Comparative Analysis of a Variety of Chili Peppers: Including

Chapter 3

Downloaded by CORNELL UNIV on November 18, 2012 | http://pubs.acs.org Publication Date (Web): November 15, 2012 | doi: 10.1021/bk-2012-1109.ch003

Comparative Analysis of a Variety of Chili Peppers: Including Components Identified in Chili Peppers for the First Time Neil C. Da Costa,* David Agyemang, Amanda M. Bussetti, Kenneth J. Kraut, and Laurence Trinnaman International Flavors & Fragrances Inc., 1515 Highway 36, Union Beach, NJ 07735 *E-mail: [email protected]

In-depth volatiles analysis was conducted on several highly pungent chili peppers, including habanero (Capsicum chinense), green Serrano, red chili (Capsicum annuum), cumari (Capsicum praetermissum) and red and green malagueta (Capsicum frutescens) varieties. Of interest amongst the many complex volatiles compositions, were several esters particularly common to chili peppers including the 4-methylpentyl analogues. In addition various capsaicinoids, macrocyclic lactones and aliphatic amides molecules are presented in this paper; some for the first time. Comparison of the compositional ratios of components in the steam distillation extracts are presented for various key components and between different liquid extraction techniques: steam distillation versus liquid/liquid extraction. In addition, for the several synthesized molecules, sensory evaluations are presented for the first time.

Introduction The genus Capsicum comprises of over two hundred varieties of chili peppers for which the “fruit” varies greatly in shape, size, color, flavor and degree of pungency. There are six main species: jalapeno, Serrano, bell (1–7) (Capsicum annuum), malagueta, Tabasco (8, 9), (Capsicum frutescens), cumari (Capsicum praetermissum), habanero, Scotch bonnet (10, 11) (Capsicum chinense), aji (Capsicum baccatum) and rocoto, manzano (Capsicum pubescens). Many © 2012 American Chemical Society In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


subspecies and hybrids stem from these. The chili pepper varieties studied and presented in this paper were habanero (Capsicum chinense), green Serrano, red chili (Capsicum annuum), cumari (Capsicum praetermissum) and red and green malagueta (Capsicum frutescens). In terms of Scoville rating units, the measure of piquancy or pungency, they can be classed as hot 10,000 to 25,000 units to ultra hot >100,000 units. Figure 1 shows the chili pepper fruits of all five varieties studied and their Scoville ratings.

Figure 1. Chili Pepper Varieties Analytically Studied. Many key components and characteristic esters have been reported in chili pepper volatiles (1, 2, 10, 11). Elmore et al (12) postulated various new esters in green Serrano chili peppers. These contained a moiety found in capsaicin and derived from 8-methyl-(E)-6-nonenoic acid (Fig. 2). They reported the ester derivatives where R = ethyl, 3-methylbutyl and 4-methylpentyl groups.

Figure 2. Capsaicin and Related Esters. 26 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Other key, high impact, trace components reported in chili peppers are αionone, β-ionone, (E)-β-damascenone and the highly potent pyrazine, 2-isobutyl3-methoxypyrazine (Galbazine); the latter having the characteristic aroma of green bell pepper (1, 2, 13). All four compounds (Fig. 3) were found to be present in trace quantities in all five chili pepper varieties studied.

Figure 3. Key Trace Components in Chili Peppers with Aroma Descriptors.

Materials and Methods Simultaneous Steam Distillation Extraction (SDE) Chili peppers (200 g) were finely chopped and charged into a LikensNickerson apparatus (5 L vessel) with 2.5 L distilled water. The peppers were extracted by steam distillation over 3 h into methylene chloride (150 mL) (all chemicals from Sigma-Aldrich, St. Louis, MO) with internal standard, diethyl phthalate. The cooled extract was dried over anhydrous sodium sulfate, filtered and concentrated to 1 mL using a Zymark Turbovap® (Caliper Technologies, Mountain View, CA).

Liquid/Liquid Extraction (L/L) Chili peppers (200 g) were finely chopped and placed in a 1 L beaker. Methylene chloride (400 mL) was added to cover the peppers and they were steeped overnight with occasional agitation. The solids were filtered off and the extract transferred to a separating funnel. A small quantity of water was separated off and the methylene chloride extract was dried over anhydrous sodium sulfate, filtered and concentrated to 2 mL using a Zymark Turbovap®. 27 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Gas-Chromatography Analysis (GC)

Each steam distillate and/or liquid/liquid extract was analyzed using an HP6890 gas chromatograph with split/splitless injection and a flame ionization detector (FID) (Hewlett Packard, Wilmington, PA). The extract was injected onto an OV-1 capillary column (50 m x 0.32 mm i.d., 0.5 μm film thickness, Restek, Bellefonte, PA) in split (split ratio 15:1) and splitless modes. Carrier gas was hydrogen with a flow rate of 1.0 mL/min. The injection port temperature was 250°C and the detector temperature 320°C. The column temperature program was from 40°C to 270°C at a rate of 2°C/min and a holding time at 270°C of 10 min. The extract was also injected into a HP6890 gas chromatograph with split/splitless injection and a flame ionization detector (FID) fitted with a carbowax capillary column (50 m x 0.32 mm i.d., 0.3 μm film thickness, Restek) using the same injection and detection parameters. GC oven temperature program was with an initial temperature of 40°C held for 10 min, ramped at 2°C/min to a final temperature of 220°C and held for 20 min. To aid in the detection of sulfur containing compounds, the extract was analyzed by HP6890 gas chromatograph equipped with an Antek chemiluminescence detector (Hewlett Packard, Wilmington, PA). The column was an OV-1 capillary column and the analysis was conducted in splitless mode. Injection and detection temperatures as well as temperature program were as described for the GC OV-1. All data was collected and stored by using HP ChemStation software (Hewlett Packard, Wilmington, PA).

Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

Identification of components in the extracts was conducted by mass spectrometry. The extracts were injected onto an HP6890 GC. The chromatographic conditions for the OV-1 column were the same as described for GC analysis. The end of the GC capillary column was inserted directly into the ion source of the mass spectrometer via a heated transfer line maintained at 280°C. The mass spectrometer was an Autospec high resolution, double-focusing, magnetic sector instrument (Micromass, Manchester UK). The mass spectrometer was operated in the electron ionization mode (EI), scanning from m/z 450 to m/z 33 @ 0.3 s per decade. For analysis on the carbowax phase (50 m x 0.32 mm i.d., 0.3 μm film thickness carbowax capillary column), the sample was introduced via an HP5890 GC into a Micromass Autospec mass spectrometer. GC oven conditions were the same as outlined above. Spectra obtained from both phases were analyzed on the MassLib data system (MPI, Mulheim/Ruhr, Germany) using an IFF in-house library and the commercial Wiley 8, NIST 98 and other libraries. The identification of flavor components was confirmed by interpretation of MS data and by GC linear retention indices based on calibration with alkanes.

28 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Gas Chromatography-Olfactometry Analysis (GC-O) and Chemical Evaluation

For the gas chromatography-olfactometry session, the extracts were injected into an HP6890 gas chromatograph equipped with flame ionization detector (FID) and odor port Model ODP-2 (Gerstel, Inc., Baltimore, MD). The FID:odor port split was 1:6. The chromatographic conditions were the same as described previously. Three trained panelists smelled through each of the five extracts, twice and recorded their aroma descriptors. From these it was possible to detect subtle aroma differences between the samples and propose new synthesis targets with potentially interesting aromas. To evaluate the synthesized compounds a panel of typically five flavorists was used. The compounds were evaluated for aroma on smelling strips and tasted in dilutions of salt and sugar water, respectively to try and give their optimum performance for flavor use. The panels used standardized flavor descriptors to record their evaluations.

Results and Discussion Figure 4 shows the total ion chromatographic profile differences between two extraction techniques for the red chili pepper variety. The liquid/liquid extract chromatogram shows the main esters and is dominated by the high concentration capsaicinoids and other higher molecular weight components. This makes it harder to detect and identify low concentration components in the baseline. In contrast the steam distillation extract gives great detail of trace components. The lack of high boiling components gives a cleaner extract and in this case lacks the higher concentration capsaicinoids. Thus these extraction techniques complement each other for quantification and quantitation purposes. Figure 5 shows a portion of the liquid/liquid chromatogram of red chilies and the homologous series of capsaicin related compounds plus their Scoville unit ratings, where known (14–21). Capsaicin and dihydrocapsaicin dominate the chromatogram and are the most pungent components detected so far in chili peppers. Note the lesser known capsaicinoids at either end of the series. Figure 6 shows another portion of the liquid/liquid chromatogram where a homologous series of aliphatic acetamides are newly reported in red chili peppers as far as the authors are aware. Not the whole series was synthesized, but could be postulated from mass spectral interpretation and predicted linear retention times.



Figure 4. Liquid/liquid extract v Steam Distillate of Red Chilis.



Figure 5. Capsaicinoids in Red Chili Peppers.



Figure 6. Aliphatic Amides in Red Chili Peppers.


Synthesis, Analytical, and Sensory


The following compounds (Fig. 7) were synthesized, based on analytical structural elucidation and interest by GC-olfactometry. Synthesis, analytical and sensory data are reported for all nine compounds.

Figure 7. Synthesized Chili Pepper Compounds.

A mixture of 4-methylpentanol (0.791 mol), aliphatic acid (0.659 mol) and sulfuric acid (0.033 mol) was heated at reflux for 1 h. The reaction mixture was cooled to room temperature, water added and stirred for 15 min. The organic layer was washed with saturated sodium bicarbonate solution followed by brine. The crude material was distilled under reduced pressure to give the product (0.499 mol). All other esters followed this synthesis route (Fig. 8).

Figure 8. Ester Synthesis Route. 33 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


4-Methylpentyl 2-methylbutyrate (1) (MW 186) C11H22O2, 500-MHz 1H NMR (CDCl3) 4.01-4.10 ppm (m, 2H), 2.32-2.40 ppm (m, 1H), 1.42-1.73 ppm (m, 5H), 1.20-1.26 ppm (m, 2H), 1.14 ppm (d, 3H, J=7.00 Hz), 0.91 ppm (t, 3H, J=7.45 Hz), 0.89 ppm (d, 6H, J=6.60 Hz), Flavor: fruity, creamy, waxy, savory, strawberry, apple, pear, soapy. 4-Methylpentyl 3-methylbutyrate (2) (MW 186) C11H22O2, 500-MHz 1H NMR (CDCl3) 4.05 ppm (t, 2H, J=6.78 Hz), 2.18 ppm (d, 2H, J=6.90 Hz), 2.06-2.15 ppm (m, 1H), 1.52-1.66 ppm (m, 3H), 1.20-1.26 ppm (m, 2H), 0.96 ppm (d, 6H, J=6.60 Hz), 0.89 ppm (d, 6H, J=6.60 Hz), Flavor: strawberry, pineapple, metallic, anisic, ripe fruit, berry, tutti frutti. 4-Methylpentyl heptanoate (3) (MW 214) C13H26O2, 500-MHz 1H NMR (CDCl3) 4.05 ppm (t, 2H, J=6.80 Hz), 2.29 ppm (t, 2H, J=7.55 Hz), 1.53-1.65 ppm (m, 5H), 1.25-1.35 ppm (m, 6H), 1.20-1.25 ppm (m, 2H), 0.89 ppm (d, 6H, J=6.65 Hz), 0.88 ppm (t, 3H, J=6.85 Hz), Flavor: solventy, ethereal, brown, green bean, metallic, slight pepper, cinnamyl, anise. 4-Methylpentyl nonanoate (4) (MW 242) C15H30O2, 500-MHz 1H NMR (CDCl3) 4.05 ppm (t, 2H, J=6.75 Hz), 2.29 ppm (t, 2H, J=7.48 Hz), 1.53-1.71 ppm (m, 2H), 1.20-1.30 ppm (m, 12H), 0.89 ppm (d, 6H, J=6.60 Hz), 0.88 ppm (t, 3H, J=7.35 Hz), Flavor: astringent, vegetative, green, slight alliaceous. 4-Methylpentyl benzoate (5) (MW 206) C13H18O2, 500-MHz 1H NMR (CDCl3) 8.04 ppm (d, 2H, J=7.30 Hz), 7.52 ppm (t, 1H, J=7.35 Hz), 7.41 ppm (t, 2H, J=7.63 Hz), 4.29 ppm (t, 2H, J=6.73 Hz), 1.72-1.79 ppm (m, 2H), 1.60 ppm (septet, 1H, J=6.63 Hz), 1.32 ppm (d, 2H, J=8.25 Hz, of t, J=7.20 Hz), 0.91 ppm (d, 6H, J=6.65 Hz), Flavor: vinyl, ethereal. Ethyl 8-methyl-(E)-6-nonenoate (6) (MW 198), C12H22O2, 500-MHz, 1H NMR (CDCl3) 5.28-5.47 ppm (m, 2H), 4.08 ppm (q, 2H, J = 7.1 Hz), 2.30 ppm (t, 2H, J = 7.3 Hz), 2.24 ppm (m, 1H), 1.97 ppm (m, 2H), 1.57 ppm (m, 2H), 1.36 ppm (m, 2H), 1.24 ppm (t, 3H, J = 7.1 Hz), 0.95 ppm (d, 6H, J = 6.8 Hz). Aroma; green, thiamine, Flavor: sulfurol like, thiamine, chicken, blue cheese, fatty, waxy, cooked, green. A mixture of pentadecan-1-amine (10.0 g, 0.044 mol) and triethylamine (4.89 g, 0.048 mol) in dichloromethane (200 mL) was cooled to -10°C. Acetyl chloride was added drop wise. After stirring for 1 h at

Comparative Analysis of a Variety of Chili Peppers: Including

Recommend Documents