Sithian Pandian

Peptidase activities in debittering and nondebittering strains of lactobacilli

Abstract Three Lactobacillus casei strains, which were isolated earlier from Cheddar cheese and which exhibited debittering and nondebittering effects during cheese ripening, were analysed for their peptide hydrolase systems. All strains exhibited an active aminopeptidase, a dipeptidase, a tripeptidase, a dipeptidyl aminopeptidase and a carboxypeptidase. The most significant difference between debittering and nondebittering strains was observed at the specific activity level of the aminopeptidase. The stability of the aminopeptidase in cheese was also investigated, and it was seen that this enzyme remained active for at least 2 months before its activity began to decrease.

Anti-DNA.RNA antibodies : an efficient tool for non-isotopic detection of Listeria species through a liquid-phase hybridization assay

This study was undertaken to evaluate the potential of a new approach using anti-DNA · RNA monoclonal antibodies to detect Listeria in both pure culture and inoculated meat and meat products. A sensitive liquid-phase assay was first developed, based on the formation in solution of a hybrid between a 784-bp DNA probe, specific for the genus Listeria, and target rRNA. Monoclonal antibody and antisera raised against hybrid nucleic acids were then used in various immunoenzymatic assays to detect specific hybrids formed in solution. System 2, using a double sandwich enzyme-linked immunosorbent assay, and system 1, using a biotinylated probe, proved to be very effective. The method using biotin-streptavidin complex, however, resulted in a higher background signal. System 2 described here, using unlabeled probe, was more effective. This strategy allowed the detection of as little as 2.5 pg target RNA from pure culture and 500 cells from inoculated meat homogenate, even in the presence of other contaminating bacteria. The assay was more sensitive and could be completed within 3 h, as opposed to several days when conventional culture methods were used.

Purification of X-prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies

Prolyl dipeptidylaminopeptidases from two subspecies of Lactobacillus casei were purified and biochemically characterized. L. casei ssp. casei UL21 (a debittering strain) and L. casei ssp. rhamnosus UL26 (a non-debittering strain) were the source bacteria for this study. Purification of the enzymes from both the sources was effected by a gel filtration step through Sephacryl S-300 followed by ion-exchange chromatography through DEAE Sephacel. This rendered an electrophoretically homogeneous enzyme preparation. The purified enzymes from both the sources showed similar temperature optimum (45 degrees C) and pH optimum (7.0). Their activity profiles on various substrates and the nature of inhibition by different inhibitors were also found to be similar, indicating that this enzyme is perhaps not significantly involved in the debittering process during the maturation of cheese.

Selected ion monitoring of tert-butyldimethylsilyl cholesterol ethers for determination of total cholesterol content in foods

Abstract A gas chromatography/mass spectrometry (GC/MS) method, using tert-butyldimethylsilyl (tBDMS) derivatives of sterols for detection in the selected ion monitoring (SIM) mode has been applied to the determination of total cholesterol in food (eggs, dairy products). Generally, comparison of results with literature data shows agreement with the amount of cholesterol determined by other chromatographic techniques, and slight underestimation if compared to the amount of cholesterol estimated by colorimetric techniques. The saponification and extraction procedure allowed for 98.6% recovery of spiked cholesterol in milk samples with a coefficient of variation of 2.1%. Amounts as low as 5 ng per 100 g food can be detected using external standards with 95% accuracy.

Effects of trichothecenes (T-2-toxin) on protein synthesis in vitro by brain polysomes and messenger RNA

The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis. Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.