The lactic acid bacteria Streptococcus thermophilus and Lactobacillus helveticus are commonly used as starter cultures in dairy product production. This study aimed to investigate the characteristics of fermented milk using different ratios of these strains and analyze the changes in volatile compounds during fermentation and storage. A 10:1 ratio of Streptococcus thermophilus CICC 6063 to Lactobacillus helveticus CICC 6064 showed optimal fermentation time (4.2 h), viable cell count (9.64 log10 colony-forming units/mL), and sensory evaluation score (79.1 points). In total, 56 volatile compounds were identified and quantified by solid-phase microextraction and gas chromatography–mass spectrometry (SPME-GC-MS), including aldehydes, ketones, acids, alcohols, esters, and others. Among these, according to VIP analysis, 2,3-butanedione, acetoin, 2,3-pentanedione, hexanoic acid, acetic acid, acetaldehyde, and butanoic acid were identified as discriminatory volatile metabolites for distinguishing between different time points. Throughout the fermentation and storage process, the levels of 2,3-pentanedione and acetoin exhibited synergistic dynamics. These findings enhance our understanding of the chemical and molecular characteristics of milk fermented with Streptococcus thermophilus and Lactobacillus helveticus, providing a basis for improving the flavor and odor of dairy products during fermentation and storage.

The lactic acid bacteria Streptococcus thermophilus and Lactobacillus helveticus are commonly used as starter cultures in dairy product production. This study aimed to investigate the characteristics of fermented milk using different ratios of these strains and analyze the changes in volatile compounds during fermentation and storage. The 10:1 ratio of Streptococcus thermophilus CICC 6063 to Lactobacillus helveticus CICC 6064 showed optimal fermentation time (4.2 h), viable cell count (9.64 log10 colony-forming units/mL), and sensory evaluation score (79.1 points). In total, 56 volatile compounds were identified and quantified by solid-phase microextraction and gas chromatography–mass spectrometry (SPME-GC-MS), including aldehydes, ketones, acids, alcohols, esters, and others. Among these, acetaldehyde, 2,3-butanedione, 2,3-pentanedione, 2-heptanone, acetoin, and 1-heptanol had the highest odor activity values and contributed to the characteristic flavor of fermented milk. The concentrations and types of key flavor compounds reached their peak at the end of fermentation. Throughout the fermentation and storage process, the levels of 2,3-pentanedione and acetoin exhibited synergistic dynamics. These findings enhance our understanding of the chemical and molecular characteristics of milk fermented with Streptococcus thermophilus and Lactobacillus helveticus, providing a basis for improving the flavor and odor of dairy products during fermentation and storage.

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.