RELEVANCE OF ETHYL LACTATE AMONG THE CACHAÇA ESTERS RELEVANCIA DEL LACTATO ETÍLICO ENTRE LOS ÉSTERES DE CACHAÇA RELEVÂNCIA DO LACTATO ETÍLICO ENTRE OS ÉSTERES DA CACHAÇA

The advancement in the technology of cachaça ─ sugarcane spirit produced in the Brazilian territory ─ has led to the search for parameters that allow the improvement of its chemical characterization and sensory specificities. Esters are important aroma agents, but their routine monitoring, as endorsed in Brazilian legislation, is restricted to the quantification of ethyl acetate, which is formed through the metabolism of yeast (Saccharomyces cerevisiae). However, being produced in a rural environment using fresh cane juice, fermentation of “cachaça de alembic” involves the presence and activity of lactic acid-producing bacteria, making the presence of ethyl lactate among the cachaça esters predictable. In this work, the levels of ethyl lactate and ethyl acetate were compared in 247 samples, corresponding to 56 brands produced in eleven Brazilian states. Ethyl lactate was found in levels that significantly alter the result of the quantitative participation of esters in the composition of cachaça. This fact is especially relevant considering that lactic acid bacteria are GRAS and widely recognized as a resource for improving the sensory quality of wines and other alcoholic beverages. Thus, studies on the contribution of lactic acid bacteria to the sensory quality of cachaça should be encouraged. Ethyl lactate, in addition to signaling a chemical specificity naturally occurring in cachaça, is a marker of other possible chemical and sensory peculiarities whose research should be stimulated.


INTRODUCTION
In addition to ethanol, distilled beverages contain a large number of secondary components, as acetaldehyde, acetic acid, propanol, isobutanol, isoamyl alcohol, ethyl acetate and other aldeydes, acids, higher alcohols and esters (Scanavini, 2010). In cachaça (the beverage produced in Brazilian territory by the distillation of fermented must from sugarcane juice) current legislation determines the quantification of these compounds by functional category, being expressed as total aldehydes, total volatile acids, total higher alcohols and total esters (Brasil, 2005).
In the last decades, however, lactic acid bacteria were recognized because of their important role in the sensory improvement of alcoholic beverages, and their intentional inoculation is a common practice (Edwards & Beelman, 1989;Revel et al., 1999;Gámbaro et al., 2002;Bauer & Dicks, 2004;Kurdys et al., 2018). Researchers highlight the effect of lactic acid bacteria on sensory enrichment, 3 which, at least in part, results from the formation of ethyl lactate by the reaction between lactic acid secreted by bacteria and ethanol secreted by yeasts (Sumby et al., 2010).
There are marked sensory differences between ethyl acetate and ethyl lactate. In alcoholic beverages, the presence of ethyl acetate can be noticed under 20 mg/L, providing a fruity aroma (Salo, 1970;Verstrepen et al., 2003). However, negative associations with glue, varnishes and solvents appear at concentrations greater than 100 mg/L. For ethyl lactate, the perception threshold is around 110 mg/L, the sensory effect is positive and it results in associations with nuts, vanilla and butter even at concentrations greater than 300 mg/L (Knoll et al., 2011;Kurdys et al., 2018).
There are no reports of intentional inoculation of lactic acid bacteria to cachaça fermentation must and this practice is not foreseen in its production process. However, ethyl lactate has already been identified in samples of cachaça (Nascimento et al., 2008;Fernandes et al., 2017;Karp et al., 2019). So far, however, the occurrence of this ester has been interpreted only as an indicator of failures in the cleaning of sugarcane juice, which should be remedied with the technological advancement of the sector.
The legislation regarding cachaça is currently under review by government agencies, with a view to advances in the scope of product characterization and inspection and operational procedures.
The present study sought to quantify the extent of occurrence of ethyl lactate in Brazilian cane spirit with a view to better discerning its role in the identity of the beverage.  4 Analytical conditions: The oven temperature was maintained at 35°C for 3 min; the oven was heated from 35 °C to 80°C at a rate of 5.0°C min -1 ; the temperature was maintained at 80°C for 3 min, and then heated from 80°C to 165°C at 6.1°C min -1 . The injector temperature was 140 °C; the detector temperature was 180°C; injection volume, 2 µL in split mode (1:1); nitrogen carrier gas (6.0 mL min -1 ).

Samples
Peak quantification was obtained using calibration curves previously prepared with standard solutions for each component.

Expression of results
The means and standard deviations for each brand and from the variable number of samples were calculated for the concentrations of ethyl acetate and ethyl lactate, always expressed in mg per 100 ml of anhydrous ethanol. In the presentation of the results, the sequence of the brands was based on specific criteria, with no correspondence between the numbers and respective brands in the different figures.

Statistical analysis
Student's t-test was applied: (a) to assess whether the quantification of esters by the ethyl acetate content was significantly altered by the increase in ethyl lactate content; (b) to compare the ranges of variation in the levels of ethyl acetate and ethyl lactate within each brand.

RESULTS AND DISCUSSION
The number of samples per brand varied between 2 and 24 (Table 1). Of the 56 brands, one was selected for separate evaluation because it involved monitoring two crops, totaling 24 samples.
The remaining 55 samples were evaluated together. The data collected is presented in Table 2. The numbering of the brands corresponds to the alphabetical order of the respective names. As can be seen in Table 2 To facilitate visualization, results of minimum and maximum levels of ethyl acetate and ethyl lactate are shown in Figure 1 for 55 brands in decreasing order of the ranges of variation (within each brand). One of the brands (brand 56) was reserved for specific evaluations (Figures 2 and 3 The occurrence of ethyl lactate in cachaça has been attributed to operational failures in fermentation and distillation (Serafim et al., 2013). It is assumed that, because they come exclusively from natural sources, lactic acid bacteria are contaminants to be combated, which can cause  8 complete reversal of the trend, with a large predominance of ethyl lactate over ethyl acetate. These data prove that the standardization procedures for annual harvests and between successive harvests are really essential, both for sensory adjustment and for chemical characterization of each brand.   9 fermented), as well as between different batches of the same producer, due to natural factors.
Therefore, monitoring ethyl lactate content is relevant and can contribute to the scope of the characterization chemical identity of the cachaça. It can also be used as a tool for standardization criteria of lots and vintages and as a reference for stimulating new advances in the scope of the appreciation of cachaça on the world stage. In addition, the analysis of ethyl lactate in cachaça does not introduce any difficulty in the analytical procedure; it is only necessary to identify and quantify an additional peak (ethyl lactate), which occurs under the same chromatographic conditions required for the analysis of acetate ethyl and other aroma components routinely analyzed.