Salvia fruticosa Modulates mRNA Expressions and Activity Levels of Xenobiotic Metabolizing CYP1A2, CYP2E1, NQO1, GPx, and GST Enzymes in Human Colorectal Adenocarcinoma HT-29 Cells.

Natural products have gained considerable interests because of their use in some industrial areas including nutrition, cosmetic, pharmacy, and medicine. Salvia fruticosa M. (Lamiaceae) is known for its antioxidant, antimicrobial, and antiproliferative activities. Phase I xenobiotic metabolizing enzymes, CYP1A2 and CYP2E1, produce reactive metabolites which are eliminated by the action of phase II enzymes, NQO1, GPx, and glutathione S-transferases (GSTs). In this study, in vitro modulatory effects of S. fruticosa and its major phenolic compound rosmarinic acid (RA) on CYP1A2, CYP2E1, NQO1, GPx, and GSTm1 mRNA expressions and enzyme activities of GPx and GSTs were investigated in HT-29 cells. An mRNA expression analysis revealed that CYP1A2 and CYP2E1 levels were decreased while those of NQO1, GPx, and GSTm1 increased after S. fruticosa and RA treatments. In parallel to gene expressions, enzyme activities of GPx and GSTs by S. fruticosa increased 1.68- and 1.48-fold, respectively. Moreover, RA increased GPx and GSTs activities 1.67- and 1.94-fold, respectively. The results of this preliminary study show that metabolism of xenobiotics may be altered due to changes in the expression and activity of the investigated enzymes by S. fruticosa.

Glassworts as Possible Anticancer Agents Against Human Colorectal Adenocarcinoma Cells with Their Nutritive, Antioxidant and Phytochemical Profiles.

In this study, the possible uses of glassworts as potential food ingredients and their antiproliferative activity against colorectal adenocarcinoma cells together with their antioxidant and phytochemical profiles were investigated for the first time. MeOH extracts of five different taxa collected from different localities were screened for their antioxidant capacities by DPPH (IC50 2.91 - 5.49 mg/ml) and ABTS (24.4 - 38.5 µmol TE/g extract) assays. Salicornia freitagii exhibited the highest DPPH radical scavenging activity. LC/MS/MS analysis displayed that vanillic acid and p-coumaric acid were two main phenolic compounds in the extract. Salicornia freitagii extracts also exhibited high antiproliferative activity against HT-29 (IC50 1.67 mg/ml) and Caco-2 (IC50 3.03 mg/ml) cells for 72 h. Mineral analysis indicated that all the species with different proportions of elemental components contained high amount of cations. These results indicate that investigated glassworts, with their high phenolic and mineral contents and also notable antioxidant and cytotoxic properties, may be utilized as a promising source of therapeutics.

Purification and characterization of a bacteriocin from an oenological strain of Leuconostoc mesenteroides subsp. cremoris.

Malolactic fermentation (MLF), which improves organoleptic properties and biologic stability of some wines, may cause wine spoilage if uncontrolled. Bacteriocins were reported as efficient preservatives to control MLF through their bactericidal effect on malolactic bacteria. Leuconostoc mesenteroides subsp. cremoris W3 isolated from wine produces an inhibitory substance that is bactericidal against malolactic bacteria in model wine medium. Treatment of the culture supernatant of strain W3 with proteases eliminated the inhibitory activity, which proved that it is a true bacteriocin and we tentatively termed it mesentericin W3. The bacteriocin inhibited the growth of food-borne pathogenic bacteria such as Enterococcus faecalis, Listeria monocytogenes, and malolactic bacteria. It was active over a wide pH range and stable to organic solvents and heat. Mesentericin W3 was purified to homogeneity by a pH-mediated cell adsorption-desorption method, cation exchange, hydrophobic interaction, and reverse-phase chromatography. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy (MS) and partial amino acid sequence analysis revealed that mesentericin W3 was identical to mesentericin Y105.

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Biophysical and microbiological study of high hydrostatic pressure inactivation of Bovine Viral Diarrheavirus type 1 on serum.

The effect of high hydrostatic pressure application on fetal bovine serum components and the model microorganism (Bovine Viral Diarrheavirus type 1 NADL strain) was studied at 132 and 220 MPa pressure for 5 min at 25°C. Protein secondary structures were found to be unaffected by an artificial neural network application on the amide I region for both untreated and HHP treated samples. FTIR spectroscopy study of both the HHP-treated and control samples revealed changes in the intensity of some bands in the finger-print region (1500-900 cm(-1)) originating mainly from lipids which are thought to result from changes in the lipoprotein structure. The virus strain lost its infectivity completely after 220 MPa HHP treatments. These results indicate that HHP can be successfully used for inactivation of pestiviruses while leaving structural and functional properties of serum and serum products unaffected.

Differentiation of mesophilic and thermophilic bacteria with fourier transform infrared spectroscopy.

In the present study the characterization and differentiation of mesophilic and thermophilic bacteria were investigated by using Fourier transform infrared (FT-IR) spectroscopy. Our results showed significant differences between the FT-IR spectra of mesophilic and thermophilic bacteria. The protein-to-lipid ratio was significantly higher for thermophiles compared to mesophiles. The absorption intensity of the CH(3) asymmetric stretching vibration was higher in thermophilic bacteria, indicating a change in the composition of the acyl chains. The higher intensity/area observed in the CH(2) symmetric stretching mode at 2857 cm(-1), and the CH(2) bending vibration band at 1452 cm(-1), indicated a higher amount of saturated lipids in thermophilic bacteria. The lipid C=O stretching vibration at 1739 cm(-1), which was observed in the mesophilic group, was not observed clearly in the thermophilic group, indicating a difference in packing that is presumably due to the decreased proportion of unsaturated acyl chains in thermophilic bacteria. In addition, the carbonyl groups become hydrogen bonded and the cellular DNA content was lower in thermophilic bacteria. Moreover, in the 1000-400 cm(-1) frequency region, the spectra of each bacterial species belonging to both the mesophilic and thermophilic bacterial groups, showed characteristic differences that were discriminated via dendrogram using cluster analysis. The current study implies that FT-IR spectroscopy could be successfully applied for the rapid comparison of bacterial groups and species to establish either similarities or discrepancies, as well as to confirm biochemical or physiological characteristics.

Use of the Weibull model for lactococcal bacteriophage inactivation by high hydrostatic pressure.

Four lactococcal bacteriophages (phiLl6-2, phiLl35-6, phiLd66-36 and phiLd67-42) in M17 broth were pressurized at 300 and 350 MPa at room temperature and their survival curves were determined at various time intervals. Tailing (monotonic upward concavity) was observed in all survival curves. The resulting non-linear semi-logarithmic survival curves were described by the Weibull model and goodness of fit of this model was investigated. Regression coefficients (R2), root mean square error (RMSE), residual and correlation plots strongly suggested that Weibull model produced a better fit to the data than the traditional linear model. Hazard plots suggested that the Weibull model was fully appropriate for the data being analyzed. These results have confirmed that the Weibull model, which is mostly utilized to describe the inactivation of bacterial cells or spores by heat and pressure, could be successfully used in describing the lactococcal bacteriophage inactivation by high hydrostatic pressure.

Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage.

Bacteria are expected to be injured or killed by high hydrostatic pressure (HHP). This depends on pressure levels, species and strain of the microorganism and subsequent storage. Injured bacteria may be repaired which could affect the microbiological quality of foodstuffs with an important safety consideration especially in low acid food products. In this study two Gram-positive (Listeria monocytogenes CA and Staphylococcus aureus 485) and two Gram-negative (Escherichia coli O157:H7 933 and Salmonella enteritidis FDA) relatively pressure resistant strains of foodborne pathogens were pressurized at 350, 450 and 550 MPa in milk (pH 6.65) and stored at 4, 22 and 30 degrees C. The results of shelf life studies indicated two types of injury, I1 and I2, for all the pathogens studied. It is obvious that I2 type injury is a major injury and after its repair (I2 to I1), the cells can form colonies on non-selective but not on selective agar. The formation of colonies on both selective and non-selective agar occurs only after full recovery of injury (I1 to AC). The results presented in this study show that even if injured cells are not detected immediately after HHP treatment, I2 type injury could be potentially present in the food system. Therefore, it is imperative that shelf life studies must be conducted over a period of time for potential repair of I2 type injury either to detectable injury (I1) or to active cells (AC) to ascertain microbiological safety of low acid food products.

Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides.

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.

Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157:H7 by differential scanning calorimetry.

Differential scanning calorimetry (DSC) was used to evaluate the relative high hydrostatic pressure (HHP) resistances of bacterial strains from Staphylococcus aureus and Escherichia coli O157:H7 in vivo. The total apparent enthalpy change and thermal stability were two DSC parameters used to compare bacterial strains of untreated control and pressure-treated bacteria. DSC thermograms indicated that ribosomal denaturation appears to be a major factor in cell death by both thermal and high pressure treatments. However, the analysis of calorimetric data for control samples as well as pressure-treated samples clearly showed that the sensitivities of bacteria to various physical stresses can be different. While S. aureus 765 had a relatively higher resistance to thermal treatment in comparison to S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be utilized in the design of processes specific to targeting certain cellular components by using different physical stresses.

Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices.

The objective of this study was to compare high pressure resistance of Listeria monocytogenes strains at 25 degrees C and 50 degrees C at 350 MPa and to use high pressure (250 MPa and 350 MPa) at 30 degrees C and 40 degrees C for the inactivation of the relatively most pressure resistant strain inoculated in pasteurized apple, apricot, cherry and orange juices. L. monocytogenes CA was found to be the relatively most pressure resistant strain and increasing pressurization from 250 MPa to 350 MPa at 30 degrees C had an additional three to four log cycle reduction in viability, still leaving viable cells after 5 min. When 350 MPa at 40 degrees C for 5 min was applied more than eight log cycle reduction in cell population of all fruit juices was achieved. This study demonstrated that low temperature (40 degrees C) high pressure (350 MPa) treatment has the potential to inactivate relatively pressure resistant L. monocytogenes strains inoculated in different fruit juices within 5 min.

Construction of an acetylcholinesterase-choline oxidase biosensor for aldicarb determination.

In this study, acetylcholinesterase and choline oxidase were co-immobilized on poly(2-hydroxyethyl methacrylate) membranes and the change in oxygen consumption upon aldicarb introduction was measured. Immobilization of the enzymes was achieved either by entrapment or by surface attachment via a hybrid immobilization method including epichlorohydrin and Cibacron Blue F36A activation. Immobilized enzymes had a long-storage stability (only 15% activity decrease in 2 months in wet storage and no activity loss in dry storage). Aldicarb detection studies showed that a linear working range of 10-500 and 10-250 ppb aldicarb could be achieved by entrapped and surface immobilized enzymes, respectively. Enzymes immobilized on membrane surfaces responded to aldicarb presence more quickly than entrapped enzymes. Aldicarb concentrations as low as 23 and 12 ppb could be detected by entrapped and surface immobilized enzymes, respectively, in 25 min.