**1. Introduction**

Given the particularity of different microclimates in Peru, the blue cabuya (*Agave americana L*.) grows wild mainly in the Regions of Huancavelica, Huánuco, Ancash, and Cajamarca where their inter-Andean valleys have mild weather between 18 C to 26 C.

In addition to this, the favorable conditions of the composition of organic nutrients and compounds of mineral origin of the soils are ideal for the development of *Agave americana L*. Thus, the maturation time of this plant fluctuates between five to ten years less than the seven to nine years required for the maturation of the same variety in Mexico.

In studies carried out in Mexico, the authenticity of parameters was determined in agricultural soils belonging to the region that grants the Denomination of Origin Tequila (DOT), characterizing 26 samples belonging to Jalisco, Michoacán, Nayarit, and Sinaloa. The characterization consisted of determining the texture, apparent density, color, pH, humidity, organic matter, total carbon, and trace metals. The trace presence of the minerals Cr, Ba, and Zr are useful as auxiliary tracers to determine the authenticity of soils and agaves belonging to the DOT region [1].

The preferable soils for growing tequilana agave from Blue Weber variety must have good drainage. The sucker roots of the agave explore the surface soil when water is available and are practically eliminated in dry seasons. For proper functioning, gas exchange is required in the soil. Therefore, flood areas must be avoided. It is essential that the pH of the soil is not very acidic, since at a pH below 5.5 the plants present nutritional imbalances that if not managed properly can cause damage such as the red ring, a disease that causes the appearance of red areas in the leaves, which form a belt around them that constricts them, many times a hardening and delay in the growth of the plant occurs [2].

Recently, new auxiliary analytical techniques have been analyzed in the current verification process for the detection of counterfeits and/or adulterations of tequila. According to the Official Mexican Standard NOM-006-SCFI-2012, tequila is a regional alcoholic beverage obtained by distillation of musts and prepared directly from the extracted material in the factory facilities of an authorized producer. Tequila is made from tequilana Weber Agave from the blue variety, cultivated in specific protected regions of Mexico that make up the geographical territory of Denomination of Origin Tequila (DOT). According to Mexican regulations, Tequila has a classification based on its category (100% agave Tequila and Tequila) and its classes (silver, gold, extraaged, and ultra-aged). Based on 2021 data from the Tequila Regulatory Council (CRT), the tequila industry produced 352 million liters and exported to 120 countries [3].

The authenticity and traceability of Tequila are established through a permanent inspection carried out by the CRT at the facilities of the tequila producers, verifying the production process in all its stages and according to the Official Mexican Standard NOM-006-SCFI- 2012. However, the consumption of tequila has increased in volume, being marketed in more countries around the world. To achieve this, certain competitive criteria must be met to demonstrate the quality, safety, and authenticity of the Tequila.

The diversity and dynamics of yeasts and bacteria during small-scale spontaneous fermentations of agave juice have been identified and described. Observing a high heterogeneity in the microbial populations and fermentation parameters that shows the bacteria have a greater diversity than the yeast. The central microorganisms identified were *Saccharomyces cerevisiae* and *Lactobacillus fermentum*. Bacterial growth and the concomitant production of lactic acid were associated with low ethanol production. Therefore, bacteria could be defined as contaminants in tequila fermentation and efforts to control them should be implemented [4].

The most important stage in tequila processing is the fermentation of sugar, mainly fructose, which is transformed into ethanol and volatile compounds that give tequila its unique characteristics [5]. Tequila companies often prefer to commercially inoculate *Saccharomyces cerevisiae* strains that provide batch-to-batch homogeneity; however, low yields are still generated as the strains are not well adapted to agave juice, a totally different environment from the origin of the strain [6].

#### *Evaluation of Ethanol Production Process by the Fermentation of Blue Cabuya Juice… DOI: http://dx.doi.org/10.5772/intechopen.111855*

The *Agave tequilana* Weber of blue var. is a semelparous plant, that is, once it blooms, it dies. Its reproduction can be sexual by seeds; or asexual by shoots of rhizomes or bulbils of the inflorescence. For Tequila production, instead of allowing the plant to develop the flower to attract pollinating birds, bats, and moths that feed on the nectar, the flower stem is cut as soon as it begins to bud. Thus, interrupting the process of flowering. The reason is that during the flowering process, the plant consumes its carbohydrate reserve and then dies [7].

The biological cycle of agave cultivation oscillates between six and eight years. During that time of its growth, the pineapple or head can weigh between 35 kg and 120 kg, although some reach up to 150 kg depending on the conditions of the cultivation process. The maturity of the plant is manifested in the appearance of the so-called "quiote", but this must be cut quickly since its permanence in the plant consumes the sugars accumulated for years [8].

Lavado, Robles, and Yenque (2015), in Peru, studied the physicochemical properties of blue cabuya juice obtained in the Huanca Huanca district, Angaraes province, Huancavelica Region [9]. **Table 1** shows the physicochemical properties of blue cabuya juice.

The water-soluble carbohydrates (WSC) contained in the agave heads were: 28,3 ± 0,1% g/100 g (fresh weight) and 86,7 ± 1,3% g/100 g (dry weight) [10]. These high values are consistent with previous reports by other researchers [11, 12].

It has been studied that the variables that influence the alcoholic fermentation process are mainly temperature, degrees Brix (°Brix), consumption of AF fermentable sugars (fructose and glucose), biomass concentration, and nitrogen concentration [13]. On the other hand, It has been studied the effect of pH and airflow on the production of biomass, ethanol, the synthesis of aromatic compounds, the consumption of reducing sugars, the yields, and speeds of production of biomass and ethyl alcohol of two strains of *Saccharomyces cerevisiae* cultivated continuously [14]. Thus, the effect of the dilution speed and the addition of nutrients on the fermentation and synthesis of volatile compounds of two native genera of *Saccharomyces cerevisiae*, S1 and S2, in continuous cultures fed with *Agave tequilana* juice have also been studied [15].

Other researchers studied the effect of temperature and pH variables on the yield of alcoholic fermentation of Agave cocui must. First, they studied the temperature: 27, 31, 33, 35, and 37 C at constant pH. Then, they evaluated the pH at values of 3.0, 4.0, and 5.0 at a constant temperature. The results obtained indicated that the maximum yield of alcoholic fermentation is at a temperature of 33 C and a pH of 4.0 with an ethanol productivity of 1.14 g/(L.h) and a yield of 81.5% [16].

For modern literature standardization purposes, fermentation is defined in microbiology as the type of metabolism of a carbon source in which energy is generated by phosphorylation at the substrate level where organic molecules function as the final acceptor of electrons (or as acceptors of reducing equivalents), generated during the decomposition of carbon-containing compounds or catabolism. As is well known,


#### **Table 1.** *Physicochemical properties of blue cabuya juice.*

the process is called respiration when the final acceptor is an inorganic compound. Respiration is called aerobic if the final acceptor is oxygen and anaerobic when it is some other inorganic compound apart from oxygen, for example, sulfate or nitrate [17].

The musts for alcoholic fermentation are inoculated within the range of 5 x 10^6 cells/ml, equivalent to 1.5 g/L to 6.0 g/L in wet weight and 0.3–1.2 g/L in dry weight. The inoculum is related to the gravity of the wort and should produce as fast fermentation as possible without compromising the quality of the beer or the size of the yeast crop. The pitching rate influences both the fermentation rate and the degree of yeast growth [18].

In this context, the research was oriented to evaluate the production process of ethanol by fermentation of the juice of the blue cabuya (*Agave americana L*.) using yeast strains (*saccharomyces cerevisiae*). First, the temperature and pH of the alcoholic fermentation process must be determined to achieve the best production yield. Then, the concentration of diammonium phosphate, which is a nutrient, will be determined. Finally, the ethanol production yield will be determined by its potential application as a blue cabuya liquor.
