Abstract
Microwave-assisted hexane or water extraction was used to extract flavor compounds from fresh red pepper fruit and also from traditionally and industrially made pepper pastes. The composition of the volatile components from each extract was analyzed using comprehensive two-dimensional gas chromatography with time of flight mass spectrometry. In total, 79 compounds were determined including 15 alcohols, 14 terpenes, 13 aldehydes, 12 ketones, 7 fatty acids, 6 fatty acid esters, and 6 browning reaction products. The major common compounds of fresh pepper and the pastes were 2-pentanone, 3-hexanol, acetic acid, oleic acid, and linoleic acid. The compounds (E)-2-undecenal, farnesol, 2-pentadecyn-1-ol, linolenic acid, and squalene were found only in the fresh pepper samples. Browning reaction products were observed in both of the pastes. However, their concentration was much higher in the industrially made one. Microwave-assisted hexane extraction from the traditionally made pepper paste gave the highest number of volatiles. Many volatiles considered to possess pleasant aroma characteristics were observed only in the traditionally made paste.
INTRODUCTION
Capsicum annuum L. (red pepper), a member of the family Solanaceae, is an important crop grown worldwide. The fruits are commonly used in foodstuffs because of their typical color, pungency, taste and/or distinctive aroma.[Citation1,Citation2] Ripe fruit of red pepper is used as a spice, a source of red pigment and as a vegetable in many countries. In addition to its use as a food, its powder disinfects the oral and gastric mucus membranes and destroys the pathogenic bacteria in the intestine.[Citation3]
Pepper paste is the most widely utilized form of red pepper in Turkey. Pepper paste is also a well-known fermented food product in Mexico, Spain, and Korea.[Citation4−Citation6] Pepper paste is added into variety of dishes as a coloring and flavoring ingredient. Other than its use as an ingredient, studies have shown that fermented pepper paste also has beneficial effects on human health such as anti-diabetic effects.[Citation7]
Two different methods exist for the production of pepper paste—traditional and industrial. Traditional pepper paste is prepared under sunlight for around four days, by mixing the paste regularly four times a day. In this traditional method, fermentation occurs under the sunlight by means of natural contaminant microrganisms and the paste then becomes rich in the desired aroma, flavors, and colors. However, this is a laborious process and with increasing consumer awareness of hygiene, an industrial method has recently been developed for pepper paste production. Industrial production involves the steps of separating the seeds and stem, washing, crushing, concentrating the paste, and finally packaging. The industrially made pepper paste is manufactured without fermentation and the desired aroma does not form properly.[Citation8] During the concentration (drying) process, some of the compounds present in the fresh pepper undergo degradation or loss by volatilization, while other new compounds appear.[Citation9] Van-Ruthet al.[Citation10] noticed the five most abundant compounds in dried red peppers were 3-methylbutanal, 2-methylbutanal, 3-methylbutyric acid, acetone, and hexanal. Cremer and Eichner[Citation11] reported the formation of hexanal, β-ionone, and 6-methyl-5-hepten-2-one in Capsicum annuum during processing.
Microwave-assisted extraction (MAE) has received great attention as a potential alternative to conventional extraction methods, mainly due to considerable savings in processing time, solvent consumption, and energy.[Citation12−Citation14] Researchers have studied the extraction of volatiles from different products using the MAE systems. However, there has been no research elucidating the volatile compounds coming from industrially and traditionally produced pepper paste.
The main aim of this study is to demonstrate the changes in the volatiles of sweet red pepper when the paste has been produced by different methods, namely traditional and industrial.
MATERIAL AND METHODS
Materials
Ripe, fresh, and undamaged fruits of red pepper (Capsicum annum L.) and industrially-produced pepper paste were purchased from a local market in Turkey. The steps involved in the industrial production of this pepper paste were known to be prewashing and sorting of peppers, separation of the seeds and stem, washing, crushing and preheating, sieving, concentration, pasteurization, and finally, packaging. The preparation of traditional homemade pepper paste involves washing, separation of seeds and stems, crushing, and then concentration (drying).[Citation8] Traditional concentration process is normally performed in large trays with regular mixing under the sunlight and it lasts around four days. Traditional paste used in this study was prepared in this way and the volatiles were extracted immediately. All chemicals were purchased from Sigma-Aldrich.
Methods
Sample preparation
The seeds and stems of fresh red peppers were separated and peppers were cut into small pieces (<5 mm). The moisture content of all the fresh and paste samples were determined by vacuum oven at 70°C and loss in weight was reported as moisture. The moisture contents of fresh pepper, industrially and traditionally produced pastes, were found to be 92.34, 75.98, and 42.38%, respectively.
Fresh pepper and the pastes produced by industrial and traditional homemade methods would be too high in water content for extraction efficiency by hexane. Samples of lower water content are necessary and also all samples should have the same water content. Therefore, further drying was needed and to avoid any deleterious effects of further drying, these samples were further dried in a desiccator to below 10% wet basis.The samples to be dried in the desiccator were homogeneously spread on glass petri dishes. The petri dishes were placed in a glass desiccator which contained PCl5 at the bottom. The glass desiccator was flushed with nitrogen gas and stored in the dark at ambient temperature (18°C) until the pepper samples were dry. The final moisture content of the pepper samples was less than 10%. Maximum drying time in the desiccator was 72 h.
MAE
Extractions were performed in an open-vessel system connected to a condenser. A Discover SP CEM microwave operating at 2450 MHz with a dynamic power control was used at atmospheric pressure. A fixed temperature was used.
The MAEs of volatiles from fresh red pepper, industrially and traditionally made pepper pastes, were carried out using optimized reaction conditions with the dynamic power mode held at fixed power (250 W) for 10 min, excluding time for heating-up and cooling-down. The samples (20 g) were subjected to open system microwave heating by adding solvent (40 mL) into 150 mL vessels at 250 W. Water and n-hexane were used as extraction solvents. The extraction temperatures were 95°C for water and 55°C for n-hexane. After microwave heating, the extraction mixtures were filtered over a Buchner to remove the remaining solid waste. In the case of extraction with n-hexane, the filtrates were dried over anhydrous MgSO4 and n-hexane was evaporated using a rotary vacuum evaporator until 2 mL hexane was left. In the case of extraction with water, the filtrates were subjected to extraction with n-hexane twice over (20 mL in each). The n-hexane phase was collected and dried over anyhdrous MgSO4. Then, the remaining n-hexane was evaporated using a rotary vacuum evaporator until 2 mL n-hexane was left. The remaining extracts were transferred into vials. The samples were kept at –18°C until further analysis. All extraction experiments were carried in duplicate.
Chromatographic Analysis
The two-dimensional gas chromatography (GC × GC)–time of flight-mass spectrometry (TOF/MS) system consisted of an HP 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph and a Pegasus III TOF-MS (LECO, St. Joseph, MI, USA). GC × GC provides increased resolution and peak capacity over single column methods. High acquisition rates of TOF-MS offer a superior separation power. Combined GC × GC with TOF/MS provides a reliable basis for the automated analysis of complex samples.[Citation15,Citation16] The first column was a non-polar BPX (50 m × 0.32 mm i.d. × 1 μm film thickness) and the second column a BPX50 (2 m × 0.10 mm i.d. × 0.10 μm film thickness). Both columns were from SGE Analytical Science (VIC, Australia). The microwave extract (1 μL) was directly injected into the GC × GC-TOF/MS. The initial temperature of the first dimension column was 60°C for 1 min and the subsequent temperature program was a heating rate of 5°C min−1 until 280°C was reached and held isothermally for a further 1 min. The initial temperature of the second dimension column was 75°C for 1 min and a 5°C min−1 heating rate was used until 295°C was reached and held isothermally for further 1 min. Helium was used as a carrier gas at a constant flow of 1.0 mL min−1. Cryogenic modulation was performed using a jet-type modulator which was installed at the front of the second dimension column. The modulation time was 5 s. Peak identification was made using TOF/MS with electron ionisation. Mass spectra were compared against the NIST 2005 mass spectral library and Kovats indices from the literature. All injections were carried in triplicate.
RESULTS AND DISCUSSION
The volatile components of fresh sweet red pepper and those from pastes prepared using different techniques (industrial and traditional) were extracted using the microwave technique and analyzed by GC × GC-TOF/MS. As has been discussed by Grimm et al.[Citation17] sample preparation is a crucial step for the further analysis of compounds. Various techniques for the extraction of volatiles from pepper have already been studied such as solid phase microextraction,[Citation2,Citation11] simultaneous distillation extraction,[Citation18] and static headspace extraction.[Citation19] However, losses of some volatile compounds, long extraction times, degradation of unsaturated or ester compounds through thermal or hydrolytic effects are the principal disadvantages of these extraction methods.[Citation20] In the last decade there has been an increasing demand for new extraction techniques, amenable to automation, with shortened extraction times and reduced organic solvent consumption, preventing pollution and reducing sample preparation costs.[Citation21] As the modern MAE techniques operate at a far higher temperature and pressure, and require shorter extraction times than traditional soxhlet or sonication technologies, they have greater extraction efficiency.[Citation22] Zhu et al.[Citation23] compared the microwave-assisted solvent extraction method (MASE) with conventional extraction methods for volatile organic acids and concluded that the proposed MASE procedure required a lower quantity of extraction solution and less time and also provided better recovery and reproducibility.
lists the compounds identified in our study from the volatiles of fresh red pepper and paste samples using the MASE technique together with their Kovats indices, corresponding percentage compositions and their standard deviations. It should be noted that the peak identification of components is based on both library mass spectra and Kovats indices. Identification was based on a mass spectral library search using similarity and reverse factors above 750 and 800, respectively. Lower values than these were counted as unknown and components having these low values were not compared for their Kovats indices. Mass spectral match factors (similarity and reverse factors) are often much higher for GC × GC compared with conventional one-dimensional GC due to better separation of components. Ozel et al.,[Citation16] Adahchour et al.,[Citation24] and Dalluge et al.[Citation25] also used similarity and reverse factors above 750 and 800, respectively.
A range of fatty acids and their esters (64.39%), ketones (10.07%), aldehydes (8.11%), alcohols (5.98%), and terpenes (2.72%) were found to be present in the volatiles of hexane extracted fresh red peppers. The major components identified in the fresh red pepper were palmitic acid (15.59%), oleic acid (14.27%), linoleic acid (10.84%), linolenic acid ethyl ester (9.03%), 9-Octadecenal (4.49%), and β-ionone (4.29%). In this study, the actual number of components for each type of extraction from each type of sample was more than 80. However, even if they were components defined by GC × GC-TOF/MS, they were discounted if they were present in a percentage of less than 0.1%. The overall number of volatile compounds (present in percentages above 0.1%) of fresh red pepper obtained by microwave-assisted hexane extraction was found to be 42. However, previous studies of the volatile compounds of fresh red pepper yielded somewhat different results. Mateo et al.[Citation18] and Vidal-Aragon et al.[Citation9] reported 55 and 59 compounds isolated from Spanish red peppers, respectively. Vidal-Aragon et al.[Citation9] found ethanol, hexane, 2-methylfuran, 2- and 3-methylbutanal, 2-pentanol, ethyl ether, 1-penten-3-ol, and hexanal as the major components while Vidal-Aragon et al.[Citation26] found 3-methylpentane, 2-methylpentane, and ethanol as the major components. These differences could be because of different varieties of pepper and extraction techniques applied. In our study, we found 9-octadecenal and β-ionone to be the major components following the fatty acids and their esters. β-ionone was present in a concentration of 4.29%. Guadayol et al.[Citation19] also found β-ionone as one of the major volatile components by simultaneous distillation extraction from paprika oleoresins. Lasekan and Abbas[Citation27] reported 9-octadecenal as the major component of steamed tropical almond nuts. In our study, hexanal, nonanal, decanal, pentadecanal and 9-octadecenal were the
TABLE 1 Effect of production methods and microwave assisted extraction techniques on the compositions of the volatiles of sweet red pepper paste
It was noted that the method of preparation of red pepper paste resulted in a change in compositions of their aromas. The number of components in the volatile fractions obtained from the fresh, industrially, and traditionally made paste samples were 42, 39, and 64 respectively in the case of microwave-assisted hexane extraction. The number of identified components common to all fresh and pepper paste samples was 18. (E)-2-undecenal, farnesol, 2-pentadecyn-1-ol, linolenic acid, and squalene were found only in the fresh pepper samples and disappeared completely with the application of the paste preparation techniques.
The major volatile components of pepper changed drastically when concentrated by the industrial and traditional techniques. Apart from fatty acids, some of the main components identified were 5-methylfurfural, furfural, 5-hydroxymethylfurfural (5-HMF), and 3-methyl- 2(5H) furanone. They are known to be the result of browning reactions. All these browning reaction products appeared only in the pastes, not in the fresh pepper and appeared in both industrially and traditionally produced pastes. However, the concentrations of the overall browning reaction products found in traditional paste (8.82%) were much lower than those found in the industrial one (21.18%). The existence of furfural and the pyrazines is attributable to Maillard reactions. Its occurrence was also suggested in hot-air dried capsicums.[Citation18] It is known that heat treatment of fruits and vegetables often reduces the number of original volatile flavor compounds, while at the same time, introducing additional ones through the auto-oxidation of unsaturated fatty acids, decomposition of amino acids, and thermal decomposition and/or initiation of caramelization and/or Maillard reactions. Benzeneacetaldehyde, citral, p-xylene, and 2,6-dimethyl pyrazine also appeared only in the paste samples and not the fresh ones. Seventeen volatile components appeared only in the traditionally produced paste and not in the industrially produced one or in the fresh samples. These were mainly fermentation and degradation products and were terpenes, ketones, alcohols, and aldehydes. Most of these compounds such as benzaldehyde, phenylethylalcohol, aromadendrene, safranal, and maltol are known to be pleasantly flavored compounds desirable in foodstuffs. Lunning et al.[Citation28] studied how hot air drying at 65°C affected the amount of volatile compounds found in peppers. They also found that some of the components (β-ocimene, hexanol, 3-hexenol, and heptyl 6-methyl-2-propenoate) had changed considerably.
Microwave-assisted water extraction of the samples, when compared with hexane, also resulted in a decrease in the number of volatile compounds and their amounts. The number of volatile compounds found by microwave-assisted water extraction of fresh pepper, industrially, and traditionally produced pastes was found to be 22, 30, and 29, respectively. In contrast, when using hexane as the solvent, these numbers were 42, 39, and 64. In the case of the traditionally produced paste, it was observed that the number of volatiles obtained by microwave-assisted hexane extraction reduced from 64 to 29 when they were obtained by microwave-assisted water extraction. This could be explained by the lower solubility of these compounds in water. However, one other reason may be their low concentration in the traditional paste.
CONCLUSIONS
The GC × GC separation chromatographically resolved hundreds components which when coupled for detection with TOF-MS gave high probability identifications. The comprehensive separation also separated a number of components which remained unresolved on a single GC column. It was found that the number of components obtained by microwave-assisted hexane extraction of fresh pepper, industrially, and traditionally produced pastes was 42, 39, and 64, respectively. When water was used as a solvent in the same technique, these numbers were lower at 22, 30, and 29. It has been shown that microwave-assisted hexane extraction as a sample preparation technique is advantageous in extracting more components even those in very low concentrations. The major components of the volatiles changed with the preparation (concentration) techniques. Traditionally produced pepper paste was better compared to the industrially produced product, in that a lesser number of degradation and browning products were produced during concentration and the components of its resulting volatile were seen to be more desirable with their pleasant flavor characteristics. The traditionally produced pepper paste was found to contain some important aromatic compounds (aldehydes, alcohols, and ketones) which were not detected in either the fresh pepper or the industrially produced paste.
FUNDING
The financial support of the UK Natural Environment Research Council is gratefully acknowledged. The authors would also like to acknowledge Gaziantep University (Turkey).
Additional information
Funding
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