Identification of Immune-Active Peptides in Casein Hydrolysates and Its Transport Mechanism on a Caco-2 Monolayer.

In this study, we investigated the transport mechanism of immune-active peptide fragments isolated from casein gastrointestinal hydrolysates via a Caco-2 monolayer. The casein gastrointestinal hydrolysates could stimulate B-lymphocyte proliferation and reduce the TNF-α level. Then, we identified the bioactive peptide fragments derived from casein gastrointestinal hydrolysis using LC-MS/MS. Our results demonstrated that the transport mechanism of five immune-active peptides at the cell level was bypass transport. In addition, the majority of peptide RYPLGYL was transported through the monolayer cell membrane as an intact form for playing immune-active functions. The KHPIK and FFSDK were mainly degraded into small fragments, except for a small amount passing through Caco-2 cells in an entire form. Overall, these results suggested that casein or its immune-active peptides might play a role in regulation of the intestinal immune system.

Bioactive peptide release and the absorption tracking of casein in the gastrointestinal digestion of rats.

Bovine casein is considered as an important source of many bioactive peptides (BAPs), which can also be produced via in vitro simulated gastrointestinal hydrolysis. To perform their physiological functions, some active peptides need to pass through the intestinal epithelial barrier and keep their structural integrity after oral administration. Owing to the complexity of in vivo digestion and absorption, there have been few studies in this area. In this study, casein was labeled with FITC to trace its digestion and absorption in Sprague Dawley (SD) rats. Gastric juice, intestinal fluid, blood, and intestinal tissue samples were collected at different time-points for preservation and analysis after intragastric administration. The results showed that CN-FITC exhibited good labeling stability in the gastrointestinal digestive juice both in vivo and in vitro, suggesting its potential to be used for the detection and tracking of casein hydrolysate. After the intra-gastric administration of FITC, the diffusion rates of fluorescent substances in serum were much higher than in the CN-FITC group. The maximum peptide content in the CN-FITC group during intestinal digestion was achieved 2 h after administration, and electrophoretic analysis of the hydrolysate composition suggested that the molecular weights of the peptides were mainly concentrated in the range of 3.4-10 kDa. The hydrolyzed peptides from CN-FITC could be absorbed into the blood just 1 h after administration. Frozen sections of rat duodenal tissue were observed under a confocal laser scanning microscope, and they showed that the CN-FITC digested products were absorbed from villi to mucosa in the rat intestines, and the casein-hydrolyzed polypeptides were accumulated significantly in tissue samples 2 h after administration. The peptides were mainly absorbed in the duodenum on the basis of absorption experiments using an everted gut sac. After intestinal digestion for 2 h, peptides with weights less than 5 kDa were enriched and identified using LC-MS-MS, and they were found to be mainly derived from ß-casein, containing potential angiotensin-I-converting enzyme inhibitory, antioxidant, dipeptidyl peptidase IV inhibitory, and morphine-like peptides. The peptides from casein hydrolysate were tracked entering the blood through the intestinal epithelial barrier in the form of complete fragments, and they might exert potential physiological activity in vivo.

Synthesis and antibacterial activity of substituted oxotriazolylphenyl oxazolidinones.

A series of 3-(4'-(2''-alkyl-3''-oxy-1'',2'',4''-triazolyl)-phenyl)-5-substituted oxazolidinones was designed and synthesized for in vitro antibacterial activity testing against fourteen Gram-negative and six Gram-positive standard organisms. The minimum inhibitory concentration (MIC) was determined by agar dilution at concentrations of 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25 microg/mL. Different alkyl groups at the 2''-position played an important role in the activity against Gram-positive organisms. (S)-3-(4'-(2''-ethyl-3''-oxy-1'',2'',4''-triazolyl)-phenyl)-5-acetamidomethyloxazolidinone was active against Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus enteridis and Streptococcus nonhemolyticus, whereas 2''-methyl, 2''-propyl and 2''-n-butyl counterparts did not show activity at 6.25 microg/mL. Modification of the 5-substitutent of oxazolidinones also affected the activity against Gram-positive organisms. (S)-3-(4'-(2''-ethyl-3''-oxy-1'',2'',4''-triazolyl)-phenyl)-5-acetamidomethyloxazolidinones was approximately two fold more potent than 5-chloroacetamido, 5-dichloroacetamido and 5-trifluoroacetamido counterparts against Streptococcus enteridis. None of these compounds showed growth inhibition against fourteen Gram-negative organisms at 6.25 microg/mL.

Synthesis and in vitro evaluation of substituted phenyl-piperazinyl-phenyl oxazolidinones against Gram-positive bacteria.

With the incidence of linezolid-resistant Enterococcus faecalis, E. faecium and Staphylococcus aureus, modification of linezolid at the 5- and/or 3-positions led to the development of a series of 3-(methoxyl-phenyl)-piperazinyl-phenyl oxazolidinone analogues. These compounds were tested in vitro against six gram-positive standard organisms (S. aureus, S. epidermidis, S. pneumoniae, S. albus, Streptococcus enteridis and S. nonhemolyticus). 5-acetylaminomethyl oxazolidinones bearing fluorine at 3'-position of phenyl ring showed activities against several gram-positive bacteria (MIC: 3.13-6.25 mug/mL). The position of methoxyl group on the phenyl ring of piperazine group affected antibacterial spectrum. 3-(4'- (para-methoxyl-phenyl)-piperazinyl)-(3'-fluoro)-phenyl-5-acetylaminomethyl oxazolidinone was found active against 5 gram-positive organisms except S. nonhemolyticus, whereas 3-(4'-(ortho-methoxyl-phenyl)-piperazinyl)-(3'-fluoro)-phenyl-5-acetylaminomethyl oxazolidinone was found active only against 2 gram-positive organisms, namely S. albus, S. enteridis.