A Study on the Stability and Carbohydrate Metabolic Traits of Starter Cultures in Response to Continuous Subculturing

The industrial application of starter cultures requires stable physiological and genetic performance. In this study, <i>Streptococcus salivarius</i> subsp. <i>thermophilus</i> and <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i> were continuously subcultured. Physiological stability was assessed through colony morphology, fermentation activity, and growth profiling. Genetic stability was evaluated through comparative genomics of carbohydrate metabolism networks and single-nucleotide polymorphism (SNP) analysis. The results showed that after 2000 generations, the cellular morphology of the strains remained intact. Additionally, the strains exhibited enhanced growth performance and fermentation capability. The Gompertz model revealed that adapted <i>S. thermophilus</i> A37 and <i>L. bulgaricus</i> B29 exhibited shortened lag phases, increased maximum specific growth rates, and high stationary-phase cell densities. Phenotypic microarray and comparative genomics revealed that <i>S. thermophilus</i> mainly used mono- and disaccharides, with impaired ribose metabolism due to the absence of the <i>rbsk</i> gene in the pentose phosphate pathway. In contrast, <i>L. bulgaricus</i> metabolized diverse oligosaccharides, sugar alcohols, and plant-derived substrates. Additionally, it effectively catabolized ribose through the phosphoketolase pathway and possessed a trehalose degradation cluster. All strains exhibited genomic stability, with SNPs revealing fewer than 21 variations per isolate. This study provides an important theoretical foundation for evaluating the stability of fermentation starter cultures.