Fermentation Time Modulates the Osteoprotective Effects of Milk Kefir in Glucocorticoid-Induced Osteoporotic Rats

Milk kefir has shown osteoprotective potential, but whether fermentation duration influences its effects remains unclear. This study evaluated the influence of fermentation time on milk kefir microbial composition and its effects on mandibular bone in glucocorticoid-induced osteoporotic rats. Milk kefir was fermented for 1, 4, or 7 days (MK1, MK4, MK7). Forty male Wistar rats were assigned to five groups (n = 8): healthy control, glucocorticoid-induced osteoporosis (GIO), and GIO treated with MK1, MK4, or MK7. Osteoporosis was induced by weekly intramuscular dexamethasone (7 mg/kg) for 5 weeks, followed by 12 weeks of daily kefir administration (0.7 mL). Microbial composition was assessed by 16S and 18S rRNA gene amplicon sequencing. Lactobacillus dominated the bacterial community, whereas Saccharomyces predominated among fungi, with no major changes across fermentation times. Prolonged fermentation (MK4 and MK7) significantly increased serum calcium, mandibular cortical thickness, interradicular bone area, collagen maturity, and fracture resistance. These findings indicate that prolonged fermentation enhances the osteoprotective effects of milk kefir, despite relative stability of the dominant microbial taxa, highlighting fermentation duration as an important factor in optimizing milk kefir for preserving mandibular bone under osteoporotic conditions.