Lactobacillus-enriched yogurt is in increasingly high demand due to its health benefits, but the product stability requires an understanding of the microbial dynamics during fermentation and storage. This study investigated the interactions between probiotic pairs (L. paracasei L9 and L. acidophilus LAC) and starter culture (HYY) through fermentation kinetics, microbial viability, organic acid profiles, and metabolomics. The results demonstrated that L. paracasei L9 significantly increased the titratable acidity from 25.20 ± 7.01 °T to 36.56 ± 3.47 °T at 3 h and reduced the fermentation time by 0.5 h, whereas L. acidophilus LAC showed minimal effects. L. paracasei L9 achieved higher viability (8.4 lg CFU/g) via the high-affinity lactose transport and Leloir pathway, whereas the L. acidophilus LAC growth remained limited (6.9 lg CFU/g). The metabolomic investigation revealed the L9 + HYY upregulated glycerophospholipid metabolism and pantothenate/CoA biosynthesis to support rapid biomass accumulation. In contrast, LAC + HYY modulated the arginine and branched-chain amino acid metabolism for acid tolerance. During 21 days of storage, there were no significant differences in final TA values and lactic acid content among the probiotic supplementation groups. L9 + HYY remained stable (>9.0 lg CFU/g) by upregulating the aromatic amino acid biosynthesis and suppressing the purine/sulfur metabolism, whereas L. acidophilus LAC decreased to 6.02 lg CFU/g. These findings demonstrate the dual role of L. paracasei L9 in accelerating the fermentation and maintaining the microbial stability through metabolic reprogramming, which guides the development of improved probiotic yogurts.

Introduction: Probiotic-fortified yogurt has gained substantial consumer preference owing to its well-documented health benefits. However, stability of probiotic yogurt necessitates a comprehensive understanding of microbial dynamics throughout fermentation and storage. Methods: This study employed an integrated approach combining fermentation kinetics, post-acidification profiling, and untargeted metabolomics to explore the complex interactions between three Bifidobacterium strains ( B. animalis 23426, B. bifidum 91, and B. longum BB68S) and starter cultures (HYY) during symbiotic fermentation. Results: The results demonstrate that Bifidobacterium supplementation notably enhanced the biomass of S. thermophilus (8.13–8.54 lg CFU/mL) after 2 h by upregulating galactose catabolism and riboflavin biosynthesis, thereby reducing fermentation time by 0.5 to 2 h. In contrast, competitive exclusion effects caused a decrease in L. bulgaricus biomass by 0.2 to 0.8 log CFU/mL. Over 21-day of refrigerated storage, the acid accumulation in Bifidobacterium -enriched yogurts was significantly lower (Δ 3.08–7.49 °T) than in HYY yogurt (Δ 9.42 °T), primarily by downregulation key metabolic pathways involving glycerophospholipid metabolism, branched-chain and aromatic amino acid metabolism, and cofactor biosynthesis, leading to reduced post-acidification. Discussion: Therefore, Bifidobacterium accelerates fermentation by promoting S. thermophilus biomass while mitigating post-acidification by inhibiting L. bulgaricus . The results provide a scientific basis for developing next-generation probiotic yogurts with controlled acidification profiles and improved shelf-life characteristics.

The addition of Bifidobacterium animalis subsp. lactis and prebiotics to fermented milk can enhance its flavor and sensory properties; however, research on the effects of their combined supplementation on flavor profiles remains limited. This study investigated the impact of simultaneously adding B. lactis IU100 and resistant starch type III (RS3) to fermented milk on flavor and texture. The results showed that co-supplementation shortened the fermentation time by 1 h. It also increased hardness by 28.8%, springiness by 1.14 mm, and water holding capacity by 12.45%, accompanied by the formation of a more continuous and dense gel network. Headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS) combined with odor activity value analysis indicated the enrichment of 115 key aromatic compounds, among which ethyl caprylate, ethyl n-butyrate, 1-octanol, and 2,3-heptanedione were identified as representative flavor compounds associated with fruity and creamy notes. KEGG pathway analysis revealed that 24 differential metabolites were predominantly enriched in purine metabolism and amino acid-related pathways. Within these pathways, coordinated enzymatic reactions convert α-keto acids and fatty acid metabolites into key flavor esters and catalyze the formation of volatile alcohols from amino acids and aromatic fatty acid precursors. Overall, this combined strategy effectively optimized fermentation efficiency, texture, and flavor through the targeted reprogramming of microbial metabolic flux.

Background: Functional constipation is a common gastrointestinal disorder that impairs quality of life. Although probiotics have shown benefits in constipation, whether live and heat-killed forms of the same strain act through similar mechanisms remains unclear. This study compared the effects of live and heat-killed Lacticaseibacillus paracasei K56 in experimental constipation. Methods: Loperamide-treated mice received live or heat-killed L. paracasei K56. Gastrointestinal motility, colonic histology, neurotransmitters, aquaporin expression, gut microbiota, short-chain fatty acids, and metabolomic profiles were evaluated. Results: Both preparations relieved constipation, as reflected by shorter defecation latency, increased fecal output and moisture, and faster intestinal transit, with live K56 producing greater improvement. Colonic injury was alleviated, accompanied by reduced Aqp2 and Aqp3 expression and normalization of 5-hydroxytryptamine, substance P, and vasoactive intestinal peptide. The treatments increased Lactobacillus and Lachnospiraceae, promoted butyrate production, and altered tryptophan metabolism. Heat-killed K56 was mainly associated with changes in phenylalanine/tyrosine- and methionine-related metabolism, whereas live K56 showed more pronounced alterations in arginine/polyamine, tryptophan, and bile acid metabolism. Conclusions: Live and heat-killed L. paracasei K56 improved constipation through shared and distinct mechanisms involving gut microbiota, microbial metabolism, enteric neurotransmission, and intestinal water homeostasis. Live K56 produced stronger effects, whereas heat-killed K56 retained substantial biological activity.

This study investigated the impact of Bifidobacterium animalis ssp. lactis (B. lactis) IU100 or/and 1.5% resistant starch type III (RS3) on fermented milk during storage. The co-supplementation with enhanced texture, increasing hardness from 10.52 g (control) to 14.88 g and springiness from 1.18 mm to 3.03 mm, and promoted a denser gel network. Volatile profiling combined with OAV analysis revealed that the addition of B. lactis IU100 significantly increased the total content of alcohols (from 1231.77 μg/L to 2841.43 μg/L), particularly promoting the accumulation of compounds such as n-butanol and 1-octen-3-ol are known to contribute fruity and mushroom-like notes in dairy systems. The individual supplementation of 1.5% RS3 markedly elevated the total aldehyde content (from 3761.05 μg/L to 7026.82 μg/L), with compounds such as 2-octenal, (2e)- is associated with distinct fatty and nutty aromas in model systems. When B. lactis IU100 was combined with RS3, the level of 1-hexanol was further elevated, enhancing a fresh green note. Untargeted metabolomics further indicated that 300 significantly differential metabolites were identified in the co-supplemented group, among which key intermediates such as dephospho-CoA and adenosine diphosphate ribose were notably upregulated. These metabolites were mainly mapped to cofactor biosynthesis, purine metabolism, and pyrimidine metabolism, suggesting coordinated roles in the formation and interconversion of flavor precursors. In summary, the combined supplementation of B. lactis IU100 and RS3 effectively enhanced the overall quality and flavor complexity of fermented milk by modulating the volatile composition and core metabolic network.

The industrial application of starter cultures requires stable physiological and genetic performance. In this study, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus 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 S. thermophilus A37 and L. bulgaricus B29 exhibited shortened lag phases, increased maximum specific growth rates, and high stationary-phase cell densities. Phenotypic microarray and comparative genomics revealed that S. thermophilus mainly used mono- and disaccharides, with impaired ribose metabolism due to the absence of the rbsk gene in the pentose phosphate pathway. In contrast, L. bulgaricus 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.