Combination of Carvacrol and Thymol: Antimicrobial Activity Against Staphylococcus aureus and Antioxidant Activity.

Plant and essential oil extracts have been used for some time as antimicrobials and antioxidants, although little is known about the interactions between the main components of these plant materials. This knowledge could help to design more potent antimicrobial and antioxidant mixtures. Carvacrol and thymol, the main components of the essential oils of the Lamiaceae family of plants, were assessed in combination to evaluate their antioxidant activity and antimicrobial effect against 19 strains of Staphylococcus aureus (S. aureus) of different origins (clinical, meat, milk, and other) and mostly (12) enterotoxin producers. The microdilution test assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the two phenolics alone and in combination. Based on the fractional inhibitory concentration index (FICI), no antimicrobial interaction (0.5 < FICI <4) between carvacrol and thymol was observed against 42% of the S. aureus strains and an antagonistic interaction (FICI >4) was observed in the rest, which indicates different behavior among strains in relation to this antimicrobial combination. Particularly, an antagonistic effect was observed in 29% of the meat origin strains and 57% of the dairy origin strains. Combinations of carvacrol and thymol were bactericidal (differences in MIC and MBC values not more than twofold) for 60% of the tested strains. At low concentrations of both components, the antioxidant effect is additive. However, at high concentrations (2.50 or 2.66 mM) of at least one of the components of the combination, it is antagonistic. The different types of interactions of the components in the combination can depend on many factors (ratio, structural characteristics, and the establishment of intermolecular complexes). The results could be used as reference to apply this combination in foods to control S. aureus, to maintain the organoleptic properties and to extend the shelf-life of them.

Stress in Phycomyces blakesleeanus by glucose starvation and acetate growth: response of the antioxidant system and reserve carbohydrates.

The objective of this study was to analyze the response of Phycomyces blakesleeanus to glucose starvation and acetate growth stress. At the onset of the exponential growth phase, the fungus shows a high tolerance to both stresses, being higher for the glucose starvation. In both stresses we have found higher activities of catalase and glutathione peroxidase, and a decrease of the pools of D-erythroascorbate (D-erythroascorbate+D-erythroascorbate monoglucoside) and glutathione (GSH+GSSG), while the intracellular GSH/GSSG redox balance becomes more reducing. Gallic acid was not detected under both stresses. Glycogen breakdown and the high levels of trehalose seem to be part of the stress response. Both stress, under the conditions of this study, seem to lead to a qualitatively similar response in P. blakesleeanus, with regard to the behavior of antioxidant system, the content of secondary metabolites and the role of the reserve carbohydrates.

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: response to oxidative stress by hydrogen peroxide.

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H2O2 (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H2O2 could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H2O2 depending on the growth phase of P. blakesleeanus.

Antibacterial activity against foodborne Staphylococcus aureus and antioxidant capacity of various pure phenolic compounds.

Six pure phenolic compounds (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, gallic acid, and octyl gallate) were tested for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against several strains of Staphylococcus aureus isolated from dairy and meat products. In addition, S. aureus reference strains (American Type Culture Collection) for antimicrobial studies and/or isolated from human infections and outbreaks of food poisoning were included in the study. Of the compounds tested, octyl gallate and hydroquinone were the most effective against S. aureus (mean MIC values of 20.89 and 103.05 µg/mL, respectively) and carvacrol and thymol the least (mean MIC values of about 413 µg/mL). The mean MBC values were 40.84, 194.37, 417.46, and 581.90 µg/mL for octyl gallate, hydroquinone, carvacrol, and thymol, respectively. Meat isolates were more resistant than those of dairy origin to hydroquinone, gallic acid, and octyl gallate, as well as to penicillin G (used as a control of the methodology used); gallic acid and penicillin G showed the highest differences in MIC values between the groups of strains (about 10 and 200 times, respectively). On the other hand, when we tested the isolates included in each group of strains (dairy, meat, and other/mixed sources) we only detected significant differences (p < 0.05) among dairy and isolates from other/mixed sources for hydroquinone and thymol, respectively. However, strains of meat origin exhibited significant differences among each other (p < 0.05) to most of the phenolic compounds tested (hydroquinone, carvacrol, gallic acid, and octyl gallate). The relationship between MICs and MBCs for each of the phenolic compounds tested suggested a bactericidal mechanism of action against S. aureus. Gallic acid and octyl gallate exhibited the highest antioxidant capacity and thymol and carvacrol the lowest. So, octyl gallate is an agent with both antimicrobial and antioxidant properties, which would be of interest to use in the food industry.

Production, stability, and antioxidative and antimicrobial activities of two L-ascorbate analogues from phycomyces blakesleeanus: D-erythroascorbate and D-erythroascorbate glucoside.

D-erythroascorbate (D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate monoglucoside (D-EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and 1084 µg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds showed UV spectral properties and ionization constants similar to those of L-AA. D-EAAG was much more stable to aerobic oxidation than D-EAA and L-AA at acidic pH. D-EAAG is synthesized from D-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate donor with K(m) = 2.5 mM and 41.3 µM for D-EAA. This glucosyltransferase activity was maximal in the stationary growth phase in parallel with maximal production of D-EAAG. The presence of D-arabinose or D-arabinono-1,4-lactone in the culture medium produces the maximal accumulation of D-EAA and D-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds showed greater antioxidant activity than L-AA and other standard antioxidants, with a capacity similar to that of L-AA to inhibit the growth of Escherichia coli.

Screening of phenolic antioxidants for their inhibitory activity against foodborne Staphylococcus aureus strains.

Seventeen phenolic compounds that are allowed to be used in the European food industry as aromatizants or antioxidants or that are naturally present in plants were tested for their ability to inhibit 19 strains of Staphylococcus aureus by using a standardized paper disc assay. Most of the strains assayed were foodborne (dairy and meat products). Human isolates and/or strains recommended for testing antimicrobial agents were also included in the study, and some of the test strains were enterotoxin producers. When the content was 200 microg/disc, various phenolic compounds had shown antimicrobial activity against all (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, octyl gallate, and tannic acid) or most (gallic acid, propyl gallate, and ellagic acid) of the S. aureus strains tested. Significant differences in the inhibition zones (p < 0.05) among strains of the same, or similar, origin and among the different origins were observed for most of the phenolic compounds that showed antimicrobial activity for all or most of the strains tested.

Glycogen and trehalose mobilization by acetic acid in Phycomyces blakesleeanus: dependence on the anion form.

Trehalose and glycogen are reserve carbohydrates that were shown to accumulate in mycelia of the filamentous fungus Phycomyces blakesleeanus. Both carbohydrates were mobilized under glucose starvation or in the presence of acetate. Glycogen was mobilized faster than trehalose in the presence of acetate. In all cases, glycogen and trehalose mobilization followed single exponential decay. There was a direct relationship between glycogen mobilization and the concentration of the dissociated form of external acetic acid. The half-life of glycogen mobilization increased as the concentration of the external acetate anion decreased, so the dissociated form of acetate was the stressor causing glycogen mobilization. Mobilization was not due to transfer to poor carbon sources, as the dissociated form of other weak acids (butyrate, lactate, pyruvate and propionate) also produced glycogen mobilization. Previous exposure of the mycelia to a lower acetate concentration decreased glycogen mobilization by subsequent exposure to a high acetate concentration. Glycogen mobilization by acetate may be involved in production of ATP necessary for acetate uptake as well as for maintenance of the internal pH homeostasis.

Different stabilities of two AMP-forming acetyl-CoA synthetases from Phycomyces blakesleeanus expressed under different environmental conditions.

The stability of acetyl-CoA synthetases (ACS1 and ACS2) from P. blakesleeanus against temperature, urea and trypsin was studied and compared. Thermal inactivation of ACS1 was biphasic, while that of ACS2 was monophasic. The thermodynamic parameters calculated from the inactivation profiles show ACS2 to be a more thermostable enzyme than ACS1. The presence of ATP and Mg(2+) exerted a protective effect on both enzymes, and led to a marked increase in the E(a), DeltaH(not =), DeltaS(not =) and DeltaG(not =) values. ACS2 is also much more stable against denaturation with urea; the estimates of DeltaG(w) (free energy change for protein unfolding at zero denaturant concentration) were 9.4 kJ mol(-1) and 18.1 kJ mol(-1) for ACS1 and ACS2, respectively. Finally, a half-life of 44.5 min for ACS2 versus the 21 min for ACS1 indicates that ACS2 is more stable than ACS1 against digestion by trypsin. These results seem to show that ACS2 is more rigid overall than ACS1, which may be essential for preserving its catalytic activity in the stress situation in which it is expressed.

Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

An acetyl-CoA synthetase not encoded by the facA gene is expressed under carbon starvation in Phycomyces blakesleeanus.

Two forms of acetyl-CoA synthetase (ACS1 and ACS2) have been detected in Phycomyces blakesleeanus. ACS1, encoded by the gene facA, was induced by acetate and repressed by glucose at the transcriptional level. ACS2, not encoded by the gene facA, was detected as a response to carbon starvation both in the wild type and in an facA(-) mutant. Both enzymes were purified and characterized. They can use acetate and propionate as substrates. ACS2 is a much more stable enzyme than ACS1. After 60 min incubation at 55 degrees C, ACS2 retained 50% of its activity whereas ACS1 only retained 3%. The optimum temperature was 50 degrees C for ACS2 and 30 degrees C for ACS1.

Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus.

D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of l-ascorbate, suggesting that D-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed.

A transplasma membrane redox system in Phycomyces blakesleeanus: properties of a ferricyanide reductase activity regulated by iron level and vitamin K3.

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.

Application of a normalised plot to the study of uni-uni enzyme-inhibitor systems.

Normalisation of kinetic data is a useful tool in the study of complex enzyme systems. In the present paper, we have applied the premises of the normalised plot to the description of uni-uni enzyme inhibition. Guidelines to the design of the experiments and to data managing using the freeware program SIMFIT (http:\\www.simfit.man.ac.uk) are offered. The treatment has a lessened demand in experimental data while ensuring biological consistence of the results. Moreover, the results are obtained without resorting to secondary plots, and the election between rival mechanisms is statistically granted. Hyperbolic mixed-type inhibition is studied as a general model for enzyme-inhibitor/activator interaction, and equations describing classical cases of linear inhibition are also considered.

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines.

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.