BCp12 is a novel casein-derived antibacterial peptide with a broad-spectrum antibacterial effect. However, its action mechanism against E. coli is unknown. In this study, the growth curve showed that BCp12 had excellent antibacterial activity against E. coli. Red (propidium iodide staining) and green (fluorescein isothiocyanate staining) fluorescence signals were detected at the edges of the E. coli cells treated with BCp12. scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that E. coli cells became rough and shrunken, and part of the cell contents leaked to form a cavity. Furthermore, the iTRAQ proteome analysis showed that 193 and 174 proteins were significantly up-regulated and down-regulated, respectively, after BCp12 treatment. Four enzymes involved in fatty acid degradation of E. coli were down-regulated, disrupting the synthesis of cell membranes. Molecular docking and gel retardation assays showed that BCp12 could bind to genes encoding four key enzymes involved in the fatty acid degradation pathway through hydrogen bonding and hydrophobic interactions, thus significantly inhibiting their activities. Overall, the results indicate that BCp12 inhibits the growth of E. coli, causing metabolic disorders, thus destroying the structure of cell membranes.

Guishan goats, a unique goat breed in Yunnan Province, have a long history and representation, but their whey protein and function remain unclear. In this study, we carried out a quantitative analysis of the Guishan and Saanen goat whey proteome using a label-free proteomic approach. A total of 500 proteins were quantified from the 2 kinds of goat whey proteins, including 463 common proteins, 37 uniquely expressed whey proteins (UEWP), and 12 differentially expressed whey proteins (DEWP). Bioinformatics analysis indicated that UEWP and DEWP were mainly involved in cellular and immune system processes, membrane, and binding. In addition, UEWP and DEWP in Guishan goats participated primarily in metabolism and immune-related pathways, whereas Saanen goat whey proteins were associated mostly with environmental information processing-related pathways. Guishan goat whey promoted the growth of RAW264.7 macrophages more than Saanen goat whey, and significantly reduced the production of nitric oxide in lipopolysaccharide-stimulated RAW264.7 cells. This study provides a reference for further understanding these 2 goat whey proteins and finding functional active substances from them.

A cysteine peptidase was previously identified from germinated Moringa oleifera seeds, but its milk-clotting properties on bovine caseins was still unclear. In this study, this novel cysteine peptidase (MoCP) showed preferential activity on κ-casein (κ-CN), with greater hydrolytic activity compared with calf rennet, whereas weak hydrolysis of α-casein and β-casein made MoCP suitable for application in cheesemaking and may yield various functional peptides. All 3 evaluated caseins were hydrolyzed to form relatively stable peptide bands within 3 h of proteolysis with MoCP. Cleavage sites were determined by gel electrophoresis, liquid chromatography mass spectrometry/mass spectrometry, and peptide sequencing, which revealed that cleavage of κ-CN by MoCP occurred at residue Ile129-Pro130 and generated a 14,895.37-Da peptide. The flocculation reaction between MoCP and κ-CN determined by 3-dimensional microscopy with super-depth of field revealed that the initial 30 min of reaction were key for milk coagulation, which may affect curd yield. Overall, the findings presented herein suggest that the cysteine peptidase from germinated M. oleifera seeds can be considered a promising plant-derived rennet alternative for use in cheese manufacture.

As unique genetic resources, the differential characteristics of whey proteins and their derived peptides between Binglangjiang and Dehong buffaloes remain unclear. This study employed an integrated proteomics and peptidomics approach to elucidate the similarities and differences in whey proteins and their derived peptides between the 2 buffalo species. Proteomic analysis identified 405 proteins in total, 15 unique proteins and 390 common proteins (6 showed significant expression differences), indicating high similarity in whey protein composition between the 2 species. A total of 75 peptides were identified from whey proteins of both buffalo species following simulated gastrointestinal digestion, with the β-LG-derived peptide TPEVDDEALEK being the most abundant. Twelve of these peptides possess potential immunomodulatory, anti-inflammatory, and antimicrobial activities in silico prediction. Furthermore, both whey proteins and their derived peptides could promote the proliferation of RAW264.7 cells and inhibit nitric oxide (NO) secretion in LPS-stimulated RAW264.7 cells, especially Binglangjiang buffalo. These 2 buffalo species demonstrated similarities in whey protein composition, derived peptides, and their activities. These findings provided important insights for understanding the nutritional value and functional properties of buffalo whey proteins.