Received: 20 May 2014 Revised: 11 December 2014 Accepted: 12 December 2014 Published online in Wiley Online Library: 24 March 2015
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1 Research article Received: 20 May 2014 Revised: 11 December 2014 Accepted: 12 December 2014 Published online in Wiley Online Library: 24 March 2015 (wileyonlinelibrary.com) DOI /jib.216 Multivariate analysis for the characterization of physico-chemical profiles of cachaça produced in copper stills over a period of six years in Minas Gerais state Wilder Douglas Santiago, 1 Maria das Graças Cardoso, 1 * Lidiany Mendonça Zacaroni, 1 Leonardo Milani Avelar Rodrigues, 2 Felipe Cimino Duarte, 2 Cleusa de Fátima e Silva Ribeiro 1 and David Lee Nelson 3 Cachaça, a traditional, popular Brazilian alcoholic drink, is increasingly appreciated in many countries. In Brazil, it is the most extensively consumed alcoholic beverage and it has great relevance for agribusiness in the country, especially for the state of Minas Gerais, which is the main centre for the production of alambic cachaça. Thus, attempts to standardize techniques in the production chain of the beverage have been made to maintain the quality required by law. The physico-chemical profile of cachaça produced in copper stills in different cities of Minas Gerais, Brazil over the years was determined by applying principal component analysis (PCA). According to the physico-chemical data, % of the samples were within the boundaries of standards identity and quality established by Brazilian legislation. According to the PCA, the composition of the cachaça produced in Minas Gerais between the years 2006 and 2011 was homogeneous. Copyright 2015 The Institute of Brewing & Distilling Keywords: legislation; sugarcane spirit; secondary compounds; contaminants; quality 244 Introduction Cachaça or distilled sugarcane spirits is a traditional beverage obtained from the fermented mash of sugarcane in Brazil. Under current law, the distinction between sugarcane spirits and cachaça is based on the alcohol concentrations at 20 C, which are 38-48% and 38-54% (v/v), respectively. Both types of beverage may contain 6 g L 1 of added sugar, expressed as sucrose (1). The cachaça agribusiness is an important contributor to the Brazilian market. It is estimated that the sector produces 1.5 billion litres of the drink annually, with a total of 30,000 manufacturing establishments generating over 450,000 direct and indirect jobs. Nationally, it is ranked as the second most extensively consumed alcoholic beverage. It ranks as the third most extensively consumed distilled beverage in other countries. Caipirinha is one of the most frequently sold beverages. However, the exportation of cachaça is still insignificant. Only 1% of the total production is exported to countries such as Germany, Italy, France, USA and Japan; so virtually all the production is destined for the domestic market (2). Cachaça production exists in almost all the states of Brazil; however, São Paulo, Pernambuco, Ceará, Rio de Janeiro, Goiás and Minas Gerais are the principal producing states. These states account for about 75% of the national production. The state of São Paulo is specialized in the production of industrial spirits in large-scale stainless steel fractionation columns, and Minas Gerais is considered to be the hub of alambic cachaça production, which is performed on a small scale using copper stills. It is estimated that 50% of the beverage produced in alambic stills is produced in this state, which has 5000 stills that produce 200 million litres of cachaça per year, especially in the north, Jequitinhonha and Rio Doce regions. These regions are responsible for about 63% of the production in the state, but only 0.3% of this production is exported. Although the processes for the production of these beverages are different, studies should be undertaken to differentiate between them on the basis of their physico-chemical properties to generate a fingerprint that would enable authorities to establish an official distinction between industrial and alambic cachaça (3,4). In spite of the traditional and economic importance of this beverage for the country, the production chain is not homogeneous. Improvements have been sought in the development of technology to improve the quality and to standardize the beverage in * Correspondence to: M. das Graças Cardoso, Department of Chemistry, Federal University of Lavras, CP 3037, Lavras, Minas Gerais, Brazil. mcardoso@dqi.ufla.br 1 Department of Chemistry, Federal University of Lavras, CP 3037, Lavras, Minas Gerais, Brazil 2 Department of Food Science, Federal University of Lavras, CP 3037, Lavras, MG, Brazil 3 College of pharmacy, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil J. Inst. Brew. 2015; 121: Copyright 2015 The
2 Multivariate analysis for the characterization of cachaça profile terms of its physico-chemical and sensory aspects (5,6). The beverage s quality must meet the Standards of Identity and Quality required by Brazilian law, as well as the sensory requirements involving colour, taste and aroma (1). All the stages of production that involve the preparation of the raw material, extraction of the sugarcane juice, fermentation and distillation must be properly controlled. According to Cardoso (7), the most important step is the fermentation stage, in which the formation of ethanol and secondary compounds such as aldehydes, methanol, higher alcohols, acids and esters occurs. Multivariate analysis has been widely used for the characterization of various food products such as cachaça and other alcoholic beverages. It is a statistical tool whose goal is to correlate the analytical results between samples (objects) through chemical analysis (chemometrics). Among the methods used, principal component analysis (PCA) transforms a set of original, intercorrelated variables into a new set of uncorrelated variables, called principal components (8,9). This study sought to evaluate the physico-chemical profile of cachaça, obtained from different cities of Minas Gerais, Brazil in the years by applying PCA to elucidate similar characteristics for the parameters analysed, correlating them and gathering data with respect to the year of production. Material and methods Samples Samples were collected from producers of alambic cachaça from different cities in the state of Minas Gerais, Brazil. The collection began in 2006 and ended in The number of cachaça samples collected during this period is presented in Table 1. Physico-chemical analysis The physico-chemical analyses were performed at the Laboratory for the Analysis of Spirit Quality, Department of Chemistry, Federal University of Lavras according to the methods recommended by the Ministry of Agriculture, Livestock and Supply (10). The parameters analysed with the respective analytical methods are described below. Alcohol concentration. This was determined by densitometry, with the result expressed in percentage by volume. The samples were re-distilled, and the alcohol content was determined by measurements at 20 C with the aid of a digital hydrometer (DensiMat Gilbertini). Volatile acidity. This was determined by volumetric titration. The volatile acids were extracted by steam distillation using a Table 1. Samples collected in the respective years Year of collection Samples collected (units) Total 396 Gilbertini Electronic Enochimico Distiller. The acids obtained were titrated with 0.1 M NaOH in the presence of 1% phenolphthalein as an indicator. The results were expressed in grams of acetic acid per 100 ml of sample. Higher alcohols. The total amount of higher alcohols was determined by the measurement of a colour reaction on a Shimadzu UV-1601 PC spectrophotometer at 540 nm. The concentrations were determined by interpolation in external calibration curves of the higher alcohols dissolved in water ethanol, 1:1. The total amount of these compounds was expressed in milligrams per 100 ml of anhydrous ethanol Aldehydes. These were analysed by direct titration of the SO 2 formed by the reactions involved in the analysis with 0.05 M iodine. The amount of aldehydes was expressed in grams of acetaldehyde per 100 ml of sample. Furfural. Quantification was accomplished by means of spectrophotometric readings at 520 nm (Shimadzu UV-1601 PC) and comparison of the absorbance observed in the samples with those of a calibration curve previously constructed with standard solutions of ethanol and furfural. The alcoholic strength of the distillate was corrected to 50 GL. The results were expressed as milligrams of furfural in 100 ml of anhydrous ethanol Esters. These were determined by titration of the carboxylic acid esters obtained by transesterification of the beverages. The concentrations were expressed in grams of ethyl acetate per 100 ml of ethanol. Methanol. Quantification was performed by colorimetric reactions and measurement at 575 nm on a spectrophotometer (Shimadzu UV-1601 PC). The concentrations of this compound were determined by the construction of calibration curves of ethanol with methanol. The results were expressed in millilitres of methanol per 100 ml of anhydrous ethanol. Copper. This analysis was performed by spectrophotometric (Shimadzu UV-1601 PC) measurements at 546 nm that were compared with the absorbance values of a calibration curve previously constructed using copper sulphate. The colorimetric determinations were performed on cachaças prior to re-distilling. The results were expressed in milligrams per litre of anhydrous ethanol. PCA Principal component analysis was used to investigate and understand the similarity amongst the samples regarding the parameters analysed in the years 2006, 2007, 2008, 2009, 2010 and The results were centred on means for further analysis. The analysis was performed using the R statistical program (11). Results and discussion The results of the physico-chemical evaluations of alambic cachaça produced in the state of Minas Gerais are presented in Fig. 1. A favourable percentage of product acceptability across the consumer market was obtained, with values ranging from 61.5 to 77.9%. These results agree with those of Pereira et al. (12), who evaluated secondary compounds in 45 cachaça samples produced in Minas Gerais and observed acceptability scores of 75.6%. In 2007, Fernandes et al. (13) evaluated 16 samples of cachaça from producers in Minas Gerais and obtained nearly 56.2% acceptability. In the same year, Miranda et al. (14) studied 245 J. Inst. Brew. 2015; 121: Copyright 2015 The wileyonlinelibrary.com/journal/jib
3 Figure 1. Result of physico-chemical analysis of cachaças of stills produced from 2006 to This figure is available in colour online at wileyonlinelibrary.com/journal/jib the chemical quality of Brazilian cachaça and spirits and observed that, amongst the 94 samples analysed, 52.0% were within the limits established by legislation. Their results were lower than those obtained in our study. The percentages of acceptability of each parameter analysed are presented in Figs. 2 and 3. The values ranged from 81.4 to 98.5% for alcohol content; from 87.6 to 94.4% for volatile acidity; from 96.8 to 100% for ester concentration; from 92.5 to 98.1% for aldehyde concentration; from 93.8 to 100% for the higher alcohols; from 96.2 to 100% for furfural; from 96.9 to 100% for methanol; and from 77.7 to 88.8% for copper concentrations. Among the compounds analysed, it was found that the concentrations of alcohol and copper were the least acceptable. W. D. Santiago et al. According to Cardoso (7), the alcohol content can be affected by the fermentation process, improper fraction cuts (head, tail and heart) during distillation or by improper packaging of the cachaça. Excess copper in cachaça is a problem that concerns the producers of cachaça in the state of Minas Gerais. The presence of this metal is mainly due to the dissolution of the basic copper carbonate [Cu 2 (OH) 2 CO 3 ] present on the inner wall of the still by acid vapours entrained by the beverage. Contamination can be prevented through careful cleaning of the stills during and between seasons, using water and lemon in the first distillation. Copper can also be removed by the use of adsorbents such as activated charcoal filters and ion exchange resins (7,15). In addition, contamination of the beverage by copper is an obstacle for exportation because the required levels abroad are much lower (2 mg L 1 ) than what is required by Brazilian law (5 mg L 1 ). According to the literature, esters can be formed by yeast during intracellular secondary metabolism during fermentation or by the esterification of alcohols and carboxylic acids during distillation and aging. Esters present at low concentrations incorporate a pleasant aroma of fruit to cachaça, but give the drink a nauseating and undesirable flavour when they are present in high concentrations (7,16,17). A high percentage of the samples contained ester concentrations within acceptable limits, thus indicating that the products were adequate for commercialization. Methanol is undesirable in a beverage because of its high toxicity. Ingestion, even in small amounts, can cause blindness or even death (18). Its formation results from the degradation of pectin, a polysaccharide present in sugarcane, during the fermentation process. The contamination of the beverage by methanol 246 Figure 2. Result of physico-chemical analysis of (A) alcoholic content, (B) volatile acidity, (C) esters and (D) aldehydes. This figure is available in colour onlineatwileyonlinelibrary. com/journal/jib wileyonlinelibrary.com/journal/jib Copyright 2015 The J. Inst. Brew. 2015; 121:
4 Multivariate analysis for the characterization of cachaça profile Figure 3. Result of physico-chemical analysis of (E) higher alcohols, (F) furfural, (G) methanol and (H) copper. This figure is available in colour online at wileyonlinelibrary.com/ journal/jib can be avoided by adequately filtering the wort and by avoiding the presence of bagasse in the fermentation process (7,19). The aldehydes are formed during the fermentation process. Their formation originates from the action of yeast during the preliminary stages of the fermentation process, and they tend to disappear in the final stages because of oxidation to acetic acid, especially during excessive aeration of the wort. The principal aldehyde formed in this stage of production of the beverage is acetaldehyde. The other aldehydes are probably obtained from the oxidation of higher alcohols. Poisoning by aldehydes can lead to serious problems related to the central nervous system (13). The concentrations of aldehydes encountered in this study were low, as the acceptability results indicate. It is worth noting that it is important to determine the concentrations of acetaldehyde because it plays a central role in the manifestation of intoxication. In wines, high concentrations of acetaldehyde result in an undesirable herbaceous aroma, and low concentrations lead to an apple aroma (20). Brazilian law limits total aldehydes in cachaça to 30 mg of acetaldehyde per 100 ml of anhydrous ethanol. From 87.6 to 94.4% of the samples contained acceptable levels of volatile acidity, which shows that most of the samples were below the limit established by law, which is 150 mg per 100 ml of anhydrous alcohol. High amounts are often associated with the practice of storing the cane and with the contamination of the wort by acetic bacteria, which occurs when the period between the fermentation process and distillation is too long (12). Furfural and hydroxymethylfurfural are organic contaminants in cachaça and should not be present because they are considered harmful to health (21). The legislation sets maximum levels of these compounds at 5 mg per 100 ml of anhydrous alcohol. These compounds are formed by dehydration of pentoses and hexoses during production (7). Masson et al. (19) quantified furfural in samples of cachaça produced from burned and unburned sugarcane. The authors concluded that the burning of sugarcane leaves previous to harvest provided a significant increase in the concentration of furfural in the cachaça. A partial dehydration occurs in a small fraction of the sugars in the cane as a result of the burning, thus making it an excellent adherent for residues from combustion of stems, solid soil particles and other minerals. These residues are transferred to the broth, and the suspension goes to the vats and then to the still. The organic matter is converted into furfural and hydroxymethylfurfural, and these substances are carried over to the final product. Barcelos et al. (22) analysed several compounds in cachaça produced from unburned sugarcane in three different regions of Minas Gerais and concluded that all of the samples contained concentrations of furfural below the limit set by Brazilian legislation. Similar results were observed in the present work. The higher alcohols are formed during the oxidation process. They can be derived from transformations of amino acids during fermentation as a result of a low degree of activity of the yeast, high temperatures and a low ph of the mash. These compounds, together with their esters, are responsible for the flavour of the cachaça (23). The low concentration of higher alcohols may be related to attentiveness in cutting the sugarcane, as well as to the time necessary for milling and fermentation. Because of the complexity of cachaça composition and the large number of samples, an exploratory statistical study of the samples using PCA was performed. The biplot graph of PC1 PC2 loadings and scores, relating the physico-chemical parameters to the production year, is shown in Fig. 4. The PCA showed that 92.7% 247 J. Inst. Brew. 2015; 121: Copyright 2015 The wileyonlinelibrary.com/journal/jib
5 W. D. Santiago et al. Figure 4. Biplot PC1 PC2 of loadings and scores for 396 samples of cachaça regarding their physico-chemical parameters of the respective studies. Figure 6. Biplot PC1 PC2 of loadings and scores for 63 samples of cachaça produced in 2007 regarding their physico-chemical parameters. of the data was described by the first and the second main components and 58.7% of the total variance was described by the first principal component. Through the analysis of the physico-chemical data generated by the PCA technique, the majority of the samples were combined into a single group to demonstrate the similarities between them. Some variables and most of the samples were concentrated near the origin of the main plane, that is, these variables have a low representation. Thus, the physical chemical properties of the cachaças in this study were homogeneous between the years 2006 and 2011, a fact that could not be concluded from Fig. 1. Because of the high number of samples, the PCA test for each year of production of artisanal cachaça was conducted to ascertain whether there was a similarity in physico-chemical parameters between years. The results are shown in Figs On the basis of the first and second principal components, it was possible to describe 91.4, 93.5, 96.1, 93.7, 92.0, and 94.7% of the data for the years 2006, 2007, 2008, 2009, 2010 and 2011, respectively (Figs. 5 10). As shown in Fig. 4, most of the variables and the samples were concentrated near the origin of the main plane, Figure 7. Biplot PC1 PC2 of loadings and scores for 68 samples of cachaça regarding their physico-chemical parameters. 248 Figure 5. Biplot PC1 PC2 of loadings and scores for 65 samples of cachaça produced in 2006 regarding their physico-chemical parameters. wileyonlinelibrary.com/journal/jib Copyright 2015 The J. Inst. Brew. 2015; 121:
6 Multivariate analysis for the characterization of cachaça profile being of homogeneous physical and chemical compositions relative to the year of production. In all of the years analysed, the dispersion observed for some samples related to the volatile acidity and higher alcohol esters parameters, but there was no significant dispersion, the number of grouped samples being lower than the total number of samples. Conclusions From 61.5 to 77.9% of the samples were within the boundaries of identity and quality established by Brazilian legislation. From the PCA, we inferred that the alambic cachaças produced in Minas Gerais between the years 2006 and 2011 were homogeneous. Figure 8. Biplot PC1 PC2 of loadings and scores for 81 samples of cachaça produced in 2009 regarding their physico-chemical parameters. Acknowledgements The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado de Minas Gerais and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for financial support, and the cachaça producers of Minas Gerais. Figure 9. Biplot PC1 PC2 of loadings and scores for 65 samples of cachaça produced in 2010 regarding their physico-chemical parameters. Figure 10. Biplot PC1 PC2 of loadings and scores for 54 samples of cachaça produced in 2011 regarding their physico-chemical parameters. References 1. Brasil, Ministério da Agricultura, Pecuária e Abastecimento (2005) Instrução normativa no. 13, de 29 de junho de APEX Brasil (2008) Agência de Promoção de Exportações e Investimentos, Available from: 3. Cançado Júnior, F. L., Paiva, B. M., and Estanislau, M. L. L. (2009) Evolução do mercado de cachaça nos anos Informe Agropecuário 30(248), Oliveira, S. G., Magalhães, M. A., and Bergerat, P. C. (2009) A luta da cachaça de alambique para se consolidar como fonte de riqueza em Minas Gerais. Informe Agropecuário 30(248), Zacaroni, L. M., Cardoso, M. G., Saczk, A. A., Santiago, W. D., Anjos, J. P., Masson, J., Duarte, F. C., and Nelson, D. L. (2011) Caracterização e quantificação de contaminantes em aguardentes de cana. Química Nova 34, Anjos, J. P., Cardoso, M. G., Saczk, A. A., Dórea, H. S, Santiago, W. D, Machado, A. M. R., Zacaroni, L. M., and Nelson, D. L. (2011) Evolution of the concentration of phenolic compounds in cachaça during aging in an oak (Quercus sp.) barrel, J. Brazil. Chem. Soc., 22, Cardoso, M. d. G. (2006) Produção de aguardente de cana, 2nd ed.,, Lavras, Editora UFLA. 8. Rencher, A. C. (2002) Methods of Multivariate Analysis, Wiley, Nova Iorque. 9. Nieuwoudt, H. H., Prior, B. A., Pretorius, I. S., Manley, M., and Bauer, F. F. (2004) Principal component analysis applied to Fourier transform infrared spectroscopy for the design of calibration sets for glycerol prediction models in wine and for the detection and classification of outlier samples. J. Agric. Food Chem. 52, Brasil, Ministério da Agricultura, Pecuária e Abastecimento (2005) Instrução normativa no. 24, de 08 de setembro de Team, R. C. (2012) R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna. 12. Pereira, N. E., Cardoso, M. G., Azevedo, S. M., Morais, A. R., Fernandes, W., and Aguiar, P. M. (2003) Compostos secundários em cachaças produzidas no Estado de Minas Gerais. Ciência Agrotecnol. 27, Fernandes, W. J., Cardoso, M. G., Vilela, F. J., Morais, A. R., Silva, V. F., and Nelson, D. L. (2007) Physicochemical quality of a blend of domestic cachaças from the South of Minas Gerais. J. Food Compos. Anal. 20, Miranda, M. B. de, Martins, N. G. S., Belluco, A. E. S., Horii, J., and Alcarde, A. R. (2007) Qualidade química de cachaças e de aguardentes brasileiras, Ciência Tecnol.Aliment. 27, Lima, A. J. B., Cardoso, M. G., Guimarães, L. G. L., Lima, J. M., and Nelson, D. L. (2009) Efeito de substâncias empregadas para remoção de cobre sobre o teor de compostos secundários da cachaça. Química Nova 32, J. Inst. Brew. 2015; 121: Copyright 2015 The wileyonlinelibrary.com/journal/jib
7 16. Boscolo, M., Bezerra, C. W. B., Cardoso, D. R., Neto, B. S. L., and Franco, D. W. (2000) Identification and dosage by HRGC of minor alcohols and esters in Brazilian sugar-cane spirit. J. Brazil. Chem. Soc. 11, Nascimento, E. P., Cardoso, D. R., and Franco, D. W. (2009) Comparação de técnicas de determinação de ésteres em cachaça. Química Nova 32, Badolato, E. S. G., and Duran, M. C. (2000) Risco de intoxicação por metanol pela ingestão de bebidas alcoólicas. Rev. Psiquiat. Clín. 27, Masson, J., Cardoso, M. G., Vilela, F. J., Pimentel, F. A., Morais, A. R., and Anjos, J. P. (2007) Parâmetros físico-químicos e cromatográficos em aguardentes de cana queimada e não queimada. Ciência Agrotecnol. 31, Azevêdo, L. C., Reis, M. M., Silva, L. A., and Andrade, J. B. (2007) Efeito da presença e concentração de compostos carbonílicos na qualidade de vinhos. Química Nova 30, W. D. Santiago et al. 21. Santiago, W. D., Cardoso, M. G., Zacaroni, L. M., Anjos, J. P., Machado, A. M. R., and Mendonça, J. G. P. (2012) Perfil físico-químico e quantificação de compostos fenólicos e acroleína em aguardentes de cana-de-açúcar armazenadas em tonéis de diferentes madeiras. Rev. Cient. 40, Barcelos, L. V. F., Cardoso, M. G., Vilela, F. J., and Anjos, J. P. (2007) Teores de carbamato de etila e outros componentes secundários em diferentes cachaças produzidas em três regiões do estado de Minas Gerais: Zona da Mata, Sul de Minas e Vale do Jequitinhonha. Química Nova 30, Vilela, F. J. V., Cardoso, M. G., Masson, J., and Anjos, J. P. (2007) Determinação das composições físico-químicas de cachaças do Sul de Minas Gerais e de suas misturas. Ciência Agrotecnol. 31(4), wileyonlinelibrary.com/journal/jib Copyright 2015 The J. Inst. Brew. 2015; 121:
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