The mathematical properties of the quasi-chemical model for microorganism growth-death kinetics in foods.

Knowledge of the mathematical properties of the quasi-chemical model [Taub, Feeherry, Ross, Kustin, Doona, 2003. A quasi-chemical kinetics model for the growth and death of Staphylococcus aureus in intermediate moisture bread. J. Food Sci. 68 (8), 2530-2537], which is used to characterize and predict microbial growth-death kinetics in foods, is important for its applications in predictive microbiology. The model consists of a system of four ordinary differential equations (ODEs), which govern the temporal dependence of the bacterial life cycle (the lag, exponential growth, stationary, and death phases, respectively). The ODE system derives from a hypothetical four-step reaction scheme that postulates the activity of a critical intermediate as an antagonist to growth (perhaps through a quorum sensing biomechanism). The general behavior of the solutions to the ODEs is illustrated by several examples. In instances when explicit mathematical solutions to these ODEs are not obtainable, mathematical approximations are used to find solutions that are helpful in evaluating growth in the early stages and again near the end of the process. Useful solutions for the ODE system are also obtained in the case where the rate of antagonist formation is small. The examples and the approximate solutions provide guidance in the parameter estimation that must be done when fitting the model to data. The general behavior of the solutions is illustrated by examples, and the MATLAB programs with worked examples are included in the appendices for use by predictive microbiologists for data collected independently.

Assessment of microwave sterilization of foods using intrinsic chemical markers.

The uniformity of microwave processing was investigated by measuring the formation of intrinsic chemical markers in disc-shaped and cylindrically-shaped whey protein gel model systems. These markers are formed as a result of thermally induced reactions of sugar and protein precursors. They were measured in samples placed in a pressurizable Teflon vessel and microwave heated to different peak temperatures using different power levels. Heating uniformity was mapped by sectioning the sample and analyzing for markers. The destruction of B. stearothermophilus spores in alginate beads was correlated with marker formation. The results show that the markers can be used to assess sterility and spatial time-temperature distributions in solid food samples.

Binding site specificity of gamma-radiation-induced crosslinking between phenylalanine and a phenylalanine-containing tetrapeptide.

The binding site specificity of crosslinking mediated by the hydroxyl radical has been investigated in a simple model system: a tetrapeptide, Gly-Gly-Phe-Leu, and 14C-labeled phenylalanine. Crosslinking leads to the tetrapeptide-phenylalanine adduct which has been isolated by gel filtration. The amino acid analysis of these adducts compared with those of gamma-radiation-induced dimers of the tetrapeptide and of the dipeptide, Gly-Phe, shows that only the phenylalanine residue is affected and that the same new peaks appear in each case. Spectrophotometric measurement indicates that the extinction coefficient at 260 nm of dimeric tetrapeptide is four times higher than that of monomeric, as is dimeric phenylalanine compared to monomeric. These observations suggest a common crosslinking mechanism in all three cases that involves the aromatic ring of phenylalanine. The appearance of several radioactive peaks in the gel filtration separation of the acid hydrolysate of the adduct suggests that the crosslinking involves more than one possible modification of the phenylalanine. Three distinct tetrapeptide-Phe species, corresponding to molecular weights of 555, 573, and 591, were observed by fast atom bombardment mass spectrometry. The partial release of radioactive phenylalanine from the tetrapeptide-phenylalanine adducts by acid hydrolysis indicates the liability of some phenylalanine-phenylalanine bonds.

Binding-site specificity of the radiolytically induced crosslinking of phenylalanine to glucagon.

The gamma-radiation-induced crosslinking of phenylalanine to glucagon, mediated by OH ., has been shown to involve a limited number of binding sites on the glucagon molecule. Glucagon-phenylalanine adducts were partially separated from other radiolysis products with Sephadex gel filtration; further isolation of adducts was achieved with reverse-phase high-performance liquid chromatography (HPLC). Amino acid analysis of the isolated adducts indicates that the aromatic residues (phenylalanine and tyrosine), basic residues (histidine and lysine), and sulfur-containing residue (methionine) of glucagon are predominantly involved in crosslinking; these are essentially the same residues implicated in glucagon-glucagon crosslinking. Acid hydrolysates and chymotryptic digests of glucagon-phenylalanine adducts were examined with HPLC. The number of amino acid-phenylalanine adducts and also chymotryptic peptides observed was much greater than would have been expected based on the amino acid analysis. This observation is best accounted for by the involvement in crosslinking of radicals formed on the glucagon with more than one possible phenylalanine-derived free radical.

Gamma-radiation-induced interactions between amino acids and glucagon.

The interaction of glucagon and phenylalanine mediated by the OH . radical causes formation of higher molecular weight products of glucagon and phenylalanine, loss of amino acid residues in glucagon, and formation of adducts of glucagon and phenylalanine. The relative yields of these products depend upon the molar ratio of phenylalanine to glucagon in solution. At low ratios, glucagon aggregation and loss of amino acid residues predominate; at high ratios, the formation of phenylalanine dimers (and possible trimers and tetramers) predominates. The formation of adducts reaches a maximum at a phenylalanine:glucagon molar ratio of 3-4, and then decreases gradually, as the molar ratio increases, but is still discernible even at high molar ratios. Mechanisms for the formation of adducts are suggested. The influence of the primary aqueous radical intermediates, OH., H., and e-aq, on adduct formation has been evaluated for several different amino acids by irradiating in the presence of specific radical scavengers. For the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), OH. is considerably more effective than e-aq for mediating adduct formation, whereas for histidine and methionine, these primary radicals are equally effective.

Modification of platelet function by radical species produced during irradiation of oxygenated water.

Gel-filtered platelets in neutral, oxygen-containing solutions irradiated to a dose of 10 krad with gamma-rays display a significant inhibition of ADP-induced aggregation. Though the superoxide anion radical (O2-.) and H2O2 are generated in water irradiated under these conditions, only catalase, but not superoxide dismutase (SOD), conveyed protection against this inhibition of platelet function. When the platelets are in the presence of 10(-3) M sodium formate, which converts the majority of the radical species formed to O2-., inhibition of aggregation was again observed. Under these circumstances, the addition of catalase significantly decreased this inhibition, whereas the addition of SOD was without effect. In order to separate the platelet-inhibition effects of the radical species generated in the medium from a direct effect of irradiation on platelet components, relatively stable quantities of O2-. were produced in alkaline solutions and reacted with gel-filtered platelets. Again, an inhibition of ADP-induced aggregation was observed. This inhibition was not significantly altered by the addition of SOD, but was abolished by the addition of catalase, either with or without SOD. Our observation would indicate that O2-. is without direct effect on platelet function, but serves as the precursor of H2O2, which can inhibit platelet reactions.

Fast electron transfer processes in cytochrome C and related metalloproteins.

Various free radicals formed on pulse radiolysis of aqueous solutions have been used to investigate the mechanisms of reduction of cytochrome(III) c by inter- and intramolecular electron transfer. The rapid formation of free radicals (t less than 1 mus) and their high reactivity with cytochrome (k approximately 10(8)(-5) x 10(10)M(-1)s(-1)) make such studies feasible. Reduction of cytochrome by free radicls is monitored by optical methods. Fast optical changes in the 1(-500)-mus region correspond to reduction of the iron center; whereas the slower changes in the 10(-500)-ms region are attributed to postreduction conformational changes. It has been concluded that the reduction path is mediated through the crevice and that no reduction intermediates are being formed.