Kefir Direct-Vat-Set Starter Producing Peptides with Angiotensin-Converting Enzyme Inhibitory and Antioxidant Activities: Formulation Optimization and Stability Evaluation

Abstract Kefir is a healthy beverage, however, traditional kefir cannot be commercially produced on a large scale due to the inconvenience of production, as well as the diversity and instability of microorganisms in kefir grains. The focus of this study is to develop a functional Direct-Vat-Set (DVS) kefir starter for commercial production. Through formulation experiments, a blend comprising five fermentation strains was determined, including 10% Lactobacillus helveticus KM1, 25.8% Limosilactobacillus fermentum M3, 23.4% Kluyveromyces lactis Y5, 18.4% Lactiplantibacillus plantarum Lp69, and 22.4% Lacticaseibacillus casei L61. Using an inoculation rate of 2.5% and fermenting at 24 °C for 23 h, the kefir exhibited a DPPH free radical scavenging rate of 79.76 ± 0.65%, an ACE inhibition rate of 78.52 ± 0.52%, and sensory evaluation scores of 65.75 ± 0.77. Subsequently, the inoculation rate was optimized to 0.9% to enhance cost-effectiveness and improve sensory perception. Throughout the storage process, the kefir maintained favourable functional properties and exhibited a gradual improvement in sensory evaluation scores.