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Chemical Characterization
of
Native Chili Peppers (Capsicum spp.)
Dissertation
to obtain the academic degree
Doctor rerum naturalium
(Dr. rer. nat.)
Faculty of Mathematics and Natural Sciences
of the
Bergische Universität Wuppertal
by
Sven Werner Meckelmann
Luedenscheid
- 2014 -
Diese Dissertation kann wie folgt zitiert werden: urn:nbn:de:hbz:468-20150204-110700-6
[http://nbn-resolving.de/urn/resolver.pl?urn=urn:nbn:de:hbz:468-20150204-110700-6]
Abstract
The genus Capsicum belongs to the botanical family of Nightshades
(Solanaceae) and is closely related to other important crops from the
“New World” like tomato, eggplant, potato or tobacco. For over 6,000
years, their fruits were used for many purposes and not only as spice
or food in the human diet. Peru and Bolivia are the supposed center
of origin of the genus Capsicum. Germplasm banks in both countries
hold more than thousand different chili pepper accessions, which
have never been characterized. This study had the aim of analyzing
the phytochemical composition and major quality traits with evaluating
the environmental impact on these traits by multi-location and year-to-
year comparison. Partner institutions in Peru and Bolivia provided the
dried and crushed chili pepper sample materials.
Improved analytical methods and a streamlined analytical
strategy were applied to analyze 362 different chili pepper
accessions. The analytical procedures included the determination of
pungency by major capsaicinoids and pattern of capsaicin,
dihydrocapsaicin and nordihydrocapsaicin. In addition, health
promoting phytonutrients and parameters such as flavonoid aglycons
(quercetin, luteolin, kaempferol and apigenin), total polyphenols
according to the Folin-Ciocalteu method, the antioxidant capacity
(TEAC assay), vitamin E by analyzing the content of α-, β- and
γ-tocopherol and vitamin C (ascorbic acid) were determined. The set
of analytical parameters was extended by the analysis of fat content,
surface and extractable color (ASTA 20.1).
The sample set included the five domesticated species
C. annuum, C. baccatum, C. chinense, C. frutescens, and
C. pubescens and some wild species belonging to C. baccatum var.
baccatum and C. eximium. Within the sample set, Capsicum
accessions were identified showing pungency from non-pungent to
extremely pungent and with outstanding content in valuable
health-related phytochemicals.
Multivariate data evaluation by principal component analysis
(PCA) and partial least square regression (PLS) did not show any
underlying structures when replanting experiments were evaluated.
However, significant influences of the environment on the
concentration and levels were observed by analysis of variance
(ANOVA) indicating the high influence of the environment on the
traits.
The obtained data allowed identifying high value accessions.
All analytical data were submitted to the project partners in Peru and
Bolivia to select high value accessions and to start market
specialization or as starting point for further breeding programs
focusing on nutrition quality. Thus, the study results add value to the
Capsicum diversity of Peru and Bolivia to generate higher income for
small-scale chili farmers. In addition, this helps conserving local
native chili peppers through their use as high value crop.
Acknowledgement - Danksagung
This work was carried out at the University of Wuppertal in the
Faculty of Mathematics and Natural Science within the research
group of Prof. Dr. Michael Petz from January 2011 until September
2014.
Mein besonderer Dank gilt Herrn Prof. Dr. Michael Petz für die
interessante Themenstellung, sowie für die Möglichkeit auch eigene
Ideen in das Forschungsprojekt einzubringen. Auch möchte ich mich
für die Teilnahme an verschiedenen nationalen und internationalen
Tagungen, die hilfreichen Diskussionen, die Unterstützung bei der
Erstellung der Publikationen und die freundschaftliche Betreuung
bedanken.
I like to thank all partners from Peru and Bolivia for preparing and
sending the chili pepper samples to Wuppertal and the realization of
the different planting experiments. Particularly, I like to thank Llermé
Ríos (†) and Karla Peña from Instituto Nacional de Innovación
Agraria, Roberto Ugas from Universidad Nacional Agraria La Molina,
Lourdes Quinonez from Centro de Investigación y Desarrollo Rural
Amazónico, Carlos Bejarano from Fundación Promoción e
Investigación de Productos Andinos, Teresa Avila from Centro de
Investigaciones Fitoecogenéticas de Pairumani und Edwin Serrano
from Instituto de Tecnología de Alimentos. For project coordination, I
thank Matthias Jäger, Maarten van Zonneveld, Xavier Scheldeman
and Marleni Ramirez from Bioversity International. My special thank is
given to Maarten van Zonneveld for helpful assistance in the
preparation of the publications.
Frau Dr. Erika Müller-Seitz danke ich für die zahlreichen Gespräche,
Diskussion und die Unterstützung bei der Erstellung der
Publikationen.
Herrn Prof. Dr. Heiko Hayen danke ich für die Unterstützung und
hilfreichen Diskussionen bei verschiedenen analytischen
Fragestellungen.
Herrn Dipl.-Ing. Dieter Riegel danke ich für die tolle Zusammenarbeit
während meiner gesamten Zeit in der Lebensmittelchemie, sowie für
die zahlreichen Analysen der Chili-Proben auf ihren ASTA-Wert, den
Fettgehalt, die Oberflächenfarbe und die NIR-Messungen.
Weiterhin danke ich Christina Schröders, Matthias Lüpertz, Désirée
Marquenie, Frederik Lessmann, Christian Jansen und Toni Regestein
für die vielfältige Unterstützung im Rahmen ihrer wissenschaftlichen
Abschlussarbeiten.
Dem gesamten Arbeitskreis der Lebensmittelchemie danke ich für die
gute Zusammenarbeit, tolle Arbeitsatmosphäre und die vielen
fachlichen als auch nicht-fachlichen Diskussionen.
Meiner Familie, besonders meinen Eltern Jutta und Peter
Meckelmann sowie meiner Großmutter Irmgard Meckelmann, danke
ich auf so vielfältige Weise, dass ich dies nicht in Worte zu fassen
vermag.
Julia, Dir danke ich für deine Unterstützung während unserer
gemeinsamen Jahre. Dein ruhiges, einfühlsames Wesen, deine
Geduld und dein Verständnis waren eine große Hilfe, wofür ich Dir
immer dankbar sein werde.
Für meine Oma
i
Table of Content
1. Chili Peppers ............................................................................... 1
1.1 History and Economy ............................................................. 1
1.2 Taxonomy and Botany ........................................................... 5
1.3 Quality Parameters ...............................................................13
1.4 Capsaicinoids and Analogs ...................................................16
1.4.1 Biosynthesis ..............................................................19
1.4.2 Physiological Properties ............................................22
1.4.3 Analysis .....................................................................23
1.5 Polyphenols ..........................................................................25
1.5.1 Biosynthesis ..............................................................28
1.5.2 Health Promoting Effects ...........................................31
1.5.3 Analysis of Polyphenols and other Antioxidants .........33
1.6 Vitamins in Chili Peppers ......................................................38
1.6.1 Ascorbic acid: Biosynthesis, Degradation and Analysis ...41
1.6.2 Tocopherols: Biosynthesis and Analysis .......................44
1.7 Color of Chili Peppers ...........................................................47
1.7.1 Carotenoids ...............................................................47
1.7.2 Extractable Color .......................................................49
1.7.3 Surface Color ............................................................50
2. Objective ....................................................................................53
2.1 General Remarks ..................................................................53
2.2 Aim and Scope .....................................................................55
3. Structure of the Results ............................................................58
4. Composition of Peruvian Chili Peppers ...................................61
4.1 Introduction ...........................................................................62
4.2 Experimental .........................................................................65
ii
4.2.1 Plant Material and Post Harvest Treatment ............... 65
4.2.2 Statistical Analysis .................................................... 66
4.3 Results and Discussion ........................................................ 68
4.3.1 Capsaicinoids and Pattern ........................................ 68
4.3.2 Specific Flavonoids ................................................... 71
4.3.3 Total Polyphenols and Antioxidant Capacity ............. 75
4.3.4 Tocopherols and Ascorbic Acid ................................. 78
4.3.5 Fat Content and Color ............................................... 81
4.4 Conclusion ........................................................................... 84
5. Phytochemicals in Peruvian C. pubescens ............................. 85
5.1 Introduction .......................................................................... 86
5.2 Experimental ........................................................................ 91
5.2.1 Plant Material and Post Harvest Treatment ............... 91
5.2.2 Statistical Analysis .................................................... 92
5.3 Results and Discussion ........................................................ 92
5.3.1 Capsaicinoids and Pattern ........................................ 93
5.3.2 Other Constituents .................................................... 97
5.4 Conclusion ......................................................................... 101
6. Environmental Impact on Phytochemicals ............................ 103
6.1 Introduction ........................................................................ 104
6.2 Experimental ...................................................................... 105
6.2.1 Plant Material and Field Experiment ........................ 105
6.2.2 Statistical Analysis .................................................. 107
6.3 Results and Discussion ...................................................... 109
6.3.1 Control Experiment ................................................. 109
6.3.2 Capsaicinoids ......................................................... 111
6.3.3 Specific Flavonoids ................................................. 113
6.3.4 Total Polyphenols and Antioxidant Capacity ........... 115
iii
6.3.5 Tocopherols............................................................. 117
6.3.6 Extractable and Surface Color ................................. 118
6.3.7 Environmental Impact .............................................. 120
6.4 Conclusion .......................................................................... 125
7. Characterization of Bolivian Chili Peppers ............................ 127
7.1 Introduction ......................................................................... 128
7.2 Experimental ....................................................................... 130
7.2.1 Plant Material and Post Harvest Treatment ............. 130
7.2.2 Statistical Analysis ................................................... 132
7.3 Results and Discussion ....................................................... 133
7.3.1 Capsaicinoids and Pattern ....................................... 133
7.3.2 Specific Flavonoids.................................................. 136
7.3.3 Total Polyphenols and Antioxidant Capacity ............ 139
7.3.4 Tocopherols and Ascorbic Acid ............................... 141
7.3.5 Fat Content ............................................................. 145
7.3.6 Extractable and Surface Color ................................. 146
7.3.7 Two-year Comparison ............................................. 146
7.4 Conclusion .......................................................................... 150
8. Analytical and Experimental Background.............................. 151
8.1 Capsaicinoid Analysis ......................................................... 152
8.2 Total Polyphenols and Antioxidant Capacity ....................... 155
8.3 Flavonoid Analysis .............................................................. 159
8.4 Analysis of Ascorbic Acid by HILIC ..................................... 161
8.5 Analysis of Tocopherols ...................................................... 163
8.6 Determination of Fat by NIR ................................................ 168
8.7 Effect of Drying on Phytonutrients in Chili Peppers ............. 171
8.8 Analytical Strategy .............................................................. 173
9. Concluding Remarks and Future Perspectives ..................... 175
iv
10. Materials and Methods .......................................................... 183
10.1 Chemicals ............................................................... 183
10.2 Sample Pretreatment .............................................. 184
10.3 Extraction and Analysis of Capsaicinoids ................ 184
10.4 Flavonoid Analysis .................................................. 185
10.5 Determination of Total Polyphenols ......................... 186
10.6 Trolox Equivalent Antioxidant Capacity (TEAC) ...... 187
10.7 Analysis of Ascorbic Acid by HPLC ......................... 187
10.8 Tocopherols by HPLC ............................................. 188
10.9 Determination of Fat Content .................................. 189
10.9.1 Gravimetric Method ................................................. 189
10.9.2 NIR Method ............................................................. 189
10.10 Determination of Extractable Color .......................... 190
10.11 Measurement of Surface Color ............................... 191
10.12 Determination of Moisture Content .......................... 191
11. List of Publications ................................................................ 192
11.1 Original Papers ....................................................... 192
11.2 Conference Contributions ....................................... 193
12. References ............................................................................. 195
13. Appendix ................................................................................ 213
Chili Peppers
1
1. Chili Peppers
1.1 History and Economy
Chili Peppers are native to South and Central America and are
originated in the arid regions of the Andean Mountains, which later
became Peru and Bolivia [1, 2]. During the pre-Columbian era,
Capsicum plants spread over South and Central America and have
been part of the indigenous cultures since almost 10,000 years [3].
Capsicum specific starch fossils found from the Bahamas to south
Peru indicate the early cultivation and domestication of the genus
6,000 years ago [4]. The native people used Capsicum fruits as food,
spice and medicine. During that time, chili peppers became important
for some regions and were one of the preferred tributes in pre-
Columbian Mexico [5].
At the end of the fifteenth century, the genus Capsicum was
still unknown in Europe. Most spices used in Europe came from India
by a long seaway around Africa. In 1492, Christopher Columbus
began his search for a shortcut to the wealth and spices of India.
Instead of finding a new trade route, he discovered the “New World”.
During his journey, he encountered several plants unknown to
Europeans. One of them mimicked the pungency of black pepper
(Piper nigrum) and due to the red pods it was called “red pepper”.
This unknown genus was classified later as Capsicum by the
taxonomist Carl Linnaeus and is not related to black pepper. The
name Capsicum is owing to its pungency and is descended from the
Latin word “capsa”, which was derived from the Greek word “kapto”
meaning to bite. On his journey back, Columbus took different plants,
Chili Peppers
2
fruits and seeds to the “Old World”. One of those was Capsicum.
Across the extensive spice trade routes of Spain and Portugal, chili
peppers started to spread around the globe and have become part of
many national cuisines [3, 5, 6].
Table 1.1: Values for selected nutrients of fresh chili peppers
Content per 100 g
Main nutrients a
Water 88.0 g
Protein 1.9 g
Lipids 0.4 g
Sugars 5.3 g
Minerals a
Potassium 322 mg
Calcium 14 mg
Magnesium 23 mg
Iron 1 mg
Vitamins b
Provitamin A 18 mg
Vitamin C 206 mg
Vitamin E 16 mg a Mean values for hot, raw, red chili peppers from United States Department
of Agriculture (USDA) - Nutrient Database [7] and b values for selected chili
peppers from Wahyuni et al. [8].
Today, chili peppers are part of the daily diet of millions of people
around the world. Chili peppers or products derived of are used as
food and spices and in various products, such as in the food industry
as colorant and spice for sauces, as medicine in ABC heat plasters,
in self-defense sprays and much more. The various compounds
found in chili peppers are the reason for the broad utilization
spectrum. Table 1.1 provides a brief overview of the general
History and Economy
3
composition of fresh chili peppers. In addition, chili peppers contain
several phenolic compounds showing antioxidant activity and the
ability to scavenge free radicals. In chili peppers flavonoids (e. g.
quercetin, luteolin or anthocyanins), different phenolic acids
(coumaric acid and caffeic acid) and capsaicinoids, a group of vanillyl
amides unique to the genus Capsicum, are found [8–10].
Chili peppers are grown in several countries of the world and are an
economical important crop. The global production of fresh and dried
chili peppers increased continuously from about 25 million tons in
2002 to about 35 million tons in 2012. In the same period, the export
values increased from 980 to 3,403 million US $ (Figure 1-1).
Therefore, Capsicum is an important economic factor for many
countries.
Figure 1-1: Global Capsicum production in 1000 metric tons obtained from
FOASTAT (Food and Agriculture Organization of the United Nations) [11]
and export values in million US $ obtained from International Trade Centre
(ITC) [12] between 2002 and 2012.
0
500
1000
1500
2000
2500
3000
3500
4000
0
5000
10000
15000
20000
25000
30000
35000
40000
[Mio
US
$]
[10
00
*t]
Global Capsicum production and export values
Production Export values
Chili Peppers
4
In 2012, China was the leading producer of fresh chili peppers.
Mexico ranged second with great distance followed by Turkey,
Indonesia and other countries. India was the leading producer of
dried chili peppers in 2012. China ranged second with great distance
followed by Peru ranged third (Figure 1-2).
In Germany, chili and paprika belong to one of the favored
spices. The percentage of the total spice imports was 9.5% in 2012.
Only pepper (Piper nigrum) with 26.4% and ginger (Zingiber
officinale) with 13.1% were imported in higher amounts [13].
Figure 1-2: Top ten pepper producing countries for fresh and dried chili peppers in 2012; (from FOASTAT [11]).
0
500
1000
1500
2000
2500
3000
[10
00
*t]
Top ten fresh chili pepper producing countries 16023
0 50
100 150 200 250 300
[10
00
*t]
Top ten dried chili pepper producing countries 1300
Taxonomy and Botany
5
1.2 Taxonomy and Botany
Taxonomy:
The genus Capsicum belongs to the botanical family of Nightshades
(Solanaceae) and is closely related to other important crops from the
“New World” like tomato (Solanum lycopersicum), eggplant (Solanum
melongena), potato (Solanum tuberosum) or tobacco (Nicotiana
tabacum) [14]. Above the species level, the taxonomy of the genus
Capsicum is well described [3]:
Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Solanales Family: Solanaceae Subfamily: Solanoideae Tribe: Solaneae Subtribe: Capsicinae Genus: Capsicum
However, taxonomic classification is discussed controversially within
the genus and several of the relationships between the different
species are not well understood [15]. Taxonomical classification is
based on three different tools. First is the morphology considering
shape of petals and leafs, color of flowers, number of flowers per
node and further more aspects of the appearances of the plants. A
second instrument for taxonomical classification is the sexual
compatibility, which takes for example into account the possibility of
producing fertile hybrids. The last and most recent tool for taxonomic
Chili Peppers
6
classification is the analysis of chromosomes, genes or proteins.
These techniques allow conclusions on phylogenetic relationships
between the Capsicum species.
The current number of different species has reached almost 40.
Eshbaugh [15] reported a number of 36, while Bosland and Votava [3]
counted currently 37 different Capsicum species, but both mentioned
that the number of species would increase by the exploration of South
America in the future. Today, it is considered that five of these
species are domesticated. They can easily be distinguished from wild
chili peppers species. Wild ones have similar fruit traits with small,
round, berry like pods and a soft peduncle while domesticated
showing different pod types with larger fruits [3].
The five domesticated and economic important species are
Capsicum annuum var. annuum, C. frutescens, C. chinense,
C. baccatum var. pendulum and C. pubescens. All 36 species
mentioned by Eshbaugh can be classified into two groups
(Figure 1-3) according to their number of chromosomes (12 or 13
diploid chromosomes) [15]. Figure 1-3 also depicts a continuing
classification of the 2n=24 – group into three complexes of closely
related Capsicum species.
The C. annuum - complex includes the three domesticated
species Capsicum annuum var. annuum, C. frutescens and
C. chinense sharing an ancestral gene pool. The complex also
contains the species C. annuum var. glabriusculum the proposed wild
ancestor of C. annuum, previously known as C. annuum var.
aviculare [15]. Because of their common gene pool, all three
domesticated species share similar morphological traits and
Taxonomy and Botany
7
Pickersgill stated that their status as distinct species is questionable
[16]. Walsh and Hoot analyzed DNA sequences from noncoding
regions of the chloroplast genome (atpB-rbcL) and five introns within
the nuclear waxy gene [1]. Their results showed that C annuum,
C. frutescens and C. chinense are very closely related, especially
C. frutescens and C. chinense, sharing very similar morphological
traits. Baral and Bosland analyzed C. frutescens and C. chinense for
morphological, sexual compatibility and phylogenetic traits to clarify
this question [17]. They reported that the similarity between
C. frutescens and C. chinense accessions was only 0.38 and that
hybridization reduced the fertility. Based on these evidences, they
concluded that both were distinct species.
The C. baccatum – complex consists of the domesticated
C. baccatum var. pendulum and its wild progenitor C. baccatum var.
baccatum. Additionally, several other species are discussed to be a
member of this group. C. chacoense was described as a sister
species of the C. annuum – complex because of morphological
analogy to the C. annuum – complex [20, 21]. But genetic studies
from Walsh and Hoot [1] and Ibiza et al. [19] identified C. chacoense
as a member of the C. baccatum – complex. C. tovarii is also
discussed as a member of this complex due to the successful
hybridization with C. baccatum [22]. Onus and Pickersgill confirmed
possible hybridization with C. baccatum [21]. Nevertheless, their
results also indicate promising hybridization with other species
outside the C. pubescens - complex (e.g. C. annuum). Genetic
studies could not explain the affiliation of C. tovarii, so the position of
this species has to be clarified [19, 23].
Chili Peppers
8
Figure 1-3: Relationship of all 36 different Capsicum species mentioned by Eshbaugh [15]; Species are classified according to their number of chromosome (
a) and the 2n=24 group into the three species complexes
(adapted and modified from [1, 8, 18, 19, 15]).
Species of the C. pubescens – complex form a very distinct group. In
contrast to other species that mostly have white flowers all three
species of this complex have purple flowers [18]. Moreover,
hybridization with other species is very difficult and typically fails or
Genus Capsicum
2n=26a
2n=24a
C. annuum - complex
C. baccatum - complex
C. pubescens - complex
C. annuum var. annum
C. chinense
C. frutescens
C. annum var. glabriusculum
C. galapagoense
C. baccatum var. pendulum
C. chacoense
C. praetermissum
C. baccatum var. baccatum
C. eximium
C. pubescens
C. cardenassii
unclassified species
C. buforumC. campylopodium
C. cornutumC. friburgense
C. lanceolatumC. mirabile
C. pereiraeC. rhomboideum
C. schottianumC. villosum
C. eshbaughii
C. flexuosum
C. parvifolium
C. tovarii
unknown
C. caballeroiC. ceratocalyx
C. coccineumC. dimorphum
C. duseniiC. geminifolium
C. hookerianumC. hunzikrianum
C. leptopodumC. minuntiflorum
C. recurvatumC. scolnikianum
Taxonomy and Botany
9
leads to completely sterile hybrids [1, 3]. The ancestral gene pool of
C. pubescens has not been identified yet. Hybrids from the two wild
species C. eximium or C. cardenasii and the domesticated
C. pubescens are often fertile and allow hypothesizing that
C. eximium and C. cardenasii are the probable ancestral gene pool.
One remarkable attribute of C. pubescens needs to be mentioned.
The seeds of C. pubescens appear brown or black, a color unknown
in all other species [24].
Botany:
Thousands of different accessions were collected and conserved in
various germplasm banks worldwide. The US National Plant
Germplasm System of the United States Department of
Agriculture (USDA) probably holds the biggest collection, of
approximately 5,000 Capsicum accessions [25]. All chili peppers
share basic botanical characteristics.
Capsicum is a dicotyledonous plant and grows under
subtropical and tropical climatic conditions. Most species do not
tolerate low temperatures. C. pubescens is the only species, which is
adapted to lower temperature and grows in the cooler elevated
regions of the Andean Mountains. The plants may live under optimal
growing conditions more than ten years. After germination of the
seeds, the plants develop a taproot with lateral roots. Most grow near
the soil surface. The plants are among the sub-shrubs and during the
growth the stem starts lignifying especially near the roots. While most
Chili Peppers
10
plants reach a typical height of 2 m, C. pubescens is described to be
able to grow up to 12 m. The leaves are arranged helically around the
stem and developed as single or as pair on opposite sides of the
stem. The leaves´ size, shape and color depend on the species and
accessions. Ovate, elliptic and lanceolate forms are described. The
typical color is green, but accessions are known with purple or yellow
leaves. None of the species has hairy stems and leafs, except
C. pubescens. The flowers grow at the axils of branches. They
usually develop solitary for example in C. annuum, but other species
like C. chinense have multiple flowers per node. The corolla has five
to seven petals (each 10-20 mm long). Their color can be white
(e.g. C. annuum or C. chinense), white with yellow spots
(C. baccatum) or purple (C. pubescens) [3, 24, 26, 27]. An example
for a Capsicum plant is given in Figure 1-4.
Taxonomy and Botany
11
Figure 1-4: Capsicum plant (C. chinense; Habanero). The plant carries
several orange fruits and white flowers. It has reached an estimated age of
more than ten years.
While wild chili peppers have only small, round and mostly red fruits,
the fruits of the domesticated species are very diverse. Botanically,
the fruits are berries and the color differs from white, purple, green,
yellow, orange, brown to red. The length varies from less than 1 cm to
Chili Peppers
12
more than 30 cm. As with the color and length of the fruits, the
variation of different fruit shapes is great. The shape of the fruits can
be round (cherry like), oblate, conical (heart-shaped), blocky or
elongated with pointed or round tips. However, among all the different
pod types, sizes and colors of all fruits, they share a very similar basic
anatomy (Figure 1-5) [3, 27, 28].
Figure 1-5: Cross section of a chili peppers fruit (C. annuum)
The fruits are connected with the node and stem by the peduncle.
The former calyx of the flowers is diverse between the fruits of
different accessions and is often very pronounced. In dependency on
the species and varieties, the calyx is immersed or jut above the
upper end of the fruit. Beginning from the calyx, the placenta is
located in the centre of the hollow and surrounded by the seeds. The
seeds are normally colorless, only in varieties of C. pubescens black
or brown seeds are found. They contain high amounts of lipids (up to
25%) [29]. The inside of the pod is separated in different chambers
through the septa. Capsaicinoid producing cells can only be found in
the placenta and septa [30, 31]. The edible part of the fruit, the
pericarp consists of three different layers. The exocarp is the outer
layer and protects the fruit against damages and drying up. It contains
PeduncleCalyx
EndocarpMesocarp
Exocarp
Pericarp
Placenta SeedSepta
Quality Parameters
13
also large amounts of pigments. The intermediate layer (mesocarp)
forms the major part of the fruit and contains high amounts of aroma
active compounds. The last layer of the pericarp is the endocarp,
which delimits the fruit inside [27, 28].
1.3 Quality Parameters
The quality parameters of Capsicum fruits can be divided according
to their use as vegetable and spice chili peppers. Both have different
quality requirements. For the vegetable use of chili peppers the
quality relies mainly on freshness, pungency and some nutrient
factors such as a high vitamin C content. For dried chili peppers used
as spice for home cooking or in the food and cosmetic industry, the
quality parameters are versatile and can be categorized to four
groups [32].
The first important quality trait is the degree of pungency. It ranged
from sweet, non- or slightly pungent varieties, usually called paprika,
to highly pungent varieties, typically named chili or chili peppers. It is
essential to know the degree of pungency to select Capsicum fruits
for specific purposes such as the use as paprika or chili powder or the
production of oleoresins. With regard to their pungency, chili pepper
powders can be classified into five groups according to Bosland and
Votava (Table 1.2) [3].
Chili Peppers
14
Table 1.2: Classification of chili peppers according to their pungency [3]
Group Class Capsaicinoids (mg/100 g)
Scoville Heat Units
I non-pungent / paprika
0 - 4.4 0 - 700
II middle pungent 4.4 - 18.8 700 - 3000
III moderately pungent
18.8 - 156.3 3000 - 25000
IV highly pungent 156.3 - 437.5 25000 - 70000
V very highly pungent
> 500 > 80000
Beside pungency, the color of paprika or chili powder is an essential
parameter in quality assessment. The typical red color, required for
industrial purposes, is caused by the content and pattern of more
than 30 different carotenoids [32]. Moreover, color is important for
pricing of paprika and chili peppers in international trade. It relies
mainly on the content of extractable carotenoids. The amount of
carotenoids is measured by the American Spice Trade Association
(ASTA) method 20.1 [33]. Sweet, non-pungent powders have
ASTA 20.1 values of 160-180 and for hot chili powders ASTA 20.1
values of 120 are reported [34]. Carotenoids are sensitive to oxidative
conditions such as low water activity. Water contents of
approximately 15% can enhance the stability of carotenoids during
storage and reduce the degradation of color [35]. However, these
rather high water contents increase the growth of bacteria and mold,
so a water content of approximately 11% for dried Capsicum powder
is recommended [32].
Quality Parameters
15
Chili peppers or paprika powders were mostly eaten as vegetables or
used as spices, so aroma is very important. Among the species and
varieties the aroma profiles differ strongly [36, 37]. The typical fruity
paprika aroma of fresh Capsicum fruits consists of more than 60
different volatile compounds. Major classes of aroma active
compounds are aliphatic alcohols, aldehydes, ketones, aromatic
components and terpenoids. Key aroma compounds of fresh
Capsicum fruits are 2-methoxy-3-isobutylpyrazine, nona-2,6-dienal,
deca-2,4-dienal, limonene and methyl salicylate [38, 39].
Technological processes like dehydration of fresh Capsicum fruits to
obtain dried fruit material lead to changes in the aroma profile. The
aroma profile of dried Capsicum fruits includes the same compounds
as found in fresh fruits. During the drying process and because of the
thermal stress various Maillard, lipid oxidation and carotenoids
degradation products can be found in dried fruits. Unsuitable raw
material, technological flaws and oxidative reactions during storage
could lead to various off-flavors. Examples for compounds, which are
responsible for off-flavors, are hexanal, 6-methyl-5-hepten-2-one and
β-ionone. Typical off-flavors are a pronounced rancidity, caramel or
hay like odor [32, 38, 40].
Mycotoxins can cause serious health damages and need to be
considered in the quality assessment [41]. Most Capsicum producing
countries are located in tropical and subtropical regions with a warm
and damp climate. In addition, these countries often have poor
agricultural practices and hygienic conditions, which can lead to the
presence of molds and a contamination with mycotoxins. Aflatoxin B1,
B2, G1 and G2 as well as ochratoxin A can be found in high numbers
Chili Peppers
16
of chili and paprika powder samples. The maximum residue level for
total aflatoxins in chili and paprika powder set by the European Union
(Regulation No. 1881/2006) is 10 µg/kg. High levels of aflatoxins
(up to 218 µg/kg total aflatoxins) are often observed and in
comparison with the maximum residue level illustrate the serious
problems with mycotoxin contaminations in chili pepper and paprika
powders [32]. Ochratoxin A in spices is currently not considered in the
regulation, but the high concentration (up to 74 µg/kg) also suggests
a problem with ochratoxin A contaminations [42, 43].
1.4 Capsaicinoids and Analogs
Fruits of the genus Capsicum are known for their hot and burning
sensation. Capsaicinoids, the pungent principle, are a complex
mixture of more than 30 different compounds unique for the genus
Capsicum. All capsaicinoids are conjugates of vanillylamine and
various alkenoic and alkanoic acids. The acyl moieties differ in the
length of the carbon chain (C7-C13), the presence or absence of an
unsaturated carbon bond, the position of this bond at the ω-3 or ω-4
carbon, the presence or absence of a methyl branch and the position
of the branch (iso or anteiso) [44, 45].
The pattern of capsaicinoids is highly inconsistent and differs
between species and varieties. Accordingly, the capsaicinoids cannot
be used for taxonomical classification [46]. Generally, three
compounds (capsaicin, dihydrocapsaicin and nordihydrocapsaicin;
Figure 1-6) dominate the composition of capsaicinoids. These major
capsaicinoids typically provide 95% of the total capsaicinoid content.
Capsaicinoids and Analogs
17
Other capsaicinoids are minor compounds and their contribution to
the pungency is limited [46].
Figure 1-6: Chemical structure of major capsaicinoids: capsaicin (8-methyl-
N-vanillyl-trans-6-nonenamide), dihydrocapsaicin (8-methyl-N-vanillyl-
nonanamide) and nordihydrocapsaicin (7-methyl-N-vanillyl-octanamide)
Watanabe et al. described “capsaicin like” substances, isolated from
a non-pungent bell peppers variety (CH-19 sweet; C. annuum) [47].
Instead of a vanillylamine being connected to the fatty acid, the new
group of “capsaicin like” substances consists of a vanillyl alcohol
esterified with fatty acids of capsaicin (8-methyl-trans-6-nonenoic
acid), dihydrocapsaicin (8-methylnonanoic acid) and nordihydro-
capsaicin (7-methyloctanoic acid) [47, 48].
These “capsinoids” are non-pungent, but they share with
capsaicinoids the same capability to act as transient receptor
NH
O
OH
O
NH
O
OH
O
NH
O
OH
O
Capsaicin
Dihydrocapsaicin
Nordihydrocapsaicin
Chili Peppers
18
potential vanilloid (TrpV1) agonist (Chapter 1.4.2) [49–51]. Later,
Watanabe et al. discovered a second class of “capsaicin like”
substances [52]. Coniferyl esters of 8-methyl-6-nonenoate
(capsiconiate) and 8-methylnonanoate (dihydrocapsiconiate) were
isolated from C. praetermissum. Capsiconoids also act as TrpV1
agonist, but to a much lesser degree compared to the activity of
capsaicinoids or capsinoids [52]. Figure 1-7 depicts the major
compounds of each class of substances.
Figure 1-7: Comparison of the chemical structures of capsaicin (8-methyl-N-
vanillyl-trans-6-nonenamide) and the structural analogs capsiate (8-methyl-
O-vanillyl-trans-6-nonenamide) and capsiconiate (8-methyl-O-coniferyl-trans-
6-nonenamide)
NH
O
OH
O
O
O
OH
O
OH
OO
O
Capsaicin
Capsiate
Capsiconiate
Capsaicinoids and Analogs
19
1.4.1 Biosynthesis
The biosynthesis of capsaicinoids and related structures is unique for
the genus Capsicum. Production of capsaicinoids represents an
evolutionary advantage. The pungent taste, the burning sensation
and the pain, when capsaicinoids are in contact with mucous
membranes, act as a deterrent against mammals. The pain is caused
by the activation of the vanilloid receptor (TrpV1). The corresponding
receptor in birds is not activated by capsaicinoids. Additionally, birds
do not digest the seeds, so they act as the preferred seed dispersers
for pungent Capsicum cultivars [53].
The capsaicinoid biosynthesis is located in the epidermis cells
of the placenta [30, 31]. The molecules are the product of an acyl
transfer reaction between medium chain fatty acids acyl CoA and
vanillylamine. The responsible gene for the production of
capsaicinoids is known as Pun1, which encodes a putative
acyltransferase and is only found in pungent chili peppers [54–56].
However, the degree of pungency is controlled by five quantitative
trait loci (QTL) [57]. Furthermore, various studies show that the
production of capsaicinoids is highly influenced by the environment
(e.g. Harvell and Bosland [58] or Gurung et al. [59, 60]).
Chili Peppers
20
OH
O
NH3
+
OH
O
OH
O
OH
PAL
Phenylalanine
Cinnamic acid
p-Coumaric acid
C4H
S
O
OH
CoA
p-Coumaroyl-CoA
4CL
S
O
OH
CoAOH
Caffeoyl-CoA
HCT
S
O
OH
CoAO
Feruloyl-CoA
COMT
O
OH
O
Vanillin
HCHL
OH
ONH3
+
Vanillylamine
pAMT
BCAT
Valine
OH
O
NH3
+
Id
-Ketoisovalerate
OH
O
O
Isobutyryl-CoA
S
O
CoA
KAS ACL
3x Malonyl-CoA
3 elongation cycles
FAT
8-Methyl-6-nonenoic acid
OH
O
ACS
8-Methyl-6-nonenoyl-CoA
S
O
CoA
CS
OH
ONH
O
Capsaicin
1 2
p-Coumaroyl shikimate O
OH
O
OHO
OHOH
C3H
p-Caffeoyl shikimate O
OH
O
OHO
OHOH
OH
HCT
CoAshikimate
Figure 1-8: Capsaicin biosynthetic pathway. 1: phenylpropanoid pathway, PAL phenylalanine ammonia lyase, C4H cinnamate 4-hydroxylase, 4CL 4-coumaroyl-CoA ligase, HCT hydroxycinnamoyl transferase, C3H coumaroyl shikimate 3-hydroxylase COMT caffeic acid O-methyl transferase, HCHL hydroxycinnamoyl-CoA hydratase/lyase, pAMT putative aminotransferase. 2: branched-chain fatty acid pathway, BCAT branched-chain amino acid transferase, Id isovalerate dehydrogenase, KAS ketoacyl-ACP synthase, ACL acyl carrier protein, FAT acyl-ACP thioesterase, ACS acyl-CoA synthetase, CS capsaicin synthase (adapted and or modified from [54, 55, 65].
Capsaicinoids and Analogs
21
The vanillylamine part of the capsaicin molecule is produced via the
phenylpropanoid pathway. In 1968, Bennett and Kirby used different
tritium (3H) labeled phenolic compounds and could show that
phenylalanine was the precursor of vanillylamine [61]. They also
identified p-coumaric acid, caffeic acid and ferulic acid as
intermediates and concluded that vanillylamine was a product of the
phenylpropanoid pathway (Figure 1-8). Leete and Lourden [62] used
14C labeling and Rangoowala [63] 15N labeling of various amino acids.
They only found phenylalanine as precursor and confirmed the results
of Bennet and Kirby. The better understanding of the phenyl-
propanoid pathway and additional radioactive tracer experiments
allowed Fujiwake et al. to postulate fundamental steps in the
biosynthesis of vanillylamine in Capsicum fruits [64]. The latest
findings of the biosynthesis of vanillylamine were summarized by
various authors to the pathway in the last years (Figure 1-8)
[54, 55, 65].
The general pathway leading to the branched 8-methyl-6-
nonenoic acid found in capsaicin is given in Figure 1-8. Various
amino acids are known as precursors for the fatty acids that can be
found in capsaicinoids. Valine is identified as the primer of the iso-
branched chains fatty acid with an even number of carbon atoms
(e.g. capsaicin). Leucine is identified for the analog fatty acids with an
odd number of carbon atoms. Isoleucine is the precursor for
capsaicinoids having an anteiso-branched fatty acid chain with an
odd number of carbon atoms and threonine for capsaicinoids with an
unbranched fatty acid, also with an odd number of carbon atoms. It
requires no special amino acid precursor for capsaicinoids with an
Chili Peppers
22
even, unbranched fatty acid moiety. The formation follows the
de novo fatty acid synthesis [62, 66].
1.4.2 Physiological Properties
The most obvious physiological property of the capsaicinoids is the
interaction with the transient receptor potential cation channel
subfamily V member 1 (TrpV1). The so called capsaicin receptor or
vanilloid receptor is an ion channel, which is highly permeable for
Ca2+ and other alkaline and earth alkaline metal ions but to a lesser
degree (permeability sequence: Ca2+ > Mg2+ > Na+ ≈ K+ ≈ Cs+) [67].
The receptor can be activated by capsaicinoids, ethanol, low pH
values and by temperatures higher than 42 °C. It is also activated by
derivates of arachidonic acid, which are inflammatory intermediates.
However, the activation of TrpV1 allows ions to flow inside the cell.
This causes a depolarization, which activates neurons leading to a
heat-like feeling or even pain [67, 68]. The fact that capsinoids are not
pungent but also activate the capsaicin receptor, can be explained by
their higher lipophilicity in comparison to capsaicinoids. Capsinoids
are absorbed to a lesser degree by the mucosa and cannot reach the
receptor [69].
Beside of the acute pain and heat perception, the activation of
TrpV1 leads to several other physiological reactions and is involved in
inflammatory processes of the gastrointestinal tract or the bladder.
Especially, the activation of TrpV1 by derivates of arachidonic acid
illustrates the important role in inflammatory processes. The
therapeutic potential by the manipulation of the capsaicin receptor
may not be restricted to a symptomatic pain therapy [68].
Capsaicinoids and Analogs
23
Today, obesity is a serious lifestyle disease, particularly in industrial
countries. It is associated with different diseases like diabetes mellitus
(type 2), coronary heart diseases, high blood pressure, sleep-
breathing disorders and cancer [70]. Capsaicinoids stimulate
thermogenesis by increasing the energy expenditure and can support
weight maintenance therefore [71–75]. Oral intake of ≥2.5 mg
capsaicinoids per meal can increase the energy expenditure
significantly. However, the oral intake of capsaicinoids is very limited
due to the tolerable pungency [75].
In addition, capsaicinoids are also discussed in cancer
therapy. The general anti-carcinogenic potential is based on the
inhibition of the cell cycle, the triggering of apoptosis and a reduction
of the proliferation of cancer cells [76–79]. On the other hand,
pro-carcinogenic effects are also reported for capsaicinoids. As an
example, long term application of capsaicinoid containing creams in
the presence of a tumor promoter (e.g. sun light) can increase skin
carcinogenesis [80]. Another important fact is the exceptional high
concentration needed for observing an anti-carcinogenic action of
capsaicin [81].
1.4.3 Analysis
Wilbur Lincoln Scoville was the first who developed a method to
estimate the content of capsaicinoids and the degree of pungency in
chili peppers. For the test, one grain (≈65 mg) dry and ground chili
pepper is extracted with 100 mL ethanol. After filtration, the extract is
diluted with a sucrose solution until no pungency is perceptible on the
tongue. The result of the test is expressed as Scoville Heat Units
Chili Peppers
24
(SHU), which represents the dilution factor until no pungency is
perceptible. Pure capsaicin has a SHU value of 16,000,000. This
means for example that 1 mg of pure capsaicin needs to be diluted
with 16,000 L of a sucrose solution until no pungency is perceptible
[82, 83].
The described organoleptic test requires six different test persons and
only allows a rough estimation of the capsaicinoid content. To
maintain a consistent quality of food, cosmetic or medical products,
the exact content of capsaicinoids is needed. Today, various methods
are available to analyze the content of capsaicinoids. Near infrared
spectroscopy and enzyme-linked immunosorbent assay (ELISA) are
methods, which allow the quantification of the total capsaicinoid
content [84, 85]. However, more recent methods are based on gas or
liquid chromatographic separation techniques to quantify the pattern
and content of individual and total capsaicinoids [86–90]. Typically,
the pungent principles of chili peppers were analyzed by reversed
phase high performance liquid chromatography (HPLC) [89-91]. The
separation is achieved by using non-polar octadecyl (C18) columns.
Binary mobile phases were used containing acetonitrile/water or
methanol/water. According to the phenolic structure of the
capsaicinoids, formic acid or acetic acid is added to the mobile phase
to enhance peak shape. The fluorescence of all capsaicinoids can be
used for detection, but UV/Vis and mass detectors can also be
applied [89-91]. Modern monolithic or fused core HPLC columns were
used as well. In comparison to fully porous silica based columns,
these columns allow a faster separation of capsaicinoids and the
analysis of crude extracts without further sample preparation [92, 93].
Polyphenols
25
Capsaicinoids can be extracted from chili peppers by various
methods with different organic solvents or by super critical fluid
extraction [87, 94].
Typical levels of capsaicinoids cannot be specified because of the
great variation within the different species and varieties. Various chili
or bell peppers do not produce capsaicinoids. On the other hand,
Bosland, Coon and Reeves analyzed the capsaicinoid content of the
hottest chili pepper by HPLC in 2012 [95]. They found concentrations
in fruits of Trinidad Moruga Scorpion (C. chinense) reaching more
than two million SHU (~12.500 mg/100 g).
1.5 Polyphenols
Foodstuffs with a high content in polyphenols are recommended for a
modern human diet and can prevent age related diseases [96, 97].
According to their wide range of occurrence in vegetables and their
implication in various cosmetic and pharmaceutical products,
polyphenols are probably the only class of bioactive phytochemicals,
the public has heard about, but the term “polyphenol” is not exactly
defined. Stéphane Quideau recently defined polyphenols as
secondary plant metabolites, which are derived from the
shikimate/phenylpropanoid pathway and/or the polyketide pathway
[98]. Accordingly, polyphenols can be substances with more than one
phenolic hydroxyl group (e.g. caffeic acid, ferulic acid, lignin or gallic
acid) or compounds with multiple benzene rings with more than one
phenolic hydroxyl group (e.g. tannins, luteolin, quercetin or
delphinidin). This broad definition thus includes many classes of
Chili Peppers
26
phenolic compounds. Table 1.3 provides examples of the major
classes of phenolic or polyphenolic compounds in plants.
The major phenolic compounds in Capsicum are hydroxycinnamates
and flavonoids [99–101]. Flavonoids are of particular importance
concerning health promoting effects and their contents in chili
peppers [96, 97, 99]. Therefore, only key aspects of flavonoids are
described here.
Table 1.3: Major classes of phenolic or polyphenolic compounds in plants (adapted from [102])
No. of C atoms
C Skeleton Compound class Compound example
6 C6 simple phenols hydroquinone
catechol
7 C6-C1 hydroxybenzoates 4-hydroxybenzoate
8 C6-C2 acetophenones
phenylacetates
4-hydroxyacetophenone
9 C6-C3 hydroxycinnamates
phenylpropenes
coumarins
caffeate
eugenol
esculetin
10 C6-C4 naphthoquinones juglone
13 C6-C1-C6 xanthones 1,3,5,6,7-hydroxyxanthone
14 C6-C2-C6 stilbenes
anthraquinones
resveratrol
emodin
15 C6-C3-C6 flavonoids quercetin
luteolin
kaempferol
18 (C6-C3)2 lignans pinoresinol
30 (C6-C3-C6)2 biflavonoids amentoflavone
n (C6-C1)n hydrolyzable tannins gallotannin
(C6-C3)n lignins guaiacyl lignins
guaiacyl-syringyl lignins
(C6-C3-C6)n condensed tannins catechin polymers
Polyphenols
27
O
A
B
C
5
2
6 310
4
76´
89
2´3´
4´
5´
Figure 1-9: Flavan skeleton (2-phenylchroman); numbering of carbon atoms
is according to [103].
All flavonoids share the same basic flavan structure (Figure 1-9).
Today, thousands of different flavonoids are known. The basic
structure of the flavan can be found in all flavonoids with differences
in the oxidation state of the pyran ring (C-ring) and degree of
hydroxylations. Accordingly, flavonoids can be categorized into
different structural classes:
flavanols -OH at pos. 3 or/and 4
flavanones C=O at pos. 4
flavanonols C=O at pos. 4 and -OH at pos. 3
flavones C=C between pos. 2 and 3, C=O at
pos. 4
flavonols C=C between pos. 2 and 3, C=O at pos.
4 and -OH at pos. 3
anthocyanins positive charge at the central oxygen
atom, double bound between O and at
pos.2 and C=C between pos. 3 and 4
Chili Peppers
28
Hydroxylations were observed particularly at position 5 and 7 of the
A-ring and at position 4´ of the B-ring. Furthermore, many of them are
methoxylated or acylated with aliphatic and aromatic acids.
Flavonoids usually occur as O-glycosides in position 3, 5 or 7 of the
A- and C-ring or in position 8 and 6 of the A-ring as C-glycosides. The
majority of glycosylations can be found at the A- and C-ring, while
sugar moieties at the B-ring are seldom. Glycosides with glucose,
galactose, rhamnose, xylose and arabinose are the most common
moieties [8, 101, 102].
In Capsicum fruits the flavonol aglycons of myricetin, quercetin,
kaempferol and the flavone aglycons of luteolin and apigenin are
predominant. In violet chili and bell peppers the anthocyanin aglycon
of delphinidin glycosides can be found. As mentioned before, most of
them were glycosylated. Typically, quercetin-3-O-rhamnoside and
quercetin-3-O-rhamnoside-7-O-glycoside were observed in a broad
range of concentrations. Luteolin often occurs as C-hexosides and
C-pentosides at position 6 and 8, but O-glycosides at position 7 are
also known. Delphinidin appears mostly as delphinidin-
3-p-coumaroyl-rutinoside-5-glucoside in violet chili and bell peppers
[100, 104, 105].
1.5.1 Biosynthesis
The flavonoid biosynthesis can be found in almost every plant.
Flavonoids are synthesized as protection against high solar and UV
radiation or as defense against pathogen stress. It is described that
different environmental conditions have strong influence on the
Polyphenols
29
biosynthesis of flavonoids. Increased stress levels caused by
pathogens, nutrient deficiency, UV radiation or wounding are factors
that enhance the production of flavonoids [106].
Beside the growing condition and maturity stage, the genotype
is an important factor influencing the content and pattern of flavonoids
[100]. In contrast to the capsaicinoid biosynthesis, which is mostly
affected by Pun1, the flavonoid biosynthesis is more complex. Many
genes are necessary to encode the regulation and the enzymes for
the polyketide pathway. Wahyuni et al. showed recently that more
than 200 QTLs influence the amount and pattern of flavonoids in chili
peppers [107]. Most of these QTLs were found in two QTL hotspots
on chromosome 9. They also concluded that the quiet large
biochemical variation in chili pepper was under control of a limited
number of chromosomal regions [55, 56, 107].
However, the principle pathway that leads to the various classes of
flavonoids has been described for Arabidopsis and also for related
species like tomato (Solanum lycopersicum), potato (Solanum
tuberosum) or tobacco (Nicotiana tabacum) [101, 103, 108].
Capsaicinoids and flavonoids share phenylalanine as precursor,
derived from the shikimate pathway and the first steps of the
phenylpropanoid pathway leading to p-coumaroyl-CoA (Figure 1-8
and Figure 1-10). p-Coumaroyl-CoA is elongated three times with
malonyl-CoA. The result is polyketo acid-CoA, the first product of the
polyketide pathway. The next step is catalyzed by the chalcone
synthase and leads to naringenin chalcone. This can be transformed
by the chalcone isomerase to naringenin and in further steps to other
flavones. Naringenin chalcone can also react to dihydrokaempferol,
Chili Peppers
30
which acts as precursor for different flavonols and anthocyanidins
[101, 102]. Figure 1-10 depicts the common polyketide pathway in
plants briefly.
OH
O
NH3
+
PAL
Phenylalanine
C4HS
O
OH
CoA
p-Coumaroyl-CoA
4CL 3x Malonyl-CoA
O
OH
S
O
CoA
SCoA
O O O O
OHPolyketo acid - CoA
+
CHS
OH
O
OHOH
OH
Naringenin chalconeOH
O
OOH
OH
Naringenin
CHI
OH
O
OOH
OH
Apigenin
FS
OH
O
OOH
OH
OH
Luteolin
F3´H
F3H
OH
O
OOH
OH
OH
DihydrokaempferolOH
O
OOH
OH
OH
Dihydroquercetin
F3´H
FLS
OH
O
OOH
OH
OH
KaempferolOH
O
OOH
OH
OH
OH
Quercetin
F3´H
FLS
DFR
OH
OOH
OH
OH
OH
Leucopelargonidin
ANS
OH
O+
OH
OH
OH
Pelargonidin
Flavones Flavonols Anthocyanidins
Figure 1-10: Polyketide pathway leading to flavonoids including the enzymes: PAL phenylalanine ammonia lyase, C4H cinnamate 4-hydroxylase, 4CL 4-coumaroyl-CoA ligase, CHS chalcone synthase, CHI chalcone isomerase, FS flavones synthase, F3´H flavonoid 3´-hydroxylase, F3H flavanone 3-hydroxylase, FLS flavonol synthase, DFR dihydroflavonol 4-reductase, ANS anthocyanidin synthase (modified from [8, 101, 102]).
Polyphenols
31
A variety of further enzymatic hydroxylations at the A- and B-ring,
leads to the different flavonoid aglycons typically found in plants.
As mentioned before, flavonoids occur as glycoside conjugates with
different mono- and/or disaccharides. Uridine diphosphate (UDP)
activation of the sugars is necessary for the function of
glycosyltranferases. But the conjugations are not restricted to
glycosylations. Numerous flavonoids carry acyl groups at the hydroxyl
groups of the flavan skeleton or at the sugar moieties. The involved
transferases use CoA acids as acyl donor [101, 102].
1.5.2 Health Promoting Effects
Flavonoids and other phenolic compounds are known to have positive
effects on the human health status. They are able to prevent cells
from oxidative damage, due to their antioxidant and radical
scavenging activity [109]. Epidemiological studies suggest that they
reduce the susceptibility to cardiovascular and other age related
diseases [96, 97, 110]. However, many flavonoids have a low
bioavailability and are metabolized by gut microbiota. Furthermore,
human enzymes are not able to hydrolyze several flavonoid
glycosides (e.g. many flavonoid rutinosides) and gut bacteria are
necessary to remove the sugar moieties before absorption of the
aglycons by the gut [111, 112]. The health promoting effect of other
flavonoid metabolites produced by gut microbiota is still unknown
[111]. Nevertheless, the general positive health effect of flavonoids is
described in several studies (e.g. [96, 97, 110]).
Chili Peppers
32
Most of the health promoting effects of flavonoids are mainly
attributed to their antioxidant and radical scavenging activity. Reactive
oxygen species (ROS) are involved in many age related diseases
such as coronary heart disease (caused by oxidized low density
lipoproteins), cellular aging, DNA damages, mutagenesis and
carcinogenesis. The reduction of ROS by antioxidants is well
described. Other protective attributes of flavonoids can be ascribed to
the radical scavenging such as the reduction of the amount of
tocopherol radicals. Additionally, flavonoids can activate antioxidant
enzymes and inhibit oxidases [109].
Flavonoids are also able to reduce the transcription factors
NF-κB and AP1. Both are involved in different cellular processes and
cellular signaling and are associated with inflammatory processes
and tumor promotion. Flavonoids and other phenolic compounds are
able to suppress the activation of both factors contributing to their
chemopreventive and anti-inflammatory effects [78]. Moreover, a
large cohort study from Knekt et al. with more than 10,000 men could
show a significant reduction of different types of cancer, Asthma and
type 2 diabetes at higher dietary flavonoid intakes [96]. Another
cohort study with ~1,300 people could also show a reduced risk of
dementia correlating with a high flavonoid intake [97]. Again, many
types of cancer, inflammation, coronary heart disease or dementia
can be associated with oxidative stress and damage. The antioxidant
activity of flavonoids and other phenolic compounds or antioxidants is
the most obvious reason for their health promoting effects [109].
Polyphenols
33
1.5.3 Analysis of Polyphenols and other
Antioxidants
Two analytical strategies can be applied to analyze polyphenols and
other antioxidants. With regard to the complex mixture of antioxidants
occurring in chili pepper fruits or generally in plant tissues, sum
parameters can be utilized for analyzing the antioxidant capacity or
the total polyphenol content. Due to the complexity of the food
composition, it is almost impossible to study each antioxidant
individually. Therefore, these assays are important in the assessment
of the general antioxidant constitutions of food. In addition, all assays
share the advantage to detect the whole mixture of antioxidants,
which includes the synergistic interactions between the antioxidant
compounds [113–115]. But there is a lack of standardized and
validated methods. Slightly changed conditions for extraction or minor
modifications in the assay procedures have strong influence on the
results of the unspecific sum parameters. So it is nearly impossible to
compare the results of different studies [114, 116].
Today, different assays are developed to detect the antioxidant
activity in biological samples. They can be classified into assays
based on a hydrogen transfer reaction such as the oxygen radical
absorbance capacity assay (ORAC) or the inhibition of the linoleic
acid oxidation assay and into assays based on an electron transfer
like the Trolox equivalent antioxidant capacity (TEAC), the total
polyphenols according to the Folin-Ciocalteu method, the diphenyl-1-
picrylhydrazyl assay (DPPH) or the ferric ion reducing antioxidant
parameter (FRAP) [113, 114]. All of these assays have their own
advantages. The ORAC assay or the inhibition of linoleic acid
Chili Peppers
34
oxidation are best suited to determine the antioxidant capacity of
lipophilic antioxidants. Other assays are applicable to aqueous
systems and are easy to perform (e.g. TEAC assay or total
polyphenols according to Folin-Ciocalteu). Especially the TEAC assay
and the total polyphenol assay were applied to a wide range of
edibles and on Capsicum. Both were used in the presented thesis to
assess the antioxidant constitution of chili pepper powders.
Miller et al. developed the TEAC assay in 1993 [117]. Later,
Re et al. improved the assay procedure [118]. Potassium persulfate
oxidizes ABTS (2,2´-azinobis-(3-ethylbenzothiazo-line-6-sulfonic
acid)) to a stable, blue-green radical in an aqueous solution
(Figure 1-11). Before testing the antioxidant capacity, the ABTS
radical solution is diluted with water, a phosphate buffer (pH 7.4) or
ethanol to an absorbance of 0.70 ± 0.02 at 734 nm to maintain a
constant concentration of the ABTS radical. The ABTS radical reacts
with the antioxidants by a single electron transfer reaction back to the
colorless ABTS. The degree of decolorization is proportional to the
amount of antioxidant compounds in the sample.
S
N
S
O-
OO N
NS
N
S
O-
OO
K2S2O8
Antioxidant
S
N
S
O-
OO N
+
NS
N
S
O-
OO
Figure 1-11: Reactions of ABTS (2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid))
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a
water soluble analogue of vitamin E and typically used as calibration
Polyphenols
35
standard. The TEAC values for many antioxidant substances are
reported. The data do not show correlations between the number of
electrons, an antioxidant can donate, and the observed TEAC values.
The TEAC values for ascorbic acid (1.05 mmol Trolox), α-tocopherol
(0.97 mmol Trolox), uric acid (1.01 mmol Trolox) and glutathione
(1.28 mmol Trolox) are almost the same. However, glutathione can
only donate one electron and should have a theoretical TEAC value
of 1 mmol Trolox, while for example ascorbic acid can donate two
electrons and should show TEAC values higher than 1 mM Trolox.
Another example for the very individual reaction of the ABTS radical
and an antioxidant are the different TEAC values for quercetin
(3.1 mmol Trolox) and kaempferol (1.02 mmol Trolox). This is rather
surprising as both share a very similar chemical structure [117, 118].
The total polyphenol assay by Folin-Ciocalteu is probably the
oldest assay to determine antioxidants. The assay was initially
developed by Folin and Ciocalteu to determine proteins because of
the reaction of the Folin-Ciocalteu reagent with the phenolic amino
acid tyrosine [119]. Later, Singelton and Rossi optimized the assay to
determine the total polyphenol content of wine [120]. The Folin-
Ciocalteu reagent consists of sodium tungstate (Na2WO4), sodium
molybdate (Na2MoO4), lithium sulfate (Li2SO4), hydrochloric acid,
phosphoric acid and water. The exact reaction mechanism of the
Folin-Ciocalteu reagent is still unknown. It is supposed that the
reagent is composed of heteropolyphospho-tungstate and
-molybdates. Sequences of reversible one and two electron transfer
reactions lead to blue species with a possible molecular formula of:
(PMoW11O40)-4 [114, 121]. Typically, the reaction is performed under
alkaline conditions (sodium carbonate solution; pH 10), leading to a
Chili Peppers
36
dissociation of phenolic protons. The phenolate anion is capable of
reducing the Folin-Ciocalteu reagent and to form the blue species
described above. With regard to the chemistry of the Folin-Ciocalteu
method, the assay detects the reducing capacity of a sample and not
the radical scavenging activity as the TEAC assay does. Obviously,
the reaction is only slightly specific for phenolic and polyphenolic
substances. Many other non-phenolic compounds as vitamin C, Fe2+
or glutathione can reduce the Folin-Ciocalteu reagent [122].
The similar chemical nature of the TEAC assay and of the
Folin-Ciocalteu method often leads to very good linear correlation.
Nevertheless, it is important to apply both, an electron transfer based
assay and an assay, which determines the reducing power to
evaluate the full antioxidant potential of a sample. One assay alone
does not cover all compounds with an antioxidant activity, which can
occur in a food sample. Carotenoids for example, can be detected by
the TEAC assay, but not by the Folin-Ciocalteu method.
Unspecific sum parameters are important to determine the overall
antioxidant constitution of a sample. Nevertheless, the analysis of
specific polyphenols (e.g. flavonoids such as quercetin) is essential
for the identification and quantification of potential health promoting
compounds. Flavonoid analysis is achieved by HPLC. Separation is
usually performed on reversed phased C18 and penta fluoro phenyl
(PFP) columns [93, 105, 123]. Modern PFP modified HPLC columns
have a strong π-π-interaction and slot selectivity. This increases the
selectivity of the chromatographic separation and leads to a better
resolution. The elution system consists of methanol, acetonitrile and
water. On C18 columns, the organic and aqueous solvents are often
Polyphenols
37
spiked with trifluoroacetic acid, which reduces peak tailing and
enhances the resolution. Due to the higher selectivity of a PFP
column, the usage of trifluoroacetic acid is not necessary [123]. The
detection method varies according to the aim of the analysis. For the
identification and quantification of flavonoid glycosids an HPLC
system coupled with a tandem mass spectrometer is necessary.
HPLC-MS/MS is needed for the identification of the flavonoid aglycon
and their sugar moieties. Electrospray ionization (ESI) in both,
positive and negative mode, is used typically for ionization. However,
only a limited number of different flavonoid glycosides or even stable
isotope labeled are commercially available. Especially stable isotope
labeled standards are needed to compensate matrix effects during
the ionization process. Accordingly, exact quantification of a wide
range of flavonoid glycosides is almost impossible. Recent studies
used for quantification of flavonoid glycosides an additional
photodiode array detector (PDA) and selected commercially available
flavonoid O-glycosides and C-glycosides as standards [100, 104].
Wahyuni et al. used for example quercetin-3-rutinoside for the
quantification of all quercetin-O-glycosides, which is possible due to
similar absorbance characteristics [100].
Relevant for the health promoting effects are the flavonoid
aglycons. Therefore, it is suitable to just analyze the concentration of
the flavonoid aglycons after hydrolysis, which allows an easier
quantification. Separation conditions are generally the same as for
the glycosides, but due to commercially available standards,
identification and detection can be performed by HPLC-PDA. Acidic,
basic or enzymatic hydrolyses can be carried out to remove the sugar
moieties. Enzymatic hydrolysis is very gentle, but the applicability of
Chili Peppers
38
organic solvents is limited. Acidic hydrolyzes with ~1.2 M hydrochloric
acid and increased temperature is very easy to apply. The hydrolyses
can be combined with the extraction of the flavonoids for a faster
sample preparation. To reduce the oxidative damage during the
extraction and hydrolyses strong antioxidants such as
tert.-butylhydroquinone are necessary [105, 123].
Typical flavonoid glycosides and aglycons, which can be found in chili
peppers, are mentioned at the beginning of Chapter 1.5. The
concentration of different flavonoid glycosides in three different fresh
C. annuum fruits ranged from <0.2 to 21 mg/100 g fresh fruit [124].
Materska and Perucka found in four different dried chili peppers
(C. annuum) levels for flavonoid glycosides between 1.8 and
36.5 mg/100 g dry matter [99]. In both studies quercetin-O-glycosides
were the major flavonoid glycosides followed by luteolin-O- and C-
glycosides and rather low amounts of apigenin and kaempferol
glycosides. Miean and Mohamed reported levels for flavonoid
aglycons in dried chili peppers for three C. annuum (green chili, red
chili, and bell pepper) and one C. frutescens (bird chili) [105].
Quercetin was found in three samples (40-80 mg/100 g), luteolin in
green chili (3.3 mg/100 g) and a remarkable high content of luteolin in
bird chili (103 mg/100 g). Apigenin was present in bell pepper (27.2
mg/100 g) and kaempferol in green chili (3.3 mg/100 g).
1.6 Vitamins in Chili Peppers
Vitamins are organic micronutrients, which are essential for the
function of the human body and cannot be synthesized or not in
Vitamins in Chili Peppers
39
sufficient amounts. Fresh Capsicum fruits contain a large set of
vitamins (Table 1.4). But from a nutrition point of view, only
provitamin A, vitamin C and E are relevant.
Table 1.4:Vitamins in fresh chili peppers and the recommended daily intake (RDI) [7, 8, 125]
Vitamin Compound name
Content in 100 g fresh chili pepper
RDI
pro-A Carotenoids 18 mg 1.0 mg
B1 Thiamine 0.07 mg 1.2 mg
B2 Riboflavin 0.09 mg 1.4 mg
B3 Niacin 1.2 mg 16 mg
B6 Pyridoxine 0.5 mg 1.5 mg
B9 Folic acid 23 µg 300 µg
C Ascorbic acid 206 mg 100 mg
E Tocopherols 16 mg 14 mg
K Phylloquinone 14 µg 70 µg
Carotenoids with a provitamin A activity such as α-carotene,
β-carotene or β-cryptoxanthin act as precursors for vitamin A, which
is produced from the precursors in the human gut wall. Vitamin A is a
complex of retinol, retinal and retinoic acid. They are implicated in the
regulation of cell growth and differentiation as well as in hormone
synthesis and immune response [126, 127]. Other important aspects
of carotenoids in chili peppers are described in Chapter 1.7.
Vitamin C is a water-soluble vitamin. It is an essential antioxidant and
an important cofactor in many enzymatic reactions. The most
prominent one is the enzymatic hydroxylation of proline to
hydroxyproline, which is part of the collagen protein, the major protein
of connective tissues. A lack in ascorbic acid supply causes the
Chili Peppers
40
deficiency disease scurvy. Intake of about 50 g fresh chili peppers
provide about 100% of the recommended daily intake. But as with
other phytochemicals, the concentration vary greatly between chili
genotypes. Additionally, the ripening stage as well as environmental
factors, affect the vitamin C content in chili peppers [8, 126, 127].
While fresh chili peppers are a rich source of vitamin C, the
content in dried fruits is degraded to residual levels of only 10% or
less [128]. High concentrations of ascorbic acid in fresh fruits together
with a suitable drying and milling technology are important for
achieving high quality chili powders. Vitamin C helps to protect and
preserve other valuable compounds. An example is the protection of
carotenoids and thereby maintaining color intensity during the drying
process and storage of chili powder [129].
Figure 1-12: Chemical structures of vitamin active tocopherols and tocotrienols
Congener R1 R2
α CH3 CH3
β CH3 H
γ H CH3
δ H H
R1
OH
O2
R2
4` 8´
R1
OH
OR2
RRR-Tocopherol
R-Tocotrienol
Vitamins in Chili Peppers
41
Four different tocopherols and tocotrienols form the lipid-soluble
vitamin E complex (Figure 1-12). Vitamin E activity is highest for the
α-congener and lowest for δ, while the antioxidant activity is inversed.
Therefore, the strongest antioxidant activity is observed for the
δ-congener. The key function of vitamin E as a lipid-soluble
antioxidant is the protection of polyunsaturated fatty acids in cell
membranes. Additionally, it protects the DNA and low-density
lipoproteins against oxidative damage. Moreover, it has functions in
the hemoglobin biosynthesis and the modulation of immune
responses [126].
The concentration of the vitamin E congeners varies between
the different chili pepper fruit compartments. α-Tocopherol is
accumulated in the pericarp, whereas γ-tocopherol is predominant in
the seeds [8]. Generally, α- and γ-tocopherol can be regarded as
major tocopherols in dried spice paprika and chili pepper. The
concentrations of α-tocopherol can easily reach levels of up to
29 mg/100 g. The concentration of the γ-congener ranges second
highest with levels of ca. 3 mg/100 g. The concentrations of β- and
δ-tocopherol are rather low and their contribution to the overall
vitamin E activity can be neglected. The reported level of β-tocopherol
in spice paprika is at 0.4 and of δ-tocopherol at 0.2 mg/100 g [130].
1.6.1 Ascorbic acid: Biosynthesis, Degradation
and Analysis
Figure 1-13 shows the biosynthesis of L-ascorbic acid in plants.
Precursor is D-glucose-6-phosphat, which is altered by several
isomerases to L-galactose. In the next step, L-galactose is oxidized
Chili Peppers
42
by NAD+ to L-galactono-1,4-lactone and finally modified to L-ascorbic
acid by the enzyme L-galactono-1,4-lactone dehydrogenase, which is
the key enzyme in the vitamin C synthesis and not present in humans
[127, 131].
O
OH
HH
H
OH
OH
H OH
H
O
P OOH
OH
O
OH
H
OH
OH
H
H
O OH
POH O
OH
O
OH
OHH
H
OH
OH
H H
H
O
POOH
OH
O
O
OHH
H
OH
OH
H H
H
OH
P
O
OHOH
D-Glucose-6-P
1 2 3
D-Fructose-6-P D-Mannose-6-P D-Mannose-1-P
4
GTP
PPi
O
O
OHH
H
OH
OH
H H
H
OH
GDP
D-Mannose-1-P
GDP-1-Mannose
5O
O
OH
H
OH
H
OH H
H
H
GDP
OH
L-Galactose-1-GDP
GMPO
O
OH
H
OH
H
OH H
H
H
P
OH
O
OHOH
L-Galactose-1-P
67O
OH
OH
H
OH
H
OH H
H
H
OH
L-Galactose
8
NAD+
NADH
O
OH
H
H
OH
H
OHO
OH
9
2H
O
OHOH
H
OHO
OH
L-Galactono-1,4-lactone L-Ascorbic acid
Figure 1-13: L-Ascorbic acid pathway in plants. Enzymes: 1 hexose phosphate isomerase; 2 phosphomannose isomerase; 3 phosphomannose mutase; 4 GDP-D-mannose pyrophosphorylase; 5 GDP-D-mannose-3,5-epimerase; 8 L-galactose dehydrogenase; 9 L-galactono-1,4-lactone dehydrogenase. Adapted and modified from Wheeler et al. [131].
As stated, ascorbic acid is degraded to residual levels of only 10% or
less during the drying process [128]. First degradation product is
dehydroascorbic acid, which can be reduced to ascorbic acid by
glutathione in the human body. Thus, it also shows vitamin C activity
[127]. The reversible redox reaction can also be use for the
simultaneous determination of ascorbic acid and dehydroascorbic
acid that can be easily reduced by reducing agents such as
Vitamins in Chili Peppers
43
dithiothreitol [93]. Vitamin C activity is lost until the lactone ring is
opened (Figure 1-14). 2,3-Diketogulonic acid reacts further on to
xylosone and 4-desoxypentosone. Both can be degraded to different
reductones, furfural and furan carboxylic acid. Another pathway that
leads to vitamin C inactive compounds is the Maillard reaction.
Dehydroascorbic acid reacts with amino acids to complex compounds
showing a red or brown color. The vitamin C degradation by the
Maillard reaction is especially important for dried fruits [132].
O
OHOH
OHO
OH
Ascorbic acid
O
OO
OHO
OH
Dehydroascorbic acid
Ox.
Red.
OO
OHO
OH
OHOH
2,3-Diketogulonic acid
H2O
Figure 1-14: Degradation of ascorbic acid [132]
HPLC with UV or PDA detection is the preferred method for the
quantification of ascorbic acid in complex food matrices. Various
chromatographic conditions can be applied for separation. With
regard to the high polar nature of vitamin C, ion pair chromatography
is a suitable method showing a good separation and retention for
polar compounds. Typically, ion pair chromatography is performed on
a non-polar column (modified with C18 or C8) and an ionic surfactant
such as sodium dodecyl sulfate for the separation of cations and for
anions cetyltrimethylammonium bromide or tetrabutylammonium
hydroxide. However, ion pair chromatography is highly affected by
changes of the pH value or temperature [133, 134]. Other methods
also use non-polar stationary phases with pure aqueous solvents to
achieve a good retention of polar compounds, but non-polar matrix
Chili Peppers
44
components remain on the separation column, so it needs to be
washed with high concentrations of acetonitrile after every injection
[100]. Hydrophilic interaction liquid chromatography (HILIC) is a very
good alternative for separation of polar compounds. Typical HILIC
columns consist of pure silica gel or polar modified silica gel (e.g.
aminopropyl or sulfobetaine). Acetonitrile and water or different
buffers were mostly used for elution and in contrast to the classical
reversed phases, water or buffers show the highest elution power
[135]. Nováková et al. described a novel method for the determination
of ascorbic acid using a sulfobetaine modified HILIC column and a
simple binary mobile phase consisting of an ammonium acetate
solution and acetonitrile [136]. This method offers a very good
opportunity for the analysis of vitamin C in complex food matrices.
1.6.2 Tocopherols: Biosynthesis and Analysis
Two different pathways provide the precursors for the biosynthesis of
tocopherols in plants. The shikimate pathway provides
p-hydroxyphenylpyruvate, which forms the chromanol backbone of
the tocopherols. The phytyl moiety is synthesized of isopentenyl
pyrophosphate, a product of the 2-C-methyl-D-erythritol-4-phosphate
(MEP) pathway and the isoprenoid pathway (Figure 1-15). The critical
step is the formation of methyl-phytyl-benzoquinone, which is
catalyzed by the homogentisate phytyltransferase (HPT) [137, 138].
Expression and activity of this enzyme are responsible for the
total tocopherol content and a mutation of homogentisate
phytyltransferase (HPT) leads to a complete deficiency of
tocopherols. Furthermore, high solar radiation, nutrition stress or
adverse environmental conditions have in general a strong influence
Vitamins in Chili Peppers
45
on the expression and lead to increased total tocopherol levels.
Organisms that are able to produce tocotrienols utilize the same
pathway with the difference that they are able to use also
geranylgeranyl pyrophosphate as substrate [137, 138].
Shikimate pathway MEP & Isoprenoid pathway
OHO
OH
O
p-Hydroxyphenylpyruvate
HPPD
OH
O
OH
OH
Homogentisic acid
HPT
CO2 + PPi
O P
OH
O
O P
OH
O
OH
Isopentenyl pyrophosphate
O P
OH
O
O P
OH
O
OH
3
Geranylgeranyl pyrophosphate
O P
OH
O
O P
OH
O
OHH
Phytyl pyrophosphate
3
OH
OH
H
3
Methyl-phytyl-benzoquinone
TC
MPBQMT
S-Adenosyl methionine
OH
OH
3-Tocopherol
-TMT
OH
OH
H
3
Dimethyl-phytyl-benzoquinone
OH
OH
3
S-Adenosyl methionine
-Tocopherol
TC
OH
OH
3-Tocopherol
-TMT
OH
OH
3
S-Adenosyl methionine
-Tocopherol
Figure 1-15: Biosynthetic pathway of tocopherols in plants. Precursor are
provided by the shikimate pathway and 2-C-methyl-D-erythritol-4-phosphate
(MEP) pathway. Enzymes: HPPD, hydroxyphenylpyruvate dioxygenase; TC,
tocopherol cyclase; HPT, homogentisate phytyltransferase; MPBQMT,
methyl-phytyl-benzoquinone methyltransferase; γ-TMT, γ-tocopherol
methyltransferase. Adapted and modified from Hussain et al. [137] and
DellaPenna and Pogson [138].
Chili Peppers
46
The determination of individual and total tocopherols is usually carried
out by normal phase or reversed phase HPLC. The fluorescence of
all vitamin E congeners is used for a sensitive and selective
detection. Column modification and elution system differ among the
methods described in the literature [100, 139–141]. Ching and
Mohamed described the determination of α-tocopherol in chili
peppers by using a C18 modified reversed phased column [140].
According to the lipophilic nature of tocopherols, elution of such non-
polar components is difficult und high contents of organic solvents
were necessary. Additionally, the separation of β- and γ-tocopherol
on C18 columns is critical. An alternative to the typical C18 stationary
phase is a C30 modification, which allows a better separation of β- and
γ-tocopherol. For elution of the tocopherols, non-polar organic
solvents such as tert.-butylmethylether were used [100]. However, the
separation on these columns often requires a very long runtime.
Grebenstein and Frank recently described a method that allows a
complete baseline separation of all four tocopherols and all four
tocotrienols [141]. They used a PFP modified fused core column to
analyze the vitamin E content in plasma samples without
saponification for triglycerides removing. The strong aromatic
interaction increases the selectivity and allows the separation of the
critical tocopherol congeners (β- and γ-tocopherol) in less than 15
minutes by using a simple binary mobile phase of methanol and water
[141]. Therefore, it can be expected, that this method should be also
applicable to other biological samples such as chili peppers.
Color of Chili Peppers
47
1.7 Color of Chili Peppers
The diverse color of Capsicum fruits is caused by the presence of
different pigments. It depends on genotype and ripening stage. The
dark green color of the most immature fruits is dominated by
chlorophyll a and b. Immature fruits of some genotypes have a black
or violet color that originates from the presence of the anthocyanin
delphinidin in combination with chlorophyll. Other anthocyanins are
not known to occur in chili peppers fruits. During ripening chlorophyll
is degraded, while the content of carotenoids increases and the color
changes from green to yellow, orange or red. Additionally, some
genotypes are capable of retaining chlorophyll in the ripe fruits and
appear brown. The typical yellow, orange or red color in fully ripe
fruits is the result of the presence and pattern of up to 30 different
carotenoids. The amount of carotenoids is important for the quality of
dried chili pepper powders and in addition, some of them show a
provitamin A activity (e.g. β-carotene) and are essential
phytonutrients [3, 142].
1.7.1 Carotenoids
Carotenoids are lipid-soluble polyunsaturated hydrocarbons. The
delocalized electron system is responsible for their intense color and
their physiological properties. Carotenoids are synthesized in both,
chromoplasts and chloroplasts. In chloroplasts, they assist in the
photosynthesis, but they are more important as photoprotectants.
Their primary function is to reduce oxidative damage during
photosynthesis by quenching excited singlet oxygen. In chromoplasts,
Chili Peppers
48
they are located in the thylakoid membrane and are responsible to
attract birds, which act as the preferred dispersers of Capsicum
seeds [3].
Figure 1-16 depicts the most common carotenoids in chili peppers.
Responsible for the red color are the three xanthophylls (oxygen
containing carotenoids) capsanthin, capsorubin and cryptocapsin.
Capsanthin is the major carotenoid in red chili peppers with up to
60% of the total carotenoid content, whereas the color in yellow fruits
mainly stems from β-carotene and violaxanthin. Orange colored fruits
contain a mixture of both, yellow and red pigments in smaller
amounts [3, 142].
The differences in the color are caused by variations of three
independent gene pairs, encoding the enzymes necessary in the
carotenoid biosynthesis. The proposed gene model consist of the y
locus, being essential for the formation of red pigments, the c2 locus
encoding the phytoene synthase, which catalyzes the first step in the
carotenoid pathway, and the c1 locus affecting level and composition
[8, 143].
The total carotenoid level and pattern are not only influenced
by the genotype but also by growing conditions and maturity stage. In
fresh unripe fruits low total carotenoid levels between 5 and
48 mg/100 g fresh weight (expressed as β-carotene equivalents)
were found [142]. Red and fully ripe fruits can contain 60 times higher
levels compared to their immature counterparts. Total carotenoids
can easily reach amounts of more than 3000 mg β-carotene
equivalents /100 g fresh weight [142].
Color of Chili Peppers
49
Figure 1-16: Common carotenoids found in chili peppers
1.7.2 Extractable Color
Carotenoids are typically analyzed by HPLC before and after
saponification, which is applied to detect also esterified carotenoids
[144]. Carotenoid analysis is very ambitious due to the high number
of free and bound carotenoids and the complex sample preparation.
The determination of the extractable color offers a simple and fast
alternative to estimate the total carotenoid content. The American
Spice Trade Association (ASTA) published the method, which is
internationally accepted for that purpose. The ASTA 20.1 method is
O
OH
OH
Antheraxanthin
-Carotene
OH
-Cryptoxanthin
OH
OH
Lutein
O
OH
OH
OViolaxanthin
OH
OH
Zeaxanthin
OH
OOH
Capsanthin
OOH
OOH
Capsorubin
OOH
Cryptocapsin
Yellow-orange
Red
Chili Peppers
50
applicable to dried Capsicum powders. For the determination of the
extractable color (so-called ASTA 20.1 value) the powder is extracted
with acetone for sixteen hours. After extraction, the absorbance is
read at 465 nm and converted by a simple equation into the
ASTA 20.1 value (see Chapter 10.10). To maintain the quality of the
measurement and to ensure the independence of the used
spectrophotometer, an instrument correction factor has to be
determined [33].
The ASTA 20.1 values are important for the quality assessment and
the pricing of spice paprika and chili pepper powders in international
trade. ASTA 20.1 values for non-pungent spice paprika range
typically between 160 and 180 ASTA units, but values above 200 are
also reported in the literature. For chili peppers values lower than 120
ASTA units are usual [34, 145].
1.7.3 Surface Color
The color measurement by the CIE L*a*b* system is best suited to
describe the surface color objectively and reproducibly. The
measurement of the surface color based on the recording of the
visible absorption spectra and the mathematical conversion of the
spectra into specific coordinates of the CIE L*a*b* color system. The
system is based on three pairs of colors: red-green, yellow-blue and
black-white. This allows a three dimensional description of the color
(Figure 1-17). Five color values are used to explain the color in full
detail: L* the brightness, a* the color stimulus specification of red and
green and b* the color stimulus specification of yellow and blue. The
Color of Chili Peppers
51
L*, a* and b* values also allow to calculate further color parameters:
the chroma C* describes the color saturation and the hue-angle h°
the shade of color [146, 147].
Figure 1-17: CIE L*a*b* color space; L*: brightness, a*/b*: color stimulus specification of red and green and of yellow and blue, C*: chroma and h°: hue-angle
- a* + a*
- b*
+ b*
C*
L*=0
L*=100
h°
Chili Peppers
52
Surface color measurement in combination with the determination of
the ASTA 20.1 values is best suited to easily describe the amount of
carotenoids and the color of Capsicum powders. The color stimulus
specification (a* and b*) are most important for the description of
Capsicum powders in combination with the hue-angle. Chili samples
lead to positive a* and b* values and hue-angles can reach values
between 0° (pure red) and 90° (pure yellow) according to the yellow,
orange or red color of the most chili pepper fruits.
Objective
53
2. Objective
2.1 General Remarks
Peru and Bolivia are supposed to be the centre of origin of the genus
Capsicum and both countries harbor a wealth of native Capsicum
varieties [15]. The international research project “Unraveling the
potential of neglected crop diversity for high-value product
differentiation and income generation for the poor: The case of chili
pepper in its centre of origin” had the aim of characterizing, protecting
and preserving the diversity of chili peppers and of generating a
higher income for poor small-scale chili farmers. The project was
conducted und organized by Bioversity1 in cooperation with the
national germplasm bank at INIA2 in Peru. To expand the set of native
Peruvian chili pepper accessions, CIDRA3 and UNALM4 also
participated and provided samples of their chili pepper germplasm
banks. Partners in Bolivia were CIFP5 and PROINPA6 with their
germplasm banks and ITA7. Beside these organizations, providing the
chili pepper samples, three German universities were involved. The
Institute for Environmental Economics and World Trade at the
University of Hannover performed market analyses and value chain
assessment and the Department of Agricultural Engineering in the
Tropics and Subtropics at the University of Hohenheim carried out the
1 Bioversity: Bioversity International: research for development in agricultural and
forest biodiversity 2 INIA: Instituto Nacional de Innovación Agraria
3 CIDRA: Centro de Investigación y Desarrollo Rural Amazónico
4 UNALM: Universidad Nacional Agraria La Molina
5 CIFP: Centro de Investigaciones Fitoecogenéticas de Pairumani
6 PROINPA: Fundación Promoción e Investigación de Productos Andinos
7 ITA: Instituto de Tecnología de Alimentos
Objective
54
improvement of agricultural practices. The Department of Food
Chemistry at the University of Wuppertal was responsible for the
chemical and sensory characterization of the Capsicum accessions.
The results of the chemical characterization are reported in this
thesis.
The three year project was founded by the Deutsche Gesellschaft für
Internationale Zusammenarbeit (GIZ) and the Federal Ministry for
Economic Cooperation and Development. In the first year, the
currently existing collections of the Peruvian and Bolivian germplasm
banks were further increased by collecting new accessions from
expeditions and including accessions from other national and
international collections to over 1,000 different chili pepper
accessions. This should increase representativity of the current
collections in both countries and help to protect the biodiversity. Out
of the approximately 1,000 Peruvian and Bolivian chili peppers,
representative core collections including about 100 accessions with
many landraces and different species were established for both
countries. Both core collections were analyzed on important chemical
traits as well as on different fruit traits (e.g. color, size, shape or
weight) and agronomical factors (e.g. yield or stress resistance).
The results provided the basis for selecting promising material
in the second year. For each country, about 30 promising accessions
were selected for replanting experiments.
A final selection of elite material was conducted according to
the different fruit traits, agronomical factors and the results of the
chemical characterization. These high value accessions were used in
combination with appropriate market strategies and in association
Aim and Scope
55
with local entrepreneurs to start the process for increasing the income
of small-scale chili farmers. At the same time, this act as incentive to
conserve local Capsicum varieties ‘through’ use.
2.2 Aim and Scope
In the last years, the demand of native chili pepper varieties has
grown because of the rising interest in ethnic food [148, 149]. In
addition, mankind is facing the climate change. The currently
neglected diversity of native chili peppers offers the opportunity to
select accessions with special attributes or good adaption to specific
climates or environments [150].
The presented thesis aimed at characterizing the biodiversity
of native Peruvian and Bolivian chili peppers by providing
compositional data for various phytochemicals and quality traits and
to gain selection criteria for high value Capsicum accessions. The
chili peppers were analyzed for the content of major capsaicinoids
and pattern of capsaicin, dihydrocapsaicin and nordihydrocapsaicin,
flavonoid aglycons (quercetin, luteolin, kaempferol and apigenin),
total polyphenols according to the Folin-Ciocalteu method, the
determination of the antioxidant capacity (TEAC assay), vitamin E by
analyzing the content of α-, β- and γ-tocopherol and vitamin C
(as sum of ascorbic acid and dehydroascorbic acid) as well as the
analysis of fat content, surface and extractable color. The sample
sets included accessions from the domesticated species as well as
typical landraces and wild chili peppers. The selection of promising
accessions was based on the compositional data, fruit and
agricultural traits.
Objective
56
Replanting experiments with the promising accessions were
conducted to identify those accessions, which were either consistent
in the production of phytochemicals and quality traits widely
independent of the location or which provided increased amounts
under specific growing conditions.
It was necessary to plant, harvest, dry, mill and analyze all
accessions under standardized and identical conditions to obtain
comparable data.
Therefore, the first objective of this thesis was the selection of
appropriate analytical methods, their standardization and
improvement to allow the analysis of large sample sets by applying
always the same methodology (Chapter 8). To comply with national
restrictions concerning the export of indigenous biological material, all
samples had to be dried and crushed before shipment to Wuppertal
to destruct fertile seeds as a measure against biopiracy. Accordingly,
the methods needed to be applicable to dried chili peppers samples.
An analytical strategy was established for an economic workflow.
The analytical procedures had to be applied on the Peruvian
and Bolivian chili pepper core collections. The results are reported in
Chapter 4, 5 and 7. Data were evaluated by applying descriptive
statistical methods. Accessions with high amounts of phytochemicals
or with a special combination of traits can be regarded as promising
accessions worth of further investigation.
The promising Capsicum accessions should be replanted on
the same test field for a year-to-year comparison and on three other
test fields to evaluate the environmental impact. The data were
analyzed by analysis of variance and by calculating an environmental
impact factor. This factor is based on the variance between the
Aim and Scope
57
results of one specific trait for an individual accession when planted in
different environments. Year-to-year and multi-location comparison
are presented in Chapter 6 and 7.
Structure of the Results
58
3. Structure of the Results
The results are presented in the following structure:
Chapter 4: Determination of major quality traits in 147 different
Peruvian chili peppers, belonging to the four
domesticated species C. annuum, C. baccatum,
C. chinense and C. frutescens.
Results are reported in the publication “Compositional
Characterization of Native Peruvian Chili Peppers
(Capsicum spp.)” Journal of Agricultural and Food
Chemistry (2013) 61 (10): 2530–2537
Chapter 5: Analysis of important phytochemicals in 32 different
C. pubescens accessions and inter-species
comparison of the five domesticated species grown in
Peru.
Analytical results are presented in the manuscript
“Phytochemicals in Native Peruvian Capsicum
pubescens (Rocoto)” Journal of Food Composition and
Analysis (2014) (submitted for publication)
Structure of the Results
59
Chapter 6: Multi-location comparison of important quality attributes
of 23 different chili peppers grown in three different
locations in Peru.
Results are reported in “Capsaicinoids, Flavonoids,
Tocopherols, Antioxidant Capacity and Color Attributes
in 23 Native Peruvian Chili Peppers (Capsicum spp.)
Grown in Three Different Locations” European Food
Research and Technology (2014) (accepted for
publication) DOI: 10.1007/s00217-014-2325-6
Chapter 7: Chemical characterization of 96 different Bolivian chili
pepper accessions and a year-to-year comparison of a
subset of twelve C. baccatum accessions grown on
identical test fields.
Results of chemical characterization and year-to-year
comparison are presented in the manuscript “Major
Quality Attributes of Native Bolivian Chili Peppers
(Capsicum spp.) Focussing on C. baccatum: A
two-year Comparison” Food Chemistry (2014)
(submitted for publication)
Chapter 8: Description of the analytical and experimental
background including the optimization of the analytical
methods and a streamlined analytical strategy.
Structure of the Results
60
Comment to the contributions of the authors
Each of the following Chapters (4, 5, 6 and 7) based on original
publications or on manuscripts submitted for publication. The
contributions of each author are stated below.
Analysis of capsaicinoids and capsaicinoid pattern, total polyphenols,
antioxidant capacity, ascorbic acid, flavonoid aglycon and flavonoid
pattern, data evaluation and manuscript preparation was performed
by the author.
Dieter Riegel was responsible for the analysis of the fat
content, extractable and surface color.
Tocopherol analysis was conducted by Christian Jansen.
Project coordination in South America, design of the field
experiments and assistance in the manuscript preparation was
carried out by Maarten van Zonneveld
Llermé Ríos, Karla Peña, Roberto Ugas, Lourdes Quinonez,
Teresa Avila, Carlos Bejarano and Edwin Serrano provided the chili
pepper samples and carried out the field experiments including drying
and crushing of the chili pepper samples.
Erika Mueller-Seitz assisted in the manuscript preparation and
statistical data analysis.
Project coordination at the University of Wuppertal and
assistance in the manuscript preparation was performed by
Michael Petz
Composition of Peruvian Chili Peppers
61
4. Composition of Peruvian Chili Peppers
based on:
Compositional Characterization of
Native Peruvian Chili Peppers (Capsicum spp.)*
Abstract:
The national Capsicum germplasm bank of Peru at INIA holds a unique
collection of more than 700 Capsicum accessions, including many
landraces. These conserved accessions have never been thoroughly
characterized or evaluated. Another smaller collection exists at UNALM and
CIDRA provided taxonomically characterized fruits from the Amazon region
of Ucayali. Out of these collections, 147 accessions have been selected to
represent the biodiversity of Peruvian C. annuum, C. baccatum, C. chinense
and C. frutescens by morphological traits as well as by agronomic
characteristics and regional origin. All fruits from the selected accessions
have been oven-dried and grinded in Peru and analyzed in Germany.
Results are reported for each accession by total capsaicinoids and
capsaicinoid pattern, total polyphenol content, antioxidant capacity, specific
flavonoids (quercetin, kaempferol, luteolin, apigenin), fat content, vitamin C,
surface color and extractable color. A wide variability in phytochemical
composition and concentration was found.
*Meckelmann SW, Riegel DW, van Zonneveld M, Ríos L, Peña K, Ugas R, Quinonez L, Mueller-Seitz E, Petz M (2013) Compositional Characterization of Native Peruvian Chili Peppers (Capsicum spp.). Journal of Agricultural and Food Chemistry 61(10): 2530–2537. DOI: 10.1021/jf304986q. Copyright 2013 American Chemical Society.
Composition of Peruvian Chili Peppers
62
4.1 Introduction
Belonging to the botanical family of Solanaceae together with other
plants like tomato, eggplant, potato or tobacco, plants of the genus
Capsicum are one of the oldest cultivated plants. For over 6000 years
their fruits were used for many purposes and not only as spice or food
in the human diet [4]. Based on taxonomic classification the genus
includes about 36 species today [3, 15, 24]. These include the five
domesticated species C. annuum, C. frutescens, C. chinense,
C. baccatum and C. pubescens.
Due to the characteristic pungency, aromas and flavors chili
pepper fruits (syn. chile, chilli, red pepper, hot pepper, spicy pepper)
are an important ingredient in millions of people’s daily diet (perhaps
even billions considering India). In addition, they are good sources of
the antioxidant vitamin C, vitamin E and provitamin A, as well as
excellent sources of other antioxidants, which counter the oxidation of
lipids via scavenging free radicals and thus are discussed as
protection against cancer, anemia, diabetes and cardiovascular
diseases [3]. The concentration and pattern of these health promoting
phytochemicals are influenced by genotype and environmental
factors as well as by processing parameters in the production of chili
powders, like sample treatment, drying conditions and milling
[129, 151]. But not only health promoting attributes are important for
the usage of chilies. The xanthophylls capsanthin and capsorubin are
the dominating carotenoids, which allow the production of natural
colorants such as oleoresins. These products are used in the food
and cosmetic industries [152, 153].
Introduction
63
According to molecular analyses of domesticated and wild species of
Capsicum, it is concluded that the genus Capsicum originated most
likely in arid regions of the Andes Mountains, in what became Peru
and Bolivia, and then migrated to tropical lowland regions of the
Americas [1, 3]. The centers of domestication are still under
discussion. C. baccatum and C. pubescens are postulated to be
domesticated in Bolivia. The putative center of crop origin of
C. annuum is currently Mexico and C. chinense and C. frutescens are
thought to have been originated in the Amazon [23]. Peru is a center
of diversification and probably the country with the highest diversity of
cultivated chili peppers in the world because of the long
pre-Columbian cultural history and the fact that this is one of the few
countries, where varieties of all five cultivated species are grown and
used in local diet. Today, Peru is also one of the leading export
countries for paprika (C. annuum) using conventional varieties
introduced to Peru more recently.
Being the world’s most important center of cultivated
Capsicum diversity, Peru holds a wealth of local Capsicum varieties,
each with specific phytochemical characteristics. More than 700
Capsicum accessions of the five cultivated species, genetic material
collected on farms and from home gardens, are kept in the Peruvian
national Capsicum germplasm bank managed by INIA (Instituto
Nacional de Innovación Agraria). Out of these ~700 Capsicum
accessions, 90 have been selected to represent the biodiversity of
native Peruvian chili peppers domiciled in the three climatic zones:
coast, Andes, Amazon. Thirty-seven accessions from UNALM
(Universidad Nacional Agraria La Molina) collection as well as 20
accessions collected in smallholder farms in the Amazon region from
Composition of Peruvian Chili Peppers
64
CIDRA (Centro de Investigación y Desarrollo Rural Amazónico) were
included to expand the set. These 147 accessions belong to the four
domesticated species C. annuum, C. baccatum, C. chinense and
C. frutescens and are commonly named as “Ajíes” in Peru, as it is
called in most other South-American countries as well. Accessions of
the species C. pubescens can easily be differentiated from other
cultivated species due to their black seeds and are commonly named
as “Rocoto” (Chapter 5).
The study characterizes the phytochemical biodiversity of native
Peruvian chili peppers (Ajíes). The following attributes have been
investigated: pungency by total capsaicinoids, capsaicinoid pattern
(capsaicin, dihydrocapsaicin and nordihydrocapsaicin), total and
specific flavonoids (quercetin, kaempferol, luteolin, apigenin), total
polyphenol content using the Folin-Ciocalteu assay, antioxidant
capacity (TEAC assay), vitamin E by analyzing the content of α- , β-
and γ- tocopherol and vitamin C (ascorbic acid) (sum of ascorbic and
dehydroascorbic acid), fat content and surface color (CIE L*a*b*) and
extractable color (ASTA 20.1). To comply with national restrictions
concerning the export of indigenous biological material, all samples
were dried and crushed in Peru and shipped for analysis via air
courier to Wuppertal/Germany.
The results of this study contribute to characterize Peruvian
Capsicum varieties for potential commercial traits. The biochemical
descriptions can be used to identify in a participatory approach with
small-scale farmers and local entrepreneurs promising material for
the development of high-value products and to start market
specialization. The results can also be a starting point to target
Experimental
65
accessions for further breeding activities. The study results thus add
value to Capsicum diversity to generate income for small-scale
farmers. At the same time, this can provide an incentive to conserve
local Capsicum varieties ‘through’ use. It would also confirm the
important role of gene banks in conservation ‘for’ use. Ex situ
conservation of Peruvian Capsicum varieties is necessary because
not all accessions have a direct commercial value.
4.2 Experimental
4.2.1 Plant Material and Post Harvest Treatment
147 different accessions provided by the three Peruvian organizations
INIA, UNALM and CIDRA were characterized. INIA collected fruits
from single plants grown in the experimental station of Donoso in the
Peruvian coastal zone, Huaral, Lima (11°31'25''S, 77°14'01''E).
CIDRA also collected fruits from single plants, which were grown by
local farmers in the community Campo Verde of Ucayali region in the
Peruvian Amazon (08°31’50’’ S, 74°04’43’’E). Samples from UNALM
were from several plants of the same accession to collect a sufficient
amount of material. Plants were grown in two experimental stations:
1) El Huerto, La Molina, Lima (12°04'60''S, 76°56'32''E ) and 2)
Casma, Ancash (09°28'54''S, 78°17'34''E) . Fully ripe fruits were
harvested in the years 2010, 2011 and 2012 (detailed information
including germplasm bank accession code (Acc. code), growing
region, taxonomical classification and harvest year are given in the
Appendix (Chapter 13, Table A 1). Peduncles were removed and
Composition of Peruvian Chili Peppers
66
fruits were oven-dried at 60 °C to constant weight for approximately
72 h, crushed and sent by air courier in sealed bags to Wuppertal.
Table 4.1: Number of Accessions per species and organization
Total C. annuum C. baccatum C. chinense C. frutescens
INIA 90 19 26 43 2
UNALM 37 2 8 27 0
CIDRA 20 0 2 15 3
Total 147 21 36 85 5
Table 4.1 shows the number of accessions per species received from
the different organizations. Plants were taxonomically classified to
belong to the four domesticated species C. annuum, C. baccatum,
C. chinense and C. frutescens [19].
4.2.2 Statistical Analysis
All determinations were carried out as duplicates (two extracts),
except for ascorbic acid. Table 4.2 shows the mean coefficients of
variation determined from 147 duplicate analyses.
Analyses were carried out on dried material. Accordingly, the
results refer to 100 g of the dry sample material as obtained after
milling. Moisture content of this material was also determined and
ranged between 0.7 and 3.2 g/100 g
Experimental
67
Table 4.2: Analytical precision data
Parameter CV* (%) Parameter CV* (%)
Capsaicinoids 4.0 TEAC 2.7
Capsaicin 4.0 Tocopherols 2.6
Dihydrocapsaicin 4.4 α-Tocopherol 2.9
Nordihydrocapsaicin 7.1 β-Tocopherol 4.3
Flavonoids 2.3 γ-Tocopherol 4.0
Quercetin 2.3 Fat content 2.5
Luteolin 2.6 Extractable color
(ASTA 20.1) 2.1
Kaempferol 2.2 Surface color (hue-angle)
0.8
Apigenin 9.0 Fat content 2.5
Total polyphenols 1.9 Moisture content 5.2
* CV= Coefficient of variation; average CV from all 147 duplicate analyses. Ascorbic acid content was only screened by single determination and is not mentioned.
Box plot analysis was done using the software tool “R 2.15.1”
(R Foundation for Statistical Computing, Vienna, Austria), freely
available at http://www.r-project.org. The box plot shows the range
minimum-maximum, 25 percentile, median, and 75 percentile.
Outliers were identified by 1.5 times of the interquartile range.
Outliers can be regarded as samples with outstanding attributes.
Statistical box plot analysis was not carried out for C. frutescens
accessions because of their small number (n=5).
Composition of Peruvian Chili Peppers
68
4.3 Results and Discussion
4.3.1 Capsaicinoids and Pattern
The pungency of chili peppers and spice paprika is an important
quality parameter. The amount of the capsaicinoids capsaicin,
dihydrocapsaicin and nordihydrocapsaicin, which are responsible for
the pungent taste, shows a wide variability among all Peruvian
species and varieties from non-pungent up to very hot (Figure 4.1
and 4.2). The highest capsaicinoid concentration was found in a
C. frutescens accession (Acc. code: AMS-M) with 1560.1 mg/100 g of
total capsaicinoids and a pattern of 68.5% capsaicin, 29.5%
dihydrocapsaicin and 1.7% nordihydrocapsaicin. This is equivalent to
ca. 250,000 SHU (Scoville Heat Units). In C. chinense the maximum
amount was 1411.6 mg/100 g (Acc. code: 175) and in C. annuum
809.0 mg/100 g (Acc. code: PER017826). C. baccatum was the least
pungent of the three species with the highest value at 711.7 mg/100 g
(Acc. code: PER017672). In two C. annuum and two C. chinense
accessions no capsaicinoids at all could be detected.
Results and Discussion
69
Figure 4-1: Individual capsaicinoid levels and pattern of 147 different
Peruvian chili pepper accessions (germplasm bank codes) sorted by
ascending capsaicinoid content. Left: accessions with capsaicinoids
between not detectable amounts and ~250 mg/100 g and right: accessions
above ~250 mg/100 g.
0 500 1000 1500
AMS-M175
LPI-PUCPER017787
EHA-UUPER017728PER006988
113SIT-PM
PER007009PER007008PER017707PER017826PER017698PER017672PER007046PER017668
AMS-CHIPER006952PER006995PER007023PER017664PER017701
44PER017784PER017712PER017732AMS-NN-1
PER006965PER006958
42AMS-NN-4
LPI-NN-3PER007021PER017667
187238
PER017665LPI-TROA
PER006942PER006990
LPI-CHAA4
AMS-CHAAPER017662PER006992PER017710
AMS-CRPER007020
157PER07026 PER007035
42PER017738PER017653
123PER017660
3LCC-TRORPER007004PER017675PER017682PER007005PER006985PER007025
75PER017849
EHA-CAPER017683
157LPI-CHAR
PER006948PER017633PER06963
LCC-CHALLPER06959 PER006954
0 50 100 150 200 250
PER006951
PER017635
60
PER006957
PER007044
PER006964
PER017721
PER017692
69
6
PER017691
132
88
222
LPI-A
EHA-CHAR
72
PER017893
2
10
PER017654
5
5
200
69
PER017621
PER006991
PER017605
7
PER017705
201
202
85
PER017671
PER017618
PER017736
PER017661
202
PER017608
PER017679
PER017625
PER017875
PER017601
PER017610
PER017833
8
PER017648
132
PER017719
252
85
PER017704
PER017699
PER017626
AMS-AD
PER017735
PER017908
PER007013
PER006984
PER017708
PER017711
PER006979
PER007040
PER017623
PER017612
AMS-RC
PER017910
PER017909
153
157
0 500 1000 1500
PER006951
PER017635
60
PER006957
PER007044
PER006964
PER017721
PER017692
69
6
PER017691
132
88
222
LPI-A
EHA-CHAR
72
PER017893
2
10
PER017654
5
5
200
69
PER017621
PER006991
PER017605
7
PER017705
201
202
85
PER017671
PER017618
PER017736
PER017661
202
PER017608
PER017679
PER017625
PER017875
PER017601
PER017610
PER017833
8
PER017648
132
PER017719
252
85
PER017704
PER017699
PER017626
AMS-AD
PER017735
PER017908
PER007013
PER006984
PER017708
PER017711
PER006979
PER007040
PER017623
PER017612
AMS-RC
PER017910
PER017909
153
157
(mg/100 g)
Capsaicin Dihydrocapsaicin Nordihydrocapsaicin
(mg/100 g)
Composition of Peruvian Chili Peppers
70
Figure 4-2: Box plot of capsaicinoid concentrations. 25 percentile, median
(thick line), 75 percentile and range minimum-maximum, outliers (•) were
identified by 1.5 times of the interquartile range. All results are expressed in
mg/100 g. A= C. annuum; B= C. baccatum; C= C. chinense.
Figure 4-3: Box plot analysis of percentage of capsaicinoid distribution.
A= C. annuum; B= C. baccatum; C= C. chinense.
A B C
0200
400
600
800
1000
1200
1400
Capsaicinoids
A B C
0200
400
600
800
1000
Capsaicin
A B C
0100
200
300
400
Dihydrocapsaicin
A B C
020
40
60
80
Nordihydrocapsaicin
A B C
05
10
15
% Nordihydrocapsaicin
A B C
020
40
60
80
100
% Capsaicin
A B C
010
20
30
40
50
60
% Dihydrocapsaicin
Results and Discussion
71
Figure 4-2 also shows the wide concentration range of each individual
capsaicinoid. It is remarkable that C. chinense samples had in
general very low nordihydrocapsaicin contents, but also the two
varieties with the highest content.
Figure 4-3 presents the pattern (percentage distribution) of
capsaicinoids. Capsaicin and dihydrocapsaicin were the dominating
capsaicinoids in all accessions. C. chinense accessions contain
smaller amounts of nordihydrocapsaicin compared to the accessions
of the other two species. Multivariate data analysis by principle
component analysis (PCA) and by partial least squares discriminant
analysis (PLS-DA) did not show any correlation between species and
pattern of the individual capsaicinoids. This has been described
before by Zewdie and Bosland [46]. One of the five C. frutescens
(Acc. code: PER007020) from this set is remarkable for the
capsaicinoid composition with 37.6% capsaicin, 43.2%
dihydrocapsaicin and 19.2% nordihydrocapsaicin, which is very
untypical with regard to nordihydrocapsaicin in comparison with the
literature data [46].
4.3.2 Specific Flavonoids
Chili peppers are a good source of flavonoids. This class of
phytonutrients has different health promoting effects. Besides their
antioxidant properties and free radical scavenging activity, they have
anti-inflammatory and anti-carcinogenic effects making them
interesting for the human diet and highly valuable compounds in chili
peppers [110]. The analysis of flavonoids was focused on the major
Composition of Peruvian Chili Peppers
72
flavones quercetin, luteolin, kaempferol and apigenin contained in the
fruits as free flavonoids and as glycosides.
Individual levels of flavonoids (sum of the four analyzed
aglycons) and each aglycon are given in Figure 4-4. Quercetin was
the dominating flavonoid and found in 141 accessions with
concentrations up to 26.6 mg/100 g. In six accessions neither
quercetin nor any other of the three flavonoids could be found.
Luteolin was the second dominating flavonoid but with much lower
concentrations between 0.4 and 5.2 mg/100 g. Kaempferol and
apigenin were found in just a limited number of chilies with
concentrations from 0.1 to 0.6 mg/100 g for kaempferol and 0.2 to
0.7 mg/100 g for apigenin.
The highest amount of an individual flavonoid was found in a
C. chinense accession (Acc. code: LPI-PUC) with 26.6 mg
quercetin/100 g, while the maximum total flavonoid concentration was
measured in a C. annuum accession with 29.5 mg/100 g (Acc. code:
PER017668). Box plot analysis of the levels of flavonoids and the two
major flavonoid aglycons, quercetin and luteolin is given in Figure 4-5.
Remarkably, 64% of all C. chinense accessions did not contain
detectable amounts of luteolin, kaempferol, or apigenin. The five
C. frutescens accessions showed in all cases rather low levels of
flavonoids with quercetin as the dominating flavonoid.
Results and Discussion
73
Figure 4-4: Individual flavonoid levels and pattern of 147 different Peruvian
chili pepper accessions (germplasm bank code) sorted by ascending
capsaicinoid content.
0 10 20 30
PER006951
PER017635
60
PER006957
PER007044
PER006964
PER017721
PER017692
69
6
PER017691
132
88
222
LPI-A
EHA-CHAR
72
PER017893
2
10
PER017654
5
5
200
69
PER017621
PER006991
PER017605
7
PER017705
201
202
85
PER017671
PER017618
PER017736
PER017661
202
PER017608
PER017679
PER017625
PER017875
PER017601
PER017610
PER017833
8
PER017648
132
PER017719
252
85
PER017704
PER017699
PER017626
AMS-AD
PER017735
PER017908
PER007013
PER006984
PER017708
PER017711
PER006979
PER007040
PER017623
PER017612
AMS-RC
PER017910
PER017909
153
157
0 10 20 30
AMS-M175
LPI-PUCPER017787
EHA-UUPER017728PER006988
113SIT-PM
PER007009PER007008PER017707PER017826PER017698PER017672PER007046PER017668
AMS-CHIPER006952PER006995PER007023PER017664PER017701
44PER017784PER017712PER017732AMS-NN-1
PER006965PER006958
42AMS-NN-4
LPI-NN-3PER007021PER017667
187238
PER017665LPI-TROA
PER006942PER006990
LPI-CHAA4
AMS-CHAAPER017662PER006992PER017710
AMS-CRPER007020
157PER07026 PER007035
42PER017738PER017653
123PER017660
3LCC-TRORPER007004PER017675PER017682PER007005PER006985PER007025
75PER017849
EHA-CAPER017683
157LPI-CHAR
PER006948PER017633PER06963
LCC-CHALLPER06959 PER006954
(mg/100 g)
0 10 20 30
PER006951
60
PER007044
PER017721
69
PER017691
88
LPI-A
72
2
PER017654
5
69
PER006991
7
201
85
PER017618
PER017661
PER017608
PER017625
PER017601
PER017833
PER017648
PER017719
85
PER017699
AMS-AD
PER017908
PER006984
PER017711
PER007040
PER017612
PER017910
153
Quercetin Luteolin Kaempferol Apigenin
Composition of Peruvian Chili Peppers
74
Figure 4-5: Box plot analysis of flavonoids (sum of the four analyzed aglycons) and the two major flavonoid aglycons, quercetin and luteolin. All results are expressed in mg/100 g; A= C. annuum; B= C. baccatum; C= C. chinense.
Miean and Mohamed analyzed three C. annuum (green chili, red chili,
and bell pepper) and one C. frutescens (bird chili) market samples
[105]. Quercetin was found in three samples (40-80 mg/100 g),
Luteolin in green chili (3.3 mg/100 g) and a remarkable high content
of luteolin in bird chili (103 mg/100 g). Kaempferol was present in
green chili (3.3 mg/100 g) and apigenin in bell pepper
(27.2 mg/100 g). As with capsaicinoids a wide range is to be
expected for flavonoids in Capsicum fruits.
A B C
05
10
15
20
25
30
Flavonoids
A B C
05
10
15
20
25
Quercetin
A B C
01
23
45
Luteolin
Results and Discussion
75
4.3.3 Total Polyphenols and Antioxidant Capacity
In the modern human diet, phytonutrients with the ability to scavenge
free radicals and with further health promoting attributes become
more and more important. The antioxidant activity and total
polyphenols are attributed to different compounds like flavonoids,
phenolic acids, capsaicinoids, vitamin C, vitamin E and other
antioxidants found in chili peppers. Assays like the determination of
the total polyphenol content using the Folin-Ciocalteu assay or the
TEAC assay are key parameters for the assessment of the health
benefit potential. The advantage is that these assays assess the
mixtures of the extracted phytonutrients in total and do not focus on a
single antioxidant or group [115]. This allows to rate chili accessions
by the degree of their antioxidant properties. However, these tests
have the disadvantage of providing only very limited comparability
with data of other studies. Slightly changed conditions for extraction
or minor modifications in the assay procedures have a strong
influence on the results of the unspecific sum parameters [116].
The results of total polyphenols and TEAC showed a wide
variation across the different accessions and species (Figure 4-6 and
4-7). The highest levels were found in a C. chinense accession
(Acc. code: PER06959) with 3.69 g gallic acid equivalents
(GAE) /100 g of total polyphenols and a TEAC value of 9.2 mmol
Trolox /100 g. The majority of samples are in the range between 1.5
and 2.0 g GAE /100 g and between 3.0 and 5.0 mmol Trolox /100 g
for TEAC value. Hervert-Herández et al. reported comparable data for
extractable polyphenols of four dried hot pepper varieties
(C. annuum) with 0.97 to 1.4 g GAE /100 g and 1.9 to 3.6 mmol
Trolox /100 g [154]. Additionally, they observed a correlation between
Composition of Peruvian Chili Peppers
76
total polyphenols and the corresponding TEAC values with R2=0.98.
A positive correlation (Figure 4-8) was also observed but with a
significant lower coefficient of correlation (R2=0.61). This could be
due to the much higher number of samples and more different
species and accessions in this study.
The accessions of this study that showed outstanding high
levels of the two sum parameters could be a good source of
antioxidants in the human diet.
Figure 4-6: Box plot of antioxidant sum parameters. Units: Total
polyphenols: g GAE/100 g, TEAC mmol Trolox /100 g. A= C. annuum;
B= C. baccatum; C= C. chinense.
A B C
1.5
2.0
2.5
3.0
3.5
Total polyphenols
A B C
24
68
TEAC
Results and Discussion
77
Figure 4-7: Results of total polyphenols and the corresponding TEAC
values. Accessions (germplasm bank code) are sorted by ascending
capsaicinoid content.
0 2 4 6 8
AMS-M175
LPI-PUCPER017787
EHA-UUPER017728PER006988
113SIT-PM
PER007009PER007008PER017707PER017826PER017698PER017672PER007046PER017668
AMS-CHIPER006952PER006995PER007023PER017664PER017701
44PER017784PER017712PER017732AMS-NN-1
PER006965PER006958
42AMS-NN-4
LPI-NN-3PER007021PER017667
187238
PER017665LPI-TROA
PER006942PER006990
LPI-CHAA4
AMS-CHAAPER017662PER006992PER017710
AMS-CRPER007020
157PER07026
PER00703542
PER017738PER017653
123PER017660
3LCC-TRORPER007004PER017675PER017682PER007005PER006985PER007025
75PER017849
EHA-CAPER017683
157LPI-CHAR
PER006948PER017633PER06963
LCC-CHALLPER06959
PER006954
0 2 4 6 8
PER006951
PER017635
60
PER006957
PER007044
PER006964
PER017721
PER017692
69
6
PER017691
132
88
222
LPI-A
EHA-CHAR
72
PER017893
2
10
PER017654
5
5
200
69
PER017621
PER006991
PER017605
7
PER017705
201
202
85
PER017671
PER017618
PER017736
PER017661
202
PER017608
PER017679
PER017625
PER017875
PER017601
PER017610
PER017833
8
PER017648
132
PER017719
252
85
PER017704
PER017699
PER017626
AMS-AD
PER017735
PER017908
PER007013
PER006984
PER017708
PER017711
PER006979
PER007040
PER017623
PER017612
AMS-RC
PER017910
PER017909
153
157
0 1 2 3 4 5 6 7 8 9 10
AMS-M
175
LPI-PUC
PER017787
EHA-UU
PER017728
PER006988
113
SIT-PM
PER007009
PER007008
PER017707
PER017826
PER017698
PER017672
PER007046
PER017668
AMS-CHI
PER006952
PER006995
PER007023
PER017664
PER017701
44
PER017784
PER017712
PER017732
AMS-NN-1
PER006965
PER006958
42
AMS-NN-4
LPI-NN-3
PER007021
PER017667
187
238
PER017665
LPI-TROA
PER006942
PER006990
LPI-CHAA
4
AMS-CHAA
PER017662
PER006992
PER017710
AMS-CR
PER007020
157
PER07026
PER007035
42
PER017738
PER017653
123
PER017660
3
LCC-TROR
PER007004
PER017675
PER017682
PER007005
PER006985
PER007025
75
PER017849
EHA-CA
PER017683
157
LPI-CHAR
PER006948
PER017633
PER06963
LCC-CHALL
PER06959
PER006954
TEAC (mmol Trolox /100 g) Total polyphenols (g GAE /100 g)
0 1 2 3 4 5 6 7 8 9 10
AMS-M
175
LPI-PUC
PER017787
EHA-UU
PER017728
PER006988
113
SIT-PM
PER007009
PER007008
PER017707
PER017826
PER017698
PER017672
PER007046
PER017668
AMS-CHI
PER006952
PER006995
PER007023
PER017664
PER017701
44
PER017784
PER017712
PER017732
AMS-NN-1
PER006965
PER006958
42
AMS-NN-4
LPI-NN-3
PER007021
PER017667
187
238
PER017665
LPI-TROA
PER006942
PER006990
LPI-CHAA
4
AMS-CHAA
PER017662
PER006992
PER017710
AMS-CR
PER007020
157
PER07026
PER007035
42
PER017738
PER017653
123
PER017660
3
LCC-TROR
PER007004
PER017675
PER017682
PER007005
PER006985
PER007025
75
PER017849
EHA-CA
PER017683
157
LPI-CHAR
PER006948
PER017633
PER06963
LCC-CHALL
PER06959
PER006954
TEAC (mmol Trolox /100 g) Total polyphenols (g GAE /100 g)
Composition of Peruvian Chili Peppers
78
Figure 4-8: Correlation between TEAC and total polyphenol levels of 147
different chili pepper accessions; R2=0.61.
4.3.4 Tocopherols and Ascorbic Acid8
The content of tocopherols was analyzed by HPLC with fluorescence
detection. The good separation allows reporting on the content of α-,
β- and γ-tocopherol, as well as the sum of the three congeners as
tocopherols content (Figure 4-9 and 4-10).
All analyzed accessions contained detectable amounts of
tocopherols. Among species, tocopherol content ranged from 0.4 to
35.3 mg/100 g. Both was found in accessions belonging to
C. baccatum (Acc. code: LPI-A and 42). C. annuum was found as the
species containing generally higher contents of tocopherols with
regard to median values.
8 Results of the tocopherol analyses were not available at the date of manuscript
submission and accordingly, not part of the original publication.
0.0
2.0
4.0
6.0
8.0
10.0
0.00 1.00 2.00 3.00 4.00
TE
AC
(m
mo
l T
rolo
x / 1
00 g
)
Total polyphenols (g GAE / 100 g)
Results and Discussion
79
Figure 4-9: Box plot analysis of the tocopherol content (sum of α-, β- and
γ-tocopherol), levels of individual tocopherols and ascorbic acid. All results
are expressed in mg/100 g; A= C. annuum; B= C. baccatum;
C= C. chinense.
Regarding the pattern of the three individual tocopherols,
α-tocopherol was the most abundant. For α-tocopherol levels up to
32.5 mg/100 g were observed (Acc. code: PER017910). This is in
accordance with data from Ching and Mohamed [140]. They found
α-tocopherol contents between 13.8 and 29.1 mg/100 g in four
different chili peppers. γ-Tocopherol was found with levels up to
7.8 mg/100 g (Acc. code: 4). In all analyzed accessions β-tocopherol
was a minor component. Only traces were determined with levels up
to 2.2 mg/100 g and several of the accessions did not show a
detectable amount of β-tocopherol.
A B C
05
10
15
20
25
30
35
Tocopherols
A B C
05
10
15
20
25
30
α-Tocopherol
A B C
0.0
0.5
1.0
1.5
2.0
β-Tocopherol
A B C
02
46
8
γ-Tocopherol
A B C
050
100
150
200
250
300
Ascorbic acid
Composition of Peruvian Chili Peppers
80
Figure 4-10: Individual tocopherol concentrations and pattern of 147
different Peruvian chili pepper accessions (germplasm bank code) sorted by
ascending capsaicinoid content.
0 10 20 30
AMS-M175
LPI-PUCPER017787
EHA-UUPER017728PER006988
113SIT-PM
PER007009PER007008PER017707PER017826PER017698PER017672PER007046PER017668
AMS-CHIPER006952PER006995PER007023PER017664PER017701
44PER017784PER017712PER017732AMS-NN-1
PER006965PER006958
42AMS-NN-4
LPI-NN-3PER007021PER017667
187238
PER017665LPI-TROA
PER006942PER006990
LPI-CHAA4
AMS-CHAAPER017662PER006992PER017710
AMS-CRPER007020
157PER07026
PER00703542
PER017738PER017653
123PER017660
3LCC-TRORPER007004PER017675PER017682PER007005PER006985PER007025
75PER017849
EHA-CAPER017683
157LPI-CHAR
PER006948PER017633PER06963
LCC-CHALLPER06959
PER006954
0 10 20 30
PER006951
PER017635
60
PER006957
PER007044
PER006964
PER017721
PER017692
69
6
PER017691
132
88
222
LPI-A
EHA-CHAR
72
PER017893
2
10
PER017654
5
5
200
69
PER017621
PER006991
PER017605
7
PER017705
201
202
85
PER017671
PER017618
PER017736
PER017661
202
PER017608
PER017679
PER017625
PER017875
PER017601
PER017610
PER017833
8
PER017648
132
PER017719
252
85
PER017704
PER017699
PER017626
AMS-AD
PER017735
PER017908
PER007013
PER006984
PER017708
PER017711
PER006979
PER007040
PER017623
PER017612
AMS-RC
PER017910
PER017909
153
157
0 500 1000 1500
AMS-M
LPI-PUC
EHA-UU
PER006988
SIT-PM
PER007008
PER017826
PER017672
PER017668
PER006952
PER007023
PER017701
PER017784
PER017732
PER006965
42
LPI-NN-3
PER017667
238
LPI-TROA
PER006990
4
PER017662
PER017710
PER007020
PER07026
42
PER017653
PER017660
LCC-TROR
PER017675
PER007005
PER007025
PER017849
PER017683
LPI-CHAR
PER017633
LCC-CHALL
PER006954
Ac
ce
ss
ion
co
de
α-Tocopherol γ-Tocopherol β-Tocopherol
(mg/100 g)
Results and Discussion
81
Fresh chili peppers are an extremely rich source of ascorbic acid
(vitamin C). Fresh fruits typically contain up to 250 mg /100 g fresh
weight. The content is influenced by the degree of ripeness [3].
During the drying process most of the ascorbic acid is degraded to
residual levels of only 10% [128]. Therefore, the ascorbic acid content
was only screened by a single determination for each accession. In
83 of the 147 accessions did not contain any ascorbic acid, while
some accessions surprisingly showed outstanding high ascorbic acid
concentrations. The maximum of ascorbic acid was found in a
C. chinense accession with 295 mg/100 g (Acc. code: PER006992).
A high resolution mass spectrometric analysis confirmed the identity
of the HPLC peak ascribed to ascorbic acid with an m/z of 175.0246
for the [M-H]- ion. Information about the individual vitamin C content is
presented in the Appendix (see Chapter 13, Table A 2).
To finally confirm this outstanding result, it will be necessary to
analyze fresh fruits of this and other exceptionally vitamin rich
accessions. Besides the health promoting effects of ascorbic acid as
vitamin and antioxidant such high concentrations help to protect and
preserve other valuable compounds, e.g. carotenoids and thereby
color intensity during the drying process and storage of chili powder
[129].
4.3.5 Fat Content and Color
The results of the fat content, the extractable and surface color are
summarized in Figure 4-11. Further information of fat content,
extractable color and surface color is given in Chapter 13 Table A 2.
Composition of Peruvian Chili Peppers
82
The fat content in the samples depends on the amount of seeds in
comparison to the content of pericarp in the powder. Fat content
ranged between 2.2 and 19.6 g/100 g (No. 131; C. chinense) with the
median for C. annuum at ca. 12 g/100 g and median values at ca.
7 g/100 g for C. baccatum and C. chinense. Because vitamin E is a
fat soluble complex of different tocopherols, correlations between
vitamin E and the fat content of the chili peppers can be expected
[3, 155]. A correlation between fat and tocopherol content in chili
peppers was described for three Capsicum varieties at different
ripening stage by Kanner et al. [155]. In the presented study with 147
accessions no correlations were observed between tocopherols and
fat content.
Figure 4-11: Box plot of fat content in g/100 g, values for the extractable
color (ASTA 20.1) and surface color (hue-angle °); A= C. annuum;
B= C. baccatum; C= C. chinense
Besides the capsaicinoids, the content of carotenoids as classified by
the ASTA 20.1 value is another important quality parameter.
Carotenoids are potent antioxidants and some have provitamin A
activity. The xanthophylls capsanthin and capsorubin in fruits of the
A B C
05
01
00
15
0 Extractable color (ASTA-20.1)
A B C
40
50
60
70
80
Surface color (hue-angle)
A B C
51
01
52
0
Fat content
Results and Discussion
83
genus Capsicum are responsible for the intense red color for a wide
range of varieties. Concentrated extracts are used as important
colorants for the food and cosmetic industry. The color measurement
by the CIE L*a*b* system is best suited to describe the surface color
objectively and reproducibly. The hue-angle can be calculated from
the L*a*b* values and describes the relation between red, orange and
yellow pigments. Because the yellowish seeds have been milled
together with the dried pericarp, the color of the powders will be
shifted into the yellow range.
In general, a hue-angle of 90° describes a pure yellow color
and 0° a pure red color, with orange in between. Most of the samples
were in the orange range with only a few appearing red. The most
intense red sample was a C. chinense accession (Acc. code:
PER007004) with a hue-angle of 36.4 and also the maximum
ASTA 20.1 value of 146 for extractable color. This is remarkable for
chili powders but far away from paprika reaching ASTA values
beyond 200. As can be seen in Figure 4-11, the median of extractable
color is significant higher in C. annuum than in C. baccatum and
C. chinense.
Nieto-Sandoval et al. noted a correlation between surface
color and the corresponding natural logarithm of the ASTA 20.1 value
with a good correlation of R2=0.97 for typical red paprika from
C. annuum [145]. Based on the surface color measurement, this
correlation was used in this study for getting an estimate of the ASTA
value. This allowed finding a suitable sample weight for the
ASTA 20.1 determination for achieving the required absorption of
0.3-0.7.
Composition of Peruvian Chili Peppers
84
4.4 Conclusion
The presented study included the compositional characterization of
147 accessions belonging to the domesticated species C. annuum,
C. baccatum, C. chinense and C. frutescens. Capsicum accessions
were identified with pungency from non-pungent to extremely
pungent, and with outstanding content in valuable health related
phytonutrients. This still under-utilized diversity of native Capsicum
varieties should be a starting point for high-value product
differentiation and income generation for poor small-scale farmers
and local entrepreneurs in Peru. Today, most consumers buy fruits
and vegetables based on appearance and not on nutritional quality.
This may be changing, however, as consumers begin to look to fruits
and vegetables as insurance against illness [3]. Due to the very
unique morphological characteristic, the results of the investigated
C. pubescens accessions were reported in a separate study.
Phytochemicals in Peruvian C. pubescens
85
5. Phytochemicals in Peruvian C. pubescens
based on:
Phytochemicals in
Native Peruvian Capsicum pubescens (Rocoto)*
Abstract:
Peru is considered a hotspot with maybe the highest diversity of
domesticated chili peppers. With regard to chemical composition
C. pubescens is the least explored compared to the other four domesticated
species. 32 different C. pubescens (Rocoto) accessions, out of the national
Peruvian Capsicum germplasm collection at the Instituto Nacional de
Innovación Agraria, were selected to investigate the diversity of
phytochemicals. After drying and milling, the fruits were analyzed for the
three major capsaicinoids (capsaicin, dihydrocapsaicin and
nordihydrocapsaicin), flavonoid aglycons (quercetin, kaempferol, luteolin,
apigenin), total polyphenol content, antioxidant capacity, tocopherols, fat
content, ascorbic acid, surface color and extractable color. The
concentrations of selected traits ranged as follows: total capsaicinoids from
55 to 410 mg/100 g, total polyphenols from 1.8 to 2.5 g gallic acid
equivalents/100 g, antioxidant capacity from 2.4 to 4.6 mmol Trolox/100 g
and tocopherols from 6.8 to 18.4 mg/100 g. Only very few of the accessions
contained detectable amounts of the major flavonoid quercetin. The results
indicate that C. pubescens is general less diverse and exhibits a lower
content of almost all analyzed traits, when compared to 147 Peruvian chili
pepper accessions belonging to the other four domesticated species.
* Meckelmann SW, Jansen C, Riegel DW, van Zonneveld M, Ríos L, Peña K, Mueller-Seitz E, Petz M (2014) Phytochemicals in Native Peruvian Capsicum pubescens (Rocoto). Journal of Food Composition and Analysis (submitted for publication)
Phytochemicals in Peruvian C. pubescens
86
5.1 Introduction
The genus Capsicum with its more than 30 different species belongs
to the Solanaceae family. Among the five domesticated Capsicum or
chili peppers, the species of Capsicum pubescens consist of relative
unknown peppers. Much of the available general knowledge in
C. pubescens is compiled in [3, 156]. It is grown extensively in
courtyards and kitchen gardens or small family plots from the
highland of Mexico to the mid-elevation Andes (between 1500 m und
3000 m) of Peru and Bolivia, where it probably originated. The
domestication of Capsicum pubescens peppers started about 6000
years ago. They are called “Rocoto” in Peru and “Locoto” in Bolivia.
“Canario” is another name, referring to the bird canary, because of
the yellow color of many C. pubescens varieties. The Guatemalans
call it either “Caballo” (horse), owing to its pungency kicking like a
horse, or “Siete caldos” because it is hot enough to season seven
soups. The fruits of these chili peppers combine the suavity and
juiciness of bell peppers with the pungency of habaneros. They are
commonly used in the Andean cuisine, mainly in fresh salsas, but
also stuffed with meat or cheese and baked in the traditional Peruvian
dish ”Rocoto relleno” or in the popular “Ceviche” with raw fish. The
demand for C. pubescens peppers has increased outside the
Americas with the interest in ethnic cuisines in the US and many
European countries, for example in Spain [157]. The fruit can also be
dried and ground into powder for use as a pepper-like spice [158].
The requirements for a cool, but freeze-free environment and long
growing season were probably responsible for the lack of success in
Introduction
87
attempts to transfer this species to the Mediterranean climate [157].
C. pubescens peppers need up to nine months to complete a fruiting
cycle, compared to four - five months for C. annuum varieties [159].
Conspicuous hairiness of the leaves, along with dark brown or
black winkled seeds help to distinguish these chili peppers from the
other Capsicum species. Although a few white flower varieties are
described from Indonesia [160], C. pubescens has typically purple
flowers with large nectarines. Fruit types vary in shape and the color
changes from green in their immature state to yellow, orange or red,
when matured. “Canario” varieties are roundish and yellow,
“Manzano” apple-shaped and red (some with pronounced neck) and
“Peron” pear-shaped and yellow. Fruits of smaller size occur in
Bolivia, suggesting that Bolivian material is closer to the ancestral
gene pool of C. pubescens, which forms a morphological and
genetical distinct complex together with the non-domesticated
species of C. eximium and C. cardenasii [3].
Pungency is the best-known trait in chili peppers. The burning
sensation is produced by capsaicinoids, with capsaicin,
dihydrocapsaicin and nordihydrocapsaicin as major compounds
besides many minor capsaicinoids. Fruits of C. pubescens contain a
higher number of individual capsaicinoids and show more diversity of
capsaicinoid profiles than other Capsicum species [161]. In a study
with five genotypes of C. pubescens, minor capsaicinoids are
described, including nornornordihydrocapsaicin, nornordihydro-
capsaicin, an isomer of dihydrocapsaicin, and homodihydrocapsaicin
[162]. Different combinations of capsaicinoids provide different
pungency characteristics. C. pubescens is characterized by a rapid
Phytochemicals in Peruvian C. pubescens
88
heat development, leading to an incredibly sharp whole mouth effect
(lips, mid-palate, throat) and one of the most lingering pungency of all
peppers as described in "The Flavor Wheel" published by the Chili
Pepper Institute of the New Mexico State University. Pungency is
typically expressed in Scoville Heat Units (SHU), which were
originally obtained by an organoleptic test [82]. Nowadays, HPLC
methods are used to determine the capsaicinoid content in mg/100 g
and converting these values to SHU with 1 mg/100 g corresponding
to 161 SHU for capsaicin or dihydrocapsaicin and 93 SHU for
nordihydrocapsaicin (AOAC method 995.03, 1995). A very limited
number of C. pubescens varieties have been studied for their
capsaicinoid content and pattern. In five accessions of the collection
of the New Mexico State University, the total capsaicinoid
concentration was between 100.3 and 545.1 mg/100 g dry weight,
with 26.1 to 44.5% for capsaicin, 33.9 to 52.9% for dihydrocapsaicin
and 21.0 to 30.0% for nordihydrocapsaicin [162]. High
nordihydrocapsaicin content and high dihydrocapsaicin/capsaicin
ratio is characteristic for C. pubescens [3]. Similar capsaicinoid
content and patterns were observed in five other studies with limited
numbers of one to five C. pubescens varieties [36, 91, 161, 163, 164].
The exotic and original aroma of fresh C. pubescens is
characterized in “The Flavor Wheel” (Chile Pepper Institute at the
New Mexico State University) by a tropical berry note and fruit-like
tones contributing to the particular flavor of many Andean recipes.
One C. pubescens variety from Guatemala and one from Ecuador
were both grown under greenhouse conditions in Spain and their fully
ripe fruits were analyzed for their aroma with identification of the
specific aroma compounds [36]. Four sensory descriptors were
Introduction
89
dominant in the odor profile of both accessions: green, cucumber,
earthy-peasy (3-isopropyl-2-methoxy-pyrazine) and paprika /bell
pepper. The predominance of sensorially relevant sulfur and nitrogen
compounds (mainly pyrazines) and cucumber-like aldehydes with a
low or nil contribution of esters and ionones are the cause for the
characteristic and powerful green/grassy aroma of the two studied
C. pubescens varieties.
Carotenoids were studied in eight accessions, collected in
Bolivia and subsequently grown in Spain to compare their
performance under open field and greenhouse growing systems
[157]. Open field conditions increased the carotenoid content of red
and yellow fruits [165]. The different color was reflected by a different
carotinoid pattern. Tristimulus color values for fresh C. pubescens
samples are reported by Ornelas-Paz et al. [166] and Vera-Guzmán
et al. [167], each describing one Mexican variety.
Chili peppers in general are one of the richest sources of
vitamin C (ascorbic acid) and contain high concentrations of
phenolics, in particular quercetin, for which health promoting
properties have been reported [96]. Eight C. pubescens varieties
provided 14.4 mg to 32 mg ascorbic acid /100 g fresh weight and
phenolics with concentrations between 88 and 166 mg/100 g [157]
when analyzed by the Folin-Ciocalteu method. Two other publications
applying the same analytical method reported 132 mg/100 g [166]
and 113 mg/100 g [167]. Rather low levels of 18 mg/100 g [167] and
23 mg/100 g ascorbic acid were analyzed in one Mexican and one
Brazilian C. pubescens accession, while in three other Mexican
varieties much higher concentrations between 238 and 455 mg/100 g
were found in fresh fruits [164].
Phytochemicals in Peruvian C. pubescens
90
The seed oil content and fatty acid composition were
examined by an NMR technique of eleven C. pubescens accessions
from the USDA/ARS Plant Germplasm Collection in Griffin/Georgia.
Seeds of C. pubescens with fat concentrations between 15 and 21%
had significant lower oil contents than those of other species. The
mean fatty acid pattern for C. pubescens is composed of 76.0%
linoleic, 10.2% palmitic, 7.6% oleic and 4.0% stearic acid [29].
Resistance to different stress situations has been
comparatively studied for the five domesticated chili pepper species
by Ou et al. [168, 169]. They found that the investigated Guatemalan
C. pubescens pepper had the strongest resistance to low
temperatures and was also most resistant to drought stress and water
logging. Therefore, they concluded that breeders should consider
cross breeding introducing elite stress tolerance genes from
C. pubescens to improve other species’ ability to resist adverse
environments.
The objective of the present study was to reveal the content and
variation of important biochemical compounds in 32 C. pubescens
chili peppers, being selected to represent the Peruvian germplasm
collection at INIA for this Capsicum species and to compare the
results with the data of 147 Peruvian chili pepper accessions of the
four other domesticated Capsicum species (C. annuum, C. baccatum,
C. chinense, and C. frutescens). All accessions were grown at the
INIA experimental station Santa Rita in Arequipa/Peru and identically
treated from raising up the plants to analysis. The fruits were
investigated as dried material using identical analytical methods for
the three major capsaicinoids capsaicin, dihydrocapsaicin,
Experimental
91
nordihydrocapsaicin, flavonoids as the aglycons quercetin, luteolin,
kaempferol and apigenin, total phenols, antioxidant capacity, α-, β-
and γ-tocopherol, ascorbic acid, fat content, surface and extractable
color. The applied analytical methods represent a compromise
between the analytical sophistication on the one hand and scope of
studied biochemical compounds, manageability of the large number
of accessions and limited sample amounts on the other hand. The
knowledge about the content of various phytochemicals and health
promoting compounds in the investigated chili pepper accessions
complements the selection criteria from morphological and
agronomical traits for future breeding programs and
commercialization.
5.2 Experimental
5.2.1 Plant Material and Post Harvest Treatment
Plants were grown at the experimental station of the Instituto
Nacional de Innovación Agraria (INIA) in Santa Rita in the Peruvian
Andes, Arequipa (16°25'28''S, 71°32'39''E) at an altitude of 2,345 m.
Thirty-two different C. pubescens accessions were grown and
harvested in 2012. Ripe fruits were collected from a single plant.
Peduncles were removed and fruits were pooled and oven-dried at
60 C° to constant weight for approximately 72 h. Dried fruits were
crushed and sent in sealed bags by air courier to Wuppertal.
Phytochemicals in Peruvian C. pubescens
92
5.2.2 Statistical Analysis
All determinations were carried out with two analytical replicates per
bulk sample. The prediction error for the fat content determined by
NIR was 10%. Precision data for the other analytical method are
reported before in Chapter 4. Analyses were carried out on dried
material. Accordingly, the results refer to 100 g of the dry sample
material as obtained after milling. Moisture content of this material
ranged from 1.4 to 3.4 g/100 g. Data of capsaicinoids and pattern,
quercetin, tocopherols and pattern, total polyphenols, TEAC,
extractable color, surface color (hue-angle) and fat content were
analyzed by their ranges and median values.
5.3 Results and Discussion
The data from the analytical characterization of the 32 C. pubescens
varieties are compiled in Table 5.1, presenting the various accessions
in the order of decreasing pungency. The results are presented as the
means of duplicate analytical determination of each trait out of one
bulk sample obtained from the ripe fruits of one single plant. Due to
logistic limitations, it was not possible to perform multiple sampling.
However, it could be demonstrated by a control experiment with
raising 10 plants each of one accession in three different positions in
the same field that the results of the investigated traits did not differ
significantly (see Chapter 6).
Results and Discussion
93
5.3.1 Capsaicinoids and Pattern
Pungency was analyzed as sum of the three major capsaicinoids and
characterized by the individual patterns of capsaicin, dihydrocapsaicin
and nordihydrocapsaicin. The range of total capsaicinoid
concentration was between 55 mg/100 g and 410 mg/100 g dry
weight. This was equivalent to ca. 8,400-60,000 SHU when applying
a conversion factor of 161 SHU per mg/100 g of capsaicin or
dihydrocapsaicin and of 93 SHU per mg/100 g of nordihydrocapsaicin
(AOAC method 995.03; 1995). Similar values were reported for
five C. pubescens samples of the collection of the New Mexico State
University with total capsaicinoids between 100.3 and 545.1 mg/100 g
or ca. 15,000 – 80,000 SHU, respectively [162]. In five other
publications dealing with pungency in C. pubescens, concentrations
of total capsaicinoids did not fall below or exceeded the range of
2,400 and 31,000 SHU [36, 91, 161, 163, 164, 170]. There seems to
be a discrepancy between the typical characterization of
C. pubescens as being “hot as habanero” or that its pungency “kicks
like a horse” and the results of this and other investigations [3].
Habanero peppers are described with pungency levels between
200,000 to 300,000 SHU [171]. The studied Peruvian C. pubescens
chili peppers are in contrast of rather low and at the most of medium
pungency.
Phytochemicals in Peruvian C. pubescens
94
Figure 5-1: Bar plots of the capsaicinoid pattern of 32 Peruvian
C. pubescens accessions for the percentage distribution of capsaicin,
dihydrocapsaicin and nordihydrocapsaicin.
0% 20% 40% 60% 80% 100%
PER 017919
PER 017925
PER 017950
PER 007302
PER 007300
PER 017948
PER 017928
PER 007255
PER 007298
PER 007230
PER 007299
PER 007215
PER 007276
PER 017971
PER 007237
PER 017927
PER 017924
PER 007291
PER 017922
PER 007283
PER 017947
PER 017961
PER 007304
PER 007303
PER 018006
PER 007219
PER 007278
PER 007295
PER 017951
PER 007234
PER 007143
PER 007139
Ge
rmp
las
m b
an
k a
cc
es
sio
n c
od
e
Capsaicin Dihydrocapsaicin Nordihydrocapsaicin
95
Table 5.1: Analytical results of 32 different C. pubescens
Accession code*
Capsaicinoids (mg/100 g)
Pattern (%)
Quercetin (mg/100 g)
Total polyphenols (g GAE /100 g)
Antioxidant capacity
(mmol /100 g)
Tocopherols (mg/100 g)
Fat (g/100 g)
Surface color
(hue-angle)
Extractable color
(ASTA 20.1) Total Ca D
b N
c
C
a D
b N
c Total α γ β
PER017919 410 111 207 92
27 51 22 1.5 2.0 3.6 11.4 11.3 nd 0.1 3.1 70 5
PER017925 367 128 190 49
35 52 13 nd 2.4 4.6 16.9 16.7 nd 0.2 6.1 44 62
PER017950 349 76 151 122
22 43 35 nd 2.2 3.3 13.5 13.3 0.1 0.0 5.0 61 3
PER007302 282 89 140 54
31 49 19 nd 2.0 3.5 9.3 8.1 1.2 nd 6.9 75 6
PER007300 255 90 113 52
35 44 21 nd 2.0 3.4 8.7 7.5 1.2 nd 7.1 48 23
PER017948 219 71 102 47
32 46 21 nd 2.1 3.0 16.9 16.7 0.1 0.1 6.6 39 50
PER017928 202 69 92 40
34 46 20 0.9 2.0 3.2 6.8 6.8 nd nd 4.8 74 7
PER007255 201 83 93 25
41 46 12 nd 2.1 3.3 12.7 11.8 1.0 nd 6.7 73 8
PER007298 183 60 88 35
33 48 19 nd 2.1 2.8 9.9 8.1 1.7 nd 6.3 51 17
PER007230 181 44 101 36
24 56 20 0.9 2.2 3.2 13.0 11.2 1.8 nd 6.8 74 8
PER007299 179 58 81 40
33 45 23 0.8 2.3 3.8 11.8 10.8 1.1 nd 5.7 50 23
PER007215 179 40 74 65
22 42 36 nd 2.1 3.1 10.3 8.5 1.7 nd 7.4 75 7
PER007276 173 43 89 41
25 51 24 0.7 2.1 3.4 6.9 5.9 1.0 nd 5.6 76 2
PER017971 171 54 92 25
31 54 15 0.7 2.1 3.0 10.6 10.6 nd nd 4.8 46 26
PER007237 170 52 101 16
31 60 10 nd 1.9 2.9 11.0 9.9 1.0 nd 9.3 72 13
PER017927 168 46 73 49
27 43 29 nd 2.1 2.7 14.4 14.4 nd 0.0 5.0 42 38
PER017924 166 52 94 19
32 57 12 nd 2.2 3.2 18.4 18.2 nd 0.1 5.5 44 66
PER007291 163 51 55 56
31 34 35 1.0 2.3 3.0 8.9 7.5 1.4 nd 6.6 69 5
PER017922 156 47 75 33
30 48 22 nd 2.2 3.0 13.2 13.0 nd 0.2 4.6 44 28
PER007283 147 45 58 44
31 39 30 nd 2.2 3.5 12.4 11.4 1.0 nd 5.9 75 9
PER017947 139 58 63 19
41 45 13 nd 1.9 2.7 8.2 7.9 0.3 nd 3.9 42 26
PER017961 130 20 60 50
15 46 39 nd 2.1 3.0 8.3 8.3 nd nd 5.0 48 26
PER007304 113 31 62 20
28 55 17 nd 2.0 2.9 10.5 9.4 1.1 nd 7.1 71 10
PER007303 105 30 59 17
28 56 16 1.2 1.9 2.8 7.5 6.3 1.1 nd 6.8 72 6
PER018006 105 33 56 16
32 53 15 1.0 1.8 2.8 8.4 8.2 0.2 nd 2.8 47 13
PER007219 102 28 53 20
27 52 20 0.7 2.0 2.4 11.9 10.7 1.2 nd 8.7 51 23
PER007278 97 30 41 26
31 42 27 nd 2.1 2.9 9.6 8.4 1.1 nd 6.0 73 5
PER007295 96 41 46 9
43 48 9 0.9 2.0 2.6 8.3 7.4 0.9 nd 6.0 72 5
PER017951 91 14 46 31
15 50 35 0.9 2.2 3.1 16.0 15.5 0.4 0.0 4.6 64 2
PER007234 65 22 27 17
33 41 26 nd 2.0 2.4 8.5 7.0 1.5 nd 5.7 53 10
PER007143 57 13 25 18
24 45 31 nd 1.8 2.5 6.8 6.0 0.8 nd 4.6 75 5
PER007139 55 19 28 9
34 50 16 nd 1.9 2.5 10.0 8.9 1.2 nd 6.0 73 7
a: capsaicin; b: dihydrocapsaicin; c: nordihydrocapsaicin; nd: not detectable;* germplasm bank accession code
Phytochemicals in Peruvian C. pubescens
96
The characterized Peruvian chili pepper accessions from other
Capsicum species, which were reported before, were all treated
identically from planting up to analysis as the C. pubescens
accessions. Table 5.2 provides the median, minimum and maximum
values for the traits of the 32 C. pubescens accessions in a
comparison with 21 accessions of C. annuum, 36 accessions of
C. baccatum , 85 accessions of C. chinense and five accessions of
C. frutescens with C. pubescens having the lowest median and also
the lowest variability in pungency.
All published studies and the results of this investigation identified a
capsaicinoid pattern typical for C. pubescens with less capsaicin,
higher contents in dihydrocapsaicin and nordihydrocapsaicin together
with many minor capsaicinoids. The capsaicinoid patterns of all 32
samples are visualized in Figure 5-1. Dihydrocapsaicin was the major
capsaicinoid in 31 of 32 C. pubescens accessions with a percentage
between 34 and 60%. Capsaicin ranged between 15 and 43% and
nordihydrocapsaicin between 9 and 39% of total capsaicinoids. The
content of nordihydrocapsaicin exceeded that of capsaicin in seven
accessions.
One accession (Acc. code: PER007291) had an interesting
capsaicinoid pattern with a nearly uniform distribution of the three
principal capsaicinoids: 35% nordihydrocapsaicin, 34%
dihydrocapsaicin and 31% capsaicin.
Results and Discussion
97
5.3.2 Other Constituents
Quercetin as the major flavonoid in most Capsicum species was
found in concentrations between 0.7 and 1.5 mg/100 g as the only
detectable flavonoid in 12 C. pubescens accessions, when applying
an HPLC method for specific analysis of quercetin, luteolin,
kaempferol and apigenin after hydrolysis of glycosides (Table 5.1 and
5.2). In none of the 20 other accessions quercetin or any of the other
flavonoids could be detected. A much higher flavonoid content of 6.4
mg/100 g in fresh ripe fruit of one Mexican C. pubescens accession
was reported after applying an unspecific colorimetric method [167].
No other reports on flavonoids in C. pubescens are published. The
observed concentrations of quercetin are very low when compared to
accessions of the other four domesticated species. There, levels up
to 27 mg/100 g were observed with only very few accessions not
containing detectable amounts (Table 5.2).
Total polyphenols were determined by the colorimetric Folin-Ciocalteu
method with gallic acid as reference. A range between 1.8 and 2.4 g
gallic acid equivalents (GAE) /100 g was found. This was above the
values for fresh fruit reported by three other studies ranging between
89 and 166 mg/100 g, even when considering a factor of 10 for water
loss [157, 166, 167]. It is described by Huang et al. that even
chemical very similar molecules can behave very differently in total
polyphenol or antioxidant capacity assays [114]. Therefore, the
analysis of concentrations and patterns of individual polyphenols is
needed to discover the reason for the unexpected high total
polyphenol content, which was not supported by high capsaicinoid
Phytochemicals in Peruvian C. pubescens
98
concentrations or high TEAC values. Although, the second most
pungent C. pubescens accession provided the highest and the
second least pungent sample, the lowest total phenol content, no
clear correlation (R2=0.16) between total phenols and capsaicinoid
content could be seen.
Antioxidant capacity of the C. pubescens accessions, which
was analyzed by the TEAC method and ranged between 2.4 and
4.6 mmol/100 g, correlated rather low with the capsaicinoid content
(R2=0.56).
α-Tocopherol in Capsicum species is preferably found in the pericarp
and γ-tocopherol is a specific constituent in the seeds [3]. All studied
C. pubescens accessions contained α-tocopherol at levels between
6.8 and 18.4 mg/100 g, but only 24 accessions had detectable
concentrations of γ-tocopherol at very low levels not exceeding 1.8
mg/100 g. β-tocopherol was present in only a few accessions at trace
levels reaching 0.2 mg/100 g at maximum. No correlation could be
found between the fat content, which ranged between 2.8 and
9.3 g/100 g, and the seed-specific γ-tocopherols. C. pubescens had
the lowest median and range in comparison with the other species.
99
Table 5.2: Comparison of median values and ranges for important traits of all five domesticated species
C. pubescens (n=32)
C. annuum* (n=21)
C. baccatum* (n=36)
C. chinense* (n=85)
C. frutescens* (n=5)
min med. max min med. max min med. max min med. max min med. max
Capsaicinoids (mg/100 g) 55
167 410 nd
336 809 52
181 712 nd
317 1411 404
1027 1560
Quercetin (mg/100 g) nd
0.0 1.5 0.9
6.4 25 0.7
6.7 25 nd
2.7 27 nd
1.5 3.4
Total polyphenols (g GAE /100 g) 1.8
2.1 2.5 1.6
1.8 2.1 1.2
1.7 2.6 1.3
1.7 3.7 1.9
2.1 2.5
TEAC (mmol /100 g) 2.4
3.0 4.6 3.1
4.0 6.5 2.7
3.7 5.4 1.8
3.8 9.2 3.4
6.0 7.3
Tocopherols (mg/100 g) 6.8
10 18 13
25 35 0.4
17 35 1.0
11 29 3.7
11 35
Fat (g/100 g) 2.8
5.9 9.3 2.6
11 17 2.2
7.5 17 2.6
7.0 20 6.2
12 17
Surface color (hue-angle) 39
67 76 36
40 71 40
50 84 34
50 75 40
48 69
Extractable color (ASTA 20.1) 2
9 66 5
75 107 1
24 66 1
25 146 3
35 60
med.: median; nd: not detectable; * Data were reported in Chapter 4
Phytochemicals in Peruvian C. pubescens
100
Vitamin C was analyzed as sum of ascorbic and dehydroascorbic
acid by a hydrophilic interaction liquid chromatographic (HILIC)
method. Despite the fact that the drying and milling process destroys
most of this vitamin it was the rationale of this analysis to identify
those accessions, still providing noteworthy amounts as an indicator
for accessions with high vitamin C content in their fresh fruits.
However, we did not detect ascorbic acid in any of the C. pubescens
accessions.
The color measurement by the CIE L*a*b* system is best suited to
describe the surface color objectively and reproducibly. The hue-
angle (h) can be calculated from the L*a*b* values and describes the
relation between red, orange and yellow pigments. In general, a hue-
angle of 90° describes a pure yellow color and 0° a pure red color,
with orange in between. The hue-angle for 18 C. pubescens
accessions was 60° or higher and resulted in a yellow color. The
remaining accessions had hue-angles between 40° and 50° and
appear orange. None of the investigated C. pubescens accession
showed the pure red and intense color often observed in chili
peppers.
A wide variability between 2 and 66 ASTA 20.1 units was
found for the extractable color. Only three of the red/orange-colored
accessions had ASTA values at 50 and above, all other were
below 30 (Acc. code: PER017925, PER017948 and PER017924).
According to Rodríguez-Burruezo et al., no red carotenoids could be
observed in yellow-fruiting C. pubescens varieties [165].
C. pubescens had the lowest median of all Peruvian Capsicum
species for extractable color.
Conclusion
101
Within the C. pubescens accessions, one combined the highest
values for total polyphenol content and antioxidant capacity with
second highest concentrations for capsaicinoids, tocopherols and
extractable color (Acc. code: PER017925). This contrasted with one
sample (Acc. code: PER007139), which was the second least
pungent with very low levels in most of the other traits.
A major result of the inter-species comparison is that the
Peruvian C. pubescens accessions had the lowest median values for
six of the seven analyzed traits and also the lowest variability,
expressed as the lowest range between minimum and maximum, in
four of the seven traits, quercetin, antioxidant capacity, tocopherols
and fat. These lower values could be originated due to a genetic
bottleneck during the domestication process of C. pubescens [156].
5.4 Conclusion
This study provides a broad dataset of important phytochemicals and
quality traits of C. pubescens. The inter-species comparison showed
that the Peruvian C. pubescens accessions had a rather low content
in capsaicinoids, quercetin, antioxidant capacity, tocopherols, fat and
extractable color when compared to accessions of other chili peppers
species and the lowest median values for six of the seven analyzed
traits (Table 5.2). Additionally, C. pubescens showed the lowest
variability in four of the seven traits (quercetin, antioxidant capacity,
tocopherols and fat). The popularity of this Capsicum species in the
Andean cuisine and the growing interest in this species outside the
Americas cannot be related to a high content in health promoting
Phytochemicals in Peruvian C. pubescens
102
compounds, but to its special aroma, its fleshy pericarp and probably
the special type of heat originating from a unique pattern of
capsaicinoids. This and the high resistance to different agronomic
stress situations makes C. pubescens an interesting species for
breeding and for further commercialization as fresh fruit, salsas and
other food products [168, 169].
Environmental Impact on Phytochemicals
103
6. Environmental Impact on Phytochemicals
based on:
Capsaicinoids, Flavonoids, Tocopherols, Antioxidant
Capacity and Color Attributes in 23 Native Peruvian Chili
Peppers (Capsicum spp.) Grown in Three Different
Locations*
Abstract:
23 Peruvian chili pepper accessions, belonging to the four domesticated
species C. annuum, C. baccatum, C. chinense and C. frutescens, were
grown under different meteorological conditions and agricultural practices in
three Peruvian locations (Chiclayo, Piura and Pucallpa). Results are
reported for powdered oven-dried bulk samples of each accession and each
location by important quality attributes (capsaicinoids, flavonoids,
tocopherols, antioxidant capacity, total polyphenols, extractable color (ASTA
20.1) and surface color). Multivariate data evaluation by principle component
analysis (PCA) and partial least square discriminant analysis PLS-DA did not
show any underlying structure. Moreover, a high influence of the
environment on the analyzed traits could be demonstrated by analysis of
variance (ANOVA). Significant differences (p≤0.001) in the accessions and
all locations were observed for all traits. Besides, significant interaction
between accessions and locations indicated that the accessions responded
differently to changes of the locations. The calculation of an environmental
impact factor allowed differing between chili peppers provided consistent
phytochemical levels widely independent of the location and those that
provided exceptional high levels for a specific trait at one of the locations.
*Meckelmann SW, Riegel DW, van Zonneveld M, Ríos L, Peña K, Mueller-Seitz E., Petz M (2014) Capsaicinoids, Flavonoids, Tocopherols, Antioxidant Capacity and Color Attributes in 23 Native Peruvian Chili Peppers (Capsicum spp.) Grown in Three Different Locations. European Food Research and Technology (accepted for publication) DOI: 10.1007/s00217-014-2325-6
Environmental Impact on Phytochemicals
104
6.1 Introduction
With regard to the influence of the environment on the content of
various phytochemicals in chili peppers, only limited information is
available. Most studies focus on the effect on the content of
capsaicinoids and carotenoids [58–60, 172–174]. The biosynthesis of
capsaicinoids is controlled by the locus Pun1 and five quantitative
trait loci (QTL) and highly influenced by the environment (for example:
temperature or drought stress) [55–57]. Interactions between
genotype and environment were also observed and indicate that
different cultivars respond differently to changes in the environment
[60]. The environmental impact on carotenoid and flavonoid
(quercetin and luteolin) content and pattern was described by Lee et
al. [172]. They observed a strong influence of the intensity of solar
radiation (MJ/m2) on the biosynthesis of carotenoids. In another study
Keyhaninejad et al. reported a decreased content of pericarp
carotenoids with increased light intensity [174]. The biosynthesis of
flavonoids follows the phenylpropanoid pathway and accordingly, a
strong impact of the environment on the production can be expected.
Increased stress levels caused by pathogens, nutrient deficiency or
UV radiation are factors that enhance the production of flavonoids
[106]. This hypothesis was confirmed by Butcher et al. for various
types of Habanero peppers [173]. Lee et al. also observed a
pronounced difference in the flavonoid content due to different
environments [172]. Munné-Bosch stated for tocopherols that stress
intensity is just one factor influencing the concentration levels. Other
factors are the physiological state of the plant and species-specific
sensitivity [175]
Experimental
105
Twenty-three accessions of native Peruvian chili peppers were
planted in three Peruvian locations. With accessions seed material
collected in a specific farm and conserved ex situ in a germplasm
bank is meant. The aim of this study was describing to which degree
differences in locations, meteorological parameters and agricultural
practices influence the content of various phytochemicals. The
outcome should help to identify accessions with a consistent
production of phytochemicals, which is important for the production of
high quality chili peppers and high value products derived thereof. On
the other hand, it could be shown, that some locations are especially
suited for obtaining outstanding high concentrations for one or more
quality traits.
6.2 Experimental
6.2.1 Plant Material and Field Experiment
All studied chili peppers are genetic stable accessions of the Instituto
Nacional de Innovación Agraria (INIA) chili pepper germplasm bank.
Seed material of each accession was obtained after regeneration in
exclusion cages to maintain the genetic integrity and to avoid cross
pollination. Of each accession, 12 seedlings were planted with a
distance of 70 cm between individuals in line. Distance was 80 cm
between rows. The two outer seedlings of each accession were not
considered in further studies to avoid boundary effects. Seedlings,
which died within two weeks, were replaced. The plants were grown
in three different environments at the experimental stations of INIA in
Chiclayo, Piura and Pucallpa. All were located in regions of
Environmental Impact on Phytochemicals
106
commercial chili pepper production. Transplanting and harvesting
dates as well as environmental information for the three growing
regions are provided in Table 6.1. In each site, the applied agricultural
management practices followed the typical site-specific crop
production scheme, contributing to the environmental conditions of
the specific locations. Table A 3 (Chapter 13) provides more details of
the environmental conditions at each location according to the
Capsicum site descriptors [26].
Table 6.1: Environmental information of the growing region
Locations Geographical coordinates
Altitude (m)
Sowing Harvest Temperature
(°C) Precipi-tation (mm)*
Sun-shine (h)** Longitude Latitude Min Max
Chiclayo -79.85 -6.76 28 05/2012 11-12 / 2012
19.4 22.7 1.4a
1230
Piura -80.32 -4.85 98 05/2012 10/2012 22.1 25.4 0.0a
1062
Pucallpa -74.57 -8.41 154 05/2012 11/2012 24.1 26.8 818.1 1177
* sum of precipitation during the growing season; **sum of sunshine during the growing season;
a irrigation
INIA staff from each location collected ripe fruits from ten plants to
acquire sufficient material per accession. The fruits were combined to
one bulk sample for each accession and location. Peduncles were
removed and fruits were oven-dried at 60 °C to constant weight for
approximately 72 h, crushed and sent in sealed bags by air courier to
Wuppertal. Detailed information for each accession (accessions code
and taxonomic classification) are given in Table A 1 (Chapter 13).
Out of the complete sample pool, one accession (Acc.
code: PER017635) was randomly chosen to evaluate whether
different positions in the test fields of 25 x 30 meter would have an
Experimental
107
influence on the content of phytochemicals and to verify the
homogeneity of seed material as well as to prove reproducible
sample treatment. This control experiment was conducted for all three
fields in Chiclayo, Piura and Pucallpa. This accession was replanted
three times in three different blocks in different parts of the test field of
each location. Only if differences are non-significant between the
blocks of one test field, differences in the analytical results obtained
for the locations can be attributed to an environmental impact.
6.2.2 Statistical Analysis
All determinations were carried out as duplicates by taking two
analytical samples from the bulked dried material of each accession
and each location. The results are given per 100 g of the dry sample
material as obtained after milling. Moisture content ranged between
0.4 and 2.6 g/100 g and is reported for each accession in the
supporting information file.
Data of the analyzed amounts of capsaicinoids, flavonoid
aglycons, tocopherols and of the values for total polyphenols, TEAC,
extractable color, and surface color (hue-angle) were evaluated by
analysis of variance (ANOVA) using The UnscramblerX 10.3 software
package, Camo Inc., Oslo, Norway. For data analysis, the sample
pool was divided into three groups according to their taxonomic
classification. The first group included four C. annuum accessions,
the second group consisted of seven C. baccatum accessions and
the third group in total of twelve accessions (eleven C. chinense and
because of the close genetic relation one C. frutescens accession)
[19]. ANOVA was used to analyze the main effects
Environmental Impact on Phytochemicals
108
environment and accession as well as the interaction
between both. Mean square values, as obtained from ANOVA, were
used to estimate the magnitude of the observed effect.
Additionally, these traits were evaluated by calculating an
environmental impact factor individually for each accession and
phytochemical or quality trait, according to a modified method
described by Roemer and reviewed by Becker and Leon [176, 177].
The environmental impact factor expresses the variance caused by
the three different environments for each accession individually. This
allows to identifying accessions that were more consistent in the
production of phytochemicals or quality traits in comparison to the
other evaluated chili peppers. To calculate the environmental impact
factor (EI) the following equation was used: ; is
the analytical result of an accession grown at a specific location; is
the mean value of the analytical results across the three locations;
i describes the location and j the accession. The results (sum of
squares) of each trait were scaled individually between 0 and 10, with
the highest obtained value assigned as 10. The lower the values for
the environmental impact factor of an accession, the higher is the
consistency of this specific quality trait in comparison to the other
accessions.
Results and Discussion
109
6.3 Results and Discussion
6.3.1 Control Experiment
A homogeneous seed and sample material are a necessary
requirement for identifying a possible influence of accession and
environment on the content of phytochemicals. Additionally, it is
important to ensure that the growing conditions within the test field of
each location are uniform. Plants of one accession (Acc. code:
PER017635; C. annuum) were grown in three different blocks of the
same field. The bulked dried and milled fruits from each block were
analyzed separately for all quality traits.
Figure 6-1 shows the results of each of the three locations and
each of the three blocks for capsaicinoids, flavonoids, total
polyphenols, antioxidant capacity (TEAC), tocopherols, extractable
color (ASTA 20.1) and surface color (hue-angle). Differences in the
individual blocks were analyzed by ANOVA for the three locations
Chiclayo, Piura and Pucallpa. The obtained p-values were between
0.079 for flavonoids and 0.842 for TEAC values. This shows that the
differences in the three blocks in Chiclayo, respectively in Piura and
Pucallpa, were not significant, indicating that the conditions within the
fields were uniform. Differences for the content of specific
phytochemicals can therefore be assigned to different environments
or different accessions.
Environmental Impact on Phytochemicals
110
Figure 6-1: Analytical results of accession PER017635 (C. annuum) being
planted in each of the three location in three different blocks on the same
field.
0
50
100
150
200
250
300
Chiclayo Piura Pucallpa
[mg
/10
0 g
]
Capsaicinoids
0
2
4
6
8
10
Chiclayo Piura Pucallpa
[mg
/100 g
]
Flavonoids
0.00
0.50
1.00
1.50
2.00
Chiclayo Piura Pucallpa
[g G
AE
/10
0 g
]
Total polyphenols
0.0
1.0
2.0
3.0
4.0
Chiclayo Piura Pucallpa
[mm
ol T
rolo
x/1
00
g]
TEAC
0
5
10
15
20
25
30
Chiclayo Piura Pucallpa
[mg
/10
0 g
]
Tocopherols
0
20
40
60
80
100
Chiclayo Piura Pucallpa
AS
TA
2
0.1
Extractable color
0
15
30
45
60
75
90
Chiclayo Piura Pucallpa
hu
e-a
ng
le [ ]
Surface color
Block 1 Block 2 Block 3
Results and Discussion
111
6.3.2 Capsaicinoids
Pungency is one of the major quality attributes of chili peppers, and
as such of high importance for breeders and industry. To develop
high value products with consistent quality, it is important to know the
level of pungency and whether an accession can be grown providing
similar capsaicinoid levels widely independent of the growing location.
Figure 6-2 shows strong differences in the capsaicinoid content
between the 23 accessions. The concentrations of capsaicinoids
ranged from 1.0 mg/100 g (Acc. code: PER006984) to
1515.5 mg/100 g (Acc. code: PER007009). Both accessions belong
to the C. chinense group. The C. annuum and C. baccatum
accessions were medium pungent in the range between 135.5 and
507.7 mg/100 g and 82.2 and 586.7 mg/100 g.
Figure 6-2: Bar plots of capsaicinoids (mg/100 g), grouped into the four
species C. annuum, C. baccatum, C. chinense and C. frutescens (from left to
right).
0
200
400
600
800
1000
1200
1400
1600
(mg
/100 g
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
Environmental Impact on Phytochemicals
112
The species-dependent source of variation is given in Table 6.2. In all
cases the type of accession had the highest effect on the
concentration. Accordingly, the genotype resulted as the most
important parameter that influenced the content of capsaicinoids. The
environment had less influence on the capsaicinoid content.
Significant interactions between accession and environment indicate
that the accessions respond differently to different environments [59].
This can be seen for example with the accessions PER007009 and
PER017787 (both C. chinense). For PER007009 the highest
capsaicinoid content was found in Chiclayo, second highest in
Pucallpa and lowest in Piura, whereas for accession PER017787, the
highest content was found in Piura, second highest in Chiclayo and
the lowest in Pucallpa. The difference, when ranking the degree of
pungency, showed that these accessions responded differently to the
environments. However, for C. annuum and C. chinense/frutescens
the amount of variance caused by the interaction was lower in
comparison to the variance caused by the environment. C. baccatum
in contrast showed a higher interaction than the environment. These
chili peppers seem to react more individually to changes in the
environment (Table 6.2). In most cases, chili peppers grown in
Chiclayo and Piura produced more capsaicinoids than plants of
identical accessions grown in Pucallpa.
Results and Discussion
113
Table 6.2: Species-dependent source of variation (Location and Accession) and significance levels for the main effects “Location” and “Accession” and their interaction expressed as mean squares as obtained from ANOVA
Species C. annuum C. baccatum C. chinense / C. frutescens
Effect L A L×A L A L×A L A L×A
Capsaicinoids 24503* 47429* 5587* 10027* 63885* 20111* 530267* 993940* 93886*
Flavonoids 49.33* 39.32* 5.63* 132.75* 20.68* 3.90* 46.67* 28.29* 5.67*
Total polyphenols
0.33* 0.13* 0.02* 0.42* 0.11* 0.04* 0.50* 0.79* 0.02*
TEAC 0.28* 1.74* 0.26* 1.17* 1.96* 0.69* 1.54* 11.39* 0.45*
Tocopherols 120.94* 89.49* 9.02* 2.46* 303.66* 1.97* 116.12* 208.10* 35.31*
Extractable color
1521* 455* 94* 909* 694* 49* 3418* 2680* 350*
hue-angle 27.89* 6.75* 0.82NS 193.80* 48.89* 6.74* 107.97* 990.98* 12.53*
C. annuum (n=4); C. baccatum (n=7); C. chinense (n=11) and C. frutescens (n=1); L: Location, A: accession, L×A: interaction between Location and Accession; NS: not significant; * significant at p≤0.001
6.3.3 Specific Flavonoids
Vegetables and fruits containing high amounts of phytochemicals with
the ability to scavenge free radicals in biological systems are
recommended for a healthy human diet.
In the present study the content of flavonoid aglycons
quercetin, luteolin, kaempferol and apigenin was analyzed by HPLC
after hydrolysis of the corresponding glycosides. Quercetin ranged
from 1.3 to 13.8 mg/100 g and luteolin from 0.6 to 3.3 mg/100 g.
Kaempferol was only found in very low concentrations of 0.4 to
0.8 mg/100 g in accession PER017826 (C. annuum). None of the
accessions contained detectable amounts of apigenin. The total
flavonoid content (sum of the four aglycons) was between 2.2 and
13.2 mg/100 g for C. annuum, between 2.1 and 12.8 mg/100 g for
Environmental Impact on Phytochemicals
114
C. baccatum and between 1.3 and 13.8 mg/100 g for C. chinense
(Figure 6-3). These are rather low concentrations in comparison with
the data of Miean and Mohamed, who investigated three C. annuum
and one C. frutescens and reported values between 8 and 160
mg/100 g for the sum of the four aglycons [105].
Figure 6-3: Bar plots of flavonoid levels (mg/100 g), grouped into the four
species C. annuum, C. baccatum, C. chinense and C. frutescens (from left to
right).
By ANOVA, it could be shown that the two main effects (accession
and location) and their interaction were significant in all cases with
p-values <0.001. The main source of variation for the content of
flavonoids was the environment (Table 6.2). The results indicated that
the amount of flavonoids in chili peppers dependents highly on the
growing condition. This is in accordance with Dixon and Paiva, who
reported, that the biosynthesis of flavonoids was influenced by
environmental factors [106]. Lee et al. showed significant differences
in the flavonoid content for some of the studied chili peppers [172].
0
2
4
6
8
10
12
14
(mg
/100 g
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
Results and Discussion
115
Butcher et al. reported that flavonoid levels of chili pepper plants were
strongly influenced by different environments [173]. This is confirmed
by these results.
6.3.4 Total Polyphenols and Antioxidant Capacity
Highest levels were found in accession PER017787 (C. chinense)
with 2.77 g gallic acid equivalents (GAE)/100 g of total polyphenols
and of 6.8 mmol Trolox/100 g. However, also the lowest level for total
polyphenols and TEAC were found in accessions belonging to the
species C. chinense with 1.34 g GAE/100 g (Acc. code: PER006991)
and 2.0 mmol Trolox/100 g (Acc. code: PER17719). It is remarkable,
that the accessions grown in Chiclayo and Pucallpa in most cases
had higher levels of total polyphenols and antioxidant capacity in
comparison with those from Piura (Figure 6-4 and 6-5). Data for total
polyphenols and antioxidant capacity were for example published by
Hervert-Hernández et al [154]. They investigated four different chili
peppers and found total polyphenol contents between 0.97 and 1.4 g
GEA/100 g and TEAC values between 1.9 and 3.6 mmol Trolox/100
g. It is, however, not very reliable to compare these sum parameter
between different studies. Already slight changes in the assay
procedure have a strong influence on the results of total polyphenols
and TEAC [116].
Environmental Impact on Phytochemicals
116
Figure 6-4: Bar plots of total polyphenols (g gallic acid equivalents (GEA)
/100 g), grouped into the four species C. annuum, C. baccatum, C. chinense
and C. frutescens (from left to right).
Figure 6-5: Bar plots of antioxidant capacity (TEAC in mmol Trolox /100 g),
grouped into the four species C. annuum, C. baccatum, C. chinense and
C. frutescens (from left to right).
Significant results were found for the two main effects and their
interaction. The most important source of variation for TEAC was in
0.00
0.50
1.00
1.50
2.00
2.50
3.00(g
GE
A /
100
g)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
0.0
2.0
4.0
6.0
8.0
(mm
ol T
rolo
x /100 g
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
Results and Discussion
117
all cases the accession (Table 6.2). For total polyphenols we found
slight different results. Accessions belonging to C. annuum and
C. baccatum were mainly affected by the environment. Accessions of
the species C. chinense seemed to be more stable against
environmental influence. Their main source of variation was the
genotype.
6.3.5 Tocopherols
As can be seen from Figure 6-6, the tocopherol levels as sum of the
three tocopherol congeners varied strongly between the 23
accessions. Levels for total tocopherols ranged from 0.23 mg/100 g
(Acc. code: PER017893, C. baccatum) to 29.1 mg/100 g
(PER017635, C. annuum). α-Tocopherol was the dominating vitamin
E congener in nearly all chili pepper accessions. Accessions
PER007026 and PER017893 (C. baccatum) were not only interesting
due to their extraordinary low contents in tocopherols, but also
because they did not contain detectable amounts of α-tocopherol and
only very low amounts of γ-tocopherol, which typically ranked second
behind α-tocopherol. β-Tocopherol was only found in a limited
number of accessions with concentrations at 0.03 and 0.54 mg/100 g.
The ratio of α- and γ-tocopherol depends on the amount of seeds in
the chili powder because α-tocopherol is found in the pericarp and
γ-tocopherol is dominating in the seed [3].
Significant results were obtained for the two main effects and
their interaction. Main source of variation for C. annuum was the
environment, but mean square values of environment and accessions
do not differ much (Table 6.2). For C. baccatum a very low
environmental influence was observed with high variability due to the
Environmental Impact on Phytochemicals
118
accession. Although a rather high impact of the environment was
obtained for C. chinense, the dominating source of variation was the
accession (Table 6.2). Interaction between the main effects was
significant for all species, but with limited influence on the content of
tocopherols.
Figure 6-6: Bar plots of tocopherols (mg/100 g), each bar represents the
sum of the three tocopherol congeners (α-, β- and γ-tocopherol), grouped
into the four species C. annuum, C. baccatum, C. chinense and
C. frutescens (from left to right).
6.3.6 Extractable and Surface Color
For dried Capsicum the amount of extractable carotenoids is
classified by the ASTA 20.1 value and usually described as
“extractable color”. ASTA 20.1 values were between 3 and 94.
Therefore, no accession is qualified for producing a colorant. The
values for surface color of the chili powder expressed as hue-angle
were between 36.6° and 72.2°. A hue-angle of 90° describes a pure
yellow color and one of 0° a pure red color. The majority had low
hue-angles and appeared red, only four of the accessions had values
0
5
10
15
20
25
30
(mg
/100 g
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
Results and Discussion
119
larger than 60° (Acc. code: PER006952, PER017707, PER017784
and PER017787) and appeared yellow-orange. Accessions grown in
Chiclayo showed higher ASTA values and lower hue-angles, when
compared to the other two locations, indicating a higher production of
red carotenoids (Figure 6-7 and 6-8).
Figure 6-7: Bar plots of the extractable color (ASTA 20.1), grouped into the
four species C. annuum, C. baccatum, C. chinense and C. frutescens (from
left to right).
Figure 6-8: Bar plots of the surface color (hue-angle), grouped into the four
species C. annuum, C. baccatum, C. chinense and C. frutescens (from left to
right).
0
20
40
60
80
100
(AS
TA
20.1
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
0
15
30
45
60
75
90
(hu
e-a
ng
le
)
0
5
10
15
20
25
30
[mg/
10
0 g
]
Chiclayo Piura Pucallpa
Environmental Impact on Phytochemicals
120
Significant differences in the ASTA values were observed for
environment, accession, and their interaction. The environment was
the main source of variation for all species, showing that carotenoid
biosynthesis was influenced by growing conditions. Significant
differences in the hue-angle for the environments and accessions
were found. Interactions were only significant for C. baccatum and
C. chinense (Table 6.2).
6.3.7 Environmental Impact
For each trait an environmental impact factor was calculated similar
to a method described by Roemer [176]. Figure 6-9 depicts the
environmental impact and shows to which degree the accessions are
susceptible to differences in the growing conditions. Accessions with
a low value exhibit a consistent production of this trait in all three
locations. This is important for maintaining a steady quality
independent of where the Capsicum fruits are grown. On the other
hand, an accession with a high impact factor may be especially suited
for one of the locations. Visualization of the environmental impact for
all traits facilitates the choice of accessions for special needs and
expectations of consumers (Figure 6-9). The degree of pungency is
an important attribute for the selection of Capsicum varieties. All
C. annuum accessions9, all C. baccatum accessions10, and most of
the C. chinense accessions11 showed a low impact factor concomitant
with low capsaicinoid content and will yield mild fruits. C. chinense
9 C. annuum accession codes: PER017635, PER017653, PER017667 and
PER017826 10
C. baccatum accession codes: PER006951, PER007026, PER017661, PER017701, PER017833, PER017849 and PER017893 11
C. chinense accession codes: PER006959, PER006984, PER006991, PER006995, PER017719 and PER017732
Results and Discussion
121
accessions PER006952 and PER017707 produced consistently fruits
with moderate pungency. C. chinense accessions PER007009 and
PER017787 and C. frutescens accession PER017728 will have very
pungent fruits depending on the location.
The production of flavonoids depends highly on the growing
conditions, but some accessions showed consistently low flavonoid
concentrations (Acc. code: PER017653, PER006952, PER006991,
PER006995, PER017719, PER017732 and PER017784).
For the sum parameters total polyphenols and antioxidant
capacity (TEAC), as well as for surface color, the analytical values do
not vary to a large extent (Figure 6-4 and 6-5). The impact factors in
Figure 6-9 for some accessions turn up as high values only due to the
scaling between 0 and 10, which is applied individually for each trait.
C. baccatum was the species with the most consistent
production of tocopherols and extractable color, whereas several
C. chinense accessions reacted more individually to growing
conditions.
Environmental Impact on Phytochemicals
122
Figure 6-9. Bar plots of environmental impact for capsaicinoids, flavonoids,
total polyphenols, antioxidant capacity (TEAC), tocopherols, extractable
color (ASTA 20.1) and surface color (hue-angle); grouped into the four
species C. annuum, C. baccatum, C. chinense and C. frutescens (from left to
right).
In most cases the replanting of the 23 selected accessions led to very
similar results of the important phytochemicals in comparison with the
first chemical characterization (Chapter 4). Due to the different
locations a year-to-year comparison was not reliable. However, some
accessions and traits are worthwhile to be discussed in more detail
(Table 6.3). These accessions either provided on the one hand rather
Results and Discussion
123
consistent levels for capsaicinoids, flavonoids or tocopherols or on
the other hand outstanding high concentrations for a specific location.
Accession PER017826 produced much lower capsaicinoid levels this
time in all three locations, compared to Loreto in the former growing
season. An especially high content was seen in fruits from accession
PER007009 only from plants grown in Chiclayo, as well as from
accessions PER017787 and PER017728 grown in Chiclayo, Piura,
Loreto, and San Martin. Accession PER017787 grown in Chiclayo
was exceptional high in flavonoids and tocopherols. Fruits from
accessions PER006951 and PER017661 were rich in flavonoids as
expected from first results but only when grown in Chiclayo. On the
other hand accession PER017833 did not show the expected high
flavonoid content in the present study, whereas fruits from accession
PER017893 had high contents this time when cultivated in Chiclayo.
Fruits from accession PER006952 showed outstanding little
influence by the environment for all phytochemicals and quality traits.
This accession is therefore a candidate for the production of fruits
with consistent concentrations of capsaicinoids, flavonoids, total
polyphenols, antioxidant capacity, tocopherols, extractable and
surface color.
Multivariate data analysis was performed by applying principle
component analysis (PCA) and partial least square regressions
discriminant analysis (PLS-DA) to the whole data set. The derived
results did not show any underlying structure.
Environmental Impact on Phytochemicals
124
Table 6.3: Detailed results of selected accessions with the potential of either very consistent concentrations independent of growing location or outstanding high concentrations for a specific phytonutrient and location. These data are compared with results of the same accession from former growing seasons (Chapter 4). Accession
Code Species
Growing location
Capsaicinoids (mg/100 g)
Flavonoids (mg/100 g)
Tocopherols (mg/100 g)
PER017826 C. annuum Chiclayo Piura
Pucallpa Loreto 2012
507 459 303 809
13.2 6.7
10.0 6.7
15.7 11.6 24.1 24.6
PER006951 C. baccatum Chiclayo Piura
Pucallpa Ucayali 2011
220 194 99
255
12.3 3.9 4.3
27.0
2.8 3.7 3.5 5.3
PER17661 C. baccatum Chiclayo Piura
Pucallpa Lambayeque
2011
187 188 166 103
12.8 5.8 5.4
12.4
22.9 18.5 20.1 22.8
PER017833 C. baccatum Chiclayo Piura
Pucallpa Loreto 2011
119 87.2 121 59.2
7.0 4.1 5.1
23.4
7.1 5.5 5.1 5.5
PER017893 C. baccatum Chiclayo Piura
Pucallpa Piura 2012
275 214 134 173
12.6 4.4 7.0 7.9
0.2 0.7 0.3 0.7
PER006952 C. chinense Chiclayo Piura
Pucallpa Ucayali 2012
721 778 630 637
3.3 2.4 3.0 2.1
7.5 6.8 7.7 9.5
PER007009 C. chinense Chiclayo Piura
Pucallpa Ucayali 2011
1515 398 684 989
4.9 1.8 2.4 4.0
12.5 9.0 18.4 14.3
PER017787 C. chinense Chiclayo Piura
Pucallpa Loreto 2012
1209 1348 821 1244
13.8 4.3 5.7 8.0
19.9 8.8 20.8 26.5
PER017728 C. frutescens Chiclayo Piura
Pucallpa San Martin
2012
1333 1210 514 1175
7.5 4.7 7.3 3.4
20.3 20.9 26.8 34.5
Conclusion
125
6.4 Conclusion
The effects of three different growing locations on the levels of
important quality attributes (capsaicinoids, flavonoids, tocopherols,
antioxidant capacity, total polyphenols, extractable color (ASTA 20.1)
and surface color) in 23 chili accessions were investigated. A high
influence on these traits could be demonstrated by ANOVA
evaluation. For all accessions a significant interaction between
location and accession was observed, showing the individual
response to changes in the growing conditions as influenced by the
environment. Finally, two accessions were identified as being very
interesting candidates for commercialization or further breeding
programs. One C. chinense accession (Acc. code: PER006952) was
very consistent in the production of phytochemicals independent of
the location and also showed very similar values for extractable
(ASTA 20.1) and surface color. Another interesting C. chinense
accession (Acc. code: PER017787) showed different phytochemical
concentrations and exceptional high values for flavonoids and
tocopherols when grown in Chiclayo. Although, the results from this
one-year study identified promising accessions, only a multi-year
cultivation at different locations combined with chemical analysis will
provide a sound basis to benefit from the full potential of these
accessions.
Environmental Impact on Phytochemicals
126
Characterization of Bolivian Chili Peppers
127
7. Characterization of Bolivian Chili Peppers
based on:
Major Quality Attributes of
Native Bolivian Chili Peppers (Capsicum spp.)
Focussing on C. baccatum: A two-year Comparison*
Abstract:
Germplasm collections of Bolivian chili peppers at CIFP and PROINPA hold
more than 500 native Capsicum accessions. 96 chili peppers including 78
accessions of C. baccatum were selected for chemical analysis and planted
in 2011. The concentrations (mg/100 g) of important quality traits ranged for
capsaicinoids 0 - 1028, quercetin 0.4 – 42.6, tocopherols 4.2 – 38.1 and
ascorbic acid 0 – 437. Quantitative data are also reported for total
polyphenols, antioxidant capacity, fat, extractable and surface color. A
subset of twelve C. baccatum accessions was selected for replanting
experiments on the identical test field in 2012. Nearly all attributes gained
higher or equal concentrations in 2012, except for fat and antioxidant
capacity. An ANOVA proved significant impact of accession, harvest year
and their interaction for all quality traits.
* Meckelmann SW, Riegel DW, van Zonneveld M, Avila T, Bejarano C, Serrano E, Mueller-Seitz E, Petz M (2014) Major Quality Attributes of Native Bolivian Chili Peppers (Capsicum spp.) Focussing on C. baccatum: A two-year Comparison. Food Chemistry (submitted for publication)
Characterization of Bolivian Chili Peppers
128
7.1 Introduction
The fruits of the genus Capsicum are one of the most important
horticultural crops. They are used as spice and vegetable and are
part of the daily diet of billions of people. The export values of the
world market for Capsicum (green and dried) have reached 3.403 Mio
US $ in 2012 [11]. Therefore, Capsicum is an important economic
factor for many countries like India, Peru, China or Spain [12].
According to their use, Capsicum peppers can be grouped into
vegetable and spice Capsicum, both with different quality
requirements. While for the vegetable use of Capsicum mainly
freshness, pungency and nutrient factors are important, the quality
requirements for dried chili peppers are more diverse [32]. The
pungency of dried chili peppers, caused by the presence of
capsaicinoids, is one of the important quality traits and reaches from
sweet non-pungent as in most bell pepper varieties to very hot in chili
peppers like “Trinidad Moruga Scorpion” [95]. Color is another
important quality attribute especial of low pungent Capsicum. The
brilliant color is the result of the presence and pattern of several
different yellow, orange and red carotenoids [8]. Chili or paprika
powders are spices and therefore the characteristic aroma is the most
important quality trait. It is described as fresh and fruity with a
pleasant aromatic smell and a fruity-sweetish, aromatic flavor. It has
to be free from off-flavors and off-odors, which are caused by
unsuitable raw material, technological steps (e.g. poor drying
conditions) or storage conditions [32].
Introduction
129
Chili peppers contain several vitamins (provitamin A, vitamin C and E)
and other antioxidants like flavonoids and are therefore a good
source of bioactive health promoting phytonutrients [8, 93, 151].
Bolivia, as a putative center of domestication of chili peppers for
Capsicum baccatum and C. pubescens [15], harbors a wealth of
native chili pepper cultivars that have never been fully characterized.
The Bolivian chili pepper collections at the Centro de Investigaciones
Fitoecogenéticas de Pairumani (CIFP) and at the Fundación
PROINPA hold together more than ~500 different chili pepper
accessions. These collections include all five domesticated species
and several wild species (C. baccatum var. baccatum, C. eximium,
C. cardenasii and C. chacoense). These wild species are locally
consumed and known as “arivivi” and “ulupica”. C. baccatum var.
baccatum, which is the progenitor of the cultivated C. baccatum, and
C. eximium are the most commonly harvested. Out of these two
collections, 96 Capsicum accessions were selected for field trials and
biochemical characterizations.
Most of the accessions characterized in this study belong to
the domesticated species C. baccatum var. pendulum. In contrast to
other domesticated species, the chemical composition of
C. baccatum accessions is rather unexplored. Jarret [178] reported
on morphological variation of C. baccatum fruits and Albrecht et al.
[179] on the genetic diversity. Phytonutrients in C. baccatum were
evaluated by Rodríguez-Burruezo et al. [157, 165]. They reported
data for the content of carotenoids, vitamin A, vitamin C and total
polyphenols in fresh fruits.
Characterization of Bolivian Chili Peppers
130
The 96 chili pepper accessions were grown in three different locations
in Bolivia in 2011. Dried fruits were analyzed for pungency by total
capsaicinoids, capsaicinoid pattern, total polyphenols, antioxidant
capacity, flavonoids, vitamin E, vitamin C, extractable color, surface
color and fat content. Twelve C. baccatum var. pendulum accessions
from one of the locations were selected as promising material and
replanted in the same field in 2012 to compare the content of
bioactive and valuable compounds of these accessions, when grown
in two consecutive years.
The results of this study are a contribution to characterize
Bolivian Capsicum varieties for unique traits with potential commercial
use and as selection criteria for Capsicum breeding programs.
7.2 Experimental
7.2.1 Plant material and Post Harvest Treatment
CIFP and PROINPA provided samples in total 96 accessions. The
number of accessions and species provided by both organizations for
the first year (2011) are given in Table 7.1.
Table 7.1: Number of accessions per species and organization
Organization CIFP PROINPA Total C. annuum 2 - 2 C. baccatum var. baccatum 7 - 7 C. baccatum var. pendulum 34 37 71 C .chinense 7 - 7 C. frutescens 2 2 4 C. pubescens 2 - 2 C. eximium 3 - 3 Total 57 39 96
Experimental
131
Environmental passport data of the growing locations are reported in
Table 7.2. Fruits of the first field trial were harvested in 2011. Twelve
different accessions were selected for further field trials and replanted
in Padilla in 2012. In all sites and both years, ripe fruits were collected
from several plants of the same accession to obtain a sufficient
amount of sample material. Fruits were first dried in open air for up to
three weeks, like it has been done in other studies [60], and then
oven-dried at 60 °C for approximately 12 hours according to a
standard protocol. After removal of the peduncles, the bulk fruit
samples were crushed and sent in sealed bags by air courier to
Wuppertal. Detailed information including accession code,
taxonomical classification and harvest date are presented in
Chapter 13 Table A 4.
Table 7.2: Environmental passport data of the growing locations
Location Organization
Geographical coordinates
Altitude (m)
Annual mean tempera-ture (°C)
Annual mean precipita-tion (mm)
Longitude Latitude
Cochabamba CIFP -66.16 -17.39 2600 17.0 516
Santa Cruz CIFP -63.17 -17.80 428 24.0 1244
Mairana CIFP -63.96 -18.12 1349 20.7 653
Padilla PROINPA -64.30 -19.30 2129 18.2 657
Characterization of Bolivian Chili Peppers
132
7.2.2 Statistical Analysis
All determinations were carried out as duplicates by taking two
analytical samples (n=2), from the bulked dried material of each
accession, except for ascorbic acid (n=1).
Analyses were carried out on dried material. Accordingly, the
results refer to 100 g of the dry sample material as obtained after
milling. Moisture content of this material ranged from 0.7 to
3.4 g/100 g
Data for capsaicinoids, flavonoids, total polyphenols, TEAC,
tocopherols, extractable color, surface color (hue-angle) and fat of the
96 accessions grown in 2011 were evaluated by box plot analysis
using the software tool “R 2.15.1” (R Foundation for Statistical
Computing, Vienna, Austria), freely available at http://www.r-
project.org. The box plots show the range minimum-maximum, 25
percentile, median and 75 percentile. Outliers were identified by 1.5
times of the interquartile range. Outlying samples with high contents
of phytonutrients can be regarded as samples with outstanding
attributes.
For year-to-year comparison results of capsaicinoids,
flavonoids, total polyphenols, antioxidant capacity (TEAC),
tocopherols, extractable color, surface color and fat content of the
twelve selected accessions were evaluated by ANOVA (The
UnscramblerX 10.3 software package; Camo Inc., Oslo, Norway).
ANOVA was used to analyze the main effects of the different harvest
years and accessions as well as the interaction between year and
accession on the content of the investigated phytochemicals. Mean
square values, as obtained from the ANOVA, were used to estimate
the magnitude of the effects.
Results and Discussion
133
7.3 Results and Discussion
7.3.1 Capsaicinoids and Pattern
Capsaicinoids are the pungent principles of Capsicum fruits. Their
content can be classified into five groups according to Bosland and
Votava; I: non-pungent or paprika (0 - 700 Scoville heat units (SHU);
0 - 4.4 mg/100 g), II: mildly pungent (700 - 3,000 SUH; 4.4 - 18.8
mg/100 g), III: moderately pungent (3,000 - 25,000 SHU; 18.8 - 156.3
mg/100 g), IV: highly pungent (25,000 - 70,000 SHU; 156.3 - 437.5
mg/100 g) and V: very high pungent (>80,000 SHU; >500 mg/100 g)
[3].
The degree of pungency among different accessions or
pepper types is usually very variable [6]. A wide range in the content
of capsaicinoids for the 96 accessions planted and harvested in 2011
was observed (Figure 7-1 and 7-2). Accessions P9, P6, P10, 319-1
and 268 were non-pungent. Accessions P1, P3, 4, P19, P14, 319-2,
637 and 543 with capsaicinoid concentrations < 4.2 mg/100 g also
belonged to the classification group I. The majority of the accessions
(n=47) showed low levels of pungency and can be classified as mildly
pungent or moderately pungent. The remaining 34 accessions were
highly pungent or very high pungent. The highest capsaicinoid
concentration of 1028 mg/100 g was found in accession 581
(C. frutescens). This is equivalent to almost 165,000 SUH and could
be regarded as very high pungent.
Characterization of Bolivian Chili Peppers
134
Figure 7-1: Individual capsaicinoid levels and pattern of 96 different Bolivian
chili pepper accessions (germplasm bank codes) sorted by ascending
capsaicinoid content. Left: accessions with capsaicinoids between not
detectable amounts and ~100 mg/100 g and right: accessions above
~100 mg/100 g.
0 250 500 750 1000
581MA 1648
Nueva ColectaProinpa 31Proinpa 35
MA 1664MA 1631MA 1628
366341
MA 1638162321
Proinpa 34109 R
514353360
MA 1657384542139
SacabaMA 1679
TM312
24320654
339 RMA 1660
517109 A
314122532300
75 A146
MA 1680582103256
8634
75 R502
48
0 20 40 60 80 100
10P17
102 R6025
102 A136
5206617
19461P526
339 A70P7P41143
P16P12108
9P11P18
80485P2
P15P8
P133
P1P3
4P19P14
319-2637543P9P6
P10319-1
268
0 250 500 750 1000
581
Proinpa 31
MA 1631
341
321
514
MA 1657
139
TM
320
MA 1660
314
300
MA 1680
256
75 R
Capsaicin Dihydrocapsaicin Nordihydrocapsaicin
(mg/100 g)
Results and Discussion
135
Individual levels for the three major capsaicinoids are shown in
Figure 7-1 and 7-2. Capsaicin was found as the major capsaicinoid
for nearly all accessions followed by dihydrocapsaicin and
nordihydrocapsaicin. Both C. pubescens samples (Acc. code: TM and
Sacaba) showed their typical pattern with high amounts of dihydro-
and nordihydrocapsaicin [46]. Interesting results were obtained for the
three C. eximium accessions (Acc. code: Proinpa 35, Proinpa 34 and
Nueva Colecta). They showed a special pattern with relative high
amounts of dihydrocapsaicin from 29% to 50% and of nordihydro-
capsaicin from 16% to 18%. In accessions Proinpa 35 and “Nueva
Colecta” the concentrations of dihydrocapsaicin were even higher
than the content of capsaicin. This unusual patterns are similar to
those usually observed in C. pubescens and can be linked to the
close genetic relationship with C. pubescens [3].
Figure 7-2: Box plot of capsaicinoid concentrations. 25 percentile, median (thick line), 75 percentile and range minimum-maximum, outliers (•) were identified by 1.5 times of the interquartile range. All results are expressed in mg/100 g.
Characterization of Bolivian Chili Peppers
136
7.3.2 Specific Flavonoids
When compared to other vegetables, chili peppers are a good source
for flavonoids [105]. Their content depends on the genotype as well
as on the growing conditions [172, 173]. For the fruits of the 96
accessions harvested in the first year (2011), a high variability in the
content of flavonoids was found. Individual flavonoid concentrations
are shown in Figure 7-3 and Figure 7-4 depicts the range for the sum
of flavonoids (quercetin, luteolin, kaempferol and apigenin) and for
quercetin and luteolin individually as the major flavonoids found in
chili peppers. All the accessions contained detectable amounts of
flavonoids.
The concentration ranged between 0.4 and 46.8 mg/100 g.
The majority of the chili peppers had concentrations < 10 mg/100 g,
which is rather low when compared with values in the literature [105].
In the previous reported of native Peruvian chili peppers the levels
ranged between not detectable and 29.5 mg/100 g. Most accessions
from Bolivia were in the same range as the Peruvian chili peppers
(Chapter 4).
Results and Discussion
137
Figure 7-3. Individual flavonoid levels and pattern of the Bolivian chili pepper
accessions (germplasm bank codes), sorted by ascending capsaicinoid
content.
(mg/100 g)0 10 20 30 40 50
581MA 1648
Nueva ColectaProinpa 31Proinpa 35
MA 1664MA 1631MA 1628
366341
MA 1638162321
Proinpa 34109 R
514353360
MA 1657384542139
SacabaMA 1679
TM312
24320654
339 RMA 1660
517109 A
314122532300
75 A146
MA 1680582103256
8634
75 R502
48
0 10 20 30 40 50
10P17
102 R6025
102 A13
652066
17
19461P526
339 A70P7P41143
P16P12108
9P11P18
80485P2
P15P8
P133
P1P3
4P19P14
319-2637543P9P6
P10319-1
268
0 10 20 30 40 50
581321TM300
Quercetin Luteolin Kaempferol Apigenin
Characterization of Bolivian Chili Peppers
138
The maximum flavonoid level was found in accession P6
(C. baccatum var. pendulum). This accession also showed the
highest content of quercetin. The highest level of luteolin was found in
accession 66 (C. baccatum var. pendulum) with 5.0 mg/100 g. For
kaempferol levels up to 0.8 mg/100 g and for apigenin up to
0.7 mg/100 g were observed. However, most chili peppers did not
contain detectable amounts of these two minor flavonoids.
Figure 7-4: Box plot analysis of flavonoids (sum of the four analyzed
aglycons) and the two major flavonoid aglycons quercetin and luteolin. All
results are expressed in mg/100 g.
Results and Discussion
139
7.3.3 Total Polyphenols and Antioxidant Capacity
Phytonutrients will become a major quality parameter for chili peppers
with the growing interest of consumers in buying fruits and vegetables
as protection against illness [3]. Across the 96 different accessions a
wide range of total polyphenols and TEAC values was observed.
Figure 7-5 depicts the results of the determination of the total
polyphenol content and the corresponding TEAC value for each
accession. For most chili peppers total polyphenol values were
between 1.4 and 1.8 g gallic acid equivalents (GAE) /100 g and
antioxidant capacity (TEAC) between 3.7 and 4.4 mmol Trolox /100 g
(Figure 7-6).
Although accession Proinpa 34 (C. eximium) was the highest
in total polyphenols (2.19 g GAE /100 g), its TEAC value of 4.4 mmol
Trolox /100 g was only medium. Lowest TEAC value was 3.0
(Acc. code: 485, C. annuum) and highest 6.3 mmol Trolox /100 g
(Acc. code: 581, C. frutescens).
Characterization of Bolivian Chili Peppers
140
Figure 7-5: Results of total polyphenols and the corresponding TEAC
values. Accessions (germplasm bank codes) are sorted by ascending
capsaicinoid content.
0 1 2 3 4 5 6
581MA 1648
Nueva ColectaProinpa 31Proinpa 35
MA 1664MA 1631MA 1628
366341
MA 1638162321
Proinpa 34109 R
514353360
MA 1657384542139
SacabaMA 1679
TM31224
320654
339 RMA 1660
517109 A
314122532300
75 A146
MA 16805821032568634
75 R50248
0 1 2 3 4 5 6
10P17
102 R6025
102 A13
652066
17
19461P526
339 A70P7P41143
P16P12108
9P11P18
80485P2
P15P8
P133
P1P3
4P19P14
319-2637543P9P6
P10319-1
268
0 1 2 3 4 5 6
581
MA 1664
MA 1638
514
542
312
MA 1660
532
582
75 R
TEAC (mmol Trolox /100 g) Total polyphenols (g GAE /100 g)
0 1 2 3 4 5 6
581
MA 1664
MA 1638
514
542
312
MA 1660
532
582
75 R
TEAC (mmol Trolox /100 g) Total polyphenols (g GAE /100 g)
Results and Discussion
141
Figure 7-6: Box plot of antioxidant sum parameters. Units: Total
polyphenols: g GAE /100 g, TEAC mmol Trolox /100 g
Compared to the Peruvian chili peppers, one accession with a total
polyphenol content of 3.69 g GAE /100 g and a TEAC value of 9.2
mmol Trolox /100 g was found. Such remarkable high values were
not found for the Bolivian chili peppers. In general, total polyphenols
and TEAC values were in the same range for both countries and
comparable to data from Hervert-Hernández et al. [154].
7.3.4 Tocopherols and Ascorbic Acid
Vitamin E is a mixture of congeners of four tocopherols and four
tocotrienols. The sufficient separation allowed quantifying and
reporting the content of α-, β- and γ- tocopherol. The vitamin E level
for each accession is shown in Figure 7-7 and the range in the
content of these three tocopherols can be seen in Figure 7-8. The
Characterization of Bolivian Chili Peppers
142
sum of these tocopherols can be considered as total vitamin E
content.
In all 96 chili peppers being investigated detectable tocopherol
concentrations (sum of α-, β- and γ- tocopherol) were present. The
majority of the samples contained tocopherol levels between 19.7 and
26.6 mg/100 g (first and third quartile). The highest tocopherol
content (38.1 mg/100 g) was observed in accession 319-2
(C. baccatum var. pendulum). This accession also showed the
highest content of α-tocopherol (31.8 mg/100 g), which was the
dominating tocopherol in 94 accessions.
γ-Tocopherol was found as second highest tocopherol and
varied from 1.28 to 7.93 mg/100 g. Only the accessions 514 and
Proinpa 31 contained larger quantities of γ- tocopherol in comparison
with the α-tocopherol content. It can be assumed that these
accessions are especially rich in seeds, since γ-tocopherol is the
major tocopherol in chili pepper seeds, while α-tocopherol is
abundant in the pericarp [180]. β-Tocopherol was found in low
concentrations up to 2.70 mg/100 g with several accessions not
containing any detectable amounts.
Ching and Mohamed reported the α-tocopherol content of 62
edible tropical plants including four Capsicum varieties [140]. They
reported α-tocopherol content was between 13.8 and 29.1 mg/100 g
dry matter. This is in accordance with the results of this investigation.
Results and Discussion
143
Figure 7-7: Tocopherol concentrations and pattern of the Bolivian accessions (germplasm bank codes), sorted by ascending capsaicinoid content.
(mg/100 g)0 10 20 30 40
581MA 1648
Nueva ColectaProinpa 31Proinpa 35
MA 1664MA 1631MA 1628
366341
MA 1638162321
Proinpa 34109 R
514353360
MA 1657384542139
SacabaMA 1679
TM312
24320654
339 RMA 1660
517109 A
314122532300
75 A146
MA 1680582103256
8634
75 R502
48
0 10 20 30 40
10P17
102 R6025
102 A13
652066
17
19461P526
339 A70P7P41143
P16P12108
9P11P18
80485P2
P15P8
P133
P1P3
4P19P14
319-2637543P9P6
P10319-1
268
0 10 20 30 40
581
MA 1628
109 R
139
654
532
256
α-Tocopherol γ-Tocopherol β-Tocopherol
Characterization of Bolivian Chili Peppers
144
Figure 7-8: Box plot analysis of the tocopherol content (sum of α-, β- and γ-
tocopherol), levels of individual tocopherols and ascorbic acid. All results are
expressed in mg/100 g.
Dependent on the stage of ripeness fresh chili peppers contained up
to 250 mg ascorbic acid/100 g fresh weight [180]. Thermal stress
during the drying process leads to degradation and to remaining
levels down to ~10% [128]. 54 of 96 analyzed dried chili peppers
powders did not contain any detectable amounts of ascorbic acid.
The other accessions contained only low concentrations below
12 mg/100 g of ascorbic acid, whereas three of the accessions had
unexpected high amounts of vitamin C (Figure 7-8). Further
information about the individual vitamin C content of the analyzed
accessions is presented in Chapter 13, Table A 5. The highest
amount of 437 mg/100 g vitamin C was found in 341 (C. baccatum
var. pendulum). The other two chili peppers showed values of 216
mg/100 g for 582 (C. chinense) and of 132 mg/100 g for 319-2
510
15
20
25
30
35
Tocopherols5
10
15
20
25
30
α-tocopherol
0.0
0.5
1.0
1.5
2.0
2.5
β-tocopherol
23
45
67
8
γ-tocopherol
0100
200
300
400
Ascorbic acidα-Tocopherol β-Tocopherol γ-Tocopherol
Results and Discussion
145
(C. annuum). These accessions have to be analyzed again as fresh
fruits to confirm the high ascorbic acid concentrations because
thermal stress during the drying process does not allow estimating
the content of vitamin C in fresh fruits.
7.3.5 Fat Content
The content of fat depends on the ratio of seeds compared to the
pericarp. Great differences were observed among the fat content of
the 96 accessions grown in 2011. Lowest content was 6.7 g/100 g
found in accession 319-2 (C. annuum). An exceptional high fat
content was found in accession 109 R (C. baccatum var. pendulum)
with 32.8 g/100 g (Figure 7-9). Chili peppers with high contents of
lipids may be useful for the production of natural chili seed oil for
cooking and industry [181]. Fat content of each accession is given in
Chapter 13 Table A 5 .
Figure 7-9: : Box plot of fat content in g/100 g, values for the extractable
color (ASTA 20.1) and surface color (hue-angle °).
Characterization of Bolivian Chili Peppers
146
7.3.6 Extractable and Surface Color
In addition to pungency and aroma, color is an important quality
attribute. Most of the accessions grown in 2011 appeared orange with
a median hue-angle of 46.6° and a median ASTA 20.1 value of 38
(Figure 7-9). Only a few of the accessions appeared red. The
maximum ASTA 20.1 value of 127 was found in accession P6
(C. baccatum var. pendulum). This is a high value for chili pepper
powders, but quite low in comparison with paprika powders reaching
typically ASTA 20.1 values above 200 (Chapter 13, Table A 5).
7.3.7 Two-year Comparison
Twelve C. baccatum var. pendulum accessions were selected for a
two-year comparison and grown on the identical test field of Padilla in
2011 and 2012. Primary selection criterion was the pungency as main
quality attribute. Further selection criteria were high amounts of
flavonoids, vitamin C and E, total polyphenols and antioxidant
capacity. Chili peppers with non, low or medium pungency were
preferred because low or medium pungency allows a better
perception of the typical aroma of the Capsicum accession.
Figure 7-10 depicts the results of the chemical
characterization of the twelve accessions for both years. Mean
square values for the two main effects year and accession and their
interaction as obtained from the ANOVA are shown in Table 7.3. Both
main effects and their interactions were significant for all analyzed
traits at a significance level of p≤0.001.
Results and Discussion
147
Comparing both harvest years, nearly all accessions grown in 2012
showed higher or equal content for capsaicinoids, flavonoids, total
polyphenols, tocopherols, extractable and surface color. This was
different for the fat content and antioxidant capacity. With the
exception of three accessions, all other had higher values in 2011.
Figure 7-10: Results of the year-to-year comparison for capsaicinoids,
flavonoids, total polyphenols, antioxidant capacity (TEAC), tocopherols, fat
content, extractable color (ASTA 20.1) and surface color (hue-angle).
0
50
100
150
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[mg
/100 g
]
Capsaicinoids
0
20
40
60
80
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[mg
/100 g
]
Flavonoids
0.0
0.5
1.0
1.5
2.0
2.5
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[g G
AE
/10
0 g
]
Total polyphenols
0.0
2.0
4.0
6.0
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[mm
ol
Tro
lox/1
00 g
] TEAC
0
10
20
30
40
50
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[mg
/100 g
]
Tocopherols
0
10
20
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[g/1
00 g
]
Fat
0
25
50
75
100
125
150
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
AS
TA
20.1
Extractable color
0
30
60
90
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
hu
e-a
ng
le
Surface color0.0
2.0
4.0
6.0
43 108 P18 3 P1 P3 4 P19 P14 P9 P6 P10
[mm
ol
Tro
lox
/10
0 g
]
TEAC
Year 1 Year 2
Characterization of Bolivian Chili Peppers
148
Table 7.3: Source of variation of the main effects “Year” and “Accession” and their interaction expressed as mean squares
Effect Year Accession
Year × Accession Trait
Capsaicinoids 2328 983 815
Flavonoids 264 911 78
Total polyphenols 0.02 0.07 0.02
TEAC 1.76 0.37 0.63
Tocopherols 141 103 18
Extractable color 2596 4899 398
hue-angle 41 648 9
Fat 4.9 12.5 3.2
n= 12 (C. baccatum var. pendulum); all results were significant at p≤0.001
The harvest year was found as the major source of variation for
capsaicinoids. The strong effect of the harvest year is mainly caused
by selecting only low-pungent accessions and that two accessions
(Acc. code: P18 and 4) especially showed very different capsaicinoid
contents in both years. Their capsaicinoid content increased from
12.9 to 81.9 mg/100 g for accession P18 and from 2.3 to
79.6 mg/100 g for accession 4. The result that the impact of the
harvest year is higher than the impact of the accession is untypical. In
most studies evaluating the capsaicinoid content in different
environments a higher impact of the accession or genotype is usually
found [60, 173]. One speaks of an interaction Y×A (Table 7.3)
between year and accession, when in consecutive years not all
accessions behave in the same way with increasing or decreasing
concentrations. For capsaicinoids this interaction can be seen for
example with the accessions 43 and P18. While the capsaicinoid
content of accession 43 decreased from 21.7 to 11.7 mg/100 g the
Results and Discussion
149
content of accession P18 increased from 12.9 to 81.9 mg/100 g
(Figure 7-10). However, the interaction is the weakest of the three
studied sources of variation.
In 2012 the content of flavonoids of the most chili peppers
reached values of at least 10-20 mg/100 g. An outstanding exception
is accession P6. This accession already had the highest flavonoid
level in 2011 (46.8 mg/100 g), which strongly increased to
78.6 mg/100 g in 2012. It is known that the biosynthesis of flavonoids
is highly effected by the growing conditions [106, 172, 173]. A
significant influence of the harvest year was also found but to a
smaller degree, when compared to the impact of accession on the
content of flavonoids (Table 7.3).
Values for total polyphenols did not vary to large extent
(Figure 7-10) and were mostly influenced by the accession. This is
different for the antioxidant capacity (TEAC). This sum parameter is
more influenced by the harvest year when compared with the
influence of the accession. Especially accession 108 showed a very
different value. While the content of total polyphenols remained
stable, the antioxidant capacity decreased from 5.8 to 3.4 mmol
Trolox /100 g.
Tocopherols were found as being highly effected and almost
showed with all accessions higher concentrations, especially
accessions P18, P3, 4 and P14.
The values for the extractable color were also in general
higher in 2012, especially accessions P3, 4, P19, P14 and P6.
Despite this pronounced effect of the year, it can be seen in
Figure 7-10 that the accessions (or genotype) was the major source
of variation.
Characterization of Bolivian Chili Peppers
150
With regard to the surface color (hue-angle) the obtained results did
not vary to a large extent. This can be seen by a low mean square
values for the year and the interaction between year and accession
when compared with the strong influence of the accession
(Table 7.3).
Out of all accession P6 needs to be mentioned as an outstanding one
with a low pungency, the highest flavonoid and extractable color and
in addition consistent values for total polyphenols, antioxidant
capacity and tocopherols. Accordingly, this accession is one of the
most promising within the whole set of investigated Bolivian chili
peppers.
7.4 Conclusion
In this study the important quality traits of 96 different chili pepper
accessions were investigated. A subset of twelve accessions was
replanted for a year-to-year comparison on the identical test field. The
results indicate a significant impact of the harvest year on the
contents of health promoting components and other valuable
attributes. One chili pepper with outstanding attributes could be
identified. Those accessions that showed high concentrations for
various phytonutrients or very consistent concentrations in both years
could help in innovating chili pepper production systems through a
better use of native varieties and are major candidates for further
investigations such as multi-year studies or impact of different
environments.
Analytical and Experimental Background
151
8. Analytical and Experimental Background
Due to the high number of chili pepper samples needing to be
analyzed throughout the project, the analytical methods needed to be
efficient, fast, robust and economical according to limited sample
amount and funding. Besides, all methods needed to be applicable to
dried chili peppers. Legal restrictions of Peru and Bolivia do not allow
the shipment of fresh non-commercial indigenous fruit material to
avoid biopiracy. To protect indigenous chili peppers only dried and
crushed fruit material, which did not contain fertile seeds, was allowed
to be shipped.
The following traits were considered to assess the quality of chili
pepper accessions according to the project aims and the scientific
literature concerning the quality of chili peppers [3, 32]:
Pungency and pattern of major capsaicinoids (capsaicin, dihydrocapsaicin and nordihydrocapsaicin)
Antioxidant and radical scavenging properties by o Total polyphenols o Antioxidant capacity o Determination of levels and composition of major
flavonoid aglycons (quercetin, luteolin, kaempferol and apigenin)
Vitamins o Ascorbic / dehydroascorbic acid (Vitamin C) o Tocopherols (Vitamin E) by analysis of individual levels
of α-, β-, γ-tocopherols
Color attributes of chili peppers by o Extractable color (ASTA 20.1) o Surface color (CIE L*a*b*)
Fat content
Analytical and Experimental Background
152
Numerous methods that were already used in the assessment of chili
pepper quality are described in the literature, but these are only rarely
applicable to a high number of samples or require large sample
amounts. Therefore, most methods had to be optimized for a higher
throughput and for handling small sample amounts due to limited chili
pepper sample material. Furthermore, the effect of the drying
procedure, which followed a strict protocol, on the content of
phytochemical was evaluated. Finally, an analytical strategy was
established, which included all methodological and organizational
aspects for the quality assessment of dried chili pepper powders.
8.1 Capsaicinoid Analysis
Chromatographic conditions:
The aim was mainly to reduce the duration of the analysis for a higher
sample throughput. Starting point was a method described by
Kirschbaum-Titze et al. [89]. They used a LiChrospher RP-18 column
(5 µm, 250 mm × 4 mm) with an isocratic elution for the separation of
major capsaicinoids. Gradient elution is unsuitable to reduce the
analysis time because of the very similar structures of all
capsaicinoids and the column re-equilibration after analysis.
Therefore, only the column dimensions were changed. Figure 8-1
shows the analysis of the same chili pepper extract under original and
optimized isocratic elution. Trace C was obtained under similar
conditions as described by Kirschbaum-Titze et al. [89]. Trace B
showed the results of applying a shorter column with smaller particles
(Luna RP-18 column; 3 µm, 150 mm × 3 mm).
Capsaicinoid Analysis
153
Figure 8-1: HPLC profiles obtained for analysis of capsaicinoids (wavelengths: 280 nm for excitation and 320 nm for detection). All three chromatograms showed the analysis of the same chili pepper extract containing nordihydrocapsaicin (1), capsaicin (2) and dihydrocapsaicin (3) under optimal chromatographic conditions with isocratic elution. A: Kinetex RP-18 column (2.6 μm, 100 mm × 3 mm) with acetonitrile / 0.5% acetic acid (38:62, v/v), 0.7 mL/min, at 50 °C; B: Luna RP-18 column (3 µm, 150 mm × 3 mm) with acetonitrile / 0.5% acetic acid (50:50, v/v), 0.5 mL/min; C: LiChrospher 100 RP-18 column (5 µm, 250 mm × 4 mm) with acetonitrile / 0.5% acetic acid (50:50, v/v), 1.2 mL/min.
This method already provided a faster separation and a better
resolution of nordihydrocapsaicin and capsaicin, but the total run time
was not reduced significantly. Thirdly, a fused core HPLC column was
used (Kinetex RP-18 column; 2.6 μm, 100 mm × 3 mm). Fused core
particles are known to increase the separation efficiency and speed of
analysis in comparison to full porous silica particles. These
advantages can be explained by the Van Deemter equation. Due to
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Minutes
A
B
C
1
2
3
1
2
3
1
2
3
Analytical and Experimental Background
154
the solid core of the particles, the mass transfer is reduced and the
narrow particle size distribution leads to a reduction of the Eddy
dispersion. Both effects increase the number of theoretical separation
plates and lead to a higher efficiency. In addition, the back pressure is
mostly lower, when compared to fully porous particles because of the
nature of the fused core particles. The lower mass transfer and the
reduced back pressure allow the application of higher flow rates of
the solvent without being detrimental for the chromatographic
separation and leading to a increase of efficiency [182]. The
application of the fused core column allowed to decrease the total run
time from 20 minutes to only 9 minutes and provided a very good
separation of the critical peak pair of nordihydrocapsaicin and
capsaicin (Figure 8-1).
Extraction procedure:
The applied extraction procedure was according to Collins et al. [91]
with slight modifications. Instead of pure acetonitrile a mixture of
acetonitrile, methanol and a phosphate buffer (0.5 M, pH 11) was
used. Methanol and the buffer were added to increase the extraction
efficiency for other antioxidants. This modifications allowed the
necessary re-use of the extract for the determination of total
polyphenols and antioxidant capacity due to the limited sample
amounts. Recovery and extraction efficiency was investigated by J.
Fang12. The applied extraction showed a full recovery rate (104%). A
comparison with the original extraction method described by
12
Mrs. Jing Fang investigated the recovery and extraction efficiency during her final thesis for the first state examination in food chemistry entitled: “Methodenetablierung und Untersuchung von Capsicum - Früchten auf wertgebende Bestandteile” in 2010
Total Polyphenols and Antioxidant Capacity
155
Collins et al. [91] showed very similar values when analyzing the
same chili peppers powder.
8.2 Total Polyphenols and Antioxidant Capacity
Both, the determination of the total polyphenol content according to
the Folin-Ciocalteu method and the analysis of the antioxidant
capacity (TEAC assay) were usually performed in a cuvette scale,
which is too laborious and time consuming to be applied for hundreds
of samples. The assay was downscaled to a 96-well microtiter plate
format to increase the number of analyses, which can be carried out
simultaneously and also to reduce the amount of reagents.
A standard procedure protocol in cuvette scale for both assays
was already established by J. Fang13. The described procedure for
the total polyphenol determination was only slightly modified by using
displacement pipettes and disposable reaction tubes. For absorbance
reading, the volume for measurement could be reduced from 2.0 mL
to 0.25 mL to fit in the cavity of the microtiter plate. The TEAC assay
was transferred completely to a microtitre plate scale. The sample
volume was 20 µL and the required volume of the ABTS solution was
reduced from 2,000 µL to 200 µl. The complete reaction was
performed in the cavities of the plate.
The modified methods were tested by analyzing five different
chili pepper powders in triplicate. Results were compared to those
obtained by applying the original cuvette scale method and are
13
Mrs. Jing Fang established the total polyphenol determination according to Folin-Ciocalteu and the TEAC assay during her final thesis for the first state examination in food chemistry entitled: “Methodenetablierung und Untersuchung von Capsicum - Früchten auf wertgebende Bestandteile” in 2010
Analytical and Experimental Background
156
presented in Table 8.1. The obtained values for the total polyphenols
determination did not show significant differences between both
procedures and values for the TEAC assay showed only minor
differences. For the microtitre plate scale slightly increased values for
the antioxidant capacity were detected. However, the results of both
miniaturized methods indicate the applicability of the microtitre scale
assay procedures, which allowed analyzing high numbers of different
chili pepper accession simultaneously.
Table 8.1 Comparison between cuvette scale and microtitre plate scale analysis of total polyphenols determination and TEAC assay
Samplea
Total polyphenols
(g GAEb/100 g ± sdev)
TEAC assay
(mmol Trolox /100 g ± sdev)
cuvette scale microtitre plate scale cuvette scale microtitre plate scale
Spice
paprika 1.32 ± 0.03 1.23 ± 0.06 2.7 ± 0.2 3.1 ± 0.1
Ají
Panca 1.23 ± 0.04 1.23 ± 0.05 2.6 ± 0.2 3.1 ± 0.3
Ají Ammarillo 1.20 ± 0.04 1.18 ± 0.02 2.0 ± 0.1 2.3 ± 0.1
Red
Pepper 1.20 ± 0.07 1.21 ± 0.06 3.2 ± 0.2 3.7 ± 0.1
Thai Red Chili 1.12 ± 0.03 1.04 ± 0.04 3.3 ± 0.1 3.7 ± 0.2
All samples were analyzed as dried and milled powders and the results represent mean values of triplicate determination and the corresponding standard deviation (sdev).
a Spice Paprika, Ají Panca and Ají Amarillo were
obtained from the Peruvian cooperative Miski S.A., Red Pepper and Thai Red Chili were obtained from Akzenta Wuppertal;
b GAE: gallic acid
equivalents.
As mentioned before, both assays are highly influenced by the assay
procedure [114]. High repeatability of the applied methodology is
necessary for reliable comparing of differences between accessions.
The repeatability of both assays was controlled during the whole
project time. For that purpose, a specific quality control sample was
always analyzed in duplicate, when the assays were applied on
Total Polyphenols and Antioxidant Capacity
157
project samples. The quality control sample was a homogenous
mixture of ca. 170 g spice paprika powder14 and ca. 20 g Red Savina
chili powder15. During the project, the quality control sample was
analyzed 34 times as duplicate for total polyphenols and antioxidant
capacity. For total polyphenols a mean value of 1.83 ± 0.05 g gallic
acid equivalents (GEA) /100 g and for the antioxidant capacity a
mean value of 3.5 ± 0.2 mmol Trolox /100 g were found. The results
are reported in a control chart (Figure 8-2). Additionally, the upper
and lower limits are included. The limits show the double standard
deviation, which are usually reported in a control chart [183].
The obtained data were normally distributed and no trend to
higher or lower values could be observed. For the determination of
total polyphenols only two values of 34 were beyond the calculated
limits and for the applied TEAC assay all values were within the limits.
This indicates the high repeatability of both assay procedures and the
applied extraction. This provided the basis for a reliable comparison
between the chili pepper accessions analyzed during the project.
14
from the cooperative Miski S.A. (Lima, Peru) 15
from Pepper-King Internet store (www.pepper-king.com)
Analytical and Experimental Background
158
Figure 8-2: Control charts for total polyphenols (n=34) and TEAC analysis
(n=34). Each data point represents the mean of duplicate analysis of the
quality control sample. Upper and lower limit represents the double standard
deviation.
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
[g G
AE
/ 1
00
g]
Control chart for total polyphenols
Total polyphenol values Upper control limit
Lower control limit Mean value
2.7
2.9
3.1
3.3
3.5
3.7
3.9
4.1
4.3
[mm
ol T
rolo
x / 1
00
g]
Control chart for TEAC
TEAC values Upper limit Lower limit Mean value
Flavonoid Analysis
159
8.3 Flavonoid Analysis
Chromatographic conditions:
Quercetin, kaempferol, luteolin, and apigenin aglycons were
chromatographically separated by a fused core column because of
the benefits described before. All four flavonoid aglycons have
comparable polarity and accordingly, a penta fluoro phenyl (PFP)
modified fused core column was used. The strong π-π-interactions
increased the selectivity of the chromatographic system for aromatic
molecules compared to other typical reversed phase columns such as
C18 modified columns. A good separation was accomplished with
methanol and water as mobile phase. Both were acidified with formic
acid to prevent peak tailing. Gradient elution was necessary since
isocratic conditioned showed only a poor separation. The final
method had a run time of 31 minutes. This included a wash step to
remove matrix components and column re-equilibration. A typical
chromatogram is shown in Figure 8-3.
Analytical and Experimental Background
160
Figure 8-3: Typical HPLC profiles obtained for flavonoid analysis recorded
at 360 nm. Separation was achieved on a Kinetex PFP column (2.6 µm,
100 mm × 3 mm). Methanol and water both with 0.1% formic acid were used
as mobile phase at 0.5 mL/min and 50 °C. A: represents a project sample
(Acc. code: PER017833) containing quercetin, luteolin, kaempferol and
traces of apigenin; B: standard solution. Peaks: 1: quercetin; 2: luteolin;
3: kaempferol; 4: apigenin.
Extraction procedure:
The extraction and hydrolysis conditions were adapted from Miean
and Mohamed [105]. The described method was only modified to
increase the number of analyses, which can be carried out
simultaneously. Thus, the extraction and hydrolysis of flavonoid
glycosides was performed in glass centrifuge tubes, which allowed
heating the samples in a laboratory oven instead of using round
bottom flasks and cooking under reflux. The solvent for extraction
was a mixture of 70% methanol, 20% water, 10% 12.5 M hydrochloric
acid and 4 g/L tert.-butylhydroquinone to prevent oxidative damages
to the flavonoids. After extraction, the samples were diluted with a
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Minutes
A
B
mA
U
1 2 3 4
Analysis of Ascorbic Acid by HILIC
161
disodium hydrogen phosphate buffer (50 mM Na2HPO4,
pH 12)/methanol solution (1:1, v/v). The alkaline buffer was used to
increase the pH value of the diluted sample extract containing high
amounts of hydrochloric acid, which was added to the extraction
solvent to hydrolyze the flavonoid glycosides and extract the aglycons
in one step. The pH shift to higher values was necessary to avoid
damages to the chromatographic column due to very low pH values.
8.4 Analysis of Ascorbic Acid by HILIC
Chromatographic conditions:
The chromatographic method was adapted from Nováková et al.
[136]. The separation of half the sample pool was performed on a
sulfobetaine ZIC-HILIC column (3.5 μm, 150 mm × 4.6 mm). A typical
chromatogram for a chili pepper accession is shown in Figure 8-4 (B)
indicating a good separation of ascorbic acid from matrix compounds.
With the availability of fused core HILIC columns, the
separations of the remaining samples were performed on a
Nucleoshell HILIC column (2.7 μm, 100 mm × 3 mm). A typical
chromatogram is shown in Figure 8-4 (A). The fused core column
allowed a higher sample throughput, increased sensitivity through
smaller peak width and a better separation from the matrix in only 4
minutes.
Analytical and Experimental Background
162
Figure 8-4: Typical HPLC profiles obtained for analysis of ascorbic acid
recorded at 260 nm. A: Bolivian chili pepper grown in 2012 (Acc. code: 542),
analyzed on sulfobetaine Nucleoshell HILIC column (fused core material)
containing 114 mg ascorbic acid/100 g. B: Bolivian chili pepper grown in
2011 (Acc. code: 542), analyzed on a sulfobetaine ZIC-HILIC column
containing 216 mg ascorbic acid/100 g
Extraction procedure:
No sample preparation procedure was reported with the
chromatographic separation conditions described by Nováková et al.
[136]. A pre-condition for a successful separation of ascorbic acid
from matrix compounds using HILIC is a high content of organic
modifier in the injected extract. Higher concentrations of water or
buffer in the injected extract can be detrimental to the
chromatographic performance. The result is a poor peak shape due
to their higher elution power on HILIC columns [135]. Consequently,
the mobile phase (70% acetonitrile and 30% of 100 mM ammonium
acetate pH 6.8) was used as extraction solvent. Additionally,
dithiothreitol was added to the extraction solvent to allow
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Minutes
Ascorbic acid
B
A
Analysis of Tocopherols
163
simultaneous determination of ascorbic acid and dehydroascorbic
acid, which still keeps vitamin C activity. Dehydroascorbic acid was
reduced to ascorbic acid by dithiothreitol. This is necessary due to the
degradation of ascorbic acid during the drying process of the chili
pepper samples [128]. To prevent further oxidative damage
tert.-butylhydroquinone was also added to the extraction solvent. For
extraction, the chili pepper powders were suspended with the
extraction solvent, shaken for two hours, centrifuged subsequently,
and filtered through a syringe filter before analysis.
The preparation method was tested by analyzing a chili
pepper sample (quality control sample; Chapter 8.2) spiked with
215 mg/100 g dehydroascorbic acid. The spiked sample was
analyzed together with a blank sample as control. Ascorbic acid
concentration in the control samples was below the limit of detection.
In the spiked sample 212 ± 31 mg /100 g were found, which represent
~99% of the dehydroascorbic acid in the spiked sample. The
developed sample preparation was used for the determination of
vitamin C in the chili pepper samples and because of the low levels
expected after drying, the content was only screened by a single
determination. However, samples that showed an unexpected high
vitamin C content were re-analyzed to confirm the result.
8.5 Analysis of Tocopherols
Chromatographic conditions:
Grebenstein and Frank reported a rapid baseline separation of all
eight tocopherols and tocotrienols by HPLC using a PFP modified
Analytical and Experimental Background
164
fused core column (2.6 µm, 150 mm × 4.6 mm) under isocratic
conditions [141]. The column used for the chili analyses had an
identical stationary phase but different column (2.6 µm, 100 mm ×
3 mm). Accordingly, the mobile phase and flow rate had to be
adjusted by reducing the concentration of organic modifier in the
mobile phase from 85% to 82% methanol and reducing the flow rate
from 0.8 to 0.3 mL/min16. These optimized conditions were used for
analysis of the major tocopherols in chili peppers (Figure 8-5).
Figure 8-5: Typical HPLC profile obtained for tocopherol analysis (wavelengths: 296 nm for excitation and 325 nm for detection) A: Peruvian chili pepper accession (PER017728, C. frutescens) grown in 2012 containing 0.18 mg/100 g β-tocopherol, 1.26 mg/100 g γ tocopherol, 25.4 mg/100 g α-tocopherol; B: standard solution; β: β-tocopherol, γ: -tocopherol, α: α-tocopherol.
16
Mr. Christian Jansen developed and improved an HPLC method for the determination of tocopherols in chili pepper powders under supervision of the author during his final thesis for the first state examination in food chemistry entitled: “Bestimmung des Tocopherolgehalts und -musters in nativen peruanischen und bolivianischen Chilipulvern mittels HPLC mit Fluoreszenzdetektion”.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Minutes
A
B
βγ
α
Analysis of Tocopherols
165
Extraction procedure:
Typically, tocopherols are extracted with non-polar solvents such as
carbon tetrachloride or dichloroethane due to the high lipophilicity of
the vitamin E congeners. These extracts, however, cannot be used
with reversed phase HPLC because of the immiscible with water.
Therefore, the first step for the development of a fast extraction
procedure was the selection of a solvent compatible with reversed
phase HPLC. Five different solvents were tested: isopropanol,
acetonitrile, methanol, acetone and as reference solvent DMA
(dichloromethane, methanol and acetone; 2:1:1; v/v/v) [184].
Figure 8-6: One step extraction efficiency of different solvents for the
extraction of tocopherols (sum of α-, β-, γ-tocopherols). Error bars represent
the standard deviation of triplicate analysis. Highest content was set as
100%. DMA: dichloromethane, methanol and acetone (2:1:1; v/v/v)
Figure 8-6 depicts the results of the extraction of a commercial chili
pepper powder using five different solvents. All tests were carried out
in triplicate under the conditions described in Chapter 10.8, except for
94
85 88 92 100
0
20
40
60
80
100
%
Analytical and Experimental Background
166
the extraction test with DMA. An aliquot of this extract was
evaporated in a nitrogen stream and the residue was dissolved in the
mobile phase. The highest total tocopherol content (sum of α-, β-, γ-
tocopherols) was found in the non-polar solvent mixture DMA and
defined as 100%. Isopropanol and acetone showed very similar
values, but the standard deviation was much higher for acetone.
Methanol and acetonitrile had the least extraction efficiency.
Accordingly, isopropanol was selected.
The extraction efficiency was further tested by multiple
extraction. Two different samples (commercial chili powders) were
analyzed after the method described in Chapter 10.8. The extraction
was performed three times for each sample. Between each
extraction, a 200 µL aliquot was removed for analysis and the
remaining solvent was decanted. The sample residue was re-
extracted and re-analyzed then.
Figure 8-7: Multiple extraction test for tocopherols of two different commercial chili pepper powders. Error bars represent the standard deviation of triplicate analysis. The sum of all three extraction steps was defined as 100%.
91
6 3
90
6 4
0
20
40
60
80
100
1 2 3
%
Extraction steps Sample 1 Sample 2
Analysis of Tocopherols
167
The results indicated that the first step extracts about 90% of the
tocopherols. In step two and three only minor levels were found.
When considering the results it should be noted that the residue may
still contained a small portion of isopropanol with dissolved
tocopherols. These contributed to the yield in the following extraction
step. So an extraction efficiency of higher than 90% was obtained in
one step. Additionally, the results of triplicate analysis showed very
similar values for both samples, which indicates the good
reproducibility of the applied method.
Samples cleanup could be restricted to dilution and filtration. It was
tested by standard addition whether matrix compounds influenced the
determination. For that purpose, the α-tocopherol content of a chili
pepper sample was determined in parallel by external calibration and
by standard addition at two different dilutions (1:5 and 1:10). The
sample was analyzed as blank and spiked with six different α-
tocopherol solutions (4, 8, 12, 16, 20, and 24 µg/mL) with results
given in Table 8.2. The higher concentrations found with standard
addition indicated small matrix effects. However, standard addition is
associated with high workload, since a calibration must be created for
each sample and is not suitable for the analysis of large sample sets.
The matrix effects were largely compensated by an extract dilution of
1:10 for the analysis of all the project samples.
Table 8.2: α-Tocopherol contents of a chili pepper sample quantified by external calibration and standard addition at two different dilutions
Method α-Tocopherol content
(mg/100 g)
External calibration 22.6
Standard addition (diluted 1:5) 24.3
Standard addition (diluted 1:10) 25.5
Analytical and Experimental Background
168
8.6 Determination of Fat by NIR17
Near infrared spectroscopy (NIR) is a fast and non-destructive
quantification technique. On the other hand, large sample sets were
necessary for calibration. The NIR-spectra were a byproduct of the
surface color measurement. The spectrometer recorded full
UV/Vis/NIR-spectra in the range of 200 nm to 2,000 nm (50,000 cm-1
to 5,000 cm-1). The Vis-range of the spectra was used for the
calculation of the surface color values according to the CIE L*a*b*
color system. During the first project year, the fat content was
analyzed by a gravimetric method to identify chili peppers with high
lipid contents, which may be used as source for the extraction of
native chili pepper seed oil. The collected data were used to develop
an NIR based method for the quantification of fat in chili peppers,
which were received later in the project. The complete data set
consisted of the NIR spectra and the reference fat contents of 330
different chili pepper samples. Reference fat content was analyzed by
a gravimetric method according to Schulte [185].
17
NIR spectra and reference analysis of fat contents were obtained under supervision of the author during the final theses for their first state examination in food chemistry by Mr. Matthias Lüpertz (Title: “Untersuchung von Capsicum-Pulvern auf den Gehalt an Capsaicinoiden und Polyphenolen sowie auf deren antioxidative Kapazität mit multivariater Datenauswertung der FT-NIR-Spektren”), Mrs. Christina Schröders (Title: “Untersuchung von Capsicum-Pulvern auf Oberflächenfarbe, Gehalt
an extrahierbarer Farbe, Fett und Wasser mit multivariater Datenauswertung der über zwei NIR-Systeme erhaltenen Spektren”) and Désirée Marquenie (Title: “Optimierung der mittels multivariater Datenanalyse von NIR-Spektren erstellten Modelle zur Untersuchung der Gehalte an wertgebenden Inhaltsstoffen von Chili-Pulvern”). Additional data were collected by Dieter Riegel. Data analysis and preparation of a prediction model was performed by the author.
Determination of Fat by NIR
169
Figure 8-8: Typical NIR spectra and the corresponding derivatives of ten
different chili pepper samples. First derivatives of the original spectra were
calculated by the Savitzky-Golay method with a third order polynomial, with
ten smoothing points left and right.
The Savitzky-Golay method was used to calculate the first derivatives
of the NIR spectra. This pretreatment was applied for baseline
correction. In addition, it accentuates the relevant information of the
spectra (Figure 8-8) [186]. Besides, all spectra were reduced to the
0
0.2
0.4
0.6
0.8
1
5000 6000 7000 8000 9000 10000
Ab
so
rba
nc
e
Wavenumber (cm-1)
NIR Spectra
-0.012
-0.008
-0.004
0.000
0.004
5000 6000 7000 8000 9000 10000
Wavenumber (cm-1)
Derivatives of the NIR Spectra
First Savitzky-Golay derivation
Analytical and Experimental Background
170
range from 5,500 cm-1 to 8,900 cm-1. This includes most of the C-H
absorption bands, which are typical for lipids (overtone and
combination oscillations mostly of C-H stretching vibration) [187]. To
determine the fat content, a partial least square (PLS) regression
model was calculated out of the pretreated spectra. For cross-
validation of the PLS, the data set was randomly divided into three
groups. Two groups were used for calculating the model and the third
one for validation. Performance data of the obtained PLS regression
model are given in Figure 8-9.
Figure 8-9: Predicted versus reference value (g fat/100 g chili powder) plot
of the third principle component and explained variance plot; both obtained
from the PLS regression used for fat quantification. *RMSE: Root Mean
Square Error
0
5
10
15
20
25
30
0 5 10 15 20 25 30
Pre
dic
ted
(g
/10
0 g
)
Reference (g/100 g)
Predicted vs. Reference
60
70
80
90
100
0 1 2 3 4 5 6 7
%
Factor
Explained Variance
Calibration Validation
Slope Offset RMSE* R2
Calibration 0,920 0,842 1,37 0,920
Validation 0,915 0,902 1,41 0,915
Effect of Drying on Phytonutrients in Chili Peppers
171
The calibration and validation showed very similar and satisfactory
values. The slope and R2 were close to one. Offset and root mean
square error were both very low. The third principle component
explained 92% of the variance and could be used for predicting the
fat content of chili pepper samples. Higher components (factor 4 - 7;
Figure 8-9) explained only the background noise of the spectra and
were not considered.
8.7 Effect of Drying on Phytonutrients in Chili
Peppers
During the whole project, only dried and milled sample material was
sent to Wuppertal. Drying and milling were performed according to a
standard operation protocol (SOP). The fruits were oven-dried at
temperatures from 55 °C to not higher than 60 °C to constant mass.
The following experiment was conducted to study the effect of the
applied drying procedure on the content of valuable compounds and
quality traits:
Approximately 1 kg fresh chili pepper fruits were divided into two
sample pools A and B (Red Pepper; C. annuum) by cutting each chili
pepper fruit into halves along the longitudinal axis. The fruit halves of
sample pool A were homogenized with a food processor and
analyzed as fresh fruits to obtain reference data. The fruit halves of
sample pool B were dried according to the drying SOP at 55 °C to
constant mass for ca. 24 hours. The dried fruit halves were crushed
with a food processor before milling to a particle size of 99%
< 850 µm.
Analytical and Experimental Background
172
The two different samples (fresh and oven dried) were
analyzed on ascorbic acid, total polyphenols, antioxidant capacity,
capsaicinoids, tocopherols and extractable color. Figure 8-10 shows
the results obtained from the experiment.
Figure 8-10: Results of the drying experiment. Error bars represent the standard deviation for duplicate analysis. Fresh fruit material was considered as 100%18.
As expected, ascorbic acid was degraded to residue levels of 10%,
which is comparable with literature data [128]. The differences for
total polyphenol content, antioxidant capacity and capsaicinoid
18
The drying experiment for tocopherols was performed by Mr. Christian Jansen during his final thesis for the first state examination in food chemistry (see footnote 16).
0
20
40
60
80
100
%
fresh oven-dried
Analytical Strategy
173
content between the fresh and dried fruit material were within the
margin of error of the applied analytical methods. Tocopherols and
extractable color were slightly degraded to remaining levels of 86%
for tocopherols and 77% for extractable color. However, in
comparison with ascorbic acid both traits could be regarded as being
widely stable. In conclusion, the applied drying method was suitable
for drying chili pepper samples without a major degradation of
important quality traits except for ascorbic acid and allowed
estimating the contents of important traits in fresh fruits.
8.8 Analytical Strategy
The improved, optimized and streamlined methods were used for the
analysis of more than 350 different chili pepper samples. The
available amount of many samples was less than 15 g. Therefore, the
re-use of extracts (same extract for the determination of
capsaicinoids, total polyphenols and antioxidant capacity) and the
continued use of sample material from non-destructive methods (NIR
and surface color analysis) was necessary. The application of the
NIR-based fat determination also helped to save sample material.
The complete analytical strategy is shown in Figure 8-11.
Each chili pepper accession was unpacked and received an
internal sample code to assure the correct identification of the
sample. Samples from Peru received a code consisting of four
different numbers starting at 0001 and samples from Bolivia a three-
digit code starting with 001. However, in all Chapters only the original
germplasm bank accession code (Acc. code) is used to clearly
specify the identity of each chili pepper accession. After unpacking
Analytical and Experimental Background
174
and internal codification, the complete sample was screen-meshed
and particles with a size of larger than 850 µm were re-milled
according to the ASTA 1.0 method [33]. Re-milling was necessary to
obtain homogenous samples with a very small particle size
distribution, which is recommended for an effective and reproducible
extraction. The streamlined analytical strategy finally allowed the
analysis of all considered quality traits with a minimum amount of
only 13 g.
Figure 8-11: Analytical strategy for the determination of different traits in chili
pepper powders.
Unpacking, registration,
internal codification
-complete sample-
Screen-meshing and milling
of all particles larger than
>850µm(according to ASTA method 1.0)
-complete sample-
Analysis of valuable
compounds
and traits
-minimum need 13 g-
Analysis of
color, fat reference
analysis
and by NIR
-total need 8 g-
NIR & CIE-L*a*b*
values
-2 x 0.5 g-
Fat determination
(reference)
-2 x 1 g-
Extractable color
(ASTA 20.1)
-2 x 0.5 g-
Water
determination
-2 x 2 g-
Analysis of
capsaicinoids,
antioxidants,
vitamin C and E
-total need 5 g-
Extraction of
capsaicinoids and
antioxidants
-2 x 1 g-
Vitamin C
determination
-1 x 1 g-
Vitamin E
determination
-2 x 0.1 g-
Flavonoid
determination
-2 x 1 g-
Analysis of major
capsaicinoids
Total polyphenols
(Folin-Ciocalteu)
Antioxidant capacity
(TEAC assay)
Concluding Remarks and Future Perspectives
175
9. Concluding Remarks and Future
Perspectives
In total, 362 different dried and milled chili pepper samples were
analyzed by applying improved and standardized methods. The
sample set included 179 different Peruvian and 96 different Bolivian
accessions. The remaining samples were obtained from the
replanting experiments conducted in both countries.
All samples were analyzed on important chemical traits
(Table 9.1) and the complete data set was evaluated by multivariate
data analysis. Unfortunately, no deeper or underlying structures were
found by applying principle component analysis (PCA) or partial least
squares (PLS) regression discriminant analysis. Due to the different
drying procedures, which were applied in Peru and Bolivia, the
analytical results were evaluated individually for of each country.
Figure 9-1 shows the score and loading plots obtained from PCA of
all Peruvian accessions. The samples are grouped according to their
taxonomical classification. As can be seen, no distinct groups are
observed. The same was found for the Bolivian samples, so a
taxonomical classification based on phytochemicals and quality traits
was not achieved, which is in accordance with Zewdie and Bosland
[46]. They reported similar results when analyzing and comparing the
capsaicinoid profiles of different chili pepper species.
Therefore, the results were analyzed by descriptive statistical
methods (Box-plot analysis) and data obtained from the replanting
experiments were evaluated by analysis of variance (ANOVA)
individually for each country. However, the whole data set provided a
sound database for the selection of high value accessions for specific
propose.
Concluding Remarks and Future Perspectives
176
Figure 9-1: Above: Score plot of a PCA of all 179 Peruvian chili peppers.
Below: Loading plot of the PCA. Data analysis included the results of:
capsaicinoids (Cap), capsaicin (C), dihydrocapsaicin (DC), nordihydro-
capsaicin (NDC), flavonoids and quercetin (Q), total polyphenols (TP)
antioxidant capacity (TEAC), tocopherols (T) and α-, β- and γ-tocopherol, fat
content, surface color values (L*, a*, b*, C* and h), and extractable color
(ASTA). Not considered were ascorbic acid, luteolin, kaempferol and
apigenin because most of the accessions did not show detectable amounts.
-6
-4
-2
0
2
4
6
-6 -4 -2 0 2 4 6 8
Fa
cto
r 2
(2
0%
)
Factor 1 (26%)
Score Plot
C. annuum C. baccatum C. chinense C. frutescens C. pubescens
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4
Fa
cto
r2
(2
0%
)
Factor 1 (26%)
h
L* b*NDC
TPDC Cap
CTEAC
Fat
γ-TC*
Q
β-T
Tα-T a*
ASTA
Loading Plot
Concluding Remarks and Future Perspectives
177
Table 9.1 summarized the concentration ranges for all analyzed traits
of the first field trials of the chili pepper accessions. A comparison
with the chemical composition of the different chili pepper accessions
grown in both countries was not performed due to the different
treatment and handling. Planting, harvesting and drying was agreed
upon to be performed according to a strict protocol. The Bolivian
partners, however, proceeded differently with all Bolivian chili pepper
accessions. Especially, the drying conditions were different. The
Bolivian partners sun-dried the chili peppers in open air for up to three
weeks followed by a final oven-drying at 60 °C for approximately 12
hours, while the Peruvian partners only applied oven-drying at 60 °C
for about 72 hours. Therefore, a data comparison was not reliable.
However, outstanding accessions were identified for each
country. Pungency ranged from non-pungent up to very highly
pungent and allowed selecting of accessions according to consumers
preference.
Two accessions, one Peruvian (Acc. code: PER017668) and
one Bolivian (Acc. code: P6), with levels of 29.5 and 46.8 mg/100 g
were identified providing an exceptionally high content in flavonoids
when compared to the other analyzed chili peppers.
The wide variability within the content of total polyphenols and
antioxidant activity offered an additional criteria for selecting high
value accessions with high contents in health promoting
phytochemicals. At least one Peruvian accession (Acc. code:
PER06959) was identified with a very high total polyphenol content
(3.69 g GEA/100 g) and antioxidant capacity (9.2 mmol Trolox/100 g)
in comparison with the other accessions
Concluding Remarks and Future Perspectives
178
With regard to the ascorbic acid content, the most accessions did not
show a detectable amount due to the applied drying procedure.
However, some Peruvian and Bolivian chili peppers showed an
unexpected high amount of vitamin C. The Peruvian accession
PER006992 showed an ascorbic acid level of 295 mg/100 g and two
Bolivian accessions showed a vitamin C content of 437 mg/100 g
(Acc. code: 341) and 216 mg/100 g (216 mg/100 g). Although more
Table 9.1: Summary of the compositional characterization of native chili peppers from Peru and Bolivia
Units
Peruvian accessions
(n=179)
Bolivian accessions
(n=96)
min mean
max min mean
max
Capsaicinoidsa
mg /100 g
nd1 312 1560 nd 144 1028
Capsaicin nd 207 1074 nd 95 824
Dihydrocapsaicin nd 89 460 nd 42 227
Nordihydrocapsaicin nd 16 122 nd 10 75
Flavonoidsb
mg /100 g
nd 5.1 29.5 0.4 8.1 46.8
Quercetin nd 4.5 26.6 0.4 6.5 42.6
Luteolin nd 0.6 5.2 nd 1.4 5.0
Kaempferol nd >0.0 0.6 nd 0.1 0.8
Apigenin nd >0.0 0.7 nd 0.1 0.7
Total polyphenols g GEA*/100 g 1.22 1.82 3.69 1.09 1.61 2.19
Antioxidant capacity mmol T#/100 g 1.8 4.0 9.2 3.0 4.1 6.2
Tocopherolsc
mg /100 g
0.4 14.0 35.3 4.2 22.6 38.1
α-Tocopherol nd 11.5 32.5 2.0 16.7 31.8
β- Tocopherol nd 0.3 2.2 nd 0.9 2.7
γ- Tocopherol nd 2.2 7.8 1.3 5.0 7.9
Ascorbic acid mg /100 g nd 7 295 nd 15 437
Fat content g /100 g 2.2 7.6 19.6 6.7 14.3 32.8
Extractable color (ASTA 20.1) 1 32 146 3 44 127
Surface color (hue-angle) 34 54 84 31 52 76 asum of capsaicin, dihydrocapsaicin and nordihydrocapsaicin;
bsum of
quercetin, luteolin, kaempferol and apigenin; c sum of α-, β- and
γ-tocopherol; 1nd: not detectable *GEA: gallic acid equivalents;
#T: Trolox
Concluding Remarks and Future Perspectives
179
than 90% of vitamin C is degraded during the drying and milling
process, concentrations up to 437 mg/100 g were fully unexpected.
Therefore, high resolution mass spectrometric analysis was applied
and confirmed the identity of the HPLC peak ascribed to ascorbic
acid. However, it will be necessary to analyze fresh fruit material of
theses accessions to finally confirm the exceptionally high content of
this vitamin.
Other outstanding accessions were found with regard to their
tocopherol contents, showing levels up to 35.3 mg/100 g
(Acc. code: 42) for the Peruvian chili peppers and 38.1 mg/100 g
(Acc. code: 319-2) for the Bolivian accessions.
The Bolivian accession 109 R showed an exceptionally high
fat content (32.8 g/100 g), which allows the production of natural chili
seed oil for cooking and industry.
The color attributes (extractable and surface color) showed a
wide variability, which allowed selecting accessions according to
customers’ preference. However, the values for extractable color are
remarkable for chili peppers reaching values of 146 ASTA 20.1 units,
but quite low in comparison with paprika powders reaching typically
ASTA 20.1 values above 200.
Concerning the Peruvian chili pepper accessions, all belonged to the
five domesticated species C. annuum, C. baccatum, C. chinense,
C. frutescens, and C. pubescens. Unfortunately, the sample set did
not include wild species. Within the sample set, Capsicum accessions
with pungency from non-pungent to extremely pungent and with
outstanding content in valuable health-related phytochemicals were
identified.
Concluding Remarks and Future Perspectives
180
Results of the Peruvian C. pubescens accessions were separately
reported due to the unique characteristics of this species. The
inter-species comparison showed that the Peruvian C. pubescens
accessions had a rather low content in capsaicinoids, quercetin,
antioxidant capacity, tocopherols, fat and extractable color, when
compared to accessions of other chili peppers species. In addition, all
analyzed C. pubescens samples showed an untypical capsaicinoid
pattern with high amount of dihydrocapsaicin and nordihydro-
capsaicin.
Replanting of 23 Peruvian accessions was conducted on the
same test field for a year-to-year comparison and on three further test
fields for a multi-location comparison to evaluate the environmental
impact. Those chili peppers that were planted on the same test field,
which was also used in the first year, unfortunately died because of
low temperatures. Therefore, it was not possible to perform a
year-to-year comparison for the Peruvian chili pepper accessions.
The evaluation of the other three test fields indicated a great
environmental impact on the content of important phytochemicals and
quality traits. Analytical data were evaluated by multivariate data
analysis PCA and PLS and by analysis of variance (ANOVA). PCA
and PLS analysis did not show underlying structures. However,
ANOVA showed significant influence on the concentrations and levels
that were observed for all traits and indicated the high influence of the
environment on the traits. Besides, significant interactions among the
accessions and locations respectively the environment were
observed, showing the individual response of accessions to changes
in the growing conditions. Furthermore, an environmental impact
factor was calculated. The factor allowed differing between
Concluding Remarks and Future Perspectives
181
accessions being consistent in the production of phytochemicals
widely independent of the growing condition and those, which
provided exceptional high levels for a quality trait at a specific
location. At least one accession (Acc. code: PER006952) provided
very consistent amounts when planted in all three locations. Other
accessions showed higher values when planted in a specific region.
This information can be used to increase the content of
phytochemicals for selected accessions grown under specific
conditions. However, the experiment was conducted only for one
year. Multi-location and long-term studies will be necessary to identify
the full potential of these accessions.
The original Bolivian sample set consisted of 114 different chili
peppers. According to a questionable taxonomic classification, it was
necessary to remove 18 accessions from the sample set and the data
of these accessions are not reported. The remaining set also included
all domesticated species. The majority of the 96 accessions belonged
to the domesticated species C. baccatum var. pendulum. In addition,
ten wild species were analyzed belonging to C. baccatum var.
baccatum (seven accessions), the ancestral of domesticated
C. baccatum var. pendulum and to C. eximium (three accessions) a
species closely related to C. pubescens. The results also indicated a
great variability in the content of phytonutrients and quality traits.
Primarily, 36 accessions were considered and replanted on
the same and two other test fields. On the two other test fields, a
completely different planting, sample and drying procedure was
applied, when compared to the first plantation. In addition, many of
the accessions died, so that a multi-location comparison could not be
Concluding Remarks and Future Perspectives
182
performed and needs to be carried out in further studies. On the
same test field, only twelve accessions produced fruits in a sufficient
amount. Due to the small number of accessions and because all
belonged to the species C. baccatum var. pendulum, the results of
the year-to-year comparison are of limited value. However, ANOVA
showed significant differences in the phytonutrient content and
between the quality traits and proved significant impact of the harvest
year and their interaction for all quality traits.
Nevertheless, the obtained data showed a high variability in the
content of phytochemicals and quality traits and offered the
opportunity to identify high value accessions and to improve food
composition databases. As an example, the nutrient database of the
United States Department of Agriculture (USDA) reported only values
for one chili pepper powder for several traits (e.g. tocopherols and
fat). All analytical data were submitted to the partners in Peru and
Bolivia. This characterized the biodiversity in the accessions of their
germplasm banks and allowed selecting high value accessions
according to their chemical composition and to start market
specialization or for further breeding programs focussing on nutrition
quality. The study results thus add value to the Capsicum diversity to
generate higher income for small-scale chili farmers. At the same
time, this can provide a chance to conserve local native chili peppers
through their use as high value crop.
Materials and Methods
183
10. Materials and Methods
10.1 Chemicals
Acetone, methanol, acetonitrile and 2-propanol HPLC grade,
disodium hydrogen phosphate, ammonium acetate and disodium
carbonate were purchased from VWR International (Darmstadt,
Germany).
Folin & Ciocalteu’s phenol reagent, formic acid, tert.-
butylhydroquinone, ABTS (2,2′-azino-bis(3-ethylbenzothiazo-line-6-
sulfonic acid) diammonium salt), gallic acid (3,4,5-trihydroxybenzoic
acid), Trolox® (6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic
acid), luteolin (3′,4′,5,7-tetra-hydroxyflavone), kaempferol (3,4′,5,7-
tetrahydroxy-flavone), apigenin (4′,5,7-trihydroxyflavone), nonanoic
acid vanillylamide, natural capsaicin (65% 8-methyl-N-vanillyl-trans-6-
nonenamide, 30% 8-methyl-N-vanillyl-nonanamide, 5% N-vanillyl-7-
methyl-octanamide), (±)-α-tocopherol, rac-β-tocopherol, (+)-γ-
tocopherol were purchased from Sigma-Aldrich (Steinheim, Germany)
Ascorbic acid, quercetin monohydrate (3,3′,4′,5,6-pentahydroxy-
flavone), acetic acid and DL-dithiothreitol (1,4-dimercapto-2,3-
butanediol), ethanol p.a. were purchased from Carl Roth (Karlsruhe,
Germany).
Water was obtained from a Milli Q Gradient A10 - System (Millipore,
Schwalbach, Germany).
Materials and Methods
184
10.2 Sample Pretreatment
Prior to analysis, all samples were sieved and material with particle
size > 850 µm was re-milled to obtain 99% < 850 µm according to
ASTA method 1.0 [33]. Milling was performed under cooling using a
knife mill (IKA Universal Mill M20 for batches > 10 g and IKA
Analytical Mill A10 for batches < 10 g, IKA-Werke Staufen, Germany).
Samples were stored in black polyethylene plastic bags at -25 °C until
analysis.
10.3 Extraction and Analysis of Capsaicinoids
The analysis of the capsaicinoid content was done by HPLC with
fluorescence detection. Two separate samples of each accession
were analyzed. For the extraction, 500 mg sample was placed in a
glass centrifuge tube. 1 mL of a disodium hydrogen phosphate buffer
(0.5 M, pH 11) and 15 mL of acetonitrile and methanol (50:50, v/v)
were added. After 16 h at 4 °C in the dark, the sample was placed in
an oven at 80 °C for 4 hours and vortexed every 30 minutes. The
crude extract was diluted with methanol/water (1:1, v/v) from 1:1 to
1:40 to fit into the calibration curve and filtered through a 0.2 µm
PVDF (polyvinylidene difluoride) syringe filter (Carl Roth, Karlsruhe,
Germany) before HPLC analysis. Separation of the capsaicinoids was
performed by injecting 10 µL into a Merck-Hitachi HPLC system
(interface L-7000, quaternary pump L-7100, autosampler L-7250,
fluorescence detector L-7485 and a CIL column oven) with a Kinetex
RP-18 column (2.6 µm, 100 mm x 3 mm) equipped with a 0.5 µm
inline filter (Phenomenex, Aschaffenburg, Germany) at 50 °C.
Materials and Methods
185
Fluorescence detector was set to 280 nm for excitation and 320 nm
for detection [89]. Separation of capsaicinoids was achieved by
isocratic elution with acetonitrile and 0.5% acetic acid (38:62, v/v) at a
flow rate of 0.7 mL/min and a total run time of 11 minutes. Nonanoic
acid vanillylamide was used as standard for an external calibration
curve for quantification because of the identical fluorescence
characteristic like other capsaicinoids and the availability in high
purity. Peak identification was done by injecting a solution of natural
capsaicin. The capsaicinoid content was calculated as the sum of
nordihydrocapsaicin, capsaicin, and dihydrocapsaicin. Minor
capsaicinoids were not considered in this study.
10.4 Flavonoid Analysis
A slightly modified method described by Miean and Mohamed was
used to analyze quercetin, kaempferol, luteolin and apigenin aglycons
[105]. For extraction and hydrolysis of the flavonoid glycosides,
750 mg of the sample was weighed into a centrifuge tube and 10 mL
of a mixture of methanol, water and 12.5 M hydrochloric acid
(70:20:10, v/v/v, containing 0.4 g/100 mL tert.-butylhydroquinone)
was added. The suspension was kept at 80 °C for 3 hours and
vortexed every 30 minutes. 500 µL of the crude extract was diluted
with a disodium hydrogen phosphate buffer (50 mM Na2HPO4,
pH 12) / methanol solution (1:1, v/v) to a final volume of 2000 µL.
After filtration through a 0.2 µm PVDF syringe filter, 10 µL was
injected in the same Merck-Hitachi HPLC system being used for
capsaicinoid determination, but with a Merck-Hitachi L 7455 photo
diode array detector and with a Kinetex PFP (penta fluoro phenyl)
Materials and Methods
186
column (2.6 µm, 100 mm x 3 mm) with a 0.5 µm inline filter
(Phenomenex, Aschaffenburg, Germany) at 50 °C. Methanol (solvent
A) and water both with 0.1% formic acid were used as mobile phase
applying the following gradient program at a flow rate of 0.5 mL/min:
0 - 5 min from 40 to 45% A, 5 - 8 min 45% A, 8 - 22 min from 45% to
95%, 22 – 22.1 min from 95% to 40% A and 22.1 - 31 min 40% A
(column re-equilibration). Quantification was performed at 360 nm for
all four flavonoids. For external calibration quercetin, kaempferol,
luteolin and apigenin were used. The sum of the four individual
flavonoids is expressed as total flavonoids.
10.5 Determination of Total Polyphenols
The method was based on the Folin-Ciocalteu procedure [120]. The
crude extract from the capsaicinoid determination was used for
analysis. 100 µL was placed in a 15 mL centrifuge tube and was
diluted with 900 µL water. 5 mL of the Folin & Ciocalteu´s phenol
reagent (1:10, v/v, diluted with water) was added. After an incubation
time between 3 and 8 minutes, 4 mL of disodium carbonate solution
(7.5 g/100 mL) was added. After 1 hour at 30 °C, 250 µL of the
solution, each, was transferred to two wells of a 96-well microtitre
plate for a duplicate reading of the absorbance at 750 nm with a
Model 680 microtitre plate reader (Bio Rad, Munich, Germany). Gallic
acid was used for external calibration. The results were expressed as
gallic acid equivalents (GAE).
Materials and Methods
187
10.6 Trolox Equivalent Antioxidant Capacity
(TEAC)
The procedure described by Re et al. was applied [118]. The crude
extract from the capsaicinoid determination was used. 100 µL of the
extract was diluted with 900 µL ethanol. 20 µL of each solution was
transferred to two wells of a 96-well microtiter plate for duplicate
measurement and 200 µL of the diluted ABTS-radical solution was
added. After incubation time of 6 minutes at 20 °C the absorbance
was read at 750 nm with the same microtiter plate reader used for
total polyphenol determination. Trolox® was used for external
calibration. The ABTS-radical stock solution was prepared by
dissolving 192 mg of ABTS in 50 mL water. The radical is produced
by adding 33 mg of potassium peroxydisulfate to the solution. The
mixture was placed in the dark at room temperature to generate the
radical for 16 hours. 1 mL ABTS stock solution was diluted with
approximately 50 mL water and absorbance was adjusted to 0.70 ±
0.02 at 750 nm before use.
10.7 Analysis of Ascorbic Acid by HPLC
500 mg of sample material was placed in a 15 mL centrifuge tube and
10 mL of a mixture of acetonitrile and an ammonium acetate buffer
(100 mM, pH 6.8) (70:30, v/v, containing 1 g/100 mL
tert.-butylhydroquinone and 1 g/100 mL dithiothreitol) was added. The
suspension was shaken at room temperature for 2 hours.
Subsequently, the solution was centrifuged at 2000 g for 10 minutes
and filtered through a 0.2 µm PVDF syringe filter before HPLC
Materials and Methods
188
analysis using hydrophilic interaction liquid chromatography (HILIC)
as described by Nováková et al. for ascorbic acid analysis [136]. 5 µL
were injected on the same Merck-Hitachi used for flavonoid
determination. The separation for half the sample pool was performed
on a sulfobetaine ZIC®-HILIC column (3.5 µm, 150 mm x 4.6 mm)
(SeQuant, Umeå, Sweden) at 35 °C. Isocratic elution was done by
using a mixture of acetonitrile and an ammonium acetate buffer (100
mM, pH 6.8) (70/30, v:v) at a flow rate of 0.5 mL/min with a run time
of 17 minutes. With the availability of core-shell HILIC columns, the
separation of the remaining samples was performed on a sulfobetaine
Nucleoshell HILIC column (2.7 µm, 100 mm x 3 mm) (Macherey-
Nagel, Dueren, Germany) at 35 °C. Elution was achieved with
acetonitrile and the same buffer (80:20, v/v) at a flow rate of 0.4
mL/min with a total run time of 9.5 minutes. Quantification was
performed in both cases at the absorption maximum of 260 nm.
Ascorbic acid was used as external standard for calibration.
Dehydroascorbic acid is reduced by dithiothreitol to ascorbic acid.
Therefore, this method detects the sum of both.
10.8 Tocopherols by HPLC
100 mg sample was placed in a 2 mL micro tube. Tocopherols were
extracted with 1 mL 2-propanol containing 2 mg/mL tert.-butylhydro-
quinone at 50 °C for two hours. Samples were agitated every 30 min.
The crude extract was diluted 1:10 with methanol and water (80:20,
v/v). After filtration through a 0.2 µm PVDF syringe filter
(Macherey-Nagel, Düren, Germany) 10 µL were injected into exactly
the same Merck-Hitachi HPLC system used for the flavonoid
Materials and Methods
189
determination. The method described by Grebenstein et al. [141] was
slightly modified and performed by isocratic elution with methanol and
water (82:18, v/v) at a flow rate of 0.3 mL/min at 50 °C and a total run
time of 17 min. The fluorescence detector was set to 296 nm for
excitation and 325 nm for emission. α-Tocopherol, β-tocopherol and
γ-tocopherol were used as standards for an external calibration curve
for quantification. δ-Tocopherol was not considered because of the
very low concentration found in chili peppers.
10.9 Determination of the Fat Content
10.9.1 Gravimetric Method
The method described by Schulte was used [185]. 1.2 g of the chili
powder was placed in a glass centrifuge tube. After addition of 10 mL
4 M hydrochloric acid and 5 mL toluene the tube was placed in an
oven at 120 °C for 2 hours and vortexed every 20 minutes. Samples
were allowed to cooling down to room temperature and centrifuged at
2800 g for 10 minutes. 1.0 mL of the toluene phase was evaporated
under a nitrogen stream at 115 °C until a constant weight for the
residue was obtained.
10.9.2 NIR Method
NIR measurements were performed on a Jasco UV/Vis/NIR-
Spektrometer V-670 (Gross-Umstadt, Germany) in the reflection
mode equipped with the PSH-001/02 powder holder. 300 mg of the
chili powder was placed in the powder holder with spectra recording
in the range between 5,000-50,000 cm-1. To determine the fat content
Materials and Methods
190
of the chili pepper a partial least-square (PLS) regression model was
calculated using The UnscramblerX 10.3 software package (Camo
Inc., Oslo, Norway). The PLS model was established by using the
spectra and reference fat contents of 330 different chili pepper
powders. The reference fat contents were gravimetrically analyzed by
the method of Schulte (Chapter 10.9.1) [185]. For cross-validation of
the PLS the data set was randomly divided into three groups. Two
groups were used for calculating the model and the third one for
validation. The third principle component was used for predicting the
fat content of the chili sample. The NIR based fat determination was
applied to all samples mentioned in Chapter 5 and to those in
Chapter 7 grown in 2012.
10.10 Determination of Extractable Color
The determination was performed according to the ASTA 20.1
method [33]. Based on the surface color data, the amount of sample
material was chosen to achieve the required absorption between 0.3
to 0.7. Typically, 70-700 mg of sample material was used and placed
in a 100 mL volumetric flask. 90 mL of acetone was added and the
flask was shaken. After 16 hours at room temperature in the dark, the
flask was filled up to the mark with acetone and shaken again. After
particles were settled, the absorbance of the clear supernatant was
measured with a Hach DR/2000 spectrophotometer (Duesseldorf,
Germany) at 460 nm and ASTA 20.1 values were calculated by the
following equitation:
Materials and Methods
191
The If value is a correction factor specific for the spectrophotometer.
Considering the If value allows a comparison with different ASTA 20.1
values. It is determined by absorbance reading of a 5% sulfuric acid
containing exactly 1,3500 g CoCl2 x 6 H2O und 0,0125 g (NH3)2Cr2O7
per 100 mL. Absorbance of the solution is read at 477 nm. The If
value is calculated by the following equitation [188]:
10.11 Measurement of Surface Color
Measurement was performed on a Jasco UV/Vis-NIR-Spektrometer
V-670 (Gross-Umstadt, Germany) in the reflection mode equipped
with the PSH-001/02 Powder holder. 300 mg of the sample was
placed in the powder holder and with subsequent spectra recording.
CIE L*, a*, b*, hue-angle and Chroma C* were calculated from the
obtained UV/Vis-spectra by the Jasco Spectramanager V.2.07.00
[146].
10.12 Determination of Moisture Content
2 g of the sample was exactly weighed into a weighing bottle and
dried in a vacuum oven at 60 °C at 100 mbar for 1 hour. The sample
was allowed to cool down in a desiccator for 1 h and weighed again.
Moisture content was calculated as difference in the sample mass
before and after drying.
List of Publications
192
11. List of Publications
11.1 Original Papers
Meckelmann SW, Riegel DW, van Zonneveld M, Ríos L, Peña K, Ugas R, Quinonez L, Mueller-Seitz E, Petz M (2013) Compositional Characterization of Native Peruvian Chili Peppers (Capsicum spp.). Journal of Agricultural and Food Chemistry 61(10): 2530–2537.
Meckelmann SW, Riegel DW, van Zonneveld M, Ríos L, Peña K, Mueller-Seitz E., Petz M (2014) Capsaicinoids, Flavonoids, Tocopherols, Antioxidant Capacity and Color Attributes in 23 Native Peruvian Chili Peppers (Capsicum spp.) Grown in Three Different Locations. European Food Research and Technology (accepted for publication) DOI: 10.1007/s00217-014-2325-6
Meckelmann SW, Jansen C, Riegel DW, van Zonneveld M, Ríos L, Peña K, Mueller-Seitz E, Petz M (2014) Phytochemicals in Native Peruvian Capsicum pubescens (Rocoto). Journal of Food Composition and Analysis (submitted for publication)
Meckelmann SW, Riegel DW, van Zonneveld M, Avila T, Bejarano C, Serrano E, Mueller-Seitz E, Petz M (2014) Major Quality Attributes of Native Bolivian Chili Peppers (Capsicum spp.) Focussing on C. baccatum: A two-year Comparison. Food Chemistry (submitted for publication)
List of Publications
193
11.2 Conference Contributions
Meckelmann S, Riegel D, van Zonneveld M, Petz M (2013) How does environment influence phytonutrients in native chili peppers? 42. Deutscher Lebensmittelchemikertag, Braunschweig, Germany.
Jansen C, Meckelmann S, Riegel D, van Zonneveld M, Petz M (2013) Tocopherolgehalte und –muster in nativen Chilipulvern. 42. Deutscher Lebensmittelchemikertag, Braunschweig, Germany.
Meckelmann S, Riegel D, Avila T, Bejarano C, van Zonneveld M, Petz M. (2012) Bioactive and valuable compounds in 114 native Bolivian chili accessions. 21st Int. Pepper Conference Naples/Florida, United States of America.
Meckelmann S, Riegel D, Avila T, Bejarano C, van Zonneveld M, Petz M. (2012) Untersuchung von 114 nativen bolivianischen Chili-Proben auf bioaktive und wertgebende Inhaltsstoffe. 41. Deutscher Lebensmittelchemikertag, Münster, Germany.
Meckelmann S, Riegel D, Müller-Seitz E, Petz M (2012) Bestimmung von Vitamin C in nativen Chilipulvern mittels hydrophiler Interaktions-chromatographie. Regionalverbandstagung NRW der Lebensmittel-chemischen Gesellschaft, Bonn, Germany.
Meckelmann S, Lüpertz M, Schröders C, Marquenie D, Riegel D, Petz M (2011) Non-destructive screening of chili powders for colour values and capsaicinoids by spectroscopic techniques. 5th Int. Symposium on Recent Advances in Food Analysis, Prague, Czech Republic.
List of Publications
194
Meckelmann S, Müller-Seitz E, Petz M (2011) Capsinoide: Die schärfefreien Strukturanaloga des Capsaicins - Analytik und Vorkommen in Chili-Varietäten. 40. Deutscher Lebensmittel-chemikertag, Halle an der Saale, Germany.
Meckelmann S, Lüpertz M, Schröders C, Marquenie D, Riegel D, Petz M (2011) Zerstörungsfreie Analytik von Chilipulvern mittels Nahinfrarotspektroskopie. 40. Deutscher Lebensmittelchemikertag, Halle an der Saale, Germany.
Riegel D, Meckelmann S, Fang J, Müller-Seitz E, Petz M (2010) Farbe von Gewuerzpaprika: Enfluss von Vermahlung und Fettgehalt. 39. Deutscher Lebensmittelchemikertag, Stuttgart-Hohenheim, Germany.
References
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Appendix
213
13. Appendix
Table A 1: Detailed information on the 147 Peruvian chili pepper accessions described in Chapter 4. Accessions are sorted according to ascending capsaicinoid content. The 23 accessions written in bold were replanted and reported in Chapter 6.
Accession code
Growing region
Harvest year
Organization Species
PER017909 Lima 2011 INIA C. annuum
PER017910 Lima 2011 INIA C. annuum
153 Loreto 2011 UNALM C. chinense
157 Loreto 2012 UNALM C. chinense
AMS-RC Ucayali 2010 CIDRA C. chinense
PER017612 Lima 2011 INIA C. annuum
PER017623 Lima 2011 INIA C. annuum
PER007040 Ucayali 2012 INIA C. chinense
PER006979 Ucayali 2011 INIA C. chinense
PER017711 San Martin 2012 INIA C. chinense
PER017708 San Martin 2012 INIA C. chinense
PER006984 Ucayali 2011 INIA C. chinense
PER007013 Ucayali 2012 INIA C. chinense
PER017908 Lima 2012 INIA C. annuum
PER017735 San Martin 2012 INIA C. chinense
AMS-AD Ucayali 2010 CIDRA C. chinense
PER017626 Lima 2012 INIA C. annuum
PER017699 Cajamarca 2012 INIA C. chinense
PER017704 San Martin 2012 INIA C. chinense
85 La Libertad 2011 UNALM C. chinense
252 Lima 2011 UNALM C. chinense
PER017719 San Martin 2012 INIA C. chinense
132 San Martín 2011 UNALM C. chinense
PER017648 Lambayeque 2012 INIA C. baccatum
8 Lambayeque 2012 UNALM C. chinense
PER017833 Loreto 2011 INIA C. baccatum
PER017610 Lima 2012 INIA C. baccatum
PER017601 Lima 2012 INIA C. baccatum
PER017875 Ayacucho 2011 INIA C. baccatum
PER017625 Lima 2012 INIA C. baccatum
PER017679 Cajamarca 2012 INIA C. baccatum
PER017608 Lima 2012 INIA C. baccatum
202 Piura 2011 UNALM C. chinense
PER017661 Lambayeque 2011 INIA C. baccatum
PER017736 San Martin 2012 INIA C. chinense
PER017618 Lima 2012 INIA C. baccatum
PER017671 Lambayeque 2012 INIA C. annuum
85 La Libertad 2012 UNALM C. chinense
202 Piura 2012 UNALM C. chinense
201 Piura 2011 UNALM C. baccatum
PER017705 San Martin 2012 INIA C. chinense
7 Lambayeque 2012 UNALM C. chinense
PER017605 Lima 2012 INIA C. baccatum
PER006991 Ucayali 2011 INIA C. chinense
PER017621 Lima 2012 INIA C. baccatum
69 La Libertad 2011 UNALM C. chinense
200 Piura 2012 UNALM C. chinense
5 Lambayeque 2012 UNALM C. baccatum
5 Lambayeque 2011 UNALM C. baccatum
PER017654 Lambayeque 2012 INIA C. annuum
Appendix
214
Accession code
Growing region
Harvest year
Organization Species
10 La Libertad 2012 UNALM C. chinense
2 Lambayeque 2012 UNALM C. baccatum
PER017893 Piura 2012 INIA C. baccatum
72 La Libertad 2011 UNALM C. baccatum
EHA-CHAR Ucayali 2010 CIDRA C. chinense
LPI-A Ucayali 2010 CIDRA C. baccatum
222 Tumbes 2011 UNALM C. chinense
88 La Libertad 2011 UNALM C. chinense
132 San Martín 2012 UNALM C. chinense
PER017691 Cajamarca 2012 INIA C. chinense
6 Lambayeque 2012 UNALM C. chinense
69 La Libertad 2012 UNALM C. chinense
PER017692 Cajamarca 2012 INIA C. baccatum
PER017721 San Martin 2012 INIA C. chinense
PER006964 Ucayali 2011 INIA C. baccatum
PER007044 Ucayali 2011 INIA C. baccatum
PER006957 Ucayali 2011 INIA C. chinense
60 Lima 2012 UNALM C. chinense
PER017635 Lambayeque 2012 INIA C. annuum
PER006951 Ucayali 2011 INIA C. baccatum
PER006954 Ucayali 2012 INIA C. baccatum
PER006959 Ucayali 2010 INIA C. chinense
LCC-CHALL Ucayali 2010 CIDRA C. baccatum
PER006963 Ucayali 2010 INIA C. baccatum
PER017633 Lambayeque 2012 INIA C. annuum
PER006948 Ucayali 2012 INIA C. baccatum
LPI-CHAR Ucayali 2010 CIDRA C. chinense
157 Loreto 2012 UNALM C. chinense
PER017683 Cajamarca 2012 INIA C. baccatum
EHA-CA Ucayali 2010 CIDRA C. chinense
PER017849 Puno 2012 INIA C. baccatum
75 La Libertad 2012 UNALM C. chinense
PER007025 Ucayali 2012 INIA C. chinense
PER006985 Ucayali 2012 INIA C. chinense
PER007005 Ucayali 2011 INIA C. chinense
PER017682 Cajamarca 2012 INIA C. chinense
PER017675 Cajamarca 2012 INIA C. annuum
PER007004 Ucayali 2012 INIA C. chinense
LCC-TROR Ucayali 2010 CIDRA C. chinense
3 Lambayeque 2011 UNALM C. annuum
PER017660 Lambayeque 2012 INIA C. annuum
123 San Martín 2011 UNALM C. chinense
PER017653 Lambayeque 2012 INIA C. annuum
PER017738 San Martin 2012 INIA C. baccatum
42 Huánuco 2012 UNALM C. baccatum
PER007035 Ucayali 2012 INIA C. chinense
PER007026 Ucayali 2010 INIA C. baccatum
157 Loreto 2011 UNALM C. baccatum
PER007020 Ucayali 2011 INIA C. frutescens
AMS-CR Ucayali 2010 CIDRA C. chinense
PER017710 San Martin 2012 INIA C. chinense
PER006992 Ucayali 2011 INIA C. chinense
PER017662 Lambayeque 2012 INIA C. annuum
AMS-CHAA Ucayali 2010 CIDRA C. chinense
4 Lambayeque 2011 UNALM C. annuum
LPI-CHAA Ucayali 2010 CIDRA C. chinense
PER006990 Ucayali 2011 INIA C. chinense
PER006942 Huanuco 2012 INIA C. chinense
LPI-TROA Ucayali 2010 CIDRA C. chinense
Appendix
215
Accession code
Growing region
Harvest year
Organization Species
PER017665 Lambayeque 2012 INIA C. annuum
238 Ucayali 2011 UNALM C. chinense
187 San Martín 2011 UNALM C. chinense
PER017667 Lambayeque 2012 INIA C. annuum
PER007021 Ucayali 2012 INIA C. chinense
LPI-NN-3 Ucayali 2010 CIDRA C. chinense
AMS-NN-4 Ucayali 2010 CIDRA C. chinense
42 Huánuco 2011 UNALM C. baccatum
PER006958 Ucayali 2011 INIA C. chinense
PER006965 Ucayali 2012 INIA C. chinense
AMS-NN-1 Ucayali 2010 CIDRA C. chinense
PER017732 San Martin 2012 INIA C. chinense
PER017712 San Martin 2012 INIA C. chinense
PER017784 Loreto 2012 INIA C. chinense
44 Huánuco 2012 UNALM C. chinense
PER017701 San Martin 2012 INIA C. baccatum
PER017664 Lambayeque 2012 INIA C. annuum
PER007023 Ucayali 2012 INIA C. chinense
PER006995 Ucayali 2011 INIA C. chinense
PER006952 Ucayali 2012 INIA C. chinense
AMS-CHI Ucayali 2011 CIDRA C. frutescens
PER017668 Lambayeque 2012 INIA C. annuum
PER007046 Ucayali 2012 INIA C. chinense
PER017672 Lambayeque 2012 INIA C. baccatum
PER017698 Cajamarca 2012 INIA C. chinense
PER017826 Loreto 2012 INIA C. annuum
PER017707 San Martin 2012 INIA C. chinense
PER007008 Ucayali 2011 INIA C. chinense
PER007009 Ucayali 2011 INIA C. chinense
SIT-PM Ucayali 2011 CIDRA C. frutescens
113 San Martín 2012 UNALM C. chinense
PER006988 Ucayali 2011 INIA C. chinense
PER017728 San Martin 2012 INIA C. frutescens
EHA-UU Ucayali 2010 CIDRA C. chinense
PER017787 Loreto 2012 INIA C. chinense
LPI-PUC Ucayali 2010 CIDRA C. chinense
175 San Martín 2012 UNALM C. chinense
AMS-M Ucayali 2011 CIDRA C. frutescens
Appendix
216
Table A 2: Ascorbic acid content, fat content, extractable color (ASTA 20.1) and surface color (hue-angle) for the 147 chili pepper accessions described in Chapter 4
Accession code
Ascorbic acid
(mg/100 g)
Fat content (g/100 g)
Extractable color
(ASTA 20.1)
Surface color
(hue-angle °) PER017909 6 12.9 22 48
PER017910 nd 19.6 27 69
153 24 10.9 25 65
157 nd 10.7 10 72
AMS-RC 12 12.8 25 52
PER017612 nd 11.9 60 40
PER017623 19 11.3 40 52
PER007040 116 15.2 47 67
PER006979 20 10.6 35 49
PER017711 7 9.4 40 44
PER017708 15 13.5 4 73
PER006984 nd 9.2 6 70
PER007013 nd 13.4 90 38
PER017908 nd 7.6 16 54
PER017735 nd 15.3 66 40
AMS-AD nd 15.5 41 45
PER017626 nd 15.1 107 36
PER017699 14 17.1 37 44
PER017704 8 11.9 4 74
85 19 17.1 20 57
252 7 9.5 40 45
PER017719 nd 16.6 92 36
132 nd 7.8 41 44
PER017648 nd 7.9 67 42
8 nd 11.0 6 73
PER017833 nd 8.4 111 34
PER017610 nd 9.2 137 36
PER017601 5 6.3 16 51
PER017875 16 5.1 63 41
PER017625 9 12.4 4 72
PER017679 nd 8.8 38 43
PER017608 9 6.3 12 70
202 8 8.8 11 63
PER017661 nd 5.0 4 54
PER017736 nd 11.5 60 39
PER017618 nd 6.0 13 50
PER017671 nd 8.0 58 42
85 nd 14.4 78 41
202 6 4.6 42 67
201 10 5.3 58 41
PER017705 17 8.9 27 54
7 9 6.6 55 68
PER017605 nd 12.1 51 45
PER006991 10 6.6 13 68
PER017621 nd 9.1 80 39
69 295 5.5 31 45
200 nd 5.9 66 41
5 6 4.8 25 47
5 5 6.2 3 69
PER017654 nd 7.7 18 51
10 14 17.1 14 47
2 nd 6.5 71 40
PER017893 nd 9.4 48 42
72 nd 7.5 37 45
Appendix
217
Accession code
Ascorbic acid
(mg/100 g)
Fat content (g/100 g)
Extractable color
(ASTA 20.1)
Surface color
(hue-angle °) EHA-CHAR nd 12.0 52 40
LPI-A nd 6.8 7 69
222 26 10.1 77 38
88 nd 11.8 45 44
132 10 5.4 21 49
PER017691 22 10.4 146 36
6 nd 12.9 75 38
69 nd 8.5 11 71
PER017692 6 7.6 18 53
PER017721 nd 7.3 55 39
PER006964 nd 6.4 18 47
PER007044 22 9.3 63 42
PER006957 nd 9.2 44 46
60 9 6.0 5 67
PER017635 nd 5.8 25 40
PER006951 nd 6.5 16 51
PER006954 5 7.8 14 53
PER06959 5 8.1 42 44
LCC-CHALL 6 11.1 82 41
PER06963 14 8.2 24 44
PER017633 5 5.5 11 56
PER006948 18 10.0 27 46
LPI-CHAR nd 8.3 17 49
157 23 5.4 1 84
PER017683 8 11.5 81 37
EHA-CA nd 10.1 72 40
PER017849 15 8.9 18 56
75 17 10.4 32 45
PER007025 6 6.6 22 45
PER006985 nd 7.2 34 45
PER007005 8 8.3 30 66
PER017682 nd 7.0 2 73
PER017675 nd 7.3 53 42
PER007004 nd 2.8 57 40
LCC-TROR nd 8.9 50 40
3 nd 6.9 3 63
PER017660 nd 6.2 4 72
123 5 7.5 16 53
PER017653 14 10.0 21 49
PER017738 nd 12.6 5 72
42 nd 6.3 10 56
PER007035 nd 8.9 36 48
PER07026 81 10.5 2 75
157 nd 11.2 43 46
PER007020 nd 13.2 32 42
AMS-CR nd 9.5 31 47
PER017710 nd 5.7 34 44
PER006992 nd 6.9 1 68
PER017662 nd 6.0 21 63
AMS-CHAA 14 6.6 21 56
4 12 6.8 18 68
LPI-CHAA 6 6.5 43 43
PER006990 nd 4.6 63 40
PER006942 nd 7.8 22 49
LPI-TROA nd 6.9 39 47
PER017665 nd 7.2 8 72
238 nd 4.6 28 65
187 5 5.2 18 70
Appendix
218
Accession code
Ascorbic acid
(mg/100 g)
Fat content (g/100 g)
Extractable color
(ASTA 20.1)
Surface color
(hue-angle °) PER017667 nd 4.3 8 69
PER007021 16 2.2 58 40
LPI-NN-3 nd 4.6 13 50
AMS-NN-4 7 4.9 27 65
42 nd 4.8 25 63
PER006958 6 4.8 14 63
PER006965 29 3.5 23 54
AMS-NN-1 nd 5.3 17 68
PER017732 nd 5.8 29 64
PER017712 66 5.3 15 73
PER017784 nd 7.2 4 69
44 7 4.1 24 63
PER017701 nd 5.8 41 42
PER017664 nd 3.3 52 40
PER007023 nd 9.1 41 49
PER006995 nd 6.5 4 68
PER006952 nd 3.1 50 40
AMS-CHI nd 2.8 24 47
PER017668 nd 4.0 27 49
PER007046 75 2.9 10 51
PER017672 nd 3.2 42 42
PER017698 nd 3.8 14 51
PER017826 nd 7.4 34 46
PER017707 21 9.5 45 54
PER007008 6 4.1 6 73
PER007009 nd 8.1 7 75
SIT-PM 36 2.6 27 47
113 nd 5.9 43 45
PER006988 nd 2.6 62 41
PER017728 5 2.8 5 71
EHA-UU 6 3.1 18 50
PER017787 nd 2.6 82 40
LPI-PUC nd 2.9 75 40
175 11 5.0 34 47
AMS-M nd 3.5 44 42
nd: not detectable
Appendix
219
Table A 3: Environmental information of the growing region
Descriptor Location
Chiclayo Piura Puccalpa
Coordinates -79.85 long.
-6.76 lati. -80.32 long.
-4.85 lati. -74.57 long.
-8.41 lati.
Sowing date 05 - 05 -2012 05 - 05 -2012 30 - 04 -2012
Transplanting date
19 – 06 - 2012 20 – 06 – 2012 22 – 06 – 2012
Harvesting date 08 -11- 2012 and
17-12 -2012 Last week of October 2012
First week of December 2012
Annual precipitation (mm)
1.4 0.0 818.1
Temperatures (ºC)
19.4-22.7 22.1-25.4 24.1-26.8
Altitude (m) 28 98 154
Fertilization
Organic, 200 kg of manure at start, a
second application after 20 days and a
third at the start of flowering
Three times: at start and second time: 11 kg urea, 7 kg diammonium phosphate, 10 kg potassium sulfate.
Third time: 6 kg urea, 3kg
diammonium phosphate and 20
kg potassium sulfate
Organic: 150 kg of poultry manure at
start. Each 15 days:
Abonofol (0.2%) until fruits started
to mature
Irrigation system Gravity through
grooves Drip irrigation Rain fed
Irrigation quantity
800 m3 in total
with intervals of 15 days (in total
10 times)
400 m3 in total
provided in irregular intervals dependent on the
water necesity
Control of pest and diseases
Integrated pest management
Integrated pest management
Integrated pest management
Parental soil material (non-consolidated
material and rock type)
In-situ weathered soil material,
limestone rock type
Alluvial soil material, unknown
rock type
Fluvial deposits, unknown rock
type.
Soil drainage Moderate High Moderate
Soil depth to groundwater table
50.1 – 100 cm > 150 cm 50.1 - 150 cm
Soil salinity 160 – 240 ppm <160 ppm <160 ppm
Soil erosion Low Low Low
Soil texture Loam Loam – Sandy
loam Loam
Appendix
220
Table A 4: Detailed information on the 96 Bolivian chili pepper accessions described in Chapter 7. Accessions are sorted according to ascending capsaicinoid content. The 12 accessions written in bold were replanted and reported in Chapter 7.
Accession code
Growing region
Harvest date
Organization Species
P9 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P6 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P10 Padilla May-2011 PROINPA C. baccatum
var. pendulum
319-1 Cochabamba May-2011 CIFP C. annuum
268 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
543 Santa Cruz May-2011 CIFP C. chinense
637 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
319-2 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
P14 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P19 Padilla May-2011 PROINPA C. baccatum
var. pendulum
4 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P3 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P1 Padilla May-2011 PROINPA C. baccatum
var. pendulum
3 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P13 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P8 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P15 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P2 Padilla May-2011 PROINPA C. baccatum var.
pendulum
485 Santa Cruz May-2011 CIFP C. annuum
80 Cochabamba Jul-2011 CIFP C. baccatum var.
pendulum
P18 Padilla May-2011 PROINPA C. baccatum
var. pendulum
P11 Padilla May-2011 PROINPA C. baccatum var.
pendulum
9 Padilla May-2011 PROINPA C. baccatum var.
pendulum
108 Padilla Jul-2011 PROINPA C. baccatum
var. pendulum
Appendix
221
Accession code
Growing region
Harvest date
Organization Species
P12 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P16 Padilla May-2011 PROINPA C. baccatum var.
pendulum
43 Padilla May-2011 PROINPA C. baccatum
var. pendulum
11 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P4 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P7 Padilla May-2011 PROINPA C. baccatum var.
pendulum
70 Mairana May-2011 CIFP C. baccatum var.
pendulum
339 A Santa Cruz Jun-2011 CIFP C. baccatum var.
baccatum
26 Padilla May-2011 PROINPA C. baccatum var.
pendulum
P5 Padilla May-2011 PROINPA C. baccatum var.
pendulum
61 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
194 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
7 Padilla May-2011 PROINPA C. frutescens
1 Padilla May-2011 PROINPA C. baccatum var.
pendulum
66 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
520 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
6 Padilla May-2011 PROINPA C. frutescens
13 Padilla May-2011 PROINPA C. baccatum var.
pendulum
102 A Mairana May-2011 CIFP C. baccatum var.
pendulum
25 Padilla May-2011 PROINPA C. baccatum var.
pendulum
60 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
102 R Mairana May-2011 CIFP C. baccatum var.
pendulum
P17 Padilla May-2011 PROINPA C. baccatum var.
pendulum
10 Padilla May-2011 PROINPA C. baccatum var.
pendulum
48 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
502 Mairana May-2011 CIFP C. baccatum var.
pendulum
75 R Mairana May-2011 CIFP C. baccatum var.
pendulum
Appendix
222
Accession code
Growing region
Harvest date
Organization Species
34 Padilla Jul-2011 PROINPA C. baccatum var.
pendulum
86 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
256 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
103 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
582 Santa Cruz May-2011 CIFP C. chinense
MA 1680 Mairana May-2011 CIFP C. baccatum var.
pendulum
146 Santa Cruz May-2011 CIFP C. chinense
75 A Mairana May-2011 CIFP C. baccatum var.
pendulum
300 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
532 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
122 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
314 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
109 A Mairana May-2011 CIFP C. baccatum var.
pendulum
517 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
MA 1660 Mairana Jun-2011 CIFP C. baccatum var.
pendulum
339 R Santa Cruz Jun-2011 CIFP C. chinense
654 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
320 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
24 Mairana Jun-2011 CIFP C. baccatum var.
pendulum
312 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
TM Cochabamba Jun-2011 CIFP C. pubescens
MA 1679 Mairana May-2011 CIFP C. baccatum var.
pendulum
Sacaba Cochabamba Jun-2011 CIFP C. pubescens
139 Santa Cruz Jun-2011 CIFP C. chinense
542 Santa Cruz Jun-2011 CIFP C. chinense
384 Mairana Jun-2011 CIFP C. baccatum var.
baccatum
MA 1657 Mairana May-2011 CIFP C. baccatum var.
pendulum
Appendix
223
Accession code
Growing region
Harvest date
Organization Species
360 Mairana Jun-2011 CIFP C. baccatum var.
baccatum
353 Cochabamba May-2011 CIFP C. baccatum var.
pendulum
514 Santa Cruz Jun-2011 CIFP C. baccatum var.
pendulum
109 R Mairana May-2011 CIFP C. baccatum var.
pendulum
Proinpa 34 Padilla May-2011 PROINPA C. eximium
321 Santa Cruz May-2011 CIFP C. chinense
162 Cochabamba Jun-2011 CIFP C. baccatum var.
pendulum
MA 1638 Santa Cruz Jun-2011 CIFP C. baccatum var.
baccatum
341 Santa Cruz May-2011 CIFP C. baccatum var.
pendulum
366 Mairana Jun-2011 CIFP C. baccatum var.
pendulum
MA 1628 Mairana May-2011 CIFP C. baccatum var.
pendulum
MA 1631 Mairana May-2011 CIFP C. baccatum var.
baccatum
MA 1664 Mairana May-2011 CIFP C. baccatum var.
baccatum
Proinpa 35 Padilla May-2011 PROINPA C. eximium
Proinpa 31 Padilla May-2011 PROINPA C. baccatum var.
baccatum
Nueva Colecta Padilla May-2011 PROINPA C. eximium
MA 1648 Mairana Jun-2011 CIFP C. frutescens
581 Santa Cruz Jun-2011 CIFP C. frutescens
Appendix
224
Table A 5: Ascorbic acid content, fat content, extractable color (ASTA 20.1) and surface color (hue-angle) for the 96 chili pepper accessions described in Chapter 7
Accession code
Ascorbic acid
(mg/100 g)
Fat content (g/100 g)
Extractable color
(ASTA 20.1)
Surface color
(hue-angle °) 268 nd 14.9 67 38
319-1 132 6.7 77 46
P10 nd 12.9 34 54
P6 nd 13.2 127 36
P9 nd 14.4 13 63
543 7 11.3 55 48
637 19 10.3 57 48
319-2 20 11.6 79 42
P14 nd 8.5 54 44
P19 nd 8.6 57 46
4 nd 11.9 74 43
P3 nd 9.7 70 42
P1 nd 11.1 9 69
3 nd 10.7 8 70
P13 nd 10.4 55 44
P8 nd 10.8 86 40
P15 nd 14.1 45 42
P2 nd 8.3 62 31
485 33 9.7 58 47
80 nd 15.1 15 65
P18 6 11.2 25 61
P11 nd 14.1 10 66
9 nd 14 60 40
108 nd 16.2 66 40
P12 nd 15 10 68
P16 nd 9.8 68 44
43 nd 15.5 68 38
11 nd 11.8 65 42
P4 nd 8.5 35 56
P7 nd 12.1 75 41
70 10 12 61 45
339 A nd 19.4 23 67
26 nd 13.1 9 68
P5 nd 12 11 66
61 nd 15.1 12 63
194 24 18.6 20 68
7 nd 15.9 61 38
1 6 13.1 25 59
66 nd 7.6 11 72
520 13 12.8 88 43
6 nd 14.8 11 71
13 nd 9.8 79 43
102 A 9 24.9 19 63
25 6 17.1 31 55
60 nd 14.3 5 70
102 R 14 19.4 57 42
P17 nd 12.1 7 71
Appendix
225
Accession code
Ascorbic acid
(mg/100 g)
Fat content (g/100 g)
Extractable color
(ASTA 20.1)
Surface color
(hue-angle °) 10 nd 8.7 29 61
48 nd 17.9 16 65
502 28 17 59 42
75 R 29 15.6 60 39
34 nd 9.9 18 72
86 nd 14.2 80 40
256 nd 13.4 101 40
103 nd 11.7 6 75
582 216 7.3 99 44
MA 1680 42 20.2 32 47
146 9 11.8 39 49
75 A 12 17.6 18 68
300 30 18.4 57 44
532 30 13.6 20 53
122 10 13.3 60 45
314 5 12.7 74 47
109 A 27 19.6 19 66
517 21 27.7 39 39
MA 1660 nd 16.5 11 70
339 R nd 14.2 35 52
654 5 11.6 59 48
320 12 9.6 95 43
24 7 16.3 57 44
312 nd 15.2 8 69
TM nd 7.3 14 66
MA 1679 16 18.9 52 43
Sacaba 7 6.9 16 62
139 49 11.8 78 48
542 45 11.8 102 45
384 nd 26.1 23 46
MA 1657 6 16.7 12 71
360 6 23.3 36 39
353 17 20.4 56 41
514 nd 13.5 22 54
109 R 31 32.8 59 33
Proinpa 34 nd 21.4 33 43
321 6 13.4 8 76
162 11 12.2 31 50
MA 1638 nd 15.7 15 55
341 437 9.3 58 47
366 nd 24.3 38 40
MA 1628 6 14.3 70 45
MA 1631 nd 21.8 32 40
MA 1664 14 13.9 84 45
Proinpa 35 nd 18.9 12 52
Proinpa 31 nd 11.8 3 75
Nueva Colecta nd 19.4 26 44
MA 1648 nd 14.8 29 53
581 19 17.1 69 44
nd: not detectable
