To elucidate the regulatory mechanism of the acid tolerance response (ATR) in Bifidobacterium longum BBMN68, this study investigated the role of response regulator BBMN68＿47 in mediating ATR through genomic and proteomic approaches. An overexpression strain of BBMN68＿47 with a 3 × FLAG tag was constructed via molecular cloning, and the recombinant plasmids (pDP152-BBMN68＿47, pDP152-3 × FLAG, and pDP152-3 × FLAG-BBMN68＿47) were validated through polymerase chain reaction (PCR), sequencing, and Western blot. Under weak acid induction (pH 4.5), chromatin immunoprecipitation sequencing (ChIP-seq) identified 118 high-confidence DNA binding peaks, over 80% of which were localized at transcription start sites (TSS). Bioinformatics analysis predicted seven candidate DNA binding motifs. Electrophoretic mobility shift assay (EMSA) analysis further confirmed specific binding of BBMN68＿47 to two conserved motifs: CTGGGCGTTT(Probe 0235) and CTGGACGAATCCTG(Probe 0075), regulating the expression of its own gene BBMN68＿47(autoregulatory) and the S-adenosylmethionine synthetase (metK) gene, respectively. These findings establish BBMN68＿47 as a central transcriptional regulator in B. longum ATR, orchestrating key acid-resistant gene networks through conserved and novel DNA motifs. This work offers a theoretical basis for elucidating the molecular mechanism of the ATR of B. longum and engineering stress resistance in probiotics.

In this study, the effect of Ligilactobacillus salivarius M18-6, with the capability to stably and rapidly ferment soybean protein and strong antioxidant capacity, on the flavor quality and antioxidant activity of fermented soymilk was analyzed. This study found that with increasing fermentation time, soybean protein and fat gradually decomposed, and the particle size of fermented soymilk showed a downward trend, making its texture more delicate and smoother. Additionally, the volatile components of soymilk before and after fermentation were analyzed by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-ion mobility spectrometry (GC-IMS). It was found that the concentration of hexanal, which primarily contributed to beany flavor, gradually decreased with fermentation time, and so did the concentration of 1-octen-3-ol. The concentrations of diacetyl, acetoin and 2-heptanone and other flavor substances associated with fruity aroma gradually increased, thereby resulting in good flavor of soymilk. Furthermore, fermented soymilk with L. salivarius M18-6 had potent hydroxyl radical scavenging activity in a Caco-2 cell model of oxidative injury induced by acrylamide. Compared with the oxidative damage model group, the activities of catalase (CAT) and superoxide dismutase (SOD) in Caco-2 cells treated with the fermented soymilk increased by 2.67 and 30.80 times, respectively, and the concentration of glutathione increased by 13.83 times. In summary, soymilk fermented by L. salivarius M18-6 has a significant repairing effect on oxidatively damaged cells.