Alpha diversity for ITS.

The dynamic changes in the physicochemical, microbiological, and metagenomic profiles of Crema de Chiapas cheese were evaluated across three ripening stages (2, 29, and 58 days). Although the main physicochemical properties —including fat content— remained remarkably stable, salt and protein levels showed noticeable variation throughout ripening. Protein content had the strongest influence on sample differentiation across ripening stages in unsupervised multivariate models, enabling the clustering of microbial diversity according to maturation time. A clear shift in microbial diversity was detected, marked by a reduction in bacterial genera and a concurrent decline in fungal and yeast populations as ripening advanced. The predominant bacterial genera throughout ripening were Streptococcus, Lactobacillus, and Lactococcus. While Streptococcus and Lactobacillus increased over time, Lactococcus exhibited the opposite trend. Metagenomic analysis revealed a decrease in Candida etchellsii and a concomitant increase in Candida tropicalis as ripening progressed. Quantitative PCR (qPCR) confirmed the presence of C. etchellsii at T1 (Ct = 7.22) and C. tropicalis at T3 (Ct = 9.84). The presence of three additional bacterial genera—Chryseobacterium, Aeromonas, and Enterobacter—identified by next-generation sequencing (NGS), was also assessed by qPCR. Chryseobacterium was detected at T2 (Ct = 3.26), whereas Aeromonas and Enterobacter were absent across all stages. Collectively, these findings suggest that potentially pathogenic microorganisms were not present at biologically relevant levels.