The evaluation of D- and L-lactic acid production by lactic acid bacteria is of critical importance, particularly for strains intended for use in infant and young child foods. Additionally, compliance with relevant regulatory standards necessitates the detection of D-lactic acid. Additionally, regulatory requirements exist in this regard. This study aimed to develop and validate a method for simultaneously measuring D-lactic acid and L-lactic acid produced by lactic acid bacteria. The method validation of the HPLC analysis was performed in terms of accuracy, precision, specificity, limit of quantification (LOQ), linearity, range, and robustness, and the measurement uncertainty was also evaluated. The method demonstrated a limit of detection (LOD) of 0.25 µg/mL and LOQ of 0.8 μg/mL for D-/L-lactic acid. For six validated bacterial strains, mean recoveries ranged from 93.50% to 105.37%, with intra-assay relative standard deviations (RSD) of 0.90–2.64% and inter-laboratory RSD of 2.56–10.16%. Excellent linearity, accuracy, and precision were observed across the concentration range of 0.8–200.0 μg/mL. Results confirmed no interference from culture media batch variations, and sample stability was maintained for 48 h. Additionally, relative expanded uncertainties were determined as 10.48% and 7.64%. The developed method was suitable for the identification and quantification of D- and L-lactic acid in lactic acid bacteria fermentation broth samples. This method was applicable for assessing the production of D-/L-lactic acid by food cultures.

Monascus species are widely used in traditional fermented foods for pigment and flavor formation, but citrinin contamination remains a major safety concern that limits broader food applications. Therefore, this study aimed to evaluate the citrinin risk of a furu-derived Monascus ruber strain, BC20, by integrating phenotypic screening across food-relevant matrices with genome-resolved analysis. After 14 days of cultivation across eight matrices, including fungal media as well as dairy-, cereal-, and bran-based substrates, citrinin was not detected by immunoaffinity cleanup combined with HPLC–FLD (LOD, 4 μg/kg; LOQ, 12 μg/kg). To investigate the genetic basis of this phenotype, we generated a chromosome-scale genome assembly for BC20 and conducted comparative analyses across a total of 19 Monascus genomes. ANI analysis and phylogenomic inference consistently placed BC20 within the ruber–pilosus clade. Comparative synteny analysis showed that the citrinin-associated locus in BC20 no longer retained an intact cluster configuration but instead exhibited a remnant-locus architecture, and similar patterns were also observed in several related genomes from the same clade. By contrast, the monacolin K (mk) locus remained syntenically conserved in BC20, supporting locus-specific structural disturbance rather than assembly-derived pseudo-absence. Additionally, its antifungal susceptibility was determined. Overall, BC20 represents a M. ruber candidate strain with undetectable citrinin, and this study provides a practical analytical framework for citrinin risk screening in food-related Monascus isolates.