Characterization and Production of Kefir from Almond Milk and Its Effect on the Colonic Luminal Microbiota of Mice

Introduction. Almond milk is among the most purchased plant-based alternatives to bovine milk and can be fermented with kefir grains, but the resulting beverage has been studied only in terms of its own properties and its effect on the gut microbiota has not been examined. Aims. This study characterized kefir produced from almond milk and examined whether it is associated with differences in the intestinal microbial community in mice. Materials and methods. Kefir grains were inoculated into commercial almond milk at 2%, 3% and 4% (w/v) and fermented at 25 °C for 22 h, and pH, titratable acidity and microbial counts on five culture media were followed over 14 days of refrigerated storage at 4 °C, with sensory evaluation on the first day of storage. The 3% product was then given by daily gavage to male C57BL/6 mice for 21 days alongside plain almond milk and an untreated control, with the fecal microbiota assessed by culture-based enumeration and the colonic luminal contents characterized by 16S rRNA gene sequencing. Results. Fermentation lowered the pH of the almond milk from 7.78 to between 4.74 and 5.27 at day 0 according to the grain concentration (p < 0.001), with titratable acidity rising in parallel (p = 0.009). Mean viable counts stayed at or above 8.00 log10 CFU/mL on four culture media throughout 14 days of refrigerated storage, and neither the counts nor the sensory scores differed between the three concentrations (p > 0.05), apart from texture, which was rated higher as the grain concentration rose (p = 0.005). In mice, fecal lactic acid bacteria were most abundant in the kefir group (p < 0.001); after adjustment for the counts recorded on day 0 the difference from the plain almond milk group remained significant (p = 0.002) while that from the untreated control did not (p = 0.071). In the colonic luminal contents, alpha diversity did not differ among groups (p > 0.05), whereas beta-diversity analysis suggested differences in community composition (PERMANOVA on Bray–Curtis distances, p = 0.004). Bacillota predominated over Bacteroidota only in the kefir group, while Lactobacillus represented 13.0% of the community compared with 4.8% and 5.8% in the almond milk and control groups, respectively. LEfSe further identified Lactobacillus as a discriminating taxon in the kefir group in this exploratory analysis. Conclusion. These findings indicate an association between almond milk kefir consumption and differences in the composition of the colonic luminal microbiota in mice, while alpha diversity remained unchanged.