Lactobacillus rhamnosus p75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted from B. subtilis was confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3–7 and maintained at 70% at 50 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics.

A pig-specific real-time PCR assay based on the mitochondrial ND5 gene was developed to detect porcine material in food and other products. To optimize the performance of assay, seven commercial TaqMan master mixes and two real-time PCR platforms (Applied Biosystems StepOnePlus and Bio-rad CFX Connect) were used to evaluate the limit of detection (LOD) as well as the PCR efficiency and specificity. The LODs and PCR efficiencies for the seven master mixes on two platforms were 0.5-5 pg/reaction and 84.96%-108.80%, respectively. Additionally, non-specific amplifications of DNA from other animal samples (human, dog, cow, and chicken) were observed for four master mixes. These results imply that the sensitivity and specificity of a real-time PCR assay may vary depending on master mix and platform used. The best combination of master mix and real-time PCR platform can accurately detect 0.5 pg porcine DNA, with a PCR efficiency of 100.49%.

We investigated the expression and stability of enhanced green fluorescent protein (eGFP) under extreme conditions using two types of high-copy-number vectors and two types of anchoring motifs (CotB and C-terminally truncated ∆CotB spore coat proteins) for the development of a spore surface display system in Bacillus subtilis. The fused cotB-gfp and ΔcotB-gfp DNA fragments were cloned into the pUB19 (pUB110-derived) and pHY300PLK vectors. Four types of expression vectors were transformed into B. subtilis 168. The expression level of eGFP on the surface of spores prepared from B. subtilis transformants was measured by flow cytometry. When pUB19 vector was used, the activities of ∆CotB-eGFP and CotB-eGFP were 17.9 and 5.6 times higher than those of the pHY300PLK vector, respectively. In addition, the activity of pUB19-∆CotB-eGFP was 1.76 times higher than that of pUB19-CotB-eGFP. Overall, the activity of eGFP was more stable under extreme conditions (heat, pH, and protease challenges) when ∆CotB was used as an anchoring motif instead of CotB. Compared to the control groups, the activities of ΔCotB-eGFP and CotB-eGFP were maintained at 56% and 41% at 80 °C and 88% and 55% at pH 10, respectively. The activities of ΔCotB-eGFP and CotB-eGFP were maintained at 62% and 41%, respectively, when treated with 0.03 U of proteinase K. In addition, the activities were maintained at 77% and 36%, respectively, when treated with 5.5 U of trypsin.

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Backgrounds The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. Results p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. Conclusions This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.