3.6. Microbial Inoculum and Prebiotic Assay

Growth stimulation of probiotic bacteria by phenolic compounds present in the extract was evaluated as previously described by Picazo et al. [55]. Bacteria strains were used to evaluate the prebiotic effect of the polyphenolic compounds on Lactobacillus brevis and Lactobacillus paracasei 25302. Both strains belong to the strain collection of the Food Research Department at the Autonomous University of Coahuila. These bacterial strains were previously activated in MRS media culture and incubated at 37 °C for 24 h. On the day of the experiment, each bacterial culture in exponential growth was diluted in sterile saline solution to a final concentration of 1.5 × 10⁸ CFU/mL. The experiments were performed in MRS broth without glucose and supplemented with rambutan peel polyphenol powder (RPP). Each well was inoculated with 4.17 µL of each bacterial suspension (1.5 × 10⁸ CFU/mL) and RPP fresh serial dilutions (0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 µg/mL) up to 250 µL. Positive (bacteria + medium) and negative (medium) controls, and a blank (PC + medium) were included. The plates were incubated for 24 h at 37 °C, and bacterial growth was then evaluated spectrophotometrically by recording the OD_620nm variations and by inoculating into MRS dishes; CFU were determined after incubation for 24 h at 37 °C. All experiments were performed by triplicate. The growth response of each strain on RPP relative to its growth response on glucose was calculated using the method of Kneifel et al. [56]. The calculation was performed using the following formula, where the growth on glucose corresponds to 100%:

where A is the mean OD_620nm of a strain on RPP substrate and B is the mean OD_620nm of the same strain grown on glucose. In this way, the results reflect the growth of a specific strain on a particular concentration of RPP relative to its growth on a glucose substrate which was fixed at 100%.