Optimizing the Formulation of Homemade Milk Kefir Drink from India: Comprehensive Microbial, Physicochemical, Nutritional, and Bioactivity Profiling

Kefir is a naturally fermented milk drink with rich probiotic content. This study aimed to develop and optimize homemade cow milk kefir (HCMK) and evaluate its microbial, chemical, nutritional, and antioxidant properties. HCMK was optimized using response surface methodology, where the independent variables were kefir grain inoculum (2–4%) and fermentation time (20 h–28 h), and the dependent variables were total bacterial count, pH, and overall acceptability. HCMK was prepared using 3% w/v Indian kefir grains inoculated into cow milk and fermented at 25 °C for 24 h. Optimized dependent variables were 2.08 × 108 ± 0.34 CFU/mL, pH 4.52 ± 0.05, and overall acceptability of 6.55 ± 0.21. Nutritional analysis revealed protein 3.6 g/100 mL, carbohydrates 2.66 g/100mL, fat 3.4 g/100 mL, iron 2.99 mg/100 mL, and calcium 29.3 mg/100 mL. Antioxidant profiling elucidated 54% radical scavenging activity and 18 mgGAE/100 mL total phenolic content. GC-MS revealed the presence of bioactive compounds with documented antioxidant, anti-inflammatory, and antimicrobial activities. This study highlights HCMK as a healthy probiotic functional food with significant antioxidant potential.