Identification and Characterization of Domains Responsible for Cell Wall Binding, Self-Assembly, and Adhesion of S-layer Protein from Lactobacillus acidophilus CICC 6074.

Lactobacillus S-layer protein (SLP) is a biologically active protein on the cell surface. To further elucidate the structures and functions of SLP in Lactobacillus acidophilus CICC 6074, this study was conducted to identify the functional domains of SLP which is responsible for cell wall anchoring, self-assembly, and adhesion. The gene (slpA) of L. acidophilus CICC 6074 SLP was amplified by polymerase chain reaction and speculated functional domains. Fusion proteins of C-terminal truncations from SLP were exogenously expressed in Escherichia coli BL21 (DE3). FITC-labeling N-terminal truncations of SLP were synthesized. The C-terminal domain was more likely to be the binding region, and the cell wall-anchored receptor of SLP was teichoic acid. Furthermore, N-terminal truncations could self-assemble to milk fat globule membrane polar lipid liposomes observed using a fluorescence microscope. Notably, SAN1 (region 32-55) of N-terminal truncations was mainly responsible for the adhesion of SLP to HT-29 cells. These results showed that SLP played a crucial role in the functions of L. acidophilus CICC 6074, which might be of significant reference value for future studies.

Production and transepithelial transportation of angiotensin-I-converting enzyme (ACE)-inhibitory peptides from whey protein hydrolyzed by immobilized Lactobacillus helveticus proteinase.

Lactobacillus helveticus LB 10 proteinases immobilized with sodium alginate were used to hydrolyze whey protein to produce angiotensin-I-converting enzyme (ACE)-inhibitory peptides. The generated hydrolysates were tested for ACE-inhibitory activity and for their ability to be transported across Caco-2 cell monolayers. Using a response surface method, we determined that a proteinase concentration of 7.55 mg/mL, sodium alginate concentration of 2.03 g/100 mL, and glutaraldehyde concentration of 0.39% were found to be the optimal immobilization conditions. Compared with free proteinase, the immobilized proteinase had significantly higher pH, thermal and storage stability, and reusability. Whey protein hydrolysates were fractionated by gel filtration chromatography and ACE-inhibitory peptide mixtures were transported across Caco-2 cell monolayers in a human intestinal-absorption model. The di- and tripeptides KA, EN, DIS, EVD, LF, AIV, and VFK (half-maximal inhibitory concentrations (mean ± standard deviation) of 1.24 ± 0.01, 1.43 ± 0.04, 1.59 ± 0.27, 1.32 ± 0.05, 1.60 ± 0.39, 2.66 ± 0.02, and 1.76 ± 0.09 mmol/L, respectively) were detected on the basolateral side of the Caco-2 cell monolayer using ultra-performance liquid chromatography-tandem mass spectrometry. These results highlight that ACE-inhibitory peptides are present on the basolateral side of the Caco-2 cell model after transportation of whey protein hydrolysate across the Caco-2 cell membrane.