## 3 Results and Discussion
### Wort Fermentation of Sugarcane Samples
#### 3.5.2 Lactic Acid Bacteria  
The population dynamics of mesophilic rod- and coccus-shaped LAB are shown in Figure 5. At the first sampling, coccus-shaped LAB cell densities (Figure 5a) ranged from 2.87 to 4.58 log CFU/mL across the trials. In the untreated trials (SC1 to SC4), microbial levels increased until day 12. The highest cell counts were recorded on day  
Figure 4: Results of microbiological analyses for _Saccharomyces_ yeast (**a**) and non-_Saccharomyces_ yeasts (**b**) of different samples. T0, T6, T12, and T18 represent the sampling times (in days) during alcoholic fermentation. Lowercase letters indicate statistically significant differences among treatments according to [PERSON]’s test (\(p\leq 0.001\)); treatments sharing the same letter are not significantly different.  
12 for spontaneous fermentation in SC1 (8.45 log CFU/mL). Overall, the trials treated with lactic acid (SC13, SC14, SC15, and SC16) showed better results. Starting from day 6, they did not contain any coccus-shaped LAB, except for the uninoculated SC13 trial, which had undetectable values from day 12. Similar results were obtained for rod-shaped LAB (Figure 5b). Microbial levels increased in the untreated trials (SC1 to SC4) up to day 6. The highest cell count was recorded on day 6 for spontaneous fermentation in SC1 (8.68 log CFU/mL). Again, the trials treated with lactic acid (SC13 to SC16) showed better results. From day 6, they did not contain any rod-shaped bacteria, except for the uninoculated SC13 trial, which had undetectable values from day 12.  
The presence of LAB during sugarcane juice fermentation is generally considered undesirable, as these microorganisms compete with yeasts for essential sugars and nutrients [16; 39]. LAB species such as _Lactobacillus_ are particularly well adapted to fermentation environments with pH values below 5.0, owing to their rapid growth rates and high ethanol tolerance [63]. The activity of lactic acid bacteria has been demonstrated to significantly  
Figure 5: Results of microbiological analyses for mesophilic cocc (**a**) and rod-shaped LAB (**b**) of different samples. T0, T6, T12, and T18 represent the sampling times (in days) during alcoholic fermentation. Lowercase letters indicate statistically significant differences among treatments according to [PERSON]’s test (\(p\leq 0.001\)); treatments sharing the same letter are not significantly different.  
influence the quality of fermentation, as lactic fermentation has been observed to reduce pH levels and inhibit the proliferation of spoilage bacteria [64]. This effect is especially relevant in uninoculated trials, where microbial competition is more pronounced.  
The concentration of AAB during the alcoholic fermentation process is shown in Figure 6, as the results follow a similar trend to LAB.  
On the first day of sampling, AAB densities in the different trials ranged from 2.49 to 4.85 log CFU/mL. Notably, microbial levels only increased in the untreated trials (SC1 to SC4) until day 6, after which they began to decrease. The trials inoculated with lactic acid (SC13 to SC16) yielded superior results, except for the uninoculated SC13 trial, where the concentration was undetectable by day 6.  
High cell densities of AAB can lead to the formation of undesirable organic compounds, including acetaldehyde, ethyl acetate, acetic acid, copper, and ethyl carbamate [65]. Overall, excessive activity of LAB and AAB negatively affects the quality of the fermented product due to the significant production of 2,3-butanedione, acids (lactic, acetic, and formic), and acetoin [8]. These bacterial species are typically considered contaminants in industrial fermentations of sugarcane ethanol [66]. Therefore, the results shown by the lactic acid-treated samples are encouraging, as they showed a total reduction in the microbial load of LAB and AAB, which could otherwise negatively affect the final product.