Chemical Characterization of Native Chili Peppers (Capsicum spp.)
to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.)
Faculty of Mathematics and Natural Sciences of the Bergische Universität Wuppertal
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-toyear 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 dihydrocapsaicin
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. 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,
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,
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
Herrn Prof. Dr. Heiko Hayen danke ich für die Unterstützung und hilfreichen
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.
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
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
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
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 iii
10. Materials and Methods .......................................................... 183 10.1
Chemicals ............................................................... 183
Sample Pretreatment .............................................. 184
Extraction and Analysis of Capsaicinoids ................ 184
Flavonoid Analysis .................................................. 185
Determination of Total Polyphenols ......................... 186
Trolox Equivalent Antioxidant Capacity (TEAC) ...... 187
Analysis of Ascorbic Acid by HPLC ......................... 187
Tocopherols by HPLC ............................................. 188
Determination of Fat Content .................................. 189
10.9.1 Gravimetric Method ................................................. 189 10.9.2 NIR Method ............................................................. 189 10.10
Determination of Extractable Color.......................... 190
Measurement of Surface Color ............................... 191
Determination of Moisture Content .......................... 191
11. List of Publications ................................................................ 192 11.1
Original Papers ....................................................... 192
Conference Contributions ....................................... 193
12. References ............................................................................. 195 13. Appendix ................................................................................ 213
Chili Peppers 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 . Capsicum specific starch fossils found from the Bahamas to south Peru indicate the early cultivation and domestication of the genus 6,000 years ago . 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 preColumbian Mexico . 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 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 a Main nutrients Water 88.0 g Protein 1.9 g Lipids 0.4 g Sugars 5.3 g Minerals a Potassium Calcium Magnesium Iron
322 mg 14 mg 23 mg 1 mg
Vitamins b Provitamin A Vitamin C Vitamin E
18 mg 206 mg 16 mg
Mean values for hot, raw, red chili peppers from United States Department b of Agriculture (USDA) - Nutrient Database  and values for selected chili peppers from Wahyuni et al. .
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 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,
(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.
40000 35000 30000 25000 20000 15000 10000 5000 0
4000 3500 3000 2500 2000 1500 1000 500 0
[Mio US $]
Global Capsicum production and export values
Figure 1-1: Global Capsicum production in 1000 metric tons obtained from FOASTAT (Food and Agriculture Organization of the United Nations)  and export values in million US $ obtained from International Trade Centre (ITC)  between 2002 and 2012.
Chili Peppers 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 .
Top ten fresh chili pepper producing countries
16023 3000 2500 2000 1500 1000 500 0
Top ten dried chili pepper producing countries 300 [1000*t]
250 200 150 100 50 0
Figure 1-2: Top ten pepper producing countries for fresh and dried chili peppers in 2012; (from FOASTAT ).
Taxonomy and Botany 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) . Above the species level, the taxonomy of the genus Capsicum is well described :
Kingdom: Division: Class: Order: Family: Subfamily: Tribe: Subtribe: Genus:
Plantae Magnoliophyta Magnoliopsida Solanales Solanaceae Solanoideae Solaneae Capsicinae Capsicum
However, taxonomic classification is discussed controversially within the genus and several of the relationships between the different species are not well understood . 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 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  reported a number of 36, while Bosland and Votava  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 . The five domesticated and economic important species are Capsicum annuum
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) . 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. 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 . Because of their common gene pool, all three domesticated
Taxonomy and Botany Pickersgill stated that their status as distinct species is questionable . Walsh and Hoot analyzed DNA sequences from noncoding regions of the chloroplast genome (atpB-rbcL) and five introns within the nuclear waxy gene . 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 . 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  and Ibiza et al.  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 . Onus and Pickersgill confirmed possible hybridization with C. baccatum . 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 C. annuum var. annum C. annum var. glabriusculum C. annuum - complex
C. chinense C. frutescens
C. galapagoense C. baccatum var. pendulum C. baccatum var. baccatum 2n=24a
C. baccatum - complex
C. chacoense C. praetermissum C. cardenassii
C. pubescens - complex
C. eximium C. pubescens C. eshbaughii
C. parvifolium C. tovarii
C. buforum C. cornutum 2n=26a
C. lanceolatum C. pereirae C. schottianum
C. campylopodium C. friburgense C. mirabile C. rhomboideum C. villosum
C. caballeroi C. coccineum unknown
C. dusenii C. hookerianum C. leptopodum C. recurvatum
C. ceratocalyx C. dimorphum
C. geminifolium C. hunzikrianum C. minuntiflorum C. scolnikianum
Figure 1-3: Relationship of all 36 different Capsicum species mentioned by Eshbaugh ; Species are classified according to their number of a chromosome ( ) 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 . Moreover, hybridization with other species is very difficult and typically fails or
Taxonomy and Botany 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. pubescens are often fertile and allow
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 .
Thousands of different accessions were collected and conserved in various germplasm banks worldwide. The US National Plant Germplasm
approximately 5,000 Capsicum accessions . 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 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
(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
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 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%) . 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
Quality Parameters 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].
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 .
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) .
Chili Peppers Table 1.2: Classification of chili peppers according to their pungency  Capsaicinoids Scoville Heat Group Class (mg/100 g) Units non-pungent / I 0 - 4.4 0 - 700 paprika II middle pungent 4.4 - 18.8 700 - 3000 moderately III 18.8 - 156.3 3000 - 25000 pungent IV highly pungent 156.3 - 437.5 25000 - 70000 very highly V > 500 > 80000 pungent 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 . 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 . 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 . Carotenoids are sensitive to oxidative conditions
approximately 15% can enhance the stability of carotenoids during storage and reduce the degradation of color . 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 .
Quality Parameters 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,
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 . 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 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 . 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].
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 . 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 Other capsaicinoids are minor compounds and their contribution to the pungency is limited .
Capsaicin O O
Dihydrocapsaicin O O
Nordihydrocapsaicin Figure 1-6: Chemical structure of major capsaicinoids: capsaicin (8-methylN-vanillyl-trans-6-nonenamide), dihydrocapsaicin (8-methyl-N-vanillylnonanamide) 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) . 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 nordihydrocapsaicin (7-methyloctanoic acid) [47, 48]. These “capsinoids” are non-pungent, but they share with capsaicinoids the same capability to act as transient receptor
Chili Peppers potential vanilloid (TrpV1) agonist (Chapter 1.4.2) [49–51]. Later, Watanabe et al. discovered a second class of “capsaicin like” substances
(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 . Figure 1-7 depicts the major compounds of each class of substances.
O O HO
Figure 1-7: Comparison of the chemical structures of capsaicin (8-methyl-Nvanillyl-trans-6-nonenamide) and the structural analogs capsiate (8-methylO-vanillyl-trans-6-nonenamide) and capsiconiate (8-methyl-O-coniferyl-trans6-nonenamide)
Capsaicinoids and Analogs 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 . 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.
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) . Furthermore, various studies show that the production of capsaicinoids is highly influenced by the environment (e.g. Harvell and Bosland  or Gurung et al. [59, 60]).
Chili Peppers 1
p-Coumaroyl shikimate O
shikimate CoA Caffeoyl-CoA
p-Caffeoyl shikimate HO HO
Feruloyl-CoA O HO
3 elongation cycles
FAT S CoA
CS O O
Figure 1-8: Capsaicin biosynthetic pathway. 1: phenylpropanoid pathway, PAL phenylalanine ammonia lyase, C4H cinnamate 4-hydroxylase, 4CL 4coumaroyl-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 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 . 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  used 14
C labeling and Rangoowala  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 phenylpropanoid pathway and additional radioactive tracer experiments allowed Fujiwake et al. to postulate fundamental steps in the biosynthesis of vanillylamine in Capsicum fruits . 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-6nonenoic 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 isobranched 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 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+) . 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 . 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 .
Capsaicinoids and Analogs 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, sleepbreathing disorders and cancer . 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 . 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 . Another important fact is the exceptional high concentration needed for observing an anti-carcinogenic action of capsaicin .
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 (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.
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 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 . They found concentrations in fruits of Trinidad Moruga Scorpion (C. chinense) reaching more than two million SHU (~12.500 mg/100 g).
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.
shikimate/phenylpropanoid pathway and/or the polyketide pathway . 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 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 ) No. of C atoms 6
10 13 14
C6-C4 C6-C1-C6 C6-C2-C6
hydroxybenzoates acetophenones phenylacetates hydroxycinnamates phenylpropenes coumarins naphthoquinones xanthones stilbenes anthraquinones flavonoids
hydroquinone catechol 4-hydroxybenzoate 4-hydroxyacetophenone
18 30 n
(C6-C3)2 (C6-C3-C6)2 (C6-C1)n (C6-C3)n
lignans biflavonoids hydrolyzable tannins lignins
caffeate eugenol esculetin juglone 1,3,5,6,7-hydroxyxanthone resveratrol emodin quercetin luteolin kaempferol pinoresinol amentoflavone gallotannin guaiacyl lignins guaiacyl-syringyl lignins catechin polymers
Polyphenols 2´ 8 7 6
Figure 1-9: Flavan skeleton (2-phenylchroman); numbering of carbon atoms is according to .
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:
-OH at pos. 3 or/and 4
C=O at pos. 4
C=O at pos. 4 and -OH at pos. 3
C=C between pos. 2 and 3, C=O at pos. 4
C=C between pos. 2 and 3, C=O at pos. 4 and -OH at pos. 3
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 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
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 biosynthesis of flavonoids. Increased stress levels caused by pathogens, nutrient deficiency, UV radiation or wounding are factors that enhance the production of flavonoids . Beside the growing condition and maturity stage, the genotype is an important factor influencing the content and pattern of flavonoids . 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 . 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 which acts as precursor for different flavonols and anthocyanidins [101, 102]. Figure 1-10 depicts the common polyketide pathway in plants briefly.
O + NH3
PAL C4H 4CL
3x Malonyl-CoA S CoA
S CoA OH O
Polyketo acid - CoA CoA S O
Naringenin chalcone CHI
F3H OH OH
ANS OH Pelargonidin
OH OH O
Figure 1-10: Polyketide pathway leading to flavonoids including the enzymes: PAL phenylalanine ammonia lyase, C4H cinnamate 4hydroxylase, 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 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
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 . 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 . Nevertheless, the general positive health effect of flavonoids is described in several studies (e.g. [96, 97, 110]).
Chili Peppers 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 . 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 . 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 . Another cohort study with ~1,300 people could also show a reduced risk of dementia correlating with a high flavonoid intake . 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 .
Polyphenols 1.5.3 Analysis of 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-1picrylhydrazyl 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 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 . Later, Re et al. improved the assay procedure . Potassium persulfate oxidizes
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.
O S O O
K 2S 2O 8 O O Antioxidant S O
O S O O
O O S O
Figure 1-11: Reactions of ABTS (2,2´-azinobis-(3-ethylbenzothiazoline-6sulfonic 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 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 . Later, Singelton and Rossi optimized the assay to determine the total polyphenol content of wine . The FolinCiocalteu 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
-molybdates. Sequences of reversible one and two electron transfer reactions lead to blue species with a possible molecular formula of: (PMoW 11O40)-4 [114, 121]. Typically, the reaction is performed under alkaline conditions (sodium carbonate solution; pH 10), leading to a
Chili Peppers 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 . 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 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 . 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 . 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 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
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 500 mg/100 g) . The degree of pungency among different accessions or pepper types is usually very variable . 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 268 319-1 P10 P6 P9 543 637 319-2 P14 P19 4 P3 P1 3 P13 P8 P15 P2 485 80 P18 P11 9 108 P12 P16 75 R 43 256 11 MA 1680 300 P4 314 P7 MA 1660 70 320 339 A TM 139 26 MA 1657 P5 514 61 321 341 194 MA 1631 7 31 1Proinpa581 66 520 6 13 102 A 25 60 102 R P17 10
20 40 60 80 100
48 502 75 R 34 86 256 103 582 MA 1680 146 75 A 300 532 122 314 109 A 517 MA 1660 339 R 654 320 24 312 TM MA 1679 Sacaba 139 542 384 MA 1657 360 353 514 109 R Proinpa 34 321 162 MA 1638 341 500 366 MA 1628 MA 1631 MA 1664 Proinpa 35 Proinpa 31 Nueva Colecta MA 1648 581
250 500 750 1000
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.
Results and Discussion Individual levels for the three major capsaicinoids are shown in Figure 7-1 and 7-2. Capsaicin was found as the major capsaicinoid for
nordihydrocapsaicin. Both C. pubescens samples (Acc. code: TM and Sacaba) showed their typical pattern with high amounts of dihydroand nordihydrocapsaicin . 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 nordihydrocapsaicin 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 .
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 7.3.2 Specific Flavonoids When compared to other vegetables, chili peppers are a good source for flavonoids . 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 . 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 268 319-1 P10 P6 P9 543 637 319-2 P14 P19 4 P3 P1 3 P13 P8 P15 P2 485 80 P18 P11 9 108 P12 P16300 TM 43321 581 11 P4 0 P7 70 339 A 26 P5 61 194 7 1 66 520 6 13 102 A 25 60 102 R P17 10
48 502 75 R 34 86 256 103 582 MA 1680 146 75 A 300 532 122 314 109 A 517 MA 1660 339 R 654 320 24 312 TM MA 1679 Sacaba 139 542 20 30 384 MA 1657 360 353 514 109 R Proinpa 34 321 162 MA 1638 341 366 MA 1628 MA 1631 MA 1664 Proinpa 35 Proinpa 31 Nueva Colecta MA 1648 581
10 20 30 40 50
10 20 30 40 50
Figure 7-3. Individual flavonoid levels and pattern of the Bolivian chili pepper accessions (germplasm bank codes), sorted by ascending capsaicinoid content.
Characterization of Bolivian Chili Peppers The maximum flavonoid
(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 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 . 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
268 319-1 P10 P6 P9 543 637 319-2 P14 P19 4 P3 P1 3 P13 P8 P15 P2 485 80 P18 P11 9 108 P12 P16 43 11 P4 P7 70 339 A 26 P5 61 194 3 7 1 66 520 6 13 102 A 25 60 102 R P17 10
48 502 75 R 34 86 256 103 582 MA 1680 146 75 A 300 532 122 314 109 A 517 MA 1660 339 R 654 320 24 312 TM MA 1679 Sacaba 139 542 384 75 R MA 1657 582 532 360MA 1660 353 312 542 514 514 109 RMA 1638 MA 1664 Proinpa 34 581 321 0 162 MA 1638 341 366 MA 1628 MA 1631 MA 1664 Proinpa 35 Proinpa 31 Nueva Colecta MA 1648 581
0 1 2 3 4 5 6
Trolox /100 g)
Total polyphenols (g GAE /100 g)
TEAC (mmol Trolox /100 g)
Figure 7-5: Results of total polyphenols and the corresponding TEAC values. Accessions (germplasm bank codes) are sorted by ascending capsaicinoid content.
Results and Discussion
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. .
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 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 . 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 . 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 268 319-1 P10 P6 P9 543 637 319-2 P14 P19 4 P3 P1 3 P13 P8 P15 P2 485 80 P18 P11 9 108 P12 P16 43 11 P4 P7 70 339 A 26 P5 61 194 7 1 66 520 6 13 102 A 25 60 102 R P17 10
48 502 75 R 34 86 256 103 582 MA 1680 146 75 A 300 532 122 314 109 A 517 MA 1660 339 R 654 320 24 312 TM MA 1679 Sacaba 139 542 384 MA 1657 360 353 514 109 R Proinpa 34 321 162 20 MA 1638 341 366 MA 1628 MA 1631 MA 1664 Proinpa 35 Proinpa 31 Nueva Colecta MA 1648 581
256 532 654 139 109 R MA 1628 581
Figure 7-7: Tocopherol concentrations and pattern of the Bolivian accessions (germplasm bank codes), sorted by ascending capsaicinoid content.
Characterization of Bolivian Chili Peppers
25 20 15
Figure 7-8: Box plot analysis of the tocopherol content (sum of α-, β- and γtocopherol), 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 . Thermal stress during the drying process leads to degradation and to remaining levels down to ~10% . 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
Results and Discussion (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 . 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 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
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 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.
43 108 P18
P19 P14 P9
1.5 1.0 0.5 0.0
43 108 P18
43 108 P18
[mmol Trolox/100 g]
Total polyphenols [g GAE/100 g]
P19 P14 P9
P19 P14 P9
P19 P14 P9
P19 P14 P9
43 108 P18
50 40 30 20
43 108 P18
[mmol Trolox/100 g]
P19 P14 P9
43 108 P18
Surface color 4
125 100 75 50
43 108 P18
P19 P14 P9
43 108 P18
P19 P14 P9
Year 1 Year 2 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).
Characterization of Bolivian Chili Peppers Table 7.3: Source of variation of the main effects “Year” and “Accession” and their interaction expressed as mean squares Year Effect Year Accession × Trait Accession Capsaicinoids
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 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 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.
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 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 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.
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. . 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
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. . Trace B showed the results of applying a shorter column with smaller particles (Luna RP-18 column; 3 µm, 150 mm × 3 mm).
9 10 11 12 13 14 15 16 17 18 19 20 Minutes 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
Analytical and Experimental Background 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 . 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.  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
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 Collins et al.  showed very similar values when analyzing the same chili peppers powder.
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 FolinCiocalteu 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 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 Sample
Spice paprika Ají Panca Ají Ammarillo Red Pepper Thai Red Chili
Total polyphenols b (g GAE /100 g ± sdev)
TEAC assay (mmol Trolox /100 g ± sdev)
microtitre plate scale
microtitre plate scale
1.32 ± 0.03
1.23 ± 0.06
2.7 ± 0.2
3.1 ± 0.1
1.23 ± 0.04
1.23 ± 0.05
2.6 ± 0.2
3.1 ± 0.3
1.20 ± 0.04
1.18 ± 0.02
2.0 ± 0.1
2.3 ± 0.1
1.20 ± 0.07
1.21 ± 0.06
3.2 ± 0.2
3.7 ± 0.1
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 a standard deviation (sdev). Spice Paprika, Ají Panca and Ají Amarillo were obtained from the Peruvian cooperative Miski S.A., Red Pepper and Thai b Red Chili were obtained from Akzenta Wuppertal; GAE: gallic acid equivalents.
As mentioned before, both assays are highly influenced by the assay procedure . 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 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 . 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.
from the cooperative Miski S.A. (Lima, Peru) from Pepper-King Internet store (www.pepper-king.com)
Analytical and Experimental Background Control chart for total polyphenols [g GAE / 100 g]
2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4
Total polyphenol values
Upper control limit
Lower control limit
Control chart for TEAC [mmol Trolox / 100 g]
4.3 4.1 3.9 3.7 3.5 3.3 3.1 2.9 2.7
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.
Flavonoid Analysis 8.3
Chromatographic conditions: Quercetin,
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
7 8 9 10 11 12 13 14 15 16 Minutes 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 . 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
Analysis of Ascorbic Acid by HILIC disodium
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.
Analysis of Ascorbic Acid by HILIC
Chromatographic conditions: The chromatographic method was adapted from Nováková et al. . 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 Ascorbic acid
8 9 Minutes
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
chromatographic separation conditions described by Nováková et al. . 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
chromatographic performance. The result is a poor peak shape due to their higher elution power on HILIC columns . Consequently, the mobile phase (70% acetonitrile and 30% of 100 mM ammonium acetate pH 6.8) was used as extraction solvent. Additionally, dithiothreitol
Analysis of Tocopherols 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 . 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.
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 fused core column (2.6 µm, 150 mm × 4.6 mm) under isocratic conditions . 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).
8 9 10 11 12 13 14 15 16 17 Minutes 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 αtocopherol; B: standard solution; β: β-tocopherol, γ: -tocopherol, α: α-tocopherol.
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”.
Analysis of Tocopherols 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) .
80 60 40 20 0
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
Analytical and Experimental Background 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-, γtocopherols) 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 reextracted and re-analyzed then. 100
80 60 40 20
Extraction steps Sample 1
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%.
Analysis of Tocopherols 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 αtocopherol 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 α-Tocopherol content Method (mg/100 g) External calibration Standard addition (diluted 1:5) Standard addition (diluted 1:10)
22.6 24.3 25.5
Analytical and Experimental Background 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
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 .
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 ChiliPulvern”). 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
NIR Spectra 1 0.8 0.6 0.4 0.2 0 5000
First SavitzkyGolay derivation Derivatives of the NIR Spectra 0.004 0.000
-0.004 -0.008 -0.012 5000
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) . Besides, all spectra were reduced to the
Analytical and Experimental Background 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) . To determine the fat content, a partial least square (PLS) regression model was calculated out of the pretreated spectra. For crossvalidation 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.
Predicted (g/100 g)
Predicted vs. Reference 30 25 20 15 10 5 0
Slope Calibration 0,920 Validation 0,915
Offset 0,842 0,902
RMSE* 1,37 1,41
R2 0,920 0,915
Reference (g/100 g)
Explained Variance 100
90 80 70 60 0
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
Effect of Drying on Phytonutrients in Chili Peppers 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.
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 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.
60 40 20 0
fresh oven-dried 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 . 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).
Analytical Strategy 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.
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
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 threedigit 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 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 . 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.
Unpacking, registration, internal codification
NIR & CIE-L*a*b* values
-2 x 0.5 gFat determination (reference) Screen-meshing and milling of all particles larger than >850µm (according to ASTA method 1.0)
Analysis of color, fat reference analysis and by NIR
-total need 8 g-
-2 x 2 g-
-2 x 1 g-
Extractable color (ASTA 20.1) -2 x 0.5 gVitamin E determination
Analysis of valuable compounds and traits
-2 x 0.1 g-
-minimum need 13 gAnalysis of capsaicinoids, antioxidants, vitamin C and E -total need 5 g-
Flavonoid determination -2 x 1 gVitamin C determination -1 x 1 g-
Extraction of capsaicinoids and antioxidants
Analysis of major capsaicinoids Total polyphenols (Folin-Ciocalteu)
-2 x 1 gAntioxidant capacity (TEAC assay)
Figure 8-11: Analytical strategy for the determination of different traits in chili pepper powders.
Concluding Remarks and Future Perspectives 9.
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 . 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 Score Plot
Factor 2 (20%)
4 2 0 -2 -4 -6 -6
0 2 Factor 1 (26%) C. chinense
Loading Plot 0.4 h
Factor 2 (20%)
-0.1 -0.2 β-T
α-T T a* ASTA
-0.4 -0.3 -0.2 -0.1
Factor 1 (26%) 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), nordihydrocapsaicin (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.
Concluding Remarks and Future Perspectives 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
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 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) mean
Capsaicin mg /100 g
Bolivian accessions (n=96) mean
mg /100 g
Kaempferol Apigenin Total polyphenols
g GEA*/100 g
mmol T#/100 g
mg /100 g
γ- Tocopherol Ascorbic acid
mg /100 g
g /100 g
sum of capsaicin, dihydrocapsaicin and nordihydrocapsaicin; sum of c quercetin, luteolin, kaempferol and apigenin; sum of α-, β- and 1 # γ-tocopherol; nd: not detectable *GEA: gallic acid equivalents; T: Trolox
Concluding Remarks and Future Perspectives 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
(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 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 nordihydrocapsaicin. 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
observed, showing the individual response of accessions to changes in the growing conditions. Furthermore, an environmental impact factor was calculated. The factor allowed
Concluding Remarks and Future Perspectives 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
increase the content
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
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 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 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
butylhydroquinone, ABTS (2,2′-azino-bis(3-ethylbenzothiazo-line-6sulfonic 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′,5,7tetrahydroxy-flavone), apigenin (4′,5,7-trihydroxyflavone), nonanoic acid vanillylamide, natural capsaicin (65% 8-methyl-N-vanillyl-trans-6nonenamide, 30% 8-methyl-N-vanillyl-nonanamide, 5% N-vanillyl-7methyl-octanamide),
tocopherol were purchased from Sigma-Aldrich (Steinheim, Germany) Ascorbic acid, quercetin monohydrate (3,3′,4′,5,6-pentahydroxyflavone), acetic acid and DL-dithiothreitol (1,4-dimercapto-2,3butanediol), 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 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 . 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 Fluorescence detector was set to 280 nm for excitation and 320 nm for detection . 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,
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 . 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 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 . 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 10.6 Trolox Equivalent (TEAC)
The procedure described by Re et al. was applied . 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,
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 analysis using hydrophilic interaction liquid chromatography (HILIC) as described by Nováková et al. for ascorbic acid analysis . 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) (MachereyNagel, 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.-butylhydroquinone 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 determination. The method described by Grebenstein et al.  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
The method described by Schulte was used . 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.
NIR measurements were performed on a Jasco UV/Vis/NIRSpektrometer 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 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) . 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 . 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 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 :
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 .
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 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 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 ChiliProben 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 Interaktionschromatographie. Regionalverbandstagung NRW der Lebensmittelchemischen 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 Meckelmann S, Müller-Seitz E, Petz M (2011) Capsinoide: Die schärfefreien Strukturanaloga des Capsaicins - Analytik und Vorkommen in Chili-Varietäten. 40. Deutscher Lebensmittelchemikertag, 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.
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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
PER017909 PER017910 153 157 AMS-RC PER017612 PER017623 PER007040 PER006979 PER017711 PER017708 PER006984 PER007013 PER017908 PER017735 AMS-AD PER017626 PER017699 PER017704 85 252 PER017719 132 PER017648 8 PER017833 PER017610 PER017601 PER017875 PER017625 PER017679 PER017608 202 PER017661 PER017736 PER017618 PER017671 85 202 201 PER017705 7 PER017605 PER006991 PER017621 69 200 5 5 PER017654
Lima Lima Loreto Loreto Ucayali Lima Lima Ucayali Ucayali San Martin San Martin Ucayali Ucayali Lima San Martin Ucayali Lima Cajamarca San Martin La Libertad Lima San Martin San Martín Lambayeque Lambayeque Loreto Lima Lima Ayacucho Lima Cajamarca Lima Piura Lambayeque San Martin Lima Lambayeque La Libertad Piura Piura San Martin Lambayeque Lima Ucayali Lima La Libertad Piura Lambayeque Lambayeque Lambayeque
2011 2011 2011 2012 2010 2011 2011 2012 2011 2012 2012 2011 2012 2012 2012 2010 2012 2012 2012 2011 2011 2012 2011 2012 2012 2011 2012 2012 2011 2012 2012 2012 2011 2011 2012 2012 2012 2012 2012 2011 2012 2012 2012 2011 2012 2011 2012 2012 2011 2012
INIA INIA UNALM UNALM CIDRA INIA INIA INIA INIA INIA INIA INIA INIA INIA INIA CIDRA INIA INIA INIA UNALM UNALM INIA UNALM INIA UNALM INIA INIA INIA INIA INIA INIA INIA UNALM INIA INIA INIA INIA UNALM UNALM UNALM INIA UNALM INIA INIA INIA UNALM UNALM UNALM UNALM INIA
C. annuum C. annuum C. chinense C. chinense C. chinense C. annuum C. annuum C. chinense C. chinense C. chinense C. chinense C. chinense C. chinense C. annuum C. chinense C. chinense C. annuum C. chinense C. chinense C. chinense C. chinense C. chinense C. chinense C. baccatum C. chinense C. baccatum C. baccatum C. baccatum C. baccatum C. baccatum C. baccatum C. baccatum C. chinense C. baccatum C. chinense C. baccatum C. annuum C. chinense C. chinense C. baccatum C. chinense C. chinense C. baccatum C. chinense C. baccatum C. chinense C. chinense C. baccatum C. baccatum C. annuum
Appendix Accession code
10 2 PER017893 72 EHA-CHAR LPI-A 222 88 132 PER017691 6 69 PER017692 PER017721 PER006964 PER007044 PER006957 60 PER017635 PER006951 PER006954 PER006959 LCC-CHALL PER006963 PER017633 PER006948 LPI-CHAR 157 PER017683 EHA-CA PER017849 75 PER007025 PER006985 PER007005 PER017682 PER017675 PER007004 LCC-TROR 3 PER017660 123 PER017653 PER017738 42 PER007035 PER007026 157 PER007020 AMS-CR PER017710 PER006992 PER017662 AMS-CHAA 4 LPI-CHAA PER006990 PER006942 LPI-TROA
La Libertad Lambayeque Piura La Libertad Ucayali Ucayali Tumbes La Libertad San Martín Cajamarca Lambayeque La Libertad Cajamarca San Martin Ucayali Ucayali Ucayali Lima Lambayeque Ucayali Ucayali Ucayali Ucayali Ucayali Lambayeque Ucayali Ucayali Loreto Cajamarca Ucayali Puno La Libertad Ucayali Ucayali Ucayali Cajamarca Cajamarca Ucayali Ucayali Lambayeque Lambayeque San Martín Lambayeque San Martin Huánuco Ucayali Ucayali Loreto Ucayali Ucayali San Martin Ucayali Lambayeque Ucayali Lambayeque Ucayali Ucayali Huanuco Ucayali
2012 2012 2012 2011 2010 2010 2011 2011 2012 2012 2012 2012 2012 2012 2011 2011 2011 2012 2012 2011 2012 2010 2010 2010 2012 2012 2010 2012 2012 2010 2012 2012 2012 2012 2011 2012 2012 2012 2010 2011 2012 2011 2012 2012 2012 2012 2010 2011 2011 2010 2012 2011 2012 2010 2011 2010 2011 2012 2010
UNALM UNALM INIA UNALM CIDRA CIDRA UNALM UNALM UNALM INIA UNALM UNALM INIA INIA INIA INIA INIA UNALM INIA INIA INIA INIA CIDRA INIA INIA INIA CIDRA UNALM INIA CIDRA INIA UNALM INIA INIA INIA INIA INIA INIA CIDRA UNALM INIA UNALM INIA INIA UNALM INIA INIA UNALM INIA CIDRA INIA INIA INIA CIDRA UNALM CIDRA INIA INIA CIDRA
C. chinense C. baccatum C. baccatum C. baccatum C. chinense C. baccatum C. chinense C. chinense C. chinense C. chinense C. chinense C. chinense C. baccatum C. chinense C. baccatum C. baccatum C. chinense C. chinense C. annuum C. baccatum C. baccatum C. chinense C. baccatum C. baccatum C. annuum C. baccatum C. chinense C. chinense C. baccatum C. chinense C. baccatum C. chinense C. chinense C. chinense C. chinense C. chinense C. annuum C. chinense C. chinense C. annuum C. annuum C. chinense C. annuum C. baccatum C. baccatum C. chinense C. baccatum C. baccatum C. frutescens C. chinense C. chinense C. chinense C. annuum C. chinense C. annuum C. chinense C. chinense C. chinense C. chinense
Appendix Accession code
PER017665 238 187 PER017667 PER007021 LPI-NN-3 AMS-NN-4 42 PER006958 PER006965 AMS-NN-1 PER017732 PER017712 PER017784 44 PER017701 PER017664 PER007023 PER006995 PER006952 AMS-CHI PER017668 PER007046 PER017672 PER017698 PER017826 PER017707 PER007008 PER007009 SIT-PM 113 PER006988 PER017728 EHA-UU PER017787 LPI-PUC 175 AMS-M
Lambayeque Ucayali San Martín Lambayeque Ucayali Ucayali Ucayali Huánuco Ucayali Ucayali Ucayali San Martin San Martin Loreto Huánuco San Martin Lambayeque Ucayali Ucayali Ucayali Ucayali Lambayeque Ucayali Lambayeque Cajamarca Loreto San Martin Ucayali Ucayali Ucayali San Martín Ucayali San Martin Ucayali Loreto Ucayali San Martín Ucayali
2012 2011 2011 2012 2012 2010 2010 2011 2011 2012 2010 2012 2012 2012 2012 2012 2012 2012 2011 2012 2011 2012 2012 2012 2012 2012 2012 2011 2011 2011 2012 2011 2012 2010 2012 2010 2012 2011
INIA UNALM UNALM INIA INIA CIDRA CIDRA UNALM INIA INIA CIDRA INIA INIA INIA UNALM INIA INIA INIA INIA INIA CIDRA INIA INIA INIA INIA INIA INIA INIA INIA CIDRA UNALM INIA INIA CIDRA INIA CIDRA UNALM CIDRA
C. annuum C. chinense C. chinense C. annuum C. chinense C. chinense C. chinense C. baccatum C. chinense C. chinense C. chinense C. chinense C. chinense C. chinense C. chinense C. baccatum C. annuum C. chinense C. chinense C. chinense C. frutescens C. annuum C. chinense C. baccatum C. chinense C. annuum C. chinense C. chinense C. chinense C. frutescens C. chinense C. chinense C. frutescens C. chinense C. chinense C. chinense C. chinense C. frutescens
Appendix 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 PER017910 153 157 AMS-RC PER017612 PER017623 PER007040 PER006979 PER017711 PER017708 PER006984 PER007013 PER017908 PER017735 AMS-AD PER017626 PER017699 PER017704 85 252 PER017719 132 PER017648 8 PER017833 PER017610 PER017601 PER017875 PER017625 PER017679 PER017608 202 PER017661 PER017736 PER017618 PER017671 85 202 201 PER017705 7 PER017605 PER006991 PER017621 69 200 5 5 PER017654 10 2 PER017893 72
6 nd 24 nd 12 nd 19 116 20 7 15 nd nd nd nd nd nd 14 8 19 7 nd nd nd nd nd nd 5 16 9 nd 9 8 nd nd nd nd nd 6 10 17 9 nd 10 nd 295 nd 6 5 nd 14 nd nd nd
12.9 19.6 10.9 10.7 12.8 11.9 11.3 15.2 10.6 9.4 13.5 9.2 13.4 7.6 15.3 15.5 15.1 17.1 11.9 17.1 9.5 16.6 7.8 7.9 11.0 8.4 9.2 6.3 5.1 12.4 8.8 6.3 8.8 5.0 11.5 6.0 8.0 14.4 4.6 5.3 8.9 6.6 12.1 6.6 9.1 5.5 5.9 4.8 6.2 7.7 17.1 6.5 9.4 7.5
22 27 25 10 25 60 40 47 35 40 4 6 90 16 66 41 107 37 4 20 40 92 41 67 6 111 137 16 63 4 38 12 11 4 60 13 58 78 42 58 27 55 51 13 80 31 66 25 3 18 14 71 48 37
48 69 65 72 52 40 52 67 49 44 73 70 38 54 40 45 36 44 74 57 45 36 44 42 73 34 36 51 41 72 43 70 63 54 39 50 42 41 67 41 54 68 45 68 39 45 41 47 69 51 47 40 42 45
Appendix Accession code
Ascorbic acid (mg/100 g)
Fat content (g/100 g)
Extractable color (ASTA 20.1)
Surface color (hue-angle °)
EHA-CHAR LPI-A 222 88 132 PER017691 6 69 PER017692 PER017721 PER006964 PER007044 PER006957 60 PER017635 PER006951 PER006954 PER06959 LCC-CHALL PER06963 PER017633 PER006948 LPI-CHAR 157 PER017683 EHA-CA PER017849 75 PER007025 PER006985 PER007005 PER017682 PER017675 PER007004 LCC-TROR 3 PER017660 123 PER017653 PER017738 42 PER007035 PER07026 157 PER007020 AMS-CR PER017710 PER006992 PER017662 AMS-CHAA 4 LPI-CHAA PER006990 PER006942 LPI-TROA PER017665 238 187
nd nd 26 nd 10 22 nd nd 6 nd nd 22 nd 9 nd nd 5 5 6 14 5 18 nd 23 8 nd 15 17 6 nd 8 nd nd nd nd nd nd 5 14 nd nd nd 81 nd nd nd nd nd nd 14 12 6 nd nd nd nd nd 5
12.0 6.8 10.1 11.8 5.4 10.4 12.9 8.5 7.6 7.3 6.4 9.3 9.2 6.0 5.8 6.5 7.8 8.1 11.1 8.2 5.5 10.0 8.3 5.4 11.5 10.1 8.9 10.4 6.6 7.2 8.3 7.0 7.3 2.8 8.9 6.9 6.2 7.5 10.0 12.6 6.3 8.9 10.5 11.2 13.2 9.5 5.7 6.9 6.0 6.6 6.8 6.5 4.6 7.8 6.9 7.2 4.6 5.2
52 7 77 45 21 146 75 11 18 55 18 63 44 5 25 16 14 42 82 24 11 27 17 1 81 72 18 32 22 34 30 2 53 57 50 3 4 16 21 5 10 36 2 43 32 31 34 1 21 21 18 43 63 22 39 8 28 18
40 69 38 44 49 36 38 71 53 39 47 42 46 67 40 51 53 44 41 44 56 46 49 84 37 40 56 45 45 45 66 73 42 40 40 63 72 53 49 72 56 48 75 46 42 47 44 68 63 56 68 43 40 49 47 72 65 70
Appendix Accession code
Ascorbic acid (mg/100 g)
Fat content (g/100 g)
Extractable color (ASTA 20.1)
Surface color (hue-angle °)
PER017667 PER007021 LPI-NN-3 AMS-NN-4 42 PER006958 PER006965 AMS-NN-1 PER017732 PER017712 PER017784 44 PER017701 PER017664 PER007023 PER006995 PER006952 AMS-CHI PER017668 PER007046 PER017672 PER017698 PER017826 PER017707 PER007008 PER007009 SIT-PM 113 PER006988 PER017728 EHA-UU PER017787 LPI-PUC 175 AMS-M
nd 16 nd 7 nd 6 29 nd nd 66 nd 7 nd nd nd nd nd nd nd 75 nd nd nd 21 6 nd 36 nd nd 5 6 nd nd 11 nd
4.3 2.2 4.6 4.9 4.8 4.8 3.5 5.3 5.8 5.3 7.2 4.1 5.8 3.3 9.1 6.5 3.1 2.8 4.0 2.9 3.2 3.8 7.4 9.5 4.1 8.1 2.6 5.9 2.6 2.8 3.1 2.6 2.9 5.0 3.5
8 58 13 27 25 14 23 17 29 15 4 24 41 52 41 4 50 24 27 10 42 14 34 45 6 7 27 43 62 5 18 82 75 34 44
69 40 50 65 63 63 54 68 64 73 69 63 42 40 49 68 40 47 49 51 42 51 46 54 73 75 47 45 41 71 50 40 40 47 42
nd: not detectable
Appendix Table A 3: Environmental information of the growing region Descriptor Coordinates Sowing date Transplanting date Harvesting date Annual precipitation (mm) Temperatures (ºC) Altitude (m)
Chiclayo -79.85 long. -6.76 lati. 05 - 05 -2012
Location Piura -80.32 long. -4.85 lati. 05 - 05 -2012
Puccalpa -74.57 long. -8.41 lati. 30 - 04 -2012
19 – 06 - 2012
20 – 06 – 2012
22 – 06 – 2012
08 -11- 2012 and 17-12 -2012
Last week of October 2012
First week of December 2012
98 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, 200 kg of manure at start, a second application after 20 days and a third at the start of flowering
Gravity through grooves
Control of pest and diseases Parental soil material (nonconsolidated material and rock type) Soil drainage Soil depth to groundwater table Soil salinity Soil erosion Soil texture
400 m in total provided in irregular intervals dependent on the water necesity Integrated pest management
Integrated pest management
In-situ weathered soil material, limestone rock type
Alluvial soil material, unknown rock type
Fluvial deposits, unknown rock type.
50.1 – 100 cm
> 150 cm
50.1 - 150 cm
160 – 240 ppm Low