**4. Discussion**

The evaluation of the variables studied and optimized responses; microwave treatment time, pH, solvent concentration (%), and solvent-to-seed ratio (X4:1), showed a significant effect (*p* < 0.05) on the extraction of the polyphenol compounds and bixin from annatto seeds. Table 2 shows the variability of the results obtained in each experimental unit, with results varying from 0.53 to 4.36 mg GA/g seeds for polyphenol compounds and from 0.03 to 0.58% for bixin. This variability is confirmed by the ANOVA (Table 3), showing that all the independent variables studied had a greater influence in the extraction process for both metabolites.

The influence of these conditions on the extraction process is mainly due to (1) the increase in the mass transfer capacity and/or the extraction power of the system, (2) the affinity of the bioactive compounds towards the solvent, and (3) the effect of the heat generated on the micro-domains of the system [25]. By increasing the solvent-to-seed ratio, the saturation point of the system is favored increasing the mobility of the seeds in the agitated system, generating a greater mass transfer of the bioactive compounds to solvent [25]. The amount of solvent in the system not only affected the extraction capability of the system, but also had a statistically significant interaction effect (X3X4) (Table 3), improving the affinity of the compounds towards the extractive solvent resulting in an enhanced mass transfer process. Polyphenol compounds present in annatto extract (i.e., apigenin, hypolaetin, and the caffeic acid derivative) possess a larger polarity as compared to bixin, which is a highly conjugated long chain molecule, also known for having a very low solubility. Therefore, increasing the concentration of ethanol favors the release of bixin by having a higher solvent content to form hydrogen bonds, and in the case of polyphenol compounds the extraction is favored by its polarity [3,26,27].

On the other hand, the effect of pH was not significant in its linear term for the extraction of the polyphenol compounds and bixin. This result is opposite to that of Rubio-Senent and collaborators, who demonstrated that the activity and solubility of polyphenol compounds and bixin were affected by pH. Further, bixin, is a carotenoid having a highly conjugated structure and a carboxyl end-group. As a result, its solubility at neutral and alkaline pH was limited. Conversely, the polyphenol compounds possess an aromatic ring structure which is attached to hydroxyl groups or other planar rings. These molecules are slightly acidic and their solubility and activity are maximized at neutral and slightly acidic pH [28]. The insignificance of pH in the system can be attributed to the powerful effect of other independent variables studied which overshadowed its effect.

The microwave treatment time was significant (*p* < 0.05) since it increased the extraction power for the polyphenol compounds and bixin. However, polyphenol compounds were more sensitive to

this extraction process than bixin (Figure 2a) since once the maximum point of extraction was reached, the content of polyphenols steadily decreased. This is explained by the heat generated during the microwave treatment. Thus, the longer the treatment, the larger the temperature reached in the system, causing solvent volatilization along with a partial degradation of the polyphenol compounds. Some authors have reported that the bioactivity of these secondary metabolites are affected by the extraction mechanism and heat treatments [7,29]. On the other hand, the optimal extraction conditions were found when a pH of 7, solvent concentration of 96%, solvent-to-seed ratio of 6:1, and a microwave time of 5 min were in place. These conditions rendered the largest yield of polyphenol and bixin compounds. It is then worthy to mention that the antioxidant and antimicrobial activities of the optimized extract were probably due to its largest content of polyphenol and bixin compounds, which in turn were responsible for their antioxidant and antimicrobial activities. These results coincide with those reported by Vasu et al. in 2010, which extracted only bixin from *B. orellana* seeds by applying microwaves for a time of 18 min as compared to a leaching extraction for 80 min. As a result, the bixin extraction raised from 8.2% to 16.3%. They also claimed that MAE is an alternative green technology and superior to conventional extraction for the extraction of bixin [12].

On the one hand, the antioxidant activity increased for the extract obtained by MAE, with a 5-and 10-fold increase of bixin and polyphenol compounds, respectively, as compared to the extract obtained by leaching. Moreover, the extract obtained by MAE presented antimicrobial activity against *B. cereus* and *S. aureus*. There were statistically significant differences (*p* < 0.05) for both activities between treatments, where MAE presented greater in vitro activities thanks to the high contents of polyphenols and antioxidant compounds. However, a direct proportional association was not appreciated between the increase of the bioactive compounds and the increments of the antimicrobial and antioxidant activities. Thus, the antioxidant activity was the most affected variable by the extraction method. The results of the antimicrobial and antioxidant activities of the lyophilized extract are comparable with those reported by some authors [1,12,30].
