ANALYSIS OF VOLATILE COMPOUNDS OF Ilex paraguariensis A . ST .-HIL . AND ITS MAIN ADULTERATING SPECIES Ilex theizans MART

The adulteration of the product Ilex paraguariensis with other Ilex species is a mAjor problem for maté tea producers. In this work, three species of Ilex were evaluated for their volatile composition by headspace solid phase microextraction coupled to gas chromatography and mass spectrum detector (HS-SPME/GC-MS). The adulterating species I. dumnosa and I. theizans Mart. ex Reissek presented a different profile of volatile organic compounds when compared to I. paraguariensis. Aldehydes methyl-butanal, pentanal, hexanal, heptanal and nonanal were detected only in the adulterating species. This result suggests that such compounds are potential chemical markers for identification of adulteration and quality analysis of products based on Ilex paraguariensis.


INTRODUCTION
Maté tea (Ilex paraguariensis A. St.-Hil.) is a plant species native to South America.Its natural dispersion covers about 540,000 km 2 , divided between the territories of Brazil, Argentina, Uruguay and Paraguay.
In Brazil, its dispersion area mainly covers the Southern region (RS, SC and PR), where it is routinely used in the preparation of maté, a stimulant drink, prepared by the infusion of its milled leaves and twigs.In 2002, Brazil produced about 500,000 tons of maté tea, and more than 48% was produced in Rio Grande do Sul (VALDUGA, 2002).
Maté undergoes a number of steps (parching, drying and roasting) during the production process that significantly influence the chemical composition of the final product and hence its sensory characteristics (VALDUGA, 2003;FILIP et al., 2010).
The maté market is often the victim of adulteration by substitution of Ilex paraguariensis leaves with other numerous plants of the same species and family, or even plants of other families.However, when added to maté, these plants modify the characteristics of the final product, and may cause health hazards to consumers (HECH et al., 2007).
In a previous study, Pires et al. (1997) observed differences in the chemical composition of two species of Ilex (dumosa and paraguariensis), especially related to the concentration of saponins.According to the authors, these differences could be used to identify adulterants in the maté tea.Caffeine is another compound that is found in significant quantities only in the species Ilex paraguariensis.In other species of this family caffeine only occurs at trace levels (FILIP et al., 1998;REGINATTO et al., 1999).
This work aimed to achieve a qualitative characterization of volatile compounds present in fresh and roasted samples of three different species of the genus Ilex.Compounds that can be used as chemical markers were identified.Such compounds would allow easy detection of adulteration in commercial maté, and also determination of the chemical transformations (responsible for the flavor of the product) that occur during processing.The samples were randomly collected.Twenty kilograms of the species Ilex paraguariensis and 5 kg of the adulterant species were collected.The samples were placed in paper envelopes after collection and stored at approximately 10° C until extraction, which was carried out using 5 g of sample for each test.

Samples of
A fraction of each sample was subjected to the process of roasting, as described by Valduga (2002).
The purpose of this step was the denaturation of the enzymes present in the leaves and to decrease the water content.
Subsequently, the fresh and roasted samples were macerated, before passing through the extraction procedure, which was carried out in duplicate.

Conditions for isolation of volatile compounds
The extraction step was carried out by solid phase microextraction.The samples (5.0 g), previously macerated, were placed in a glass flask sealed with rubber septum.The polydimethylsiloxane fiber (100 mm, Supelco, USA) was previously conditioned following the instructions of the manufacturer.After conditioning the fiber was introduced into the flask and the temperature was gradually increased up to 60° C, and the fibers held at this temperature for an exposure period of 20 minutes.Subsequently, the fiber was collected and subjected to desorption in the gas chromatograph injector.

Instrumental Analysis
T he sam ples wer e an a lyzed usi ng a ga s chromatograph coupled to a mass spectrum detector (GC-MS -Shimadzu, QP5050A, USA), operating at 1.2 kV impact energy.The fragments were collected between 40 and 400 Da.The chromatographic analysis was carried out using an initial temperature of 45° C, which was kept constant for 5 minutes, followed by an increase up to 190° C at 10° C min -1 , where it remained for more 10 minutes.The pressure used in the column was 25 kPa, with a flow rate of carrier gas (He) 0.7 mL min -1 and split ratio of 1 / 10.The chromatographic column used was a DB-5 (30m x 0.25 mm x 0.25 mm, J & W Scientific, USA), with nonpolar characteristics.The temperature employed in the injector, responsible for desorption of the compounds of the fiber, was 230° C. The interface temperature was 250° C. With the exception of linalool, acetic acid, ethanol, butanal, hexanal, propanoic acid and acetaldehyde, which were compared to external standards, the other compounds were identified by comparing their mass spectra with the database of the libraries of mass spectra (Wiley 229, Nist 21 and 107).In t hi s case, t h ese com poun ds wer e consi dered qualitatively identified.

RESULTS AND DISCUSSION
Compounds qualitatively identified using the libraries of mass spectra, with a probability (match quality) exceeding 90%, are presented in Table 1.Ciênc. agrotec., Lavras, v. 35, n. 6, p. 1166-1171, nov./dez., 2011 Table 1 -Volatile compounds qualitatively identified in fresh and roasted samples of the three species Ilex.The order of the compounds listed in the table corresponds to the order of elution.
a Compounds identified only in adulterants (roasted samples).The sample of roasted Ilex paraguariensis presented an increase in the number of compounds extracted in relation with the fresh sample.This same behavior was also observed for the adulterants species studied (Table 1).Regardless of the species analyzed, significant reductions in the intensity (area) of peaks corresponding to the roasted sample were observed, when compared to the fresh samples.Both of these features (increase in the number of signals and reduction of their respective areas) have been linked to Maillard reactions (KAWAKAMI; KOBAYASHI, 1991) (presence of furfural in the roast sample of the Ilex dumosa species) and oxidative changes occurring during roasting.The high temperatures (  350° C) associated with the oxidizing atmosphere employed during roasting favors the oxidative cleavage of compounds of long carbon chains into compounds of smaller carbon chains, rather than the gradual oxidation of functional groups (alcohol  aldehyde  carboxylic acid) present in these compounds.Both changes lead to the formation of compounds with lower boiling points, favoring the volatilization and consequently its extraction.The presence of acetic acid only in the roasted sample of the Ilex paraguariensis species was linked to oxidative transformation of ethanol during the step of roasting.
Many alcohols and aldehydes qualitatively identified in the adulterant species (fresh and roasted) were observed by Kawakami and Kobayashi (1991) as volatile constituents of green and roasted maté.
Considering the percentage areas (Table 2) of the identified compounds, there was a predominance of alcohols and aldehydes in the three studied species (Ilex paraguariensis, Ilex theizans Mart.ex Reissek and Ilex dumosa).Among these two classes of compounds, a predominance of alcohols over aldehydes in the fresh samples was observed.Aldehydes prevail in the roasted samples.
This trend (aldehyde > alcohols) observed for the roasted samples was related with the conditions employed during the step of roasting (high temperature and oxidizing atmosphere), which favors the oxidation of alcohols to aldehydes and even to carboxylic acids.Similar results were observed by different authors studying green coffee and roasted coffee (MERRITT et al., 1985;SHIBAMOTO ;YEO, 1992;SHIBAMOTO;YAMAGUCHI, 1981).
The oxidative cleavage of the double bonds of fatty acids leads to the formation of aldehydes and ketones (VARNAM; SUTHERLAND, 1997).When lipids are exposed to heat in the presence of oxygen, they may decompose into secondary products, mainly aldehydes and alcohols (SHIBAMOTO; YAMAGUCHI, 1981).
The 2,4-heptadienal, a product of fatty acid degradation, was observed only for the roasted samples of Ilex theizans Mart.ex Reissek.This compound was also reported by Machado et al. (2007) and Kawakami and Kobayashi (1991) in the composition of volatiles in maté tea.
Some compounds identified in this study, such as linalool and hexanal, are employed by some authors to indentify teas of superior quality (SHIBAMOTO; KATO, 2001;GUYOT et al., 1982).
Other compounds were present only in the roasted samples of adulterant species (Table 1), like 5 to 8-carbon aldehydes.The presence of specific compounds in one or more species of the same family allows its distinction from the other species and can be used to certify the quality of a product.
Chemical indicators have been employed by Guyot et al. (1982) to differentiate a healthy green Arabica coffee from a coffee that was considered defective.The compounds ethyl isobutanoate, ethyl butanoate, ethyl 2methyl-butanoate and n-hexyl acetate were observed only in the volatile fraction of the defective coffee.Similar studies were conducted by Merritt Junior et al. (1985) to identify certain types of coffee, considering the presence of some esters of high molecular weight alcohols, only found in Colombian coffee beans.

CONCLUSIONS
The proposed methodology was suitable to characterize, in a quick and simple manner, volatile compounds present in three species of Ilex, in fresh and roasted samples.
The qualitative differences observed between the volatile fractions of three species of Ilex allowed them to be differentiated from each other.The aldehydes methyl butanal, pentanal, hexanal, heptanal, octanal and nonanal were only observed in the adulterant species I. theizans Mart.ex Reissek and I. dumosa.They were defined as plausible chemical markers for use in the identification of adulterants during quality control of products derived from maté tea I. paraguariensis.
It is noteworthy that the conditions employed during the roasting stage (high temperatures and an oxidizing atmosphere) lead to oxidative transformations of the compounds present in Ilex samples, increasing the occurrence of aldehydes in relation to alcohols, what can improve the flavor of the final product.

Table 2 -
Contribution as a percentage of the area, linked to the compounds identified as alcohols and aldehydes present in fresh and roasted samples.