*2.5.2. Microorganisms*

instead of aqua vitae occurs in 1660. Considering the three ethnic groups that formed the Brazilian nation (Native Brazilians, Africans, and Europeans), scarce information is available about the real contribution of each group to the initial production of fermented sugar‐ cane. However, *cachaça*, which is the result of the distillation of fermented sugarcane juice, was certainly "discovered" by Europeans, the most technologically advanced group, who had knowledge and equipment to do so [77]. Nowadays, *cachaça* is the typical and exclusive denomination to Brazilian spirit produced from sugarcane juice with alcohol content ranging 38%–48% (v/v) at 20°C (68°F), which present unique characteristics (Table 2) [78]. Brazil has an estimated installed capacity of *cachaça* production ranging from 1.2 to 1.5 billion litters/year; however, the production is less than 800 million litters/year. Accord‐ ing to the Brazilian Institute of Geography and Statistics (IBGE), almost 15,000 establish‐ ments are currently producing *cachaça*. In 2014, 10.2 million liters of *cachaça* were exported

\*Sum of isobutyl (2-methyl-1-propanol), isoamyl (2-methyl-1-butanol and 3-methyl-1-butanol), and n-propyl (1-

The main raw material for the production of *cachaça* is the juice of sugarcane (*Saccharum* spp.). The first step to prepare of the fermentation medium is the extraction of sugarcane juice. Small producers extract the juice by crushing the sugarcane using a mill. Yet, the large producers use a more complex system of extraction: (i) crushing system, (ii) cutting machines, and (iii) shredders [80]. The resulting sugarcane juice is an opaque (color ranging from brown to dark green), viscous, and sweet liquid. The color is due to different pigments such as chlorophyll

38-48

to 66 countries, generating US\$ 18.33 million in revenue [79].

Source: Ministry of Agriculture, Livestock and Supply - Brazil

propanol) alcohols.

120 Food Production and Industry

*2.5.1. Production*

+ 5-(Hydroxymethyl)furfural

**Compounds Units Limits** Copper mg/L 5.0 Ethyl carbamate μg/L 210.0 Volatile acidity mg/100mL anhydrous ethanol 150.0 Total esters mg/100mL anhydrous ethanol 200.0 Aldehydes mg/100mL anhydrous ethanol 30.0 Total higher alcohols\* mg/100mL anhydrous ethanol 360.0 Furfural+HMF+ mg/100mL anhydrous ethanol 5.0 Methanol mg/100mL anhydrous ethanol 20.0 Acrolein mg/100mL anhydrous ethanol 5.0 Particles in suspension - Absent Dry extract g/L 6.0 Total sugars g/L 6.0

**Table 2.** Components present in *cachaça* and its limits in accordance with the Brazilian law.

As important as the preparation of the medium is the preparation of microorganisms that will ferment the sugarcane juice, the so-called foot-of-vat. Traditionally, *S. cerevisiae* is the most used yeast, but some other species are also utilized, namely, *Pichia* sp. [11, 84]. The cell concentration usually found at the beginning is about 106 –107 cells/mL and about 108 cells/mL at the end of the fermentation. Traditionally, the majority of the *cachaça* is produced through self-inoculation, using different protocols. Usually, a homemade mixture is prepared: (i) diluted sugarcane juice (carbon source), (ii) rice bran and/or corn bran (nutrient sources), and (iii) lemon/orange juice (to reduce initial pH) [85]. All these ingredients are mixed and kept at rest for 24 hours, during which wild yeast present in sugarcane juice will multiply—microor‐ ganism multiplication is verified through foaming. At the end of the first 24 hours, a new volume of diluted sugarcane juice is added, and the process is restarted [85]. The process is repeated until it reaches 20% of the volume of the fermentation vat. At the end of the process, a wild yeast community is obtained, which is adapted to the physical and chemical conditions of the fermentation. Producers, which aim to improved organoleptic characteristics, tend to use selected yeast in fermentative process. There are two basic types of selected yeast: (i) yeast that are used to produce ethanol or other beverages without being specific for *cachaça* production and (ii) yeast selected from their own vats of *cachaça* fermentation. Yeast selected in their own fermentation tanks have advantages over the first because they are more adapted to the *cachaça* fermentation specific conditions and are able to provide unique features to the product [86, 87]. There are numerous advantages to using selected yeast, including (i) rapid and (ii) homogeneity of the fermentation, (iii) higher fermentation yields, (iv) higher quality of the final product, (v) lowest risk of contaminations, and (vi) highest resistance to stress [88]. The improved aroma obtained in the final product is another advantage of using the selected yeast [86]. Given that *cachaça* fermentation develops in an environment with few controls, these vats represent a unique ecological niche. Interesting phenotypes, such as the production of flavor and aroma compounds, can be assessed by the detailed characterization of isolated strains [89]. The use of yeast strains producing aroma compounds, as esters (ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate) and higher alcohols (propyl, isoamyl, and isobutyl alcohol), can significantly increase the final quality of *cachaça*. Thus, such profile can be a differential characteristic between several *cachaça* producers or even distinct producing regions—if all producers in a particular region start using a selected yeast strain.
