*2.1.3 Obtaining cabuya juice*

The development of organoleptic properties such as sweetness, acidity, aromas, and flavorstakes place in the state of complete maturity of the blue cabuya; therefore, it is important to extract the juice when the plant is fully mature. The blue cabuya completes its life cycle after flowering, so the juice is extracted before the floral scape comes out.

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


#### **Table 2.**

*Experimental design of the first stage of alcoholic fermentation of blue cabuya juice with a substrate concentration of 60 g/L.*

Some physical characteristics that indicate that the plant is mature according to the ancestral knowledge of the inhabitants of the La Merced district, Junín region, Peru are described below:


Once the mature plant has been selected, the external leaves are cut in such a way that there is free access to the trunk, leaving the rest of it with its respective leaves. Then, a hole is made approximately 30 centimeters (cm) deep and 20 to 25 cm in diameter in the upper part of the pineapple or trunk to accumulate the juice [21].

Depending on the size of the mature plants, the juice is collected between two to three months, with the collection time being longer in larger plants. The collection is carried out two to three times a day, depending on the size of the plant: the first extraction is carried out between 4 and 5 in the morning; the second, at noon; and the third extraction, between 3 and 5 in the afternoon. In each extraction, about three liters of juice obtained. After each extraction, the walls of the hole are scraped so that they do not seal and prevent the juice from accumulating in the hole, while it is cleaned to avoid fermentation of the juice.

#### *2.1.4 Determination of the physicochemical properties of blue cabuya juice*

The blue cabuya juice was characterized to determine the following physicochemical parameters: °Brix, density, pH, and viscosity. The following laboratory instruments were used in the measurements: portable refractometer, series X0015RURCN; pycnometer; Milwaukee brand potentiometer, MW 102, pH/Meter, USA, and QUIMIS viscometer, Brazil. The results are shown in **Table 3**.

#### *2.1.5 Alcoholic fermentation process*

To carry out the alcoholic fermentation process, the yeast *Saccharomyces cerevisiae* variety ellipsoideus, D 47 - LALVIN - Canada was used. This yeast is lyophilized and comes in 5 g sachets, so it was necessary to reproduce it up to the concentration necessary to inoculate 20 L of cabuya juice.

To obtain the inoculum to 3 L of blue cabuya juice, 6 g of DAP was added as a nutrient. Subsequently, the solution was sterilized at 110 C and transferred to a 5 L flask where the aeration system was installed. The solution was cooled to the incubation temperature of 24 C, the yeast strains were added, and sterile air was supplied at a flow of 1 vvm for 24 h until reaching a concentration of 1,15 g of biomass/L. This biomass concentration is equivalent to 2875 x 107 cells/mL, which is within the range required for must fermentation that considers 1,0 g of cells ≅ 1,4 x 107 cells [22].

The experimental tests of alcoholic fermentation were carried out in the environmental microbiology and biotechnology laboratories of the Faculty of Biological Sciences and Unitary Operations of the UNMSM. A 120 L capacity bioreactor equipped with automatic temperature control and a 0.5 HP motor was used, with a stirring speed of 90 rpm and a supply of sterile air with a variable volumetric flow rate of 20 L/min. at 60 L/min. For each experimental test, 20 L of cabuya juice (minimum amount of bioreactor operation) was used. The cabuya juice was sterilized at 110 C in the bioreactor before carrying out the experimental tests of the alcoholic fermentation process at different temperatures, pH, and DAP nutrient concentrations.

The tests were carried out under the following conditions: constant substrate concentration, AR of 60 g/L, pH of 3.5 and 4.5 [23], at temperatures of 24 C, 28 C, and 32 C, and DAP nutrient concentration of 1 and 2 g/L. To 20 L of cabuya juice, previously sterilized, the yeast inoculum was supplied at a concentration of 1.15 g/L (dry basis), sterile air at a flow of 1vvm during the first 8 h of the process and the agitation speed was 90 rpm. The process was monitored by taking samples every 2 h for 26 h for the respective analysis of biomass growth, sugar consumption and ethanol production.

#### *2.1.6 Measurement of reducing sugars, ethanol, and microbial biomass*

Reducing sugars were determined by Miller's method using 3,5-Dinitrosalicylic acid (DNS), and ethyl alcohol concentration was determined by spectrophotometry using potassium dichromate, at a maximum wavelength of 580 nm.

To measure the concentration of microbial biomass, a 10 ml sample is taken from the broth during fermentation and centrifuged at 4000 rpm for 15 minutes. The sediment is deposited in a crucible and 10 mL of 0,85% w/v alkaline NaCl solution is


**Table 3.** *Physicochemical properties of blue cabuya juice.* *Evaluation of Ethanol Production Process by the Fermentation of Blue Cabuya Juice… DOI: http://dx.doi.org/10.5772/intechopen.111855*

added. Then, this solution is dehydrated in the oven at a temperature of 80 C for 6 h until it reaches a constant weight. The amount of biomass is determined by the difference in weight between the crucible with dry biomass and the crucible without sample.

### **2.2 Results**

**Table 4** indicates the tests carried out in the investigation at different temperatures, pH, and DAP concentrations for a substrate concentration of 60 g/L of the blue cabuya juice to determine the conditions in which the highest ethanol yield is obtained.

**Figure 1** shows the biomass yields of the experimental tests from the blue cabuya juice alcoholic fermentation process. As shown below, test 8 has the highest biomass yield.

**Figure 2** shows the graphs of Ln (biomass) Vs time in hours of the alcoholic fermentation process experimental tests. It is observed that the kinetics of microbial growth is greater at a temperature of 28 C, pH 3.5, and DAP concentration of 2 g/L (test 8).

In **Figure 3**, the AR substrate consumption of the experimental tests of the alcoholic fermentation of cabuya azul juice is shown. It is observed that the speed of consumption of AR is greater in the conditions of test 8.

In **Figure 4**, the ethanol production of the experimental tests from the alcoholic fermentation process is observed. The highest yield of ethanol is obtained in test 8.

From the experimental results, it can be appreciated that test 8 at a temperature of 28 C, pH 3.5, Diammonium phosphate (DAP) nutrient concentration of 2 g/L, and substrate concentration (AR) of 60 g/L represents the best conditions in ethanol and biomass yield with Yp|s = 0.4739 and Yx|s = 0.166, respectively, as shown in **Figure 5**.

#### *2.2.1 Analysis of the results of the consumption of reducing sugars*

**Table 5** shows the statistical treatment of the experimental tests performed on reducing sugar consumption.


#### **Table 4.**

*Experimental tests performed at different temperatures, pH, and DAP concentrations for a substrate concentration of 60 g/L.*

**Figure 1.** *Biomass production of alcoholic fermentation process in the first stage of experimental tests.*

**Figure 2.**

*Microbial kinetics from the 12 experimental tests of the alcoholic fermentation process.*

In **Table 5** of inter-subject effects, the significant value of 0.000 ˂ 0.05 is observed. Therefore, Ha is accepted. This value indicates that there is a significant difference between the groups (Test and time).

**Figure 6** of estimated marginal means shows that test 8 has the highest consumption of reducing sugars.
