alcohol, so they are stable in non-polar solvents such as ethyl acetate, but they decompose
easily in polar solvents such as water, methanol, and so on [
49
]. This is why, if the highest concentration
of these compounds of interest and their health benefits are to be attained, green peppers should be
eaten raw. Since these compounds are unstable in water, and also when subjected to high temperatures,
cooking should be avoided in order to keep the largest possible content of capsinoids. Another factor
to keep in mind is that as these fruits mature and turn from green into red color, their present a smaller
capsinoid content [50].
Although it has been seen that, in general, the accumulation pattern of CTE content in pepper
fruit during its ripening stages followed similar trends, it is also true that, depending on the pepper’s
genotype, as well as on the growing conditions or environmental factors, such content may vary and
result differently accordingly. In this sense, it would be necessary to monitor every detail of each
crop’s cultivation conditions, including all the possible environmental factors, since they may greatly
influence the final product and its composition [
51
,
52
]. For this reason, it would be necessary to
complete deeper studies where a greater number of varieties and under a wider range of different
conditions would be analyzed. The present work intends to be a preliminary study that can be used as
a starting point to demonstrate that there is no fixed pattern with regards to the variations in capsinoid
Agronomy 2020,10, 1337 9 of 14
content during the fruit ripening, so that each variety reaches a maximum concentration of these
beneficial compounds at different stages of development.
3.2. Comparison of Capsiate and Capsaicinoids Contents
A comparison between the results obtained in this work with respect to the capsaicinoid
accumulation patterns previously reported has been carried out in order to determine any similarities
or differences in both compound families (capsinoids and capsaicinoids), both with similar
pharmacological properties but different pungent capacities. It should be noted that the fruits
have been grown in acclimatized greenhouses and under the same conditions (they are the same
samples as the ones used for our previous work on capsaicinoids [
35
,
36
]), which should allow a more
reliable comparison. It should also be noted that both families of compounds share part of their
biosynthetic pathway.
3.2.1. ‘Habanero’ pepper
The maximum capsaicinoid content in ‘Habanero’ peppers was obtained on the 33th dpa
(
≈1400 µg g−1
of DW) [
35
]. A slight reduction in their content (8.5%) was observed from the 34th
until the 48th dpa, which was associated to the effect of the peroxidases. After that, the capsaicinoid
content remained practically constant over the rest of the ripening process. The evolution of the
CTE content followed a similar trend. However, it reached its maximum concentration (137.84
µ
g
g
−1
of FW) a few days earlier, specifically on the 27th dpa, possibly as a consequence of the greater
degradability and instability of this compound [
53
]. Furthermore, it was notable that such reduction
was substantially more drastic (88.47%) than that in capsaicinoids and continued decreasing until
the end of the maturation process. This correlation between the accumulation patterns from each
compound family seems to indicate that the environmental factors have had similar effects on both
biosynthetic pathways.
3.2.2. ‘Habanero Roxo’ pepper
As can be seen in our previous work [
35
], each family of compounds present distinctive evolution
patterns, even when capsaicinoid and capsinoid contents have been determined for the same plant that
had been grown under the same environmental conditions. It can then be said that the differences in
content between the two families seem to be due to genetic factors inherent to this variety. The maximum
capsaicinoid content was registered on the 41st dpa, which coincided, in this case, with a change
of color in the peppers from green to violet. From that moment, there was a period over which the
concentration remained practically stable until the 55th dpa. After that, the capsaicinoid concentration
increased substantially until the over-ripening stage was reached. On the contrary, the maximum CTE
content was registered on the 27th dpa, and then a drastic reduction by 77.66% took place between the
27th and 48th dpa. From then on, the CTE concentration remained practically stable until the end of
the fruit’s ripening process.
3.2.3. ‘Bode’ pepper
In our previous studies on this pepper variety [
35
], a similar behavior was observed with regards
to the accumulation pattern of the two families of compounds. Both the total capsaicinoid and CTE
content raised from the first point of harvest until the end of the ripening period. A substantial increase
in the content of these compounds was observed in the early stages of the fruit development up to the
33rd dpa for capsaicinoids and to the 20th dpa for CTE. At the end of the fruit’s maturation, two more
moderate increments were registered (the former took place between the 48th and the 69th dpa, and
the latter between the 76th and 83rd dpa), which could be reasonably associated with genetic factors
since the plant had been cultivated under the same environmental conditions and, therefore, we should
assume that both families of compounds had been equally affected. The specific growing conditions
that were controlled in the greenhouse were temperature, humidity, irrigation, and fertilization, and
Agronomy 2020,10, 1337 10 of 14
these controlled conditions resulted in some increment in the amount of both compounds. It was
also observed that halfway through the development of the fruit, a decrease in the amount of these
compounds took place as a result of the action of the peroxidase enzymes in the peppers [48].
3.2.4. ‘Malagueta’ pepper
The fruit accumulation pattern for capsaicinoids during the ripening period of this variety followed
the same trend as the ‘Bode’ variety [
36
]. Thus, in general, there was a concentration increment over
the ripening, even if, as previously mentioned, there was a series of increases and decreases throughout
the process. Nevertheless, CTE evolution followed a particular evolution pattern where the maximum
concentration was reached on the 27th dpa, after which a drastic reduction in the content (83.96%) took
place. This was followed by a content stability period until the end of the maturation process.
Firstly, it should be noted that for all of the pungent varieties that have been analyzed, capsaicinoids
were found in peppers in substantially larger concentrations that capsinoids. The main capsaicinoids
found in peppers are capsaicin and dihydrocapsaicin, both with considerably larger concentrations
than that of CTE, the only capsinoid detected. Nevertheless, the concentration of CTE in the varieties
that have been analyzed was slightly above the concentration levels registered for other capsaicinoids
(nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin). Moreover, no similar concentration
pattern or trend has been encountered that could equally be applied to the different varieties under
study. This indicates, as mentioned above, that the genetic factors that are inherent to each variety play
a significant role with regards to accumulation patterns or compound contents. Furthermore, since
environmental factors seem to have a considerable influence on such contents and patterns, every
possible detail with regards to growing conditions should be closely monitored in order to determine
their influence on the fruit final composition [
50
,
51
]. Thus, and considering the greater degradability
of capsinoids, growing conditions should be carefully contemplated and implemented. Future studies
that intend to deepen not only in the study of capsinoid accumulation patterns in a greater number
of varieties, but also in their comparison with those of capsaicinoids in the same varieties should
be covered.
4. Conclusions
The current work has demonstrated, for a number of pepper varieties, that the bioactive content
of their fruits with regards to the bioactive compounds responsible for pepper pungency, capsaicinoids,
and capsinoids, may vary widely depending on their genotype, the fruit developmental stage, and
the specific growing conditions. Since drastic changes in CTE content have been observed over the
ripening period, determining how maturity stages may affect the composition of the peppers with
regards to such biologically interesting bioactive compounds is of the utmost interest. This study
intends to determine the optimal harvesting moment based on the moment of the greatest CTE content,
which would be on the 27th dpa for ‘Habanero’, ‘Habanero Roxo’, and ‘Malagueta’ peppers, and on the
55th dpa for ‘Bode’ peppers (the later increase has not been taken into account since they are overripe
peppers that are not suitable for consumption due to their organoleptic properties). This study also
has deepened knowledge of the accumulation patterns for CTE content over the fruit development
and their correlation with pepper capsaicinoid content. For the four varieties under study, different
accumulation patterns of the two families of compounds of interest have been determined. Since no
definitely clear pattern has been established, an in-depth study with a greater number of varieties and
fruit-development monitoring points would be necessary. The variations that our study has registered
with regards to bioactive compound contents, and that can be attributed to genetic factors, constitute a
practical foundation for the improvement of the nutritional qualities of pepper products.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4395/10/9/1337/s1.
Figure S1. Mass spectrum of capsiate obtained by UHPLC-QToF-MS. Figure S2. Representative chromatograms for
the four varieties of peppers studied ((A) ‘Habanero’; (B) ‘Habanero Roxo’; (C) ‘Bode’; (D) ‘Malagueta’) obtained by
Agronomy 2020,10, 1337 11 of 14
UHPLC-PDA (280 nm) at the point of maximum capsiate content. (1) Nor-dihydrocapsaicin (n-DHC); (2) Capsaicin
(C); (3) Dihydrocapsaicin (DHC); (4) Homo-capsaicin (h-C); (5) Homo-dihydrocapsaicin (h-DHC); (6) Capsiate (CTE).
Author Contributions:
Conceptualization, G.F.B. and A.G.-C.; methodology, M.V.-E.; software, M.F.-G.; formal
analysis, M.V.-E., O.F., and A.V.G.-d.-P.; investigation, M.V.-E.; resources, M.P., and A.G.-C.; data curation, M.F.-G.,
O.F., and E.E.-B.; writing—original draft preparation, M.V.-E.; writing—review and editing, G.F.B., E.E.-B., and
O.F.; supervision, G.F.B.; project administration, A.G.-C. and G.F.B.; funding acquisition, A.G.-C. and G.F.B.
All authors have read and agreed to the published version of the manuscript.
Funding:
This work is part of the RTA2015-00042-C02-01 project funded by the National institute for Agriculture
and Food Research and Technology (INIA, Spain) and cofinanced by the European Fund for Regional Development
(FEDER). It was also supported by A11-17R project and V. la Andaluza and University of Cadiz by the project
OT2016/046. The authors thank the Spanish Ministry of Education, Culture, and Sport for the predoctoral contract
(FPU17-02962) granted to Mercedes Vázquez-Espinosa.
Acknowledgments:
The authors are grateful to the Instituto de Investigaci
ó
n Vitivin
í
cola y Agroalimentaria
(IVAGRO) for providing the necessary facilities to carry out the research. A special remark goes to Carmelo Garc
í
a
Barroso (in memoriam) for his contribution to the scientific community in the area of phenolic compounds and
oenology and his important inputs to this research.
Conflicts of Interest: The authors declare no conflict of interest.
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