Received: 13 September 2016 Revised: 31 December 2016 Accepted: 1 January 2017 Published online in Wiley Online Library: 6 April 2017

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1 Research article Received: 13 September 2016 Revised: 31 December 2016 Accepted: 1 January 2017 Published online in Wiley Online Library: 6 April 2017 (wileyonlinelibrary.com) DOI /jib.414 Cachaça stored in casks newly constructed of oak (Quercus sp.), amburana (Amburana cearensis), jatoba (Hymenaeae carbouril), balsam (Myroxylon peruiferum) and peroba (Paratecoma peroba): alcohol content, phenol composition, colour intensity and dry extract Wilder Douglas Santiago, 1 Maria das Graças Cardoso 1 * and David Lee Nelson 2 Aging is the last stage of the cachaça production process. The containers used for aging are wooden barrels and casks, which act as a semi-permeable membrane, allowing the passage of alcohol and water vapours. This passage is a function of the relative humidity and the temperature of the storage location. The wood traditionally used in Brazil is oak. However, various native woods of Brazilian origin have been used in the manufacture of barrels. The principal compounds extracted from wood by the distillates are volatile compounds, phenolic compounds, tannic substances, sugars, glycerol and non-volatile organic acids. The objectives of this study were to evaluate and compare the phenolic substances found in cachaça stored in recently constructed oak, amburana, Jatoba, balsam and peroba casks. We evaluated some physicochemical parameters that changed during the storage process. After 12 months of storage, we observed a decrease in alcohol content and an increase in dry extract. An increase in total phenolic compounds and colour intensity was observed, and there was a strong linear correlation between total phenolic compounds, solids and colour intensity. According to the results for the phenols analysed by HPLC, a progressive increase in all the compounds and a heterogeneity in all woods used for the storage of beverages were observed. Copyright 2017 The Keywords: alcoholic beverage; aging; organic compounds; HPLC Introduction Cachaça, widely consumed in Brazil, is a beverage produced in all regions of the country. The estimated annual production is 1.4 billion L, 4000 brands are registered and there exist about 40,000 legalized and illegal producers. According the Brazilian legislation, 90% of these are small farmers and commercial cachaça is found in more than 960,000 points of sale, including bars, supermarkets and restaurants, generating about 600,000 direct jobs, plus an annual turnover of over $600 million. An average annual consumption of 11.5 L per capita is estimated. However, the exportation of cachaça is still insignificant and only 1% of the total production is exported to countries like Germany, Italy, France, the USA and Japan, so almost all of the production is destined for the domestic market (1,2). The alembic cachaça production process can be divided into four basic stages: obtaining of the must; fermentation; distillation; and aging (3). Aging is the last step of the production process, but it is not mandatory. It is an important step in the manufacture of cachaça that enables the producer to increase the value of his beverage. The newly distilled spirit has sensory characteristics that are a little aggressive, and it has a strong taste of alcohol. These characteristics can be smoothed by aging (4). According to the law, for the beverage to be considered aged, it must fit the denomination proposed in which the aged sugarcane liquor or cachaça refers to the beverage that contains at least 50% of sugar cane liquor or cachaça aged in an appropriate wooden container with a maximum capacity of 700 L for a period not less than 1 year. In addition to being considered aged, during the years of storage, aged cachaça receives other denominations. Premium sugarcane liquor or cachaça refers to a beverage that contains 100% distilled sugarcane spirits/cachaça aged in an appropriate wooden container with a maximum capacity of 700 L for a period not less than 1 year. Extra premium distilled sugarcane spirits/cachaça refers to a beverage that contains 100% of distilled sugarcane spirits/cachaça aged in an appropriate wooden * Correspondence to: Maria das Graças Cardoso, Department of Chemistry, Federal University of Lavras, C.P. 3037, Lavras, Minas Gerais, Brazil. mcardoso@dqi.ufla.br 1 Department of Chemistry, Federal University of Lavras, C.P. 3037, , Lavras, Minas Gerais, Brazil 2 Pro-Rector of Research and Graduate Program, Federal University of the Jequitinhonha and Mucuri Valleys, Diamantina, MG, Brazil J. Inst. Brew. 2017; 123: Copyright 2017 The

2 Cachaça aged in wood casks and glass containers container with a maximum capacity of 700 L for a period not less than 3 years (5). The containers used for aging are wooden barrels and casks, which act as a semipermeable membrane that allows the passage of water and alcohol vapours. This transfer depends on the conditions of relative humidity and temperature of the storage location. However, the losses over the year depend on many other factors, such as expansion and contraction of the sugar cane liquor in the barrel, leakage and expulsion of liquor through cracks and consequent suction of air because of contraction on cooling. This expansion and contraction can occur successively as a result of inappropriate facilities or environment and the mode of storage (6). Oak (Quercus sp.) is the wood traditionally used for aging of cachaça and other spirits in Brazil, but several native Brazilian woods are also used make barrels and casks for aging cachaça, such as peanut (Plerogyne nitens), amburana (Amburana cearensis), cedar (Cedrela fissilis), jatoba (Hymenaeae carbouril), ipé (Tabebuia sp), freijó (Cordia goeldiana), garapa (Apuleia leiocarpa), balsam (Myroxylon peruiferum), yellow peroba (Plathynemia foliosa), peroba (Paratecoma peroba) and jequitibá (Carinian legalis) (6,7). The use of other types of wood has occurred because of the high cost of oak, which is a wood typical of the Northern Hemisphere. However, there is still a great need for systematic studies to characterize the compounds extracted from each type of wood during the aging process (3,8,9). The principal reactions occurring during the aging process are the reactions among the secondary compounds from the distillation, the direct extraction of wood components, the decomposition of macromolecules from wood (cellulose, hemicellulose and lignin) and the subsequent incorporation of these compounds into the beverage. Reactions between the compounds from the wood and the compounds present in the original distillate might also occur (3,4,10). The principal compounds extracted from wood by the distillates are volatile oils, phenolic compounds, tannic substances, sugars, glycerol and non-volatile organic acids. Among these compounds, the presence of phenolic compounds in aged cachaça is important because they can act as antioxidants that are important for human health (11,12). Several studies have been made to evaluate the chemical and sensory quality of aged beverages. Numerous aldehydes and phenolic acids, such as vanillin, syringaldehyde, coniferaldehyde and sinapaldeído, probably resulting from the acidic alcoholysis of lignin at room temperature, have been identified in alcoholic distillates aged in oak barrels. Other phenolic acids have also been identified: gallic acid, p-hydroxybenzoic acid, p-coumaric acid, cinnamic acid, vanillic acid and syringic acid (13,14). The objectives of this study were to evaluate the development of phenolic compounds in cachaça stored in barrels newly manufactured from oak (Quercus sp.), amburana (Amburana cearensis), jatoba (Hymenaeae carbouril), balsam (Myroxylon peruiferum) and peroba (Paratecoma peroba) woods and to evaluate some physicochemical parameters that change during the aging process. 2. Material and methods 2.1. Construction of the casks The casks were constructed in the town of Ponte Nova, MG, Brazil, located in the Zona da Mata Mineira region. The amburana, balsam, oak, jatoba and peroba woods were acquired by the cooperage in charge of the manufacture of the barrels. The preparation was accomplished with new or virgin wood except for oak. The oak wood was obtained from a 200 L European oak cask, which was dismantled and sanded. This procedure was performed because it was not a native Brazilian wood and had previously been used for the aging of other beverages. The other types of wood were acquired from specialized vendors. All of the casks used in the study had a final volume of 20 L each. They were produced with the following dimensions: 40 cm length, 30 cm height, 102 cm central radius and 1.8 cm thick walls. The barrels were tanned with distilled water, and the water was changed every 5 days for 30 days Production and collection of samples The samples were produced in the alembic of the Cachaca Artisanal João Mendes ( JM), located in the city of Perdões, MG, in the period of the 2014 harvest The variety of cane used was RB The fermentation process was accomplished using cornmeal and Saccharomyces cerevisiae was employed as the yeast. The preparation of the yeast was performed for 5 days, and the fermentation of the sugar cane broth lasted 18 h. The initial Brix was 20. When the Brix reached 0, the must was distilled in a 1000 L copper still. The distillation yielded 15 L of head fraction, 180 L of heart fraction and 144 L of tail fraction. The heart fraction was transferred to oak, amburana, balsam, jatoba and peroba casks and 20 L of each beverage was stored in each cask. The casks were kept in a closed shed without control of the temperature and humidity (room temperature, ~25 C). The casks were placed in a horizontal position to allow greater contact of the beverage with the wood. Every 2 months for a period of 12 months, 2-L samples were collected and sent for the physicochemical and chromatographic analyses, which were conducted in the Laboratório de Análises de Qualidade de Aguardentes of the Departamento de Química, Universidade Federal de Lavras Physicochemical analysis The physicochemical and chromatographic analyses were performed periodically in triplicate. The determinations of alcohol and dry extract contents were performed according to the specifications established by the Normative Instruction no. 24, of 8 September 2005 of the Ministério Agricultura, Pecuária e Abastecimento (15) Colour intensity The determination of the color intensity was accomplished by reading at 420 nm (λ max ), in a Shimadzu UV-1601 PC spectrophotometer using quartz cuvettes (6,16,17) Total phenolic compounds The analysis of total phenolic compounds was achieved using the modified Folin Ciocalteau method (6,18 20) Phenolic compounds by HPLC Phenolic compounds were evaluated on a Shimadzu HPLC equipped with two model SPD-M20A high-pressure pumps, a 233 J. Inst. Brew. 2017; 123: Copyright 2017 The wileyonlinelibrary.com/journal/jib

3 model DGU-20A3 degasser, a model CBM-20A interface, a model SIL-10AF autosampler and a diode array detector (DAD). An Agilent-Zorbax Eclipse XDB-C 18 column ( mm, 5 μm) connected to an Agilent-Zorbax Eclipse XDB-C 18 pre-column ( mm, 5 μm) was used. The analysis of 12 phenolic compounds in cachaça stored in amburana, balsam, oak, peroba and jatoba casks was performed according to the method proposed by Anjos et al. (6) and Santiago et al. (14). The phenolic compounds analysed were gallic acid, catechin, vanillic acid, phenol, syringic acid, vanillin, syringaldehyde, p-coumaric acid, sinapic acid, coumarin, 4-methylumbelliferone and o-coumaric acid. The standard compounds were purchased from Sigma-Aldrich Chemical Company or Acros Organics. The mobile phase was composed of HPLC-grade methanol (Merck), glacial acetic acid ( J.T. Baker) and type I water obtained from a Milli-Q system. Solvent A was composed of 2% acetic acid in water, and solvent B was composed of methanol water acetic acid (70:28:2 v/v). The samples and standards were eluted with the following gradients: 0 25 min (0 40% B); min (40 55% B); min (55 100% B); min (100 0% B). The wavelength used was 280 nm, the flow rate was 0.8 ml min 1, and the volume injected was 20 μl. The samples and standards were filtered through a 0.45 μm polyethylene membrane (Millipore) and injected directly into the chromatographic system. The injections of standards and samples were performed in triplicate, and the identities of the analytes were confirmed by retention time and peak profile of the samples compared with the standards. The external standardization method was used for quantification. For the construction of calibration curves, dilutions of an intermediate solution containing a mixture of all the standards were performed, and this solution was prepared by dilution of previously prepared stock solutions. The concentrations of the standards in the intermediate solution were 6.80 mg L 1 ( gallic acid), mg L 1 (catechin), 6.73 mg L 1 (vanillic acid), 3.76 mg L 1 (phenol), 7.93 mg L 1 (syringic acid), 6.08 mg L 1 (vanillin), 7.29 mg L 1 (syringaldehyde), 6.56 mg L 1 (p-coumaric acid), 8.97 mg L 1 (sinapic acid), 5.85 mg L 1 (coumarin), 7.05 mg L 1 (4-methylumbelliferone) and 6.56 mg L 1 (o-coumaric acid). To ensure the analytical quality of the results, procedures for validation of the method were used, and the following parameters were evaluated: linearity, detection limit, quantification limit and accuracy (6,14) Statistical analysis A completely randomized design in split plot in space was employed. The data were submitted to analysis of variance and the averages were compared by the Schott Knott test at 95% of confidence using the SISVAR (21) statistical program. A principal component analysis was applied to investigate possible similarities between the kinds of wood with respect to the composition of phenolic compounds. The results were mean centered for later analysis. The analysis was performed using the program CHEMOFACE (22). 3. Results and discussion W. D. Santiago et al. The results obtained for the physical and chemical evaluations (alcoholic and total solids contents) of the heart fraction and the beverages stored in different types of casks are presented in Table 1. After 12 months of storage, the cachaças stored in casks constructed of different types of wood presented different alcohol contents. A significant decrease compared with that of the heart fraction was observed. According to Miranda et al. (17), Anjos et al. (6) and Santiago et al. (14), the decrease in the alcohol content of cachaça during aging is caused by the loss of alcohol through the pores of the wooden cask and the by reaction of ethanol with other substances present in the beverage. Various physicochemical parameters of cachaças are modified during aging because of the partial evaporation of ethanol and water. Table 1. Alcohol and total dry extract contents of the heart fraction and the beverages stored in casks made from different kinds of wood 234 Cask Alcolhol concentration (% v/v) Heart (time zero): ± Storage time (months) A aa ac ab ad ae af B ba cb ec dd ee ef O ba BC db cd ce cf J aa BC cb cd de df P aa ab bb BC be bd Tonel Dry extract ( g L 1 ) Heart (time zero): ± Storage time (months) A af ae ad ac ab aa B bf be bd BC bb ba O cf ce cd cc cb ca J de dd dc db db da P ee ed ed ec eb ea Means followed by the same lower case letter in a column and the same upper case letter in a row do not differ by the Scott Knott test at a level of 5% of probability (with regard to the parameter being studied). A, Amburana; B, balsam; O, oak; J, jatoba; P, peroba. wileyonlinelibrary.com/journal/jib Copyright 2017 The J. Inst. Brew. 2017; 123:

4 Cachaça aged in wood casks and glass containers A gradual increase in the concentration of phenolic compounds throughout the storage process can be seen, and there were distinct diferences with respect to the incorporation of compounds extracted from wood. According to Dias et al. (8),Mori et al. (7) and Santiago et al. (23), the longer the storage time, the higher is the degree of direct extraction of the wood components and this extraction results in an increase in the dry matter content. This fact was observed in this study, because the mass of dry extract increased with increasing aging time in the cachaças stored in all the casks used in the study. Miranda et al. (24) explained that this increase occurs because of the degradation of lignin to liberate aromatic compounds, leading to their incorporation into the beverage. It is believed that these aromatic compounds are tannins and phenolic compounds, which represent up to 40% of the extract (25). Thus, it is expected that the evolution of dry extract in the beverage will be proportional to the extraction of phenolic compounds and, consequently, the intensity of its colour. The results for the monitoring of the total phenolic compounds in cachaça stored in the casks are shown in Fig. 1. There was a progressive increase in total phenolic content of the cachaça during the storage period in all the casks. However, there was no similarity in the phenolic composition of the cachaças stored in the different types of cask. The total phenolic composition in the cachaça ranged from to mg L 1 in the amburana cask; from to mg L 1 in balsam; from to mg L 1 in oak; from to mg L 1 in jatoba; and from to mg L 1 in the peroba cask. For Cardoso (4) and Santiago et al. (23), various factors may determine the extraction of compounds from the wood during the aging period, such as size and pretreatment of the cask, environmental conditions, duration of the aging and the amount of alcohol, so that different quantities of phenolic compounds are extracted from the wood during the aging process. However, these assumptions are not very valid for the current results for extraction because all these parameters were similar for all the casks, so that differences in the composition of phenols must be explained by the chemical characteristics of each type of wood. The colour intensities are depicted in Fig. 2. There was a progressive increase in the colour of the beverage during the storage period in the respective wooden casks. The development of a yellow colouration in the cachaças stored in the amburana, Figure 1. Evolution of the total phenolic composition during the storage period of cachaça. Figure 2. Evolution of colour intensity as a function of storage time cachaças in different barrels study. oak, peroba and jatoba casks and a reddish colour in the cachaça stored in the balsam cask during the aging period was observed. According to Miranda et al. (24), the main agents responsible for the progressive darkening or intensification of the yellow-orange colour in beverages during the maturation in wood are the tannins and their oxidation products. A linear correlation test (ρ) was applied to the results for the dry extract, total phenolic compounds and colour intensity to assess any relationship between them in the samples stored in each type of wood. According to Callegari-Jacques (26), the correlation coefficient can be qualitatively evaluated as follows: if 0 < ρ < 0.3, there is a weak correlation; if 0.3 < ρ < 0.6, there is moderate correlation; if 0.6 < ρ < 0.9, there is a strong correlation; and if 0.90 < ρ < 1.0, there is a very strong correlation. A strong positive linear correlation between the parameters total phenolic compounds, solids and colour intensity was observed in the samples aged in all the casks in the study. The coefficients ranged from to in amburana; from to in balsam; from to in oak; from to jatoba; and from to in peroba. It appears from the data obtained that variations in the increase in one of the parameters lead to an increase in another parameter during the aging period. The chromatogram of the standard solution of the 12 phenolic compounds analysed by HPLC-PDA is presented in Fig. 3. The test compounds were well separated under the chromatographic conditions employed. These values corroborate those found by Anjos et al. (6) and Santiago et al. (20). At the end of the run, there was an increase in the baseline which may be explained by the presence of oligomers and polymers. In the literature, studies that correlate phenols using reverse-phase liquid chromatography reported that the presence of many oligomers and polymers of flavan-3-ol only caused changes in the base line of the chromatogram (27,28). The linearity was evaluated by the correlation coefficients (r 2 ). Coefficients ranged from to These figures demonstrate the strong linear correlation between the concentration of the compounds analysed and the peak areas, and values > 0.99 are recommended in the literature (29,30). The limits of detection and quantification were estimated using the parameters of the analytical curve. The detection and quantification limits of the phenolic compounds ranged from 235 J. Inst. Brew. 2017; 123: Copyright 2017 The wileyonlinelibrary.com/journal/jib

5 W. D. Santiago et al. 236 Figure 3. Chromatogram of standard solution of phenolic compounds with spectrophotometric detection. Concentration of standard: mol L 1. The average retention times obtained for each of the compounds analysed were ± min (1, gallic acid), ± min (2, catechin), ± min (3, vanillic acid), ± min (4, phenol), ± min (5, syringic acid), ± min (6, vanillin), ± min (7, syringaldehyde), ± min (8, p-coumaric acid), ± min (9, sinapic acid), ± min (10, coumarin), ± min (11, 4-methylumbelliferone) and ± min (12, o-coumaric acid) to and from to mg L 1, respectively. The limits of detection and quantification of the 12 phenolic compounds were as follows: gallic acid, and mg L 1 ; catechin, and mg L 1 ; vanillic acid, and mg L 1 ; phenol, and mg L 1 ; syringic acid, and mg L 1 ; vanillin, and mg L 1 ; syringaldehyde, and mg L 1 ; p-coumaric acid, and mg L 1 ; sinapic acid, and mg L 1 ; coumarin, and mg L 1 ; 4-methylumbelliferone, and mg L 1 ;ando-coumaric acid, and mg L 1.The values are lower than those found by Aquino et al. (9) and Santiago et al. (31). However, they approximate those found by Anjos et al. (6), Zacaroniet al. (13) and Santiago et al. (14). Santiago et al. (31) found values ranging from to and from to mg L 1, respectively, for the limits of detection and quantification. Anjos et al. (6) found values ranging from to and from to mg L 1 for the limits of detection and quantification, respectively. Santiago et al. (14) found values ranging from to and from to mg L 1 for the respective limits of detection and quantification. A high sensitivity of the method was obtained, and according to literature, the differences in these parameters can be the result of different chromatographic conditions (equipment and methods used for the quantification of the compounds) (32,33). Accuracy was another parameter analysed, and it was expressed in recovery tests. The average results for the percentage recoveries were 90% for gallic acid, 86% for catechin, 101% for vanillic acid, 82% for phenol, 93% to syringic acid, 99% vanillin, 90% for syringaldehyde, 98% for p-coumaric acid, 81% for sinapic acid, 104% for coumarin, 91% for 4-methylumbelliferone and 82% for o-coumaric acid. These values are acceptable (32,33) and are close to those obtained by Aquino et al. (9),Anjoset al. (6), Zacaroni et al. (13) and Santiago et al. (14). The results obtained for quantification of the 12 phenolic compounds by HPLC-DAD during storage in amburana, balsam, oak, peroba and jatoba casks are shown in Tables 2 6, respectively. Table 2. Concentration of the 12 phenolic compounds (mg L 1 ) during the period of storage of the cachaça in amburana casks Storage times (months) Increase Compounds Heart fraction (times) Gallic acid ND <LD <LD <LD <LD <LD <LD Catechin ND <LD <LD <LD <LD <LD <LD Vanillic acid ND ± a ± a ± a ± a ± a ± a Phenol ND <LD <LD <LD <LD <LD <LD Syringic acid ND <LD <LD <LD <LD <LD <LD Vanillin ND <LD <LD <LD <LD <LD <LD Syringaldehyde ND ± ± ± ± ± ± p-coumaric acid ND ± ± ± ± ± ± Synapic acid ND ± ± ± ± ± ± Coumarin ND ± a ± a ± a ± a ± a ± a Methylumbelliferone ND ± a ± a ± a ± a ± a ± a o-coumaric acid ND <LD <LD <LD <LD <LD <LD Total ND, Not detected; <LD, lower than the detection limit; <LQ, lower than the limit of quantification. a Sample diluted 10-fold in 40% ethanol for quantification. wileyonlinelibrary.com/journal/jib Copyright 2017 The J. Inst. Brew. 2017; 123:

6 Cachaça aged in wood casks and glass containers Table 3. Concentration of the 12 phenolic compounds (mg L 1 ) during the period of storage of the cachaça in balsam casks Storage times (months) Increase Compounds Heart fraction (times) Gallic acid ND ± ± ± ± ± ± Catechin ND <LD <LD <LD <LD <LD <LD Vanillic acid ND ± ± ± ± ± ± Phenol ND ± ± ± ± ± ± Syringic acid ND ± ± ± ± ± ± Vanillin ND ± ± ± ± ± ± Syringaldehyde ND ± ± a ± a ± a ± a ± a p-coumaric acid ND <LD <LD <LD <LD <LD <LD Synapic acid ND <LD <LD <LD <LD <LD <LD Coumarin ND ± ± ± ± ± ± Methylumbelliferone ND <LD <LD <LD <LD <LD <LD o-coumaric acid ND <LD <LD <LD <LD <LD <LD Total a Sample diluted 10-fold in 40% ethanol for quantification. Table 4. Concentration of the 12 phenolic compounds (mg L 1 ) during the period of storage of the cachaça in oak casks Storage times (months) Increase Compounds Heart fraction (times) Gallic acid ND ± ± a ± a ± a ± a ± a Catechin ND <LD <LD <LD <LD <LD <LD Vanillic acid ND ± a ± a ± a ± a ± a ± a Phenol ND <LD <LD <LD <LD <LD <LD Syringic acid ND ± ± ± ± ± ± Vanillin ND <LQ ± ± ± ± ± Syringaldehyde ND ± ± ± ± ± ± p-coumaric acid ND <LD <LD <LD <LD <LQ <LQ Synapic acid ND ± ± ± ± ± ± Coumarin ND <LD <LD <LD <LD <LD <LD 4-Methylumbelliferone ND <LD <LD <LD <LD <LD <LD o-coumaric acid ND ± ± ± ± ± ± Total a Sample diluted 10-fold in 40% ethanol for quantification. 237 J. Inst. Brew. 2017; 123: Copyright 2017 The wileyonlinelibrary.com/journal/jib

7 W. D. Santiago et al. Table 5. Concentration of the 12 phenolic compounds (mg L 1 ) during the period of storage of the cachaça in jatoba casks Storage times (months) Compounds Heart fraction Increase (times) Gallic acid ND ± ± ± ± ± ± Catechin ND <LD <LD <LD <LD <LD <LD Vanillic acid ND ± ± ± ± ± ± Phenol ND <LD <LD <LD <LD <LD <LD Syringic acid ND ± ± ± ± ± ± Vanillin ND <LD <LD <LD <LD <LD <LD Syringaldehyde ND ± ± ± ± ± a ± a p-coumaric acid ND <LQ <LQ <LQ <LQ <LQ ± Synapic acid ND ± ± ± ± ± ± Coumarin ND <LD <LD <LD <LD <LD <LD 4-Methylumbelliferone ND <LD <LD <LD <LD <LD <LD o-coumaric acid ND ± ± ± ± ± ± Total a Sample diluted 10-fold in 40% ethanol for quantification. Table 6. Concentration of the 12 phenolic compounds (mg L 1 ) during the period of storage of the cachaça in peroba casks Storage times (months) Compounds Heart fraction Increase (times) Gallic acid ND <LD <LD <LD <LD <LD <LD Catechin ND <LD <LD <LD <LD <LD <LD Vanillic acid ND ± a ± a ± a ± a ± a ± a Phenol ND <LD <LD <LD <LD <LD <LD Syringic acid ND ± ± ± ± ± ± Vanillin ND <LD <LD <LD <LD <LD <LD Syringaldehyde ND ± ± ± ± ± ± p-coumaric acid ND <LQ ± ± ± ± ± Synapic acid ND ± ± ± ± ± ± Coumarin ND <LD <LD <LD <LD <LD <LD 4-Methylumbelliferone ND <LD <LD <LD <LD <LD <LD o-coumaric acid ND <LD <LD <LD <LD <LD <LD Total a Sample diluted 10-fold in 40% ethanol for quantification. 238 wileyonlinelibrary.com/journal/jib Copyright 2017 The J. Inst. Brew. 2017; 123:

8 Cachaça aged in wood casks and glass containers A progressive increase in the concentration of phenolic compounds in all the casks used for storage of the beverage was observed. This increase can be observed from the sum of the concentrations of phenolic compounds after 12 months of aging. The concentrations ranged from to mg L 1 for the cachaça stored in the amburana cask; from to mg L 1 for the balsam cask; from to mg L 1 for the oak cask; from to mg L 1 for the jatoba cask; from and to mg L 1 for the peroba cask. The beverage stored in the amburana cask contained the highest concentration of phenol. This fact is not consistent with the result of the analysis of the total phenolic composition, where beverages stored in jatoba had the highest concentration. This difference is explained by the different techniques (spectrophotometric and chromatographic) employed. The spectrophotometric analysis utilized the Folin Ciocaulteau test to determine the total phenolic content by measuring the reducing capacity of the samples. Therefore, other compounds, in addition to phenols, might react with the reagent. Overestimation of the phenolic content can occur because various non-phenolic interferences are known that can react, including vitamin C, compounds with hydroxyl groups, sugars, proteins and other compounds (34 36). Although distinct phenolic concentrations were observed, the wooden casks behaved similarly with respect to the extraction of phenolic compounds when 2 and 12 months of storage were compared. In the amburana cask, there was a fold increase in the concentration of phenolic compounds; in balsam, the concentration increased fold; in oak, it increased fold; in jatoba, it increased fold; and in peroba, it increased fold. The predominance of certain phenolic compounds was observed in the beverage stored in each type of cask: a predominance of vanillic acid, coumarin and 4- methylumbelliferone in the cachaça stored in the amburana cask; a predominance of gallic acid, phenol, syringic acid and syringaldehyde in the balsam cask; a predominance of gallic acid, vanillic acid and syringaldehyde in the oak cask; a predominance of gallic acid and syringaldehyde in the jatoba cask; and a predominance of vanillin and syringic acid in the cachaça stored in the peroba cask. These predominances corroborate some studies found in the literature, but they do not corroborate others (6,8,13,14,31,37).Dias et al. (8) showed that ellagic acid and vanillic acid predominated in cachaça stored in oak barrels after six months of aging; vanillin and ellagic acid predominated in a balsam barrel; vanillic acid and sinapaldeído predominated in the amburana barrel; and coniferaldehyde predominated in the jatoba barrel. These results differ from those found in the present work. Anjos et al. (6) reviewed the evolution of phenolic composition in beverages stored in an oak barrel during 12 months. In this study, syringaldehyde and gallic acid were found to predominate. Zacaroni et al. (13) evaluated aged cachaça in different wooden casks and found a predominance of coumarin and eugenol in the cachaça stored in an amburana cask; a predominance of coumarin in a jatoba cask; a predominance of syringaldehyde, ellagic acid, and p-coumaric acid in an oak cask; and a predominance of syringic acid, ellagic acid and eugenol in a balsam barrel. Santiago et al. (14) evaluated the phenolic composition of cachaça aged in oak and amburana casks and found gallic acid, vanillic acid, syringaldehyde and sinapic acid to be the predominant compounds in cachaça aged in an amburana cask, and gallic acid, syringic acid and syringaldehyde in cachaça stored in an oak cask. A similarity in the presence of some major compounds found in these studies was observed, whereas a difference in the composition can be explained by the fact that new casks were used in the present study. Gallic acid and syringaldehyde were the predominant compounds found in most of the casks. Gallic acid is a phenolic compound derived from the degradation of tannins, which account for approximately half of the dry matter in the bark of many trees (38). According to Queiroz et al. (39), these tannins are the second most common source of polyphenols, and they are responsible for the astringency and bitterness in beverages. They are extracted from the casks and are gradually hydrolysed during the maturation process. Syringaldehyde is one of the key markers of aging, and it is derived from lignin (8,40). Because its structure is composed of guaiacyl and syringyl units, two different groups of compounds are formed from lignin. One of these groups include sinapaldehyde, syringaldehyde and syringic acid, which originated from the syringyl structure. Mechanisms involving the extraction of the counterparts from lignin are proposed in two possible pathways. One involves the simple extraction of phenolic compounds present in wood to be incorporated into the beverage and the other involves the extraction of lignin from wood by the action of ethanol (41,42). The presence of coumarin at high concentrations in amburana is not desirable because of the degree of toxicity observed in humans. In Europe, use as a flavouring in the food industry is controlled and the maximum concentration of coumarin permitted in alcohol and certain caramels is 10 mg.kg 1 (13). There was a progressive increase in the concentration of phenolic compounds in the cachaça stored in different casks. Although complex, the mechanism of this gradual increase in the levels of acids and aldehydes seems to follow the pathway: cinnamic aldehydes (coniferaldehyde and sinapaldeído), benzoic aldehydes (vanillin and syringaldehyde) and benzoic acids (vanillic acid and syringic acid) (4,6,8). Figure 4 is a biplot graph of PC1 PC2 loadings and scores in which the phenolic compounds of the cachaças stored in different Figure 4. Graph biplot PC1 PC2 loadings and scores of phenolic compounds of cachaça stored in amburana, balsam, oak, jatoba and peroba casks. [Colour figure can be viewed at wileyonlinelibrary.com] 239 J. Inst. Brew. 2017; 123: Copyright 2017 The wileyonlinelibrary.com/journal/jib

9 W. D. Santiago et al. 240 tanks are correlated. It was possible to describe 93.82% of the data with the first and second principal components, and 67.55% of the total variance was described by the first principal component. Beverages stored in amburana were differentiated by coumarin, and a similarity of the samples stored in oak and peroba casks with respect to vanillic acid was observed and samples stored in jatoba and balsam were similar with respect to the other compounds. 4. Conclusion After 12 months of storage in casks made with different types of wood, the cachaças contained different concentrations of alcohol and dry extract. A significant decrease in alcohol content and an increase in dry extract was observed in all the samples as well a progressive increase in total phenolic content and colour intensity during the storage period. There was also a strong correlation between the quantities of dry extract, colour intensity and phenolic compounds, in such way that an increase in one of the parameters lead to an increase in the others. There was also verified a progressive increase in the concentrations of phenolic compounds analysed by HPLC in all of the beverages. The sum of the concentrations of phenolic compounds in the cachaças stored in casks made from different types of wood showed distinct values but behaved similarly with respect to the extraction of phenolic compounds, when comparing their variation from month 2 to month 12 of storage. 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