Identification of volatile compounds in cultivars barker , collinson , fortuna and geada of avocado ( Persea americana , Mill . ) fruit

Flavor is one of the most important attributes of foods in determining consumer acceptance and there has been an increasing interest to study its impact on quality of a food product. In the case of tropical fruits, aroma constituting of volatile components is one of the most appreciated characteristics, which determines its quality and it is particularly sensitive to compositional alterations (Ibáñez et al., 1998; Barreto et al., 2013). In addition, tropical fruits are often inexpensive, extremely rich in vitamins and can be used in a wide range of food products (Pino et al., 2001). Among the tropical and subtropical fruits, the avocado (Persea americana, Mill.) is very much appreciated, and it occupies a prominent place in the market due to its nutritional value, especially fibers and lipids. Moreover the fruit has soft flavor and low sugar content (about 10 g/kg of pulp), and thus is even recommended for diabetic suffering people since it serves as high-energy food (Sinyinda & Gramshaw, 1998; Maitera et al., 2014), maintain skin elasticity (Athar & Nasir, 2005) and reduce the coronary heart risk (Berasategi et al., 2012).


Introduction
Flavor is one of the most important attributes of foods in determining consumer acceptance and there has been an increasing interest to study its impact on quality of a food product.In the case of tropical fruits, aroma constituting of volatile components is one of the most appreciated characteristics, which determines its quality and it is particularly sensitive to compositional alterations (Ibáñez et al., 1998;Barreto et al., 2013).In addition, tropical fruits are often inexpensive, extremely rich in vitamins and can be used in a wide range of food products (Pino et al., 2001).Among the tropical and subtropical fruits, the avocado (Persea americana, Mill.) is very much appreciated, and it occupies a prominent place in the market due to its nutritional value, especially fibers and lipids.Moreover the fruit has soft flavor and low sugar content (about 10 g/kg of pulp), and thus is even recommended for diabetic suffering people since it serves as high-energy food (Sinyinda & Gramshaw, 1998;Maitera et al., 2014), maintain skin elasticity (Athar & Nasir, 2005) and reduce the coronary heart risk (Berasategi et al., 2012).
Botanically, avocado fruit is classified in three varieties: West Indian, Mexican and Guatemalan races (Morton, 1987).
The Collinson and Fortuna cultivars are Guatemalan x West Indian hybrids, while Barker and Geada are of West Indian race (Brasil, 1971;Medina, 1980).In Brazil, the fruit is classified in two groups (Brasil, 1971): the first group, in which Barker, Collinson and Geada cultivars are included, are suitable for long-distance transport and hence are commercialized as whole fruit, while the second group, in which Fortuna cultivar belongs, is delicate due to its consistency having soft pulp texture and it is more suited for oil extraction.

Materials
Avocado fruits of four cultivars, Barker, Collinson, Fortuna and Geada were obtained from a farm located in Goiana city in the Pernambuco state of Brazil.The fruits were transported to the laboratory in the city of João Pessoa in the standard cardboard boxes currently used for export packaging and did not have any application whatsoever of inhibitor or accelerator for the control of maturation.Fruits free from any apparent skin damage were selected for analysis.The ripe mature fruit, after being washed with distilled water, was cooled to 2 o C. The skin and kernel were separated manually by using a stainless steel knife and the pulp macerated.The solvents and authentic standard flavor compounds were of more than 99.5% purity and belonged to the firms, Merck and Sigma-Aldrich, respectively.

Volatiles isolation
The volatile compounds from the pulp of avocado fruit were extracted by simultaneous distillation and extraction technique using a modified procedure Likens and Nickerson's apparatus (Likens & Nickerson, 1964).The extraction conditions were initially optimized by varying the parameters such as: solvent (hexane or a mixture of pentane and ethyl ether in proportion 2:1) and extraction period (40, 60, 70 or 80min), with the objective to obtain a large number of compounds on chromatographic analysis.The weight of pulp (150 g), the volume (200 mL) of water added and the volume (20 mL) of the solvent were fixed.The extracts were concentrated to a final volume of 0,3 mL under flow of nitrogen gas (Narain & Galvão, 2004).

High resolution gas chromatography/mass spectrometry
A combined system of Varian gas chromatograph (GC 3800) coupled with mass spectrometer (Saturn 2000R) and its processing workstation was used.Two microliters of the concentrated volatile extract was injected in the column in split (1:20) mode.Capillary GC investigations were carried out on a 30 m (length) x 0.25 mm (internal diameter) polyethylene glycol innophase bondable polar capillary column (HP-INNDWax; 0.25 μm film thickness; Hewlett Packard, Inc., Palo Alto, USA).The carrier gas was helium (99.995% pure) and column head pressure was maintained at 11.5 psi having a flow rate of 1 mL/min.The oven temperature was programmed: initiation at 30 o C for 5 min, increased at 7 o C/min to 100 o C, maintained at 100 o C for 5 min, increased at 1 o C/min to 130 o C, followed by a later increase of 10 o C/min to 195 o C wherein it was maintained for 45 min.The temperatures of the injection port and the GC/MS interface were 190 o C and 210 o C, respectively.The mass spectrometer was operated in the electron ionization mode with an electrical energy of 70 eV and an ion source temperature of 250 o C. The mass spectrum was scanned between 33 and 450 atomic mass units at 0.3 sec interval.

Compounds identification
The linear retention index (LRI) values for unknowns were determined based on retention time and index data obtained by analyzing a series of normal alkanes (C 6 -C 25 ).Volatile components were positively identified by matching their RI values and mass spectra with those of standards, also run under identical chromatographic conditions in the laboratory.The identification was also based on matching an unknown mass spectrum with the spectra available on the NIST (National Institute of Standards and Technology, USA) and mass spectral data system or the literature (Jennings & Shibamoto, 1980;Adams, 1995;Kondjoyan & Berdagué, 1996).

Statistical analysis
All determinations were obtained from triplicate measurements and results are expressed as means ± standard deviations.All results were processed using the SAS software (SAS Institute, Cary, NC) Version 9.1.3.Significant differences between the mean values of different characteristics were determined by applying Tukey's test for multiple comparisons at the probability of 5% (p ≤ 0.05).

Results and discussion
Based on the analysis of total ion chromatograms of the volatile extracts obtained from pulps of different cultivars (Collinson, Barker, Fortuna and Geada) of avocado extracted with different solvents, Table 1 summarizes the data on number of peaks in different chromatograms and its quantitative representation classified according to the peak area.
The data presented in Table 1 reveal the capture of a larger number (84) of volatile compounds when the extraction was performed using the pulp of cultivar Collinson, with the solvent mixture pentane-ether (2:1) for 80 min.However, for Barker and Geada cultivars a better extraction was obtained after 60 minutes, using hexane and pentane-ether (2:1) as a solvent, respectively.The best condition for extraction from Fortuna cultivar was the one realized for 80min, regardless of the solvent, when it was possible to separate 50 volatile compounds.It was also observed that increasing the extraction time from 40 to 80 min did not result in any major change in number of compounds in the extract obtained from Collinson and Geada cultivars.The extraction time which showed better efficiency in capturing the volatiles from Barker and Geada cultivars was 60min which is lower than that for Fortune and Collinson varieties (80 min).Chaintreau (2001) reported that the higher the lipid content of food matrix greater is the time required for volatiles extraction as lipid-containing matrix strongly increases the required time.Thus, the ideal conditions of extraction for a large number of volatile compounds depend on the cultivar of avocado and its lipid content (Galvão et al., 2014).
Table 2 lists the volatiles compounds identified in pulp of different cultivars -Collinson, Barker, Fortuna and Geada of avocado along with their characteristic odor notes.
An earlier work (Whitfield et al., 1980) reported a maximum of 90 volatiles compounds identified in avocado pulp.However, in the present study, the higher number of compounds recorded could be related to two factors such as selection of specific avocado varieties and the method of extraction employed when compared to other studies.Furthermore, the experimental conditions were also optimized which contribute to the achievement of better results.Although there was such difference seen in the number of peaks, the majority classes of compounds in all published work were seen to be those of terpenes and aldehydes.All terpenic compounds identified in this work were previously reported.However, among the 13 compounds belonging to aldehydes class found in this study, only one compound reported first time in this work was (E)-2-pentenal, which possesses fruity aroma and was found only in the cultivar Fortuna.
Among the volatile compounds identified in this study, hexanal represented over 25% of the total chromatogram in Fortuna, Collinson and Barker cultivars, this result is coherent with most of the work published (Yamaguchi et al., 1983;Guzmán-Gerónimo et al., 2008, Pereira et al., 2013;Dbenland et al., 2012).Hexanal (linoleic acid derivative) has been reported to be a volatile compound present in avocado pulp together with [E] 2-hexenal, octanal and nonanal.This compound is one the most abundant volatiles present in avocado and is mostly associated with low maturity fruit.The changes that occur in this compound also are known to characterize a green or grassy aroma.Dne of the clearest associations of aroma volatiles with flavor is related to the changes that occur in hexanal, 2-hexenal and 2,4-hexadienal, three aldehydes which characterize for a green or grassy aroma which is associated with ripening (El-Mageed, 2007, Pereira et al., 2013;Dbenland et al., 2012).
According to Haiyan et al. (2007), hexanal arises from linoleic acid, whereas octanal and nonanal are oleic acid derivatives.Hexanal levels were generally lower in avocado in which there was a higher oleic acid content which was also observed in our results.These results correspond to the physical and chemical parameters presented earlier, as well as to some studies on avocado oil (Haiyan et al., 2007;Villa-Rodríguez et al., 2011;Galvão et al., 2014).
The difference in concentration of some volatile compounds among the cultivars could be explained by several factors including the lipid concentration of the pulp for example the high concentration of hexanal in Fortuna cultivar could be related to the higher lipid content (16 g/100 g) of this pulp.Ethyl acetate was present in higher content in Collinson cultivar and this compound has an appreciative and fresh fruity flavor which could be responsible for high acceptability of this cultivar.
Among the fruits of the West Indian race (Barker and Geada), 4 compounds such as 2-octanol, tridecanol, benzaldehyde and propyl decanoate were present only in these cultivars However in hybrid (West Indian vs Guatemala) fruits belonging to Collison and Fortuna cultivars the compounds viz.2,3-butanediol and (E)-2-pentenal were specific and presente in both these cultivars.
The most prominent difference among the cultivars was in their total terpenes content which was 20% in Geada and lower concentrations of 3, 3.15 and 13% in Collison, Barker and Fortuna cultivars, respectively.Although Geada cultivar is characterized for its low lipid content (3g/100g) and early maturing of fruit (Gayet, 1995), it has the highest terpenes which characterizes from aroma standpoint this cultivar to be very different from other 3 cultivars.Benzaldehyde which possesses characteristic fruity and nutty odor note was present at a higher concentration (4,3%) in only Geada cultivar and in traces in Barker cultivar but it was not detected in Collinson and Fortuna cultivars.Among all these cultivars, the cultivars Geada and Fortuna cultivars are largely produced and commercialized in Brazil (Francisco & Baptistella, 2005;Cabia et al., 2014).According to Souza (2008), Brasil is a country where avocado production is mainly for the domestic market where Fortuna cultivar occupies good produtivity, good resistance to diseases and its higher pulp yield (Campos, 1984).However, this cultivar is considered to be of low aroma intensity and is mostly utilized for oil extraction while the cultivar Geada is much appreciated for its pleasing aroma.

Conclusions
This work studied the volatile composition of Barker, Collinson Geada and Fortuna cultivars of avocado fruit.The ideal conditions of extraction for capture of a large number of volatile compounds depend on the cultivar of avocado.The higher concentrations the some volatile compounds in Fortuna cultivar was related to higher lipid content.Hexanal was the main compound for Barker, Collinson and Fortuna cultivars while β−caryophyllene was for Geada cultivar.The aromatic profile of Barker cultivar was quite similar to that of Collinson variety which could be related primarily due to the almost same lipid content of these pulps.Benzaldehyde which possesses characteristic fruity and nutty odor note was present at a higher concentration (4.3%) in only Geada cultivar and in traces in Barker cultivar but it was not detected in Collinson and Fortuna cultivars.
The main cultivars of avocado grown In Brazil, are Fortuna, Hass, Fuerte, Geada and Yard.The Experimental Station of Itambé (IPA -region in the Pernambuco, Brazil) recommends the cultivation of Collinson, Barker, and Fortuna cultivars especially in the northeast region of Brazil where these are largely grown.Thus the objective of this work was to initially obtain volatile extracts, following a modified simultaneous distillation and extraction technique, from avocado pulp of different cultivars viz.Barker, Collinson, Fortuna and Geada by varying the extraction conditions such as use of different solvents and extraction time in order to evaluate the number of compounds present in these volatile extracts followed by identification and quantification of the volatile compounds present in the pulps of different cultivars.

Table 1 .
Distribution of number and peak area of volatile components in chromatograms resulting from different extracts obtained by varying extraction conditions of pulp from different cultivars of avocado fruit.