Research Article Received: 18 September 2013

Revised: 5 February 2014

Accepted article published: 30 April 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6718

Comparison of techniques for the isolation of volatiles from cashew apple juice Karina L Sampaio,a* Aline C T Biasotoa,b and Maria Aparecida A P Da Silvaa Abstract BACKGROUND: The aim of this study was to compare the performance of the following techniques on the isolation of volatiles of importance for the aroma/flavor of fresh cashew apple juice: dynamic headspace analysis using PorapakQ® as trap, solvent extraction with and without further concentration of the isolate, and solid-phase microextraction (fiber DVB/CAR/PDMS). RESULTS: A total of 181 compounds were identified, from which 44 were esters, 20 terpenes, 19 alcohols, 17 hydrocarbons, 15 ketones, 14 aldehydes, among others. Sensory evaluation of the gas chromatography effluents revealed esters (n = 24) and terpenes (n = 10) as the most important aroma compounds. CONCLUSION: The four techniques were efficient in isolating esters, a chemical class of high impact in the cashew aroma/flavor. However, the dynamic headspace methodology produced an isolate in which the analytes were in greater concentration, which facilitates their identification (gas chromatography–mass spectrometry) and sensory evaluation in the chromatographic effluents. Solvent extraction (dichloromethane) without further concentration of the isolate was the most efficient methodology for the isolation of terpenes. Because these two techniques also isolated in greater concentration the volatiles from other chemical classes important to the cashew aroma, such as aldehydes and alcohols, they were considered the most advantageous for the study of cashew aroma/flavor. © 2014 Society of Chemical Industry Keywords: dynamic headspace analysis; solvent extraction; solid-phase microextraction; mass spectrometry; gas chromatography

INTRODUCTION Cashew apple (Anacardium occidentale L.) is a tropical fruit whose concentrated juice generates an annual revenue close to US$ 44 million for Brazil, surpassed only by orange and grape juices, products of great economic importance for the country.1 The popularity of cashew apple juice amongst consumers can be attributed mainly to the exotic aroma and flavor of the beverage. Since the composition of the volatile fraction of a food or beverage defines its aroma and has a great influence on the quality of its flavor,2,3 various studies have focused on the identification of the volatile compounds present in cashew apple, its juice and other by-products.4 – 13 One of the first investigations on the composition of the volatiles of fresh cashew apple juice used simultaneous extraction–distillation to isolate the volatiles.4 This technique, developed by Nickerson and Likens,14 is based on the separation of the volatile compounds of a food matrix by vacuum distillation, with the simultaneous concentration of the evaporated compounds by their condensation in a small amount of pure solvent. Macleod and Troconis,4 using 2-methyl butane to isolate the volatiles, identified 35 compounds in fresh cashew apple juice, the majority of which were hydrocarbons (14), followed by aldehydes (eight), terpenes (six), esters (three), alcohols (two) and ketones (two). Bicalho et al.,7 also employing simultaneous extraction–distillation and dichloromethane as the solvent, identified 68 volatiles in fresh cashew apple, the majority being esters (27), followed by terpenes (14), hydrocarbons (nine), acids (six), aldehydes (five), alcohols (four), lactones (two) and one ketone. J Sci Food Agric (2014)

Dynamic headspace methods are also commonly employed to isolate the volatile compounds present in cashew apple and its derived products. These methods use a flow of an ultra-pure gas such as nitrogen or helium, amongst others, to strip the volatiles present in the headspace of the sample to a trap, where the analytes are then trapped. The trap can consist of a cryogenic system2 or of porous polymers such as Tenax®, Chromosorb® and Porapak®15,16 packed into a small glass tube. Desorption of the captured volatiles can be carried out by eluting them with a solvent or by heating the polymer directly in the chromatograph injector. Maciel et al.5 used dynamic headspace analysis and a flow of nitrogen to strip the volatiles from a sample of fresh cashew apple juice into a trap containing Tenax (60/80 mesh), and identified 40 volatile compounds, of which 21 were esters, seven alcohols, five terpenes, two ketones, two acids, two sulfur compounds and one aldehyde. The esters were the major chemical class of the sample. In dynamic headspace methods, the gas used to strip the volatiles present in the headspace of the sample to the trap can

∗

Correspondence to: Karina L Sampaio, Department of Food and Nutrition, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, CEP 13083-862, Campinas, SP, Brazil. E-mail: [email protected]

a Department of Food and Nutrition, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, CEP 13083-862, Campinas, SP, Brazil b Brazilian Agricultural Research Corporation – EMBRAPA Tropical Semi-Arid, Rodovia BR-428, Km 152, CEP 56302-970, Petrolina, PE, Brazil

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www.soci.org be substituted by the use of a low vacuum produced by a water siphon17,18 or a vacuum pump,13 which in the latter case promotes economic advantages. The technique has been shown to be efficient in isolating the volatile compounds from various tropical fruits, such as umbu-caja (Spondias citherea), camu-camu (Myrciaria dubia), Brazilian guava (Eugenia spititata), cupuaçu (Theobroma grandiflorum),19 passion fruit,20 cashew apple9 and cashew apple by-products.13 Using the dynamic headspace analysis as modified by Franco and Rodrigues-Amaya,18 to isolate volatiles from fresh cashew apple juice, Garruti et al.9 identified 48 volatile compounds of which 24 were esters, nine were alcohols, seven aldehydes, three acids, two ketones, two lactones and 2-butoxy-ethanol, a polyfunctional compound. The esters represented the major chemical class, corresponding to approximately 50% of the total area of the chromatogram, followed by the aldehydes with 32%. Solvent extraction is one of the most traditional techniques used to analyze the totality of the volatile compounds present in foods and beverages.21 It is based on partition of the volatile compounds between two immiscible liquids, usually water and an organic solvent. Solvent extraction with dichloromethane was used by Sampaio et al.12 in the identification of the volatile compounds present in the water phase generated during the industrial concentration of cashew apple juice. Of the 71 compounds identified, 27 were esters, 21 alcohols, 11 acids, four aldehydes, four ketones, three lactones and one hydrocarbon. The alcohols represented 42% of the total area of the chromatogram, and were therefore the major chemical class in the water phase, followed by the esters which represented 21% of the total area of the chromatogram. Another technique that can be used to analyze the total volatiles present in a liquid sample is sorptive extraction.2 As proposed by Pawliszyn,22 solid-phase microextraction (SPME) involves the adsorption of the volatile compounds onto an inert fused silica fiber, coated with a stationary phase, following heat desorption of the volatiles directly in the gas chromatograph injector. For liquid samples, the volatiles can be isolated either by immersion of the fiber directly in the product or by exposition of the fiber in the sample headspace, a technique known as HS-SPME.23 Using the solid-phase microextraction methodology (HS-SPME) and the fiber polydimethylsiloxane (PDMS; 100 μm), Cardeal et al.10 identified 18 volatile compounds in processed cashew apple juice, of which seven were esters, four terpenes, three acids, two furans and one ketone. In turn, when evaluating the performance of four different SPME fibers in the isolation of volatile compounds from processed cashew apple pulp using the HS-SPME technique, Carasek and Pawliszyn11 showed that the fiber divinylbenzene/carboxen/PDMS (DVB/CAR/PDMS) presented better performance than the others [CAR/PDMS, 75 μm; PDMS, 100 μm and polyacrylate (PA), 85 μm], isolating 15 volatiles of which nine were esters, five terpenes and one lactone. According to these authors, PA (85 μm) offered the worst performance, isolating only six volatiles and demonstrating that for the HS-SPME technique, the type of fiber has a strong influence on the efficiency of the isolation of the volatiles from cashew apple pulp. The identification of the volatile compounds present in a food aims to understand the factors that positively and negatively influence the aroma and flavor of the product. However, different volatiles have different impact on the food’s aroma and flavor, with some volatiles not showing odor impact on the sample even when present at high concentration. Thus, in recent decades, research in flavor chemistry incorporated gas chromatography–olfactometry

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KL Sampaio, ACT Biasoto, MAAP Da Silva (GC-O) techniques in which, through sensory evaluation of chromatographic effluents, it is possible to identify the qualitative and quantitative contribution of each volatile on the aroma and flavor of the product.9,12,13,24 – 27 The GC-O techniques require the dilution of the volatiles separated by the chromatographic column in a stream of air at the exit of the column in order to carry the compounds to the judge’s nose. Therefore, on GC-O studies, the technique of isolation should produce not only an isolate representative of the sample’s aroma but also an isolate containing the volatiles in sufficiently high concentrations so that they can be perceived sensorially in the chromatography effluent after its dilution in a flow of air. Some GC-O techniques, such as Charm24 and aroma extract dilution analysis (AEDA),25 require the assessment of the isolate in several successive dilutions, which increases the need to obtain an isolate containing the volatiles at high concentrations. Thus, isolation techniques considered suitable for studies where the purpose is merely the identification of the volatile compounds present in the sample, may be inappropriate for GC-O studies where sensory evaluation of the volatiles separated by the chromatography column is also required. Despite the great interest in determining the composition of the volatiles of cashew apple and its derived products, and of the great impact that the isolation method has on the results, no studies were found in the literature that, using a single sample, directly compared the efficiency of the various techniques in the isolation of aroma volatiles of cashew apple. Thus the objective of the present study was to compare the techniques of dynamic headspace, solvent extraction and solid-phase microextraction (HS-SPME) in the isolation of volatiles of importance for the aroma/flavor of fresh cashew apple juice.

MATERIALS AND METHODS Raw material Pseudofruits of the early dwarf cashew apple, clone CCP 76, cultivated in the year of 2009 in the State of São Paulo, Brazil, on a property situated in the municipality of Artur Nogueira (22∘ 34′ 23′′ S and 47∘ 10′ 21′′ W, 588 m altitude) were used in this experiment. The mature material, after removal of the peduncle, was washed, ground in a blender and filtered through cotton cloth in order to produce fresh cashew juice. Extraction of the volatiles The following four isolation techniques were used to extract the volatiles: (1) the dynamic headspace with a PorapakQ® trap (HSD-PorapakQ®) employed by Garruti et al.9 for cashew apple and by Sampaio et al.13 for cashew water phase; (2) the solid-phase microextraction (HS-SPME) used by Valim et al.8 and by Carasek and Pawliszyn11 for processed cashew apple juice; (3) the liquid–liquid extraction with dichloromethane and extract concentration (CLLE) used by Sampaio et al.12 for cashew apple water phase; and (4) the liquid–liquid extraction with dichloromethane and no subsequent extract concentration (LLE). To compare the efficiency of these techniques, which are the most used to extract aroma volatiles from cashew apple and by-products, they were used as described by their respective authors, and therefore have distinct concentration factors, i.e. their quantitative relation ‘juice extracted:isolate obtained’ differ. Dynamic headspace analysis Three hundred grams of juice were placed in a 1000 mL glass flask and 90 g of NaCl added (Merck, Darmstadt, Germany) to avoid the

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Techniques for the isolation of volatiles from cashew apple juice occurrence of enzymatic reactions. The volatile compounds of the cashew apple juice were captured using the dynamic headspace technique developed by Franco and Rodrigues-Amaya18 and optimized by Garruti et al.9 to isolate the volatiles from fresh cashew apple juice. The trap consisted of a 10 cm long borosilicate glass tube with an internal diameter of 3 mm, filled with 100 mg of PorapakQ® (polymer of ethyl vinyl benzene–divinyl benzene), 80–100 mesh (Supelco, Bellefonte, PA, USA), which occupied 4 cm of the length of the trap. The volatiles were captured for 2 h under a vacuum of 70 mmHg with magnetic stirring. After capture, 300 μL of acetone (chromatographic grade; Mallinckrodt, Paris, KY, USA) were used to elute the volatiles from the trap. The procedure was carried out with three replications to obtain three isolates; each one being analyzed by gas chromatography–mass spectrometry (GC-MS). Solid-phase microextraction Based on the methodology described by Valim et al.,8 5 g of juice were placed in a 20 mL glass flask with a stopper equipped with a Teflon septum, and the flask covered with aluminium foil to avoid exposure of the juice to sunlight. The sample was stirred at 500 rpm and 25 ∘ C for 10 min using a magnetic stirrer. The volatiles present in the headspace of the juice were then isolated by solid-phase microextraction (SPME) using a 50/30 μm DVB/Carboxen/PDMS fiber (Supelco), which, according to the manufacturers, is ideal for a broad range of analyte polarities. Carasek and Pawliszyn11 showed that for processed cashew apple juice, this fiber was much more efficient in extraction when compared to the other fibers tested: PA (85 μm), CAR/PDMS (75 μm) and PDMS (100 μm). Preliminary trials were carried out in order to optimize the following variables, associated with the isolation of the volatiles: (1) capture time (30, 45 and 60 min), (2) exposure time of the fiber in the injector (3 and 5 min), (3) injector temperature (200, 220 and 250 ∘ C), and (4) initial temperature of the chromatographic column (45, 50 and 55 ∘ C). After an evaluation of the number of volatiles isolated and the resolution of the chromatographic peaks and their abundances, a capture time of 45 min was chosen, with desorption in the injector at 200 ∘ C for 5 min and an initial column temperature of 50 ∘ C. The procedure was carried out with three replications to obtain three (HS-SPME) isolates, all of which were analyzed by GC-MS. Liquid–liquid extraction The volatiles present in the cashew apple juice were also extracted using dichloromethane (chromatographic grade; Merck), a solvent showing good affinity for esters, terpenes, alcohols and other volatiles usually showing major presence in cashew apple and its derived products, as observed in the results obtained by Sampaio et al.12 The volatiles were extracted from 40 mL of cashew apple juice using three consecutive extractions with 5 mL dichloromethane. The extracts were then combined and centrifuged at 10 000 × g and 4 ∘ C for 5 min to separate the liquid fraction containing the dichloromethane and the extracted volatiles. The extract was concentrated to up to 0.5 mL in a flow of ultra-pure nitrogen, transferred to a hermetically sealed amber flask and stored at −18 ∘ C until analysis. The procedure was carried out with three replications to obtain three isolates (CLLE), each of which was analyzed by GC-MS. A second type of isolate was also obtained following the above procedures, but without the final concentration of the extract with nitrogen. This isolate was denominated as LLE and J Sci Food Agric (2014)

www.soci.org was also produced with three replications, each being analyzed by GC-MS. Sensory evaluation of the isolates To help identifying the technique that isolated the aroma volatiles in similar proportion they are usually found in cashew apple juice, a panel of 10 trained judges evaluated the intensity of cashew apple aroma present in the isolates generated by the techniques of dynamic headspace (HSD-PorapakQ®) and extraction with dichloromethane, with and without concentration (CLLE and LLE). The judges had previous experience in descriptive sensory analysis and were trained as described in Sampaio et al.12 For the evaluation, an aliquot of 10 μL of each isolate was transferred with the aid of a micropipette to a paper strip suitable for sensory evaluation of essences (IFF International Flavors & Fragrances, SP, Brazil) and submitted to the judge after 5 s, the time required for evaporation of the solvent. After smelling the strip, the judge rated the intensity of cashew apple aroma using an unstructured scale of 9 cm anchored at the left end to the term ‘weak’ and the right end to the term ‘strong’. The judges were also asked to describe the additional aromatic notes perceived in the strip. All isolates were evaluated in three replicates by all the judges. Intensity data was analyzed by ANOVA and Tukey test (P = 5%). The additional aromatic notes perceived by the panelists in the isolates were computed as % of citation and expressed on a polar coordinates graph. The sensory evaluation of the volatiles adsorbed in the SPME fiber requires their simultaneous thermal desorption from the fiber, followed by an immediate evaluation by the judge in a flow of air.28 This method is very different from the sensory technique employed for the other isolates, making their results inadequate for comparison. Furthermore the sensory evaluation of the HS-SPME isolate was not performed. Identification of the isolated volatiles The volatiles present in the isolates were identified in an Agilent 7890/5975 gas chromatograph mass spectrometer (Agilent, Palo Alto, CA, USA) operating under an ionization voltage of 70 eV in the scan mode, with an m/z range from 35 to 350. A DB-Wax (JW Scientific, Folsom, CA, USA) fused silica capillary column (30 m × 0.25 mm, 0.25 μm) was used to separate the volatiles, using the conditions previously optimized by Garruti et al.9 Initially the column temperature was maintained at 50 ∘ C for 8 min and then raised to 110 ∘ C at 4 ∘ C min−1 , then to 200 ∘ C at 16 ∘ C min−1 and finally maintained at 200 ∘ C for 20 min, giving a total run time of approximately 48 min. The injector was set in the splitless mode and maintained at 200 ∘ C, and helium was the carrier gas at a linear velocity of 41.8 cm s−1 . The injection volume was 2 μL. To help with identification of the volatile compounds, retention indices were calculated for the GC-MS data and compared with the indices reported in the literature.8,9,12,13,29 – 31 The retention index of each compound was calculated used the non-isothermal retention indices from temperature programming proposed by Van Den Dool and Kratz32 using a C7–C28 series of straight-chain alkanes (Polyscience 211 C kit; Polyscience, Chicago, IL, USA). The individual compounds were identified by: (1) comparing the experimentally obtained mass spectra of the compound with those provided by the NIST library (NIST/EPA/NIH Mass Spectral Library, version 2.0, 2008); (2) comparing the volatile retention index obtained using the DB-Wax column with the retention indices published in the literature for columns with the

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www.soci.org same polarity;8,9,12,13,29 – 31 and (3) comparing the volatile mass spectra with the mass spectra of pure compounds analyzed in the same GC-MS using the same methodological conditions. The compounds were considered identified when their mass spectra matched those available in the computerized library and their retention indices were similar to those reported in the literature. The compounds were considered positively identified if, in addition, their mass spectra and retention indices were similar to those of the pure standards. When the identification of the volatiles was based solely on their mass spectrometric data, the compounds were considered tentatively identified. In order to evaluate the repeatability of each technique (HSD-PorapakQ®, HS-SPME, CLLE and LLE), the mean abundances and coefficients of variation were calculated for each peak present on the chromatogram. The performance of each of the four isolation techniques was evaluated by comparing the number of volatiles isolated by each technique, the proportion of each chemical class present in each isolate and the values of the coefficients of variation of each chromatographic peak. Gas chromatography–olfactometry The odoriferous importance of each volatile present in the isolates generated through the techniques of dynamic headspace (HSD-PorapakQ®) and extraction with dichloromethane with and without further concentration with nitrogen (CLLE and LLE) was determined by using a gas chromatograph (Agilent model 7890) modified for olfactometry as described in Sampaio et al.12 and Sampaio et al.13 and the GC-O technique known as Osme.33 For the analysis, volatiles upon leaving the chromatographic column were mixed with synthetic air previously filtered in charcoal and humidified. Two trained judges evaluated the effluents into two replicates. Using both a time-intensity software-type and a 9 cm scale allocated on a computer monitor, the subject recorded the intensity of each aroma perceived in the effluents and described to the researcher, the quality of the odor perceived (floral, fruity, etc.).The collected data were integrated via a software named SCDTI13 producing an aromagram for each of the three isolates analyzed: dynamic headspace (HSD-PorapakQ®) and with dichloromethane extraction (CLLE) and without further concentration with nitrogen (LLE). Only volatiles perceived in at least two sensory evaluations of the GC effluents were included in the aromagram. Comparisons amongst the aromagrams and the chromatograms of the isolates allowed the identification of the odor impact volatiles present in each isolates. The chromatographic conditions were identical to those described above for the GC-MS analysis, except for the carrier gas, which in this case was hydrogen instead of helium.

RESULTS AND DISCUSSION Aroma profile of the isolates Table 1 shows the mean intensities of fresh cashew apple aroma perceived by the panelists in the isolates obtained by dynamic headspace (HSD-PorapakQ®) and by extraction with dichloromethane with and without subsequent concentration (LLE and CLLE). As can be seen, the three techniques isolated compounds important for the cashew apple aroma, since they all showed the fruit aroma at intensities ranging from moderate to weak. However, the isolates obtained with dichloromethane presented the cashew aroma at higher intensity (P ≤ 0.05) than the isolate obtained with the dynamic headspace (HSD-PorapakQ®).

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KL Sampaio, ACT Biasoto, MAAP Da Silva

Table 1. Mean intensityaof fresh cashew apple aroma perceived by the trained panel in the isolates analysed Aroma characteristic of cashewa

Isolate

5.24a 5.05a 2.05b

CLLE LLE HSD a

Intensity scale: 0 = none, 4.5 = moderate, 9 = strong. Means with letters in common do not differ significantly (P = 0.05) from each other according to Tukey’s test. HSD, dynamic headspace-PorapakQ®; LLE, liquid–liquid extraction; CLLE, liquid–liquid extraction followed by concentration with N2 .

paperboard

alcoholic

sweety/vanilla 35% 30 25 20 15 10 5 0

fermented/acid

HSD LLE

fruity

CLLE grassy/citric

fresh fruity

wood

floral

Figure 1. Percentage of citation of aromatic notes perceived by the trained panelists in the isolates obtained by dynamic headspace (HSD-PorapakQ®), by dichloromethane extraction without concentration (LLE) and by dichloromethane extraction followed by concentration with nitrogen (CLLE).

Figure 1 shows the aroma notes additional to ‘characteristic of cashew’ perceived by the panelists in the isolates. It is noted that the isolate obtained with dichloromethane and without subsequent concentration (LLE) stood out from the others by presenting additional aromatic notes described as ‘green/citrus’ and ‘fresh fruit’. In turn, in the dichloromethane isolate submitted to further concentration with N2 , aroma notes described as ‘sweet/vanilla’ were perceived more frequently; and in the isolate generated by the dynamic headspace technique, aroma notes similar to ‘woody’ were perceived with greater frequency. Each one of the techniques used to generate the isolates listed in Table 1 presents a different concentration factor in capturing the volatiles. While in the technique of dynamic headspace the volatiles contained in 300 g of sample were isolated in 300 μL of solvent, which promoted a concentration factor of 1000:1, in the extraction with dichloromethane followed by concentration of the isolate, the concentration factor was of 80:1 (40 g of sample in 0.5 mL of solvent) and in the dichloromethane isolate that was not concentrated, the concentration factor was of approximately 3:1 (40 g of sample in 15 mL solvent). Thus, the results shown in Table 1 and Fig. 1 are very interesting because they suggest that the dichloromethane techniques, which have a lower concentration factor to capture the volatiles, produced isolates showing better sensory quality that the technique of dynamic headspace, notably the dichloromethane isolate obtained without further concentration. However, it must be considered that the two solvents present in the isolates – acetone and dichloromethane – due to differences relative to their affinity regarding the volatiles extracted, their

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Techniques for the isolation of volatiles from cashew apple juice 30 esters terpenes aldehydes alcohols ketones lactones acids hydrocarbons miscellaneous

20

10

0 HSD n=79

HS-SPME n=63

CLLE

LLE

n=84

n=82

Figure 2. Total number of volatile compounds identified by each isolation technique, segmented according to chemical class. HSD, dynamic headspace–PorapakQ®, HS-SPME, solid-phase microextraction; CLLE, liquid–liquid extraction followed by concentration with N2 ; LLE, liquid–liquid extraction. n is the total identified volatiles.

volatility, among others, when evaporating from the paper strip before the sensory evaluation, might have carried different types and proportions of volatile compounds; this would also explain the differences observed in Table 1 and Fig. 1. Thus, the efficiency of the isolation techniques can not be judged solely on the basis of the sensory analysis of the isolates; the information to be presented and discussed below should also be considered. Profile of the volatiles in the isolates Figure 2 shows the total number of volatile compounds identified in the fresh cashew apple juice by the techniques of dynamic headspace with a PorapakQ® (HSD-PorapakQ®) trap, solid-phase microextraction with the fiber DVB/CAR/PDMS (HS-SPME), liquid–liquid extraction (LLE) and liquid–liquid extraction followed by concentration of the isolate (CLLE). For each isolate, Fig. 2 also presents the number of volatiles identified in each chemical class existing in the sample. As discussed above, each isolation technique exhibits a different concentration factor in capturing the volatiles, this being 1000:1 for the dynamic headspace technique (PorapakQ-HSD®), of 80:1 for the dichloromethane extraction with subsequent concentration (CLLE), and about 3:1 for the dichloromethane isolate that has not undergone concentration (LLE). Despite these differences, the number of volatiles identified in the HSD-PorapakQ®, LLE and CLLE were similar and varied between 79 and 84, an amount greater than the number of volatiles previously isolated from fresh cashew apple juice by simultaneous extraction–distillation techniques,4,6,7 dynamic headspace analysis5,9 and solid-phase microextraction.10,11 For the HS-SPME technique, in which the volatiles of 5 g of sample were adsorbed on the SPME fiber DVB/CAR/PDMS and desorbed directly in the chromatographic column, the number of volatiles identified (n = 63) was slightly lower than in the other techniques (Fig. 2). The major chemical class, numerically speaking, in the isolates obtained using the four methodologies evaluated in the present study was the class of esters. This is in agreement with various studies developed previously with fresh cashew apple juice or pulp5,7,9 – 11 and confirms the great importance of this chemical class in the aroma of cashew apple. The number of esters identified in the four isolates varied between 19 (LLE) and 24 (CLLE), suggesting that the four techniques studied presented similar performance in the isolation of esters. These values were similar to those identified in fresh J Sci Food Agric (2014)

www.soci.org cashew apple juice by Bicalho et al.,7 who used simultaneous extraction–distillation as the isolation technique (27 esters), by Garruti et al.,9 who used dynamic headspace with a PorapakQ® trap (25 esters) and by Maciel et al.5 who used dynamic headspace with a Tenax® trap (21 esters). Figure 2 shows that, numerically speaking, the terpenes constituted the second largest chemical group in the isolates of fresh cashew apple juice obtained by liquid–liquid extraction without concentration (LLE) and by dynamic headspace (HSD-PorapakQ®). This was also the case in the fresh cashew apple juice isolate obtained by Bicalho et al.7 by simultaneous extraction–distillation, and in the cashew apple isolate obtained by Carasek and Pawliszyn11 using solid-phase microextraction. Only five terpenes were identified in the isolate extracted with dichloromethane and subsequently concentrated with N2 (CLLE), a number well below the 17 terpenes identified in the non-concentrated dichloromethane isolate (LLE). These results are very important because they show that, in this case, the concentration of the isolate using nitrogen flow was inadequate, promoting a significant loss of terpenes. Thus, for this concentration, a more adequate procedure is recommended, such as Dufton columns or Kuderna–Danish columns.34 – 36 The alcohols constituted the second largest class in the CLLE isolate and third in the LLE isolate, 14 alcohols being identified in the former and 12 in the latter. On analyzing the fresh cashew apple juice isolate obtained by the HSD-PorapakQ® technique, Garruti et al.9 also found the alcohols to be the second largest chemical class in the isolate, numerically speaking, having identified nine alcohols. Finally, the acids constituted the second largest chemical group in the isolate obtained using the DVB/CAR/PDMS (HS-SPME) fiber (Fig. 2), different from that occurring in the other isolates obtained in this study and in the cashew apple isolates obtained by Carasek and Pawliszyn11 with the fibers CAR/PDMS, DVB/CAR/PDMS, PA and PDMS. Table 2 presents the volatiles identified in each of the four isolates obtained from the fresh cashew apple juice, reporting the retention index and the abundance value for the chromatographic peak area (arbitrary units) of each volatile. In addition, for each chemical class identified in the isolates – esters, terpenes, aldehydes, etc. – Table 2 exhibits the total abundance and the % relative area of the class on the chromatogram. It can be seen that with the exception of the isolate LLE, the esters represent the chemical class present in greater proportions in the isolates, corresponding to 53.7% of the total area of the chromatogram of the HSD-PorapakQ® isolate, 43.9% of the DVB/CAR/PDMS (HS-SPME) fiber isolate and 32% of the CLLE isolate. It can also be seen (Table 2) that, in general, the abundance of the esters in the chromatogram of the HSD-PorapakQ® isolate was greater than those obtained for the other isolates, indicating that amongst the isolation techniques studied, the dynamic headspace analysis associated with the use of PorapakQ® as the trap produced an isolate containing esters at highest concentration. Among other factors, this can be attributed to the fact that the technique has a higher concentration factor (1000:1) to capture the volatiles compared to the others techniques; it is an advantage in studies where further dilution of the volatiles is needed for the sensory evaluation of the chromatographic effluents by GC-O techniques. Various studies carried out with fresh cashew apple juice also reported the esters as being the chemical class present in greater proportions in the isolate.5,7,9 This class exerts a great impact on

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Table 2. Volatile compounds of cashew apple juice isolated by different techniques (HSD= dynamic headspace-PorapakQ®, LLE= liquid-liquid extraction, CLLE = liquid-liquid extraction followed by concentration with N2 and HS-SPME = headspace solid phase micro-extraction) and grouped by chemical classes Isolation techniques Dynamic headspace– PorapakQ® Compound Esters Ethyl propanoateb n-Propyl acetatea Methyl butanoateb Methyl 2-methyl butanoateb Methyl isovalerateb Ethyl butanoatea Ethyl 2-methyl butanoateb Ethyl isovaleratea Butyl acetateb Methyl trans-2-butenoateb Isoamyl acetatea Methyl 3-methyl pentanoateb Ethyl trans-2-butenoatea Ethyl-3-methyl pentanoateb Methyl hexanoateb Methyl 2-methylene butanoateb Ethyl hexanoatea Ethyl trans-2-pentenoatec Isoamyl butanoate b Amyl isovalerateb Ethyl trans-3-hexenoateb Propyl tiglateb Ethyl heptanoateb Ethyl trans-2-hexenoateb Allyl hexanoateb Ethyl 3-hydroxy-3-methyl butanoateb n-Butyl tiglateb Ethyl octanoatea Ethyl 3-hydroxybutanoateb Ethyl 2-hydroxy-4-methlypentanoateb Ethyl trans-2-octenoateb Ethyl benzoateb Ethyl 4-acetylbutyratec Ethyl phenylacetateb Dodecyl acrylatec Ethyl cinnamatea Methyl hexadecanoateb Isopropyl hexadecanoateb Ethyl hexadecanoateb Ethyl 9-hexadecanoateb Methyl isostearatec Methyl oleateb Ethyl octadecanoateb Ethyl cis-9-octadecenoateb Total area % area Terpenes 𝛽-Myrcenea

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RId Area × 106

980 992 999 1020 1028 1036 1056 1075 1082 1115 1131 1139 1167 1176 1192 1193 1241 1263 1271 1295 1298 1321 1340 1347 1372 1408 1427 1438 1519 1550 1553 1661 1717 1750 1999 2148 2222 2250 2259 2304 2428 2450 2464 2485 – –

CV (%)

n = 23 177.40 12.50 13.53 10.15 80.19 292.84 118.06 491.10 20.10 6.30 58.39 15.30 336 23.04 3.34 5.27 294.56 5.52 – – 2.51 – – 7.48 tr – – 6.56 – – – – – – – tr – – – – – – – – 1980.14 53.72

1 41 39 8 6 1 19 14 26 51 39 12 20 13 34 25 16 27 – – 30 – – 78 – – – 70 – – – – – – – – – – – – – – – – – –

n = 16 CV (%) RId Area × 106 1170 – –

Liquid–liquid extraction Area × 106

CV (%)

Liquid–liquid extraction followed by concentration with N2 Area × 106

CV (%)

n = 19 – – – – – – – – – – 25.30 25 5.19 56 15.21 1 – – – – – – – – 16.19 1.6 1.73 28 – – – – 10.82 10 – – – – – – – – – – – – 1.58 11 – – 3.13 18 – – 2.84 38 5.07 27 2.35 7 – – – – 6.02 17 – – 26.82 12 3.34 – 5.69 14 – – 10.29 12 – – – – 7.42 28 6.45 22 15.50 9 168.10 – 23.91 –

– – – – 20.69 40.85 13.00 20.13 19.64 – – – 30.86 2.74 – 0.96 16.02 – – – – – – 1.87 – 6.06 – 2.87 11.65 1.82 – – 12.49 – 74.50 6.39 14.23 19.05 9.32 9.05 5.38 – 7.26 24.42 371.25 31.95

n = 17 Area × 106 CV (%) 0.11 –

Area × 106 –

© 2014 Society of Chemical Industry

n = 24 – – – – – – – 14 – – – – 6 20 – 15 28 – – – – – – 31 – 25 – 25 23 – – – 30 – 25 19 21 22 – – – – 20 11 – – n=5 CV (%) –

Headspace solid phase microextraction Area × 106

CV (%)

n = 20 – – – – – – – 3.10 tr – – – 22.29 47.32 – tr 92.03 4.76 12.61 6.50 2.09 0.66 0.49 2.20 tr – 2.04 8.54 – – 2.71 0.44 tr tr – – – – – – – – – – 281.78 43.91

– – – – – – – – – – – – 32 115 – – 3 82 78 19 70 8 55 14 – – 114 26 – – 129 26 – – – – – – – – – – – – –

n=5 Area × 106 CV (%) – –

J Sci Food Agric (2014)

Techniques for the isolation of volatiles from cashew apple juice

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Table 2. Continued Isolation techniques Dynamic headspace– PorapakQ® Compound

RId Area × 106

CV (%)

Liquid–liquid extraction Area × 106

CV (%)

Liquid–liquid extraction followed by concentration with N2 Area × 106

CV (%)

Headspace solid phase microextraction Area × 106

CV (%)

D-Limonenea Cineoleb 𝜌-Cymeneb Linalool oxideb Menthoneb Linalool pyran oxideb 𝛼-Copaeneb 𝛼-Cubebeneb 𝛽-Elemeneb 4-Terpineolb 𝛽-Terpineolb 𝛼-Terpineolb 𝛽-Selineneb 𝛼-Selineneb 𝛿-Cadineneb Epoxy linaloolb cis-Geraniolb trans-Geraniolb trans-Geranyl acetoneb Total area % area

1189 1210 1269 1452 1455 1471 1479 1481 1586 1599 1638 1699 1710 1716 1732 1746 1800 1826 1875 – –

Ketones

n=8 CV (%) RId Area × 106 993 8.62 26 994 6.54 33 1135 76.25 40 1136 – – 1282 4.65 9 1294 8.10 – 1337 5.45 57 1361 ND – 1421 – – 1428 – – 1531 74.75 27 1593 – – 1643 – – 2102 – – 2239 – – – 184.36 – – 5.0 –

n=7 Area × 106 CV (%) – – – – – – 2.67 5 56.37 26 – – 6.13 – – – 9.94 17 tr – – – 0.91 18 – – – – 0.25 – 76.27 – 10.85 –

Area × 106 – – – 3.90 164.29 – – 2.84 27.60 tr – 2.95 6.42 – tr 208.0 17.90

CV (%) – – – – 22 – – 15 20 – – 14 0.2 – – – –

n=3 Area × 106 CV (%) – – – – – – – – 7.66 39 – – – – – – – – – – – – – – 1.60 62 2.58 – – – 11.84 – 1.84 –

n=4 CV (%) RId Area × 106 1206 – – 1220 42.36 32 1329 – – 1366 25.78 – 1388 – – 1404 – – 1472 – – 1495 12.32 – 1569 – –

n = 12 Area × 106 CV (%) – – 9.77 8 2.44 26 2.10 21 0.56 20 0.64 23 – – 15.66 30 – –

n = 14 Area × 106 CV (%) – – 18.89 8 1.43 22 3.52 16 0.79 24 1.89 23 13.81 23 33.95 5 2.72 28

n=6 Area × 106 CV (%) 0.87 92 – – – – tr – – – – – – – 3.00 110 2.63 87

2,3-Butanedionea 2-Pentanoneb 3-Penten-2-oneb 2,3-Hexanedioneb 3-Hydroxy-2-butanoneb 1-Hydroxy-2-propanoneb 6-Methyl-5-hepten-2-oneb 4-Hydroxy-4-methyl-2-pentanonea 2-Cyclohexen-1-oneb 5-Hydroxy-4-octanonec 4-Hydroxy-2-butanoneb 2-Hydroxy cyclohexanonec Acetophenoneb 1,3-Dihydroxy-2-propanonec 2-Heptadecanoneb Total area % area Alcohols Isoamyl alcoholb 3-Methyl-1-butanola cis-2-Penten-1-olb Hexanola cis-3-Hexen-1-ola Cyclohexanolb 2-Cyclohexen-1-olb 2-Ethyl-1-hexanolb Octanolb

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30.07 11.94 0.74 183.62 – – 18.11 19.06 29.65 11.04 0.01 321.94 24.84 18.46 – 32.84 27.05 43.55 7.56 780.48 21.17

58 26 40 19 – – 8 14 39 37 – 22 25 54 – 28 24 21 29 – –

0.14 5.02 tr 45.43 – 9.58 3.45 – 1.79 2.81 1.60 80.40 4.27 4.0 3.72 7.91 4.41 11.20 – 185.84 26.43

– 20 – 19 – – 17 – 21 75 15 19 23 26 10 15 7 10 – – –

© 2014 Society of Chemical Industry

9.31 – – 6.25 – – – – – – – 21.30 – – 8.67 – – 3.58 – 49.11 4.22

9 – – 27 – – – – – – – 29 – – 7 – – 23 – – – n=8

24.76 – – – 1.54 tr – – – 0.21 – – – – 2.61 – – – – 30.03 4.68

54 – – – 84 – – – – 4 – – – – 97 – – – – – –

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Table 2. Continued Isolation techniques Dynamic headspace– PorapakQ® Compound

RId Area × 106

CV (%)

Liquid–liquid extraction Area × 106

CV (%)

Liquid–liquid extraction followed by concentration with N2 Area × 106

2-Tetradecanolc Benzyl alcohola 2-Phenyl ethanola Dodecanolb Phenolb 2-Methyl-1-hexadecanolb 3,4-Dimethyl-5-hexen-3-olb Glycerolb Hexadecanolb Octadecanolb Total area % area

1769 1793 1925 1975 2017 2069 2210 2330 2385 2592 – –

Aldehydes

n = 10 CV (%) RId Area × 106 1088 21.03 59 1162 5.80 22 1188 3.93 31 1198 30.97 47 1287 2.95 13 1329 tr – 1393 6.27 58 1501 13.28 47 1509 – – 1527 – – 1584 22.82 – 1570 9.86 34 2352 – – 2379 – – – 116.91 – – 3.17 –

n=5 Area × 106 CV (%) 2.59 14 – – – – – – – – 16.61 25 1.34 19 – – 2.99 34 – – – – – – – – 3.39 9 26.92 – 3.82 –

Area × 106 3.22 – – – – 46.98 3.17 – 4.83 – – – 9.44 7.27 74.91 6.45

n=3 CV (%) RId Area × 106 1458 125.40 45 1644 – – 1680 15.78 26

n=5 Area × 106 CV (%) 8.82 43 – – 23.31 31

Hexanala 2-Methyl-2-pentenalb Heptanalb 3-Methyl-2-butenalb Octanalb 2-Ethenyl-2-butenalb Nonanalb Decanala Benzaldehydea trans-2-Nonen-1-alb trans-2-cis-6-Nonadienalb cis-4-Decenalb Octadecanalb cis-13-Octadecenalb Total area % area Carboxylic acids Acetic acidb Butyric acida 3-Methyl butanoic acidb, e + 2-methyl butanoic acida, e Hexanoic acidb cis-5-Dodecenoic acidc 2-Ethyl hexanoic acidb Octanoic acidb cis-Vaccenic acidc Total area % area Lactones 𝛾-Hexalactoneb 𝛾-Crotonolactoneb 𝛿-Hexalactoneb 𝛾-Octalactoneb 𝛿-Octalactoneb

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1784 1795 1971 2085 2458 – –

– 19.09 – – – – – – – – 99.55 2.70

– – – – – 141.18 3.83

– 79 – – – – – – – – – –

– – – – – – –

n=1 RId Area × 106 CV (%) 1692 – – 1727 – – 1751 – – 1925 – – 1983 – –

– 12.85 – 6.0 – 7.82 – 8.97 6.65 8.62 82.08 11.67

– 17 – 24 – – – – 1 12 – –

4.71 tr – – – 36.84 5.24

5 – – – – – –

n=6 Area × 106 CV (%) 3.89 – – – 2.34 11 4.11 21 13.42 14

© 2014 Society of Chemical Industry

CV (%)

tr 8.19 – 22.06 – – – 7.22 11.06 9.70 135.23 11.64

– 10 – 15 – – – – 23 14 – – n=6

Headspace solid phase microextraction Area × 106 – – – – 5.89 – tr – – – 12.39 1.93

CV (%) – – – – 116 – – – – – – –

CV (%) 8 – – – – 24 22 – 13 – – – 26 21 – –

n=5 Area × 106 CV (%) – – – – – – – – 6.17 17 – – 3.53 48 0.84 42 2.10 98 6.77 113 – – – – – – – – 19.41 – 3.02 –

Area × 106 6.86 – 72.49

CV (%) 17 – 24

n=7 Area × 106 CV (%) 74.15 111 2.30 94 43.34 119

tr – – – tr 79.35 6.83

– – – – – – –

n=5

n=5 Area × 106 11.39 – – 11.90 30.45

CV (%) – – – 2 26

5.38 – 0.10 tr – 125.27 19.52

95 – – – – – –

n=5 Area × 106 CV (%) – – 0.62 80 – – – – 1.85 88

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Techniques for the isolation of volatiles from cashew apple juice

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Table 2. Continued Isolation techniques

Dynamic headspace– PorapakQ® Compound

RId Area × 106

CV (%)

Liquid–liquid extraction Area × 106

CV (%)

Liquid–liquid extraction followed by concentration with N2 Area × 106

CV (%)

𝛾-Nonalactonea 2-Hydroxy-𝛾-butyrolactonec 𝛿-Decalactoneb 𝛾-Dodecalactonea Total area % area

2086 2188 2218 2392 – –

Hydrocarbons

n=7 RId Area × 106 CV (%) 1004 5.91 – 1127 61.21 74 1130 13.38 30 1141 9.05 – 1183 6.91 30 1256 17.82 52 1298 – – 1400 5.04 57 1499 – – 1600 – – 1773 – – 1786 – – 1900 – – 1937 – – 2107 – – 2172 – – 2393 – – 2501 – – – 119.32 – – 3.23 –

n=7 Area × 106 CV (%) – – 19.00 32 4.68 24 2.60 24 – – – – – – 3.82 9 6.24 40 – – – – – – 8.35 5 – – 1.03 – – – – – – – 45.72 – 6.50 –

n = 13 Area × 106 CV (%) – – 25.81 6 – – 3.97 10 3.04 13 – – – – 6.30 15 19.28 8 0.21 – tr – 0.37 – 9.84 2 7.82 30 – – 3.10 – 9.23 28 13.04 12 102.01 – 8.78 –

n=7 CV (%) RId Area × 106 705 11.68 8 1079 10.19 9 1160 – – 1271 0.30 – 1383 – – 1401 28.5 – 1574 – – 1597 49.00 25 1609 – – 1643 164 22 1667 – – 1953 – – 1986 – – 2046 – – 2158 – – 2182 tr –

n=4 Area × 106 CV (%) – – – – 0.647 – – – – – – – – – – – 2.43 4 – – – – 12.30 8 – – – – 17.14 10 – –

Area × 106 – – 0.86 – tr – – – 5.89 – – – – – 41.78 –

Decanea Ethyl benzeneb m-Xyleneb p-Xyleneb o-Xyleneb Styreneb Cardeneb Tetradecanea Pentadecanea Hexadecanea 9-Octhyl heptadecaneb 2,6,10-Trimethyl pentadecaneb Nonadecanea 6-Phenyl dodecaneb Heneicosyl cyclopentaneb Dodecyl benzeneb Tetracosanea Pentacosanea Total area % area Miscellaneous Methyl sulfidea Methyl disulfidea Cyclohexene epoxydeb 3,4-Epoxy-2-pentanoneb 2-Acetoxy-3-butanonec 2-Butoxy ethanolb Dimethyl sulfoxideb 5,5-Dimethyl-2(5H)-furanoneb 2,3-Epoxy cyclohexanonec 2,5-Dihydro-3,5-dimetil-2-furanonec 2-Furan methanolb 2,6-Dimethyl-3,7-octadiene-2,6-diolb 2,5-Furan dicarboxaldehydeb Furaneolb 2-Phenoxy ethanolb Ethyl 4-ethoxy benzoatec

J Sci Food Agric (2014)

0.26 – – – 0.26 0.01

– – – – – –

– – 6.49 18.52 48.77 6.94

– – 4 43 – –

© 2014 Society of Chemical Industry

– – 16.77 23.08 93.59 8.05

– – 10 20 – –

n=4 CV (%) – – – – – – – – 20 – – – – – 12 –

Headspace solid phase microextraction Area × 106 4.75 10.76 – 3.48 21.46 3.34

CV (%) 112 100 – 37 – –

n=2 Area × 106 CV (%) – – – – – – – – – – 57.82 74 0.91 – – – – – – – – – – – – – – – – – – – – – – – 58.73 – 9.01 – n = 10 Area × 106 CV (%) – – – – – – – – – – – – tr – – – – – – – 50.04 124 – – 5.73 130 tr – – – – –

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Table 2. Continued Isolation techniques

Dynamic headspace– PorapakQ® Compound 3,5-Dihydroxy-6-methyl-2,3dihydro-4H-pyran-4-one b 5-[Hydroxymethyl]dihydro-2(3H)-furanoneb 2-Hydroxymethyl-5-furfuralb 1-(2-Furyl)-1,2-ethanediolc 4-Hydroxydihydro-2(3H)-furanoneb 3,5-Dihydroxy-6-methyl-2,3-dihydro4H-pyran-4-one b Total area % area

RId Area × 106

CV (%)

Liquid–liquid extraction Area × 106

CV (%)

Liquid–liquid extraction followed by concentration with N2 Area × 106

CV (%)

Headspace solid phase microextraction Area × 106

CV (%)

2289

–

–

–

–

–

–

5.04

96

2491

–

–

–

–

–

–

2.49

38

2508 2584 2599 2289

– – – –

– – – –

– – – –

– – – –

– – –

– – – –

4.55 3.72 5.04 5.04

119 96 96 96

– –

263.67 7.15

– –

32.52 4.62

– –

48.53 4.18

– –

81.65 12.72

– –

a Positively identified compound. b Identified compound. c Tentatively identified. d RI, retention index in the DB-wax column. e Co-eluted compounds.

ND, not detected. tr, detected in trace amounts.

the aroma of fresh cashew apple5,7,9,12,13 and of many other tropical fruits.20,37,38 In the isolate produced by solvent extraction with no subsequent concentration (LLE), the esters were the second chemical class in greater proportions, representing 23.9% of the total area of the chromatogram. In this isolate (LLE), the terpenes occupied 26.4% of the total area of the chromatogram, and were thus the chemical class present in the greatest proportion. These results confirm that, although the concentration of the dichloromethane isolate using a N2 flow (CLLE) was efficient in concentrating the extracted esters, it caused losses of terpenes showing that for this concentration, a more adequate procedure is recommended, such as Dufton columns or Kuderna–Danish columns.34 – 36 In the study conducted by Macleod and Troconis,4 all the terpenes identified by the authors showed an odoriferous impact on the chromatographic effluent, and their odors were described as ‘fruity’, ‘sweet’, ‘floral’ and ‘green’. Thus, the loss of the terpenes could explain the fact that the isolate that was not concentrated (LLE) showed higher aroma notes of ‘fresh fruit’ and ‘green’ compared to that isolate submitted to concentration (Fig. 1). The terpenes were also present in great proportions in the HSDPorapakQ® isolate, where they represented 21.2% of the total area of the chromatogram and were the second largest chemical group in this isolate. Table 2 shows that the abundance of terpenes in the HSD-PorapakQ® isolate was greater than that observed in the LLE isolate. On the other hand, the values for abundance and % relative area of the terpenes present in the isolate extracted with the SPME fiber were as low as those found in the CLLE isolate. Thus for the extraction of terpenes, the results shown in Table 2 suggest that the techniques of dynamic headspace using PorapakQ® as the trap and dichloromethane extraction with no subsequent concentration of the isolate (LLE) were the most effective techniques, notably if the isolates will be further submitted to GC-O. In the

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research carried out by Macleod and Troconis,4 the terpenes represented the major chemical class present in the fresh cashew apple extract obtained by simultaneous distillation–extraction, representing 38% of the total area of the chromatogram. The values for abundance and % relative area shown in Table 2 also indicate that the extraction of the volatiles from fresh cashew apple juice with dichloromethane, followed by concentration of the extract using N2 (CLLE) isolated at highest concentration, the chemical classes of alcohols, ketones and lactones. For its part, the technique HS-SPME with the use of the fiber DVB/CAR/PDMS was the least efficient in isolating these chemical classes. Differently to what occurred with the isolates obtained by HSD-PorapakQ®, LLE and CLLE, the acids and hydrocarbons represented the major chemical classes in the isolate obtained by HS-SPME, corresponding to 19.52% and 9.01%, respectively, of the total area of the chromatogram. In the studies of Maia et al.6 and of Bicalho et al.,7 the acids also represented a major chemical group in the fresh cashew apple pulp, and according to Garruti et al.9 and Maciel et al.,5 the acids 3-methyl butanoic and 2-methyl butanoic have an odoriferous impact on the aroma of fresh cashew apple juice. In the present study the HS-SPME methodology associated with the DVB/CAR/PDMS fiber also stood out from the other isolation techniques for capturing the greatest proportion of compounds classified as ‘miscellaneous’, amongst which various furans and pyranones. Together these compounds represented more than 12% of the total area of the chromatogram (Table 2). As suggested by Lestremau et al.39 it is possible that some of these compounds, such as 2-furan-methanol and 2-hydroxy-methyl-5-furfural, represent artifacts formed by thermal oxidation during desorption of the volatiles from the fiber in the GC-MS injector. These two volatiles were not identified by other authors who worked with fresh cashew apple juice, with the exception of Cardeal et al.,10 who also used the SPME technique and

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J Sci Food Agric (2014)

Techniques for the isolation of volatiles from cashew apple juice desorption at 220 ∘ C, identified 2-furan-methanol in fresh cashew apple. In the current study, a total of 181 volatile compounds were identified, of which 44 were esters, 20 terpenes, 19 alcohols, 17 hydrocarbons, 15 ketones, 14 aldehydes and nine acids, amongst other compounds. Of the esters identified, the following 16 had not previously been identified in cashew apple peduncle or its derivatives: propyl acetate, amyl isovalerate, 2-methyl butyl isovalerate, propyl 2-methyl crotonate, ethyl 2-hydroxy isovalerate, methyl 2-methyl crotonate, ethyl 3-hydroxy butanoate, dodecyl acrylate, methyl hexadecanoate, isopropyl hexadecanoate, ethyl 9-hexadecenoate, methyl isostearate, methyl oleate, ethyl octadecanoate, ethyl oleate and ethyl trans-9-octadecenoate. More volatile esters, such as ethyl propanoate, propyl acetate, methyl butanoate, methyl 2-methyl butanoate and methyl trans-2-butenoate were only identified in the isolate produced by the technique HSD-PorapakQ®. As a counterpart, this technique was the least efficient in the isolation of less volatile esters, notably when compared to dichloromethane extraction followed by concentration of the extract with N2 (CLLE). With the exception of ethyl cinnamate, none of the 11 esters with retention indexes above 1500, identified in the CLLE isolate, were found in the HSD-PorapakQ® isolate. Thus the results of the present study suggest that the extraction of the volatile compounds from fresh cashew apple juice with dichloromethane, followed by concentration of the extract in a N2 flow, can be considered a good choice of methodology for the capture of the less volatile esters, such as ethyl hexadecanoate, dodecyl acrylate and ethyl octadecanoate. In all, 20 terpenes were identified in the present study, of which the following 10 had not been identified in previous studies with the cashew apple peduncle or its derivatives:4 – 11 𝛽-myrcene, 𝜌-cymene, menthone, 4-terpineol, 𝛽-elemene, 𝛽-terpineol, 𝛼-terpineol, 𝛼-selinene, cis-geraniol and trans-geraniol. In addition to the esters and terpenes, volatile compounds from other chemical classes were found in the isolates in the present study which, according to previous studies, are important in the aroma of fresh cashew apple. These include 2-methyl butanoic acid and 3-methyl butanoic acid, whose odors were described in earlier studies as cheese-like/stinky,9,12 the aldehydes hexanal (grassy), benzaldehyde (green/fruity/cashew),4,9 2-methyl-2-pentenal (grassy/herbal);9 the alcohols 3-methyl-1butanol (smokey/over-ripe cashew), cis-3-hexenol (fruit/grass)9,12 and 2-ethyl-hexanol (grass, herb);12 and the ketone acetophenone (over-ripe cashew/cheese/acid).9,12 The efficiency with which the mentioned compounds were isolated from the juice varied from technique to technique. Apart from the alcohols, aldehydes, acids and ketones, the sulfur compounds are also important in the cashew apple aroma.5,8,9 In the present study, although no specific detector for the analysis of sulfur compounds was used, as did Valim et al.8 and Maciel et al.,5 two sulfur compounds (methyl sulfide and methyl disulfide) were identified by the HSD-PorapakQ® technique. In general, in the present study, the techniques LLE, CLLE and HSD-PorapakQ® showed the best repeatability. More than 50% of the volatiles identified in the isolates generated by the HSD-PorapakQ®, LLE and CLLE techniques presented coefficients of variation (CV) equal or below 30%. On the other hand, in the extraction by HS-SPME, more than 70% of the volatiles identified presented CV > 50%. These results are different from those reported by Madruga et al.,40 who used HS-SPME (fiber of Carboxen-PDMS, 75 μm) to analyze the volatile compounds from cooked goat meat, and obtained CV values below 30% for J Sci Food Agric (2014)

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Table 3. Number of odor compounds found in the isolates obtained by the techniques HSD (dynamic headspace–PorapakQ®) (HSD), liquid–liquid extraction (LLE) and liquid–liquid extraction followed by concentration with N2 (CLLE) and segmented by chemical class Isolates Chemical class Esters Terpenes Aldehydes Ketones Acids Lactones Alcohols Sulfur compounds Others Not detecteda Not identified TOTAL

HSD

LLE

18 6 3 5 0 2 2 1 2 2 3 44

9 8 2 2 2 3 3 0 4 0 8 41

CLLE 9 2 2 3 3 1 5 0 2 0 10 37

a Compound not detected in the flame ionization detector of the gas chromatograph although it was sensorially detected in chromatographic effluents.

most of the volatiles identified. For the authors, the technique of dynamic headspace associated with Tenax TA® showed worse repeatability than the HS-SPME technique, since over 50% of the volatiles isolated by the Tenax TA® presented CV greater than 50%. In turn, Dirinck et al.,41 on isolating the volatiles from cured hams by dynamic headspace associated with the polymer Tenax TA®, reported that the majority of the compounds identified presented CV values below 50%, results similar to those displayed in Table 1. Odor volatiles in the isolates Table 3 presents the odor volatiles perceived by the trained sensory panel in chromatographic effluents of the isolates obtained by dynamic headspace (HSD-PorapakQ®) and with dichloromethane with (CLLE) and without concentration (LLE). There was no need to conduct olfactometry studies with the isolate obtained by the HS-SPME technique to conclude that it was the least advantageous of the four techniques analysed because: (1) in previous studies4 – 9,12,13 its isolate showed a lower number and concentration of the volatiles that were considered important for the aroma and flavor of cashew; and (2) it is laborious and time-consuming to use GC-O studies because, for each judge, in each repetition, it is necessary to perform a new extraction of volatiles from the headspace of the sample before the compounds can be sensorially evaluated in the chromatographic effluents. As can be seen in Table 3, in the isolate obtained by dynamic headspace (HSD-PorapakQ®) 44 odorous compounds were detected, a number slightly higher than that detected in the isolates obtained with dichloromethane without further concentration (n = 41 volatile) and concentrated (n = 37). But considering that the concentration factor during the isolation of the volatiles was 1000:1 for the technique HSD-PorapakQ®, of 80:1 for the dichloromethane isolate that underwent concentration (CLLE), and of 3:1 for the dichloromethane isolate that did not undergo concentration (LLE), there was no major advantage of the technique HSD-PorapakQ® with respect to the total odor volatiles that

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Table 4. Odor active esters and terpenes identified in each isolate, their odor quality perceived by the panelists in the chromatographic effluents, and respective abundance from the chromatogram

Dynamic headspace –PorapakQ® Compound

Area × 106

Det.

Liquid–liquid extraction followed by concentration with N2

Liquid–liquid extraction Area × 106

Det.

Area × 106

Det.

Odor description

Esters Ethyl propanoateb Methyl butanoateb Methyl 2-methyl butanoateb Methyl isovalerateb Ethyl butanoatea Ethyl 2-methyl butanoateb Ethyl isovaleratea Butyl acetateb Methyl trans-2-butenoateb Isoamylacetatea Ethyl trans-2-butenoatea Ethyl-3-methyl pentanoateb Methyl hexanoateb Ethyl hexanoatea Ethyl trans-2-pentenoatec Ethyl trans-3-hexenoateb Ethyl trans-2-hexenoateb Ethyl 3-hydroxy-3-methyl butanoateb Ethyl octanoatea Ethyl 3-hydroxybutanoateb Ethyl 2-hydroxy-4-methlypentanoateb Ethyl hexadecanoateb Methyl oleateb Ethyl cis-9-octadecenoateb

177.40 13.53 10.15 80.19 292.84 118.06 491.10 20.10 6.30 58.39 336 23.04 3.34 294.56 5.52 2.51 7.48 – 6.56 – – – – –

X X X X X X X X X X X X X X X X X – X – – – – –

– – – – 25.30 5.19 15.21 – – – 16.19 1.73 – 10.82 – – 1.58 3.13 2.84 5.07 2.35 10.29 7.42 15.50

– – – – X – X – – – Xd – – X – – X – – X – X X X

– – – 20.69 40.85 13.00 20.13 – – – 30.86 2.74 – 16.02 – – 1.87 6.06 2.87 11.65 1.82 9.32 – 24.42

– – – – X – X – – – X X – – – – – X X X X – – X

Sweet, fruity Fruity Fruity, cashew Fruity, cashew Sweet, fruity Fruity, sweet Grassy, fruity Red fruits Fruity Sweet, banana Fruity, vanilla Floral, sweet Fruity, sweet Fruity, sweet Vanilla, sweet Fruity, vanilla Sweet, grassy Grassy, pungent Grain, earth Sweet, pungent Sweet, pungent Pungent, gas Toasted, refreshing Vanilla, grassy

Terpenes 𝛽-Myrcenea Linalool oxideb 𝜌-Cymeneb 𝛼-Cubebeneb 𝛽-Elemeneb 4-Terpineolb 𝛽-Terpineolb 𝛼-Terpineolb cis-Geraniolb trans-Geraniolb

– 183.62 0.74 19.06 29.65 11.04 0.01 321.94 27.05 43.55

– X X X – X – X – X

0.11 45.43 – – 1.79 2.81 1.60 80.40 4.41 11.20

Xd X

– 6.25 – – – – – 21.30 – 3.58

– X – – – – – X – –

Fruity, green, grassy Sweet, green, grassy Sweet, citric Floral, refreshing pungent Green, grassy, floral Sweet, fruity Grassy, refreshing Oil, popcorn Fruity, pungent

X X X X X X

a Positively identified compound. b Identified compound. c Tentatively identified. d Co-eluted compounds in GC-O. Det., Detection by GC-O; X, compound detected by GC-O; –, compound not detected by GC-O.

could be assessed by olfaction in the chromatographic effluents. However, having produced an isolate in which the esters were in a much higher concentration than in the other isolates (Table 2), the number of esters sensorially detected in the chromatographic effluents of the HSD-PorapakQ® isolate was twice as high compared to the other isolates (Table 3). In turn, the isolation with dichloromethane without further concentration (LLE) was more efficient than the HSD-PorapakQ® technique in the isolation of terpenes, since a greater number of these compounds were smelled in the chromatographic effluents.

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Table 3 also shows that the concentration of the dichloromethane isolate with N2 flow increased only the number of alcohols sensorially detected in the GC effluents, indicating no advantage in the concentration of the dichloromethane isolate in the way it was performed in the current study. Finally, Table 4 shows for the three isolates, the esters and terpenes sensorially perceived in the chromatographic effluents, presenting their abundance from the chromatogram (Table 2) and their odor quality as perceived by the sensory panel in the chromatographic effluents. As noted in previous studies4,5,8,9,12,13

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Techniques for the isolation of volatiles from cashew apple juice most of the esters were described by the panelists as having fruity and sweet aroma. In turn, several of the terpenes showed an aroma described as green grassy, fruity or floral (Table 4), which justifies the trained judges reporting an aroma note of ‘green/citrus’ in the dichloromethane isolate obtained without concentration (Fig. 1). Thus, for studies where cashew volatiles will be sensorially accessed in the chromatographic effluents, Table 4 shows very clearly that while the technique HSD-PorapakQ® was more efficient in isolating the esters, concentrating this class of compounds well above those levels shown by the other techniques, the extraction with dichloromethane without further concentration of the isolate was superior for the isolation of terpenes.

CONCLUSIONS In the current study, the analysis of the values obtained for abundance and % relative area of the chromatograms showed that the four techniques studied were efficient in isolating the esters, a chemical class of great impact in the characteristic aroma of fresh cashew apple. However, the dynamic headspace methodology (HSD-PorapakQ®) was more efficient than the others, producing an isolate with a greater concentration of the analytes, facilitating the detection, identification and sensory evaluation of these compounds in the chromatographic effluents. This technique isolated the more volatile esters better, but was less efficient in isolating the less volatile esters, notably when compared with dichloromethane extraction, followed by concentration of the extract using N2 (CLLE). Thus in studies where esters are the chemical class of interest, it is an advantage to use these two techniques in a complementary way. In the extraction of terpenes, a chemical class that also shows great impact on the aroma of fresh cashew apple and of tropical fruits in general, the dichloromethane extraction without concentration of the isolate (LLE), was the most efficient technique, followed by the dynamic headspace technique with PorapakQ® as the trap (HSD-PorapakQ®). The technique HS-SPME with the fiber DVB/CAR/PDMS was efficient in isolating esters, aldehydes and acids, and was inefficient in isolating terpenes and alcohols, important chemical classes for cashew apple aroma. Future studies should explore the performance of other fibers in the isolation of the volatiles from cashew apple, including the fiber Carbowax/DVB, which has a more polar nature.

ACKNOWLEDGEMENTS The authors are grateful for the financial support of CAPES (AUX-PE-PNPD-1470/2008, PNPD99082), FAPESP (2008/55986-0) and CNPq (476588/2008-1; 302009/2009-4).

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