The number of live lactic acid bacteria (LAB) is an important quality indicator for yogurt, the quantitative testing of LAB has become an important task in the evaluation of product quality and function. By analyzing and comparing the performance of 16S rRNA gene and tuf gene used in absolute quantification, the tuf gene with copy number 1 was selected as the target gene of six LAB. By drawing a standard curve to achieve qualitative and quantitative detection of six strains of LAB, the detection range was found to be 1 × 103–1 × 108 copies/µL. The traditional plate colony count and Flow Cytometry (FCM) were compared with the method of qPCR, which was used in this experiment. Meanwhile, the confocal laser microscope combined with STYO 9 and propidium iodide dyes was used to determine that the content of viable bacteria in the yogurt was more than 90%, which proved that the detection result using qPCR method was closer to the true level of LAB in yogurt. Compared with the existing methods, the method in this study allowed the qualitative and quantitative detection of the six kinds of LAB in yogurt, and the distribution of live and dead bacteria in yogurt could be calculated.

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

Superoxide dismutase (SOD) is an important antioxidant enzyme with different physiological functions, which can be used as a nutritional fortifier in food. Cereal-based fermented products are becoming popular worldwide. In this study, novel millet-based flavored yogurt enriched with SOD was developed. Lactiplantibacillus plantarum subsp. plantarum was screened, which manufactured SOD activity of 2476.21 ± 1.52 U g−1. The SOD content of millet yogurt was 19.827 ± 0.323 U mL−1, which was 63.01, 50.11, and 146.79% higher than that of Bright Dairy Yogurt 1911, Junlebao and Nanjing Weigang, respectively. Fifty-four volatile flavor substances and 22,571 non-volatile flavor substances were found in yogurt. Compared to traditional fermented yogurt, 37 non-volatile metabolites in yogurt with millet enzymatic fermentation broth were significantly upregulated, including 2-phenyl ethanol, hesperidin, N-acetylornithine and L-methionine, which were upregulated by 3169.6, 228.36, 271.22, and 55.67 times, respectively, thereby enriching the sensory and nutritional value of yogurt. Moreover, the manufacture of unpleasant volatile flavor substances was masked, making the product more compatible with consumers' tastes.

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The function of the autoinducer-2 exporters (AI-2E) family transporter protein of Lactobacillus acidophilus is still unclear. The phylogenetic analysis was used to analyze the relationship between the AI-2E protein of the L. acidophilus CICC 6074 strain and other AI-2E family members. Escherichia coli KNabc strain was used to verify whether the protein has Na+ (Li+)/H+ antiporter activity. The AI-2E protein overexpression strain was constructed by using the pMG36e expression vector, and the overexpression efficiency was determined by real-time quantitative PCR. The vitality and AI-2 activity of L. acidophilus CICC 6074 strains were determined. The results showed that the AI-2E protein of Lactobacillus formed a single branch on the phylogenetic tree and was closer to the AI-2E family members whose function was AI-2 exporter group I. The expression of AI-2E protein in the E. coli KNabc strain did not recover the resistance of the bacteria to the saline environment. Overexpression of AI-2E protein in L. acidophilus CICC 6074 could promote the AI-2 secretion of L. acidophilus CICC 6074 strain and enhance their survival ability in intestinal juice.