In vitro study of fermented bovine milk digestion: Validated method for the isolation of indigenous probiotics

Probiotic bacteria have gained popularity due to increasing scientific evidence supporting their health benefits. However, although some lactic acid bacteria (LAB) demonstrate good survival in vitro during digestion simulations, their survival is influenced by various factors and may decrease when incorporated into food products. Therefore, in this study, in vitro digestion simulations of fermented bovine milk and acid-curd cheese samples (control and those with isolated Lacticasei bacillus paracasei A11) were performed using the standardized static method. The counts of A11 were analyzed in cheese pre- and post-digestion to assess survival. The probiotic properties of the strain were evaluated in acid (2.5 pH; 0, 3 h) and bile salt (0.3, 0.5, and 1%; 0, 24 h) conditions. Counts of this strain in cheese samples were determined using 16S rRNA gene amplicon sequencing (MiSeq, Illumina). Results indicated that A11 tolerated acid (8.61 ± 0.06 log CFU/g after 3 h) and bile salts (6.66 ± 0.2 log CFU/g after 24 h with 1% bile salts), highlighting its probiotic potential. A significant decrease in LAB counts was observed in cheese without A11 post-digestion (approximately 4.46 log CFU/g), while cheese with A11 maintained higher LAB stability (around 6.08 log CFU/g) post-digestion (p < 0.05). Predigestion, A11 counts were 6.95 ± 0.05 log CFU/g on day 1 and 6.89 ± 0.03 log CFU/g on day 10. Postdigestion, counts remained stable at 6.7 ± 0.44 log CFU/g on day 1 and 6.84 ± 0.1 log CFU/g on day 10, indicating a 98% survival rate. This study demonstrated that A11 is highly tolerant of acid and bile salts, confirming its probiotic potential. Cheese without A11 showed a significant reduction in LAB counts post-digestion, while cheese with A11 maintained greater stability. The strain exhibited a 98% survival rate during digestion, supporting its viability as a probiotic.