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Adenosine triphosphate (ATP) assay and electrical measuring techniques were assessed for their ability to produce an estimate of total colony count. ATP assay could be used for estimating the microbial flora in foods, provided that non-microbial ATP was removed from the sample first. Linear relationships between log[10] microbial ATP content and log[10] colony count were obtained. Electrical measuring techniques provided an inverse linear relationship between electrical detection time and log[10] colony count. The capacitance element of the impedance signal was found to be superior to the conductance element for the estimation of pure cultures and the flora of two foods, since faster detection times and higher correlations with colony count were obtained. Capacitance detection times at elevated temperatures (35° and 25°C) provided a good estimate of psychrotrophic colony count at 15°C. The use of electrical measuring techniques was also investigated for the rapid detection of salmonellae. Most salmonellae tested could be detected electrically in pure culture by the production of a specific signal from the fermentation of dulcitol or by an inhibition of this signal in the presence of a Salmonella-specific bacteriophage. However, when these two tests were used for artificially contaminated foods, an unacceptably high number of false-negative results was obtained. Therefore, further tests were investigated for their potential in the electrical detection of salmonellae. These were: effect of antiserum, lysine decarboxylation and hydrogen sulphide production. The final section of this thesis was concerned with the use of conventional and rapid microbiological techniques in the analysis of bacterial growth/temperature relationships. A Square Root plot (√r = b(T-T[o])) was found to be preferable to the Arrhenius plot (k = Ae[-muu/RT]) in describing the growth of psychrotrophic food spoilage bacteria at chill temperatures. The Square Root plot was also applicable to mixtures of organisms and to ATP and capacitance detection time data. The relevance of such work to the investigation of food spoilage was discussed.

Microbial thermal inactivation survivor curves (log10 numbers plotted against time) have long been described as maintaining a strictly linear rate of decline. However, much evidence exists which suggests deviation from log-linear kinetics does occur, and that this is not purely the result of experimental procedure as contended by some authors. Here, the shape of inactivation kinetics in Salmonella enteritidis was investigated. A heat challenge method was developed which, as far as could be ascertained, was free from methodological artefacts influencing the shape of survivor curves. High initial cell densities allied to sensitive enumeration resulted in biphasic survivor curves at 60°C. Tailing survivors accounted for approximately 1 in 105 of the initial population and possessed roughly four times the heat resistance. At temperatures 50 to 65°C, the presence of tailing prevented the use of D-values to accurately describe death rates. However, describing survivor curves using a log-logistic model increased data-fit at all temperatures investigated. The biphasic nature of survivor curves was studied closely between 49 and 60°C. It was observed that the extent of tailing was temperature-dependent; as temperature decreased, linearity increased such that at 51°C, survivor curves had no tailing. Studies using S. typhimurium and S. senftenberg 775W revealed similar kinetics. In these salmonellas, survivor curves demonstrated linearity at 54 and 57°C, respectively. The influences of culture age and growth rate on the shape of 60°C-inactivation curves were also investigated. Batch-cultured S. enteritidis cells of various maturities gave rise to survivor curves of differing heat sensitivities. Exponentially growing cells were shown to be the most heat sensitive, while late-stationary phase cells were the only populations to result in non-tailed survivor curves. Carbon-limited continuously cultivated cells demonstrated similar biphasic inactivation kinetics. Predictably, the slowest dilution rate corresponded to the greatest heat resistance. Starved cells produced linear inactivation kinetics that were virtually identical to those of late-stationary phase batch-cultured cells. That tailing in batch cultures was similar to chemostat populations, indicated that possible differences in growth rates in batch-cultured cells could not account for tailing. Furthermore, growth was necessary for tailing to be observed. Investigations into the cause of tailing revealed that these cells were not genetically distinct from the majority population. Instead, it is believed that tailing cells arise following the expression of heat-shock proteins during heating. Partial inhibition of de novo protein synthesis during heating resulted in much reduced levels of tailing. It is proposed that the temperature of inactivation determines the proportion of cells capable of expressing a heat-shock response, such that the temperature at which linearity is achieved corresponds to the point at which all cells are fully heat-shock protected.

Pretreatment of stainless steel (AISI 304 & AISI 316) surfaces with skimmed UHT milk significantly reduced the numbers of attaching Staphylococcus aureus and other Gram positive bacteria, although the same effect was not observed with the Gram negative Pseudomonas fragi and Escherichia coli. Milk constituents with molecular weights below 1000 Daltons did not show the observed effect. Droplet contact angle experiments showed that the stainless steel surface became only marginally more hydrophobic when milk was adsorbed. The attachment of S. aureus onto stainless steel was found to be dependent on the physico-chemical properties of the bacterial surface, the stainless steel surface and of the suspending solution. SDS-polyacrylamide gel electrophoresis showed that proteins are adsorbed onto the stainless steel surface in approximately the same proportions as those in the bulk milk. Of the milk proteins, the caseins were more effective at inhibiting attachment. This was found to be true even when the proteins were used at identical concentrations, rather than in the proportions found naturally in milk, k-Casein was the most effective at inhibiting attachment.

The lactic acid fermentation of cheese whey permeate by Lactobacillus helveticus was studied. Precipitate formation during autoclaving of whey permeate was examined. Precipitation was found to be pH and temperature dependent. Qualitative analysis suggested that the precipitate was a calcium-phosphate complex. Solubilisation was achieved both by acidification and use of the sequestering agent EDTA. Optimisation of L. helveticus growth in whey permeate was carried out using factorial design, as opposed to a traditional univariate approach. Using this technique, the variation of specific growth rate with pH, temperature and stiirer speed was assessed. Cell growth and lactic acid formation in whey permeate containing various supplements, were investigated. Yeast extract was the most effective nitrogen/growth factor supplement. Maximum lactic acid production was achieved in permeate containing yeast extract (0.75% w/v), Tween 80 (0.1% v/v) and sodium acetate (0.05% w/v). Optimisation of lactic acid production in supplemented whey permeate was performed using factorial design. Optimum conditions for both acid formation and cell growth were pH 5.9, temperature 42°C and stirrer speed 200 rpm. Fourier transform infrared spectroscopy was applied to the on line and off line quantitative analysis of lactose and lactic acid during the fermentation process. This technique enabled substrate and product levels to be assessed quickly and simply, with no sample pre-treatment. Continuous culture of L. helveticus in MRS medium and supplemented whey permeate was carried out. Substrate conversion and lactic acid productivity decreased with increasing dilution rate. Maximum productivity corresponded to a dilution rate of 0.3 h" 1, whereas minimum residual substrate occured at a dilution rate of 0.1 h' 1 . Translation of the fermentation process from bench scale (11) to pilot scale (161) appeared to be successful. Completion times, productivity and lactose utilisation compared favourably with bench scale results.

Chiroptical, rheological and thermodynamic studies have been undertaken to investigate temperature-induced changes in the ý molecular organisation of gelatin. From the results obtained, a unified model for gelation and melting has been developed, and tested using Monte Carlo computer simulation. The temperature at which gelatin gels are formed has a major influence on the properties of the resulting network, with higher curing temperatures conferring increased thermal stability. In particular, gels formed by sequential curing at two different temperatures show biphasic melting. This is explained in terms of a temperature-dependence of helix length within the junction zones of the gel, and quantified by considering end-effects in the thermodynamics of helix stability. Measurements of 'initial slope' kinetics, performed over a broad concentration range, showed first-order kinetics at low gelatin concentrations, while at higher concentrations a second-order process was also evident. The results are interpreted as triple-helix nucleation at metastable 'hairpin turns' in one chain (bringing two chain segments into close proximity) together with a third strand from either the same chain (first order) or a different chain (second order). From simple geometric considerations, the maximum length of intermolecular helices ( which contribute to the gel network) is greater than that of twasted 9 intramolecular structures, giving a qualitative explanation of the increased strength of gels formed by precuring at higher temperatures (where only long helices are stable) over those quenched directly to low temperature. Monte Carlo simulation incorporating an initial assumption that helix propagation is rapid and proceeds to geometric limits gave unrealistic helix lengths and simulated melting profiles, and was replaced by the assumption that cis-trans isomerisation of peptide bonds is the controlling factor in helix propagation. Using the latter assumption, most aspects of the observed behaviour were successfully reproduced using program variables set within realistic ranges or, where possible, fixed at experimentally-determined values. In particularg the co-operativity of the simulated melting process was critically dependent on the value of a parameter x (the number of triplet units within each helix incapable of participating in bonding, due to end-effects), with a value of x=1 giving the best fits with experiment (consistent with accepted bonding patterns for the collagen triple helix). Other key parameters were the midpoint temperature for melting of the parent collagen, which gave best agreement when set at 37-38"C, and t6e proportion of cis peptide residues present in disordered gelatin chains, with an optimum lower limit of 0.15. Using these values, the simulation reproduced, with excellent precision, the helix fraction and melting profile of gels formed over a wide range of quench temperatures, and gave an acceptable approximation to the form of reaction progress curves obtained for helix formation. The biphasic melting of samples held at intermediate temperature before final quenching was also modelled realistically.

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Lactic acid fermented fruit and vegetables are normally obtained following a natural spontaneous fermentation in which no starter cultures are added. It could be expected that a suitable starter culture would help standardise production. Several lactic acid bacteria were selected for a series of physiological studies, in a defined medium (MRS broth) and in carrot juices, under varying conditions of growth temperature, salt concentration and carbohydrate source. Based on these, the homofermenter Lactobacillus pentosus and the heterofermenter Leuconostoc mesenteroides were tested as potential starters, in single and mixed cultures, for the fermentation of carrots (Daucus carota), as a novel fermentable substrate, and cabbage (Brassica oleracea) into sauerkraut. Fermentations were performed in the presence of the natural microflora. Sugar catabolism and acid production were monitored through H.P.L.C. In the fermentation of carrots Leuconostoc mesenteroides played a major role, with no homofermenters present. For sauerkraut, the mixed starter culture composed of Leuconostoc mesenteroides and Lactobacillus pentosus gave the closest resemblance to the product normally obtained following a natural commercial fermentation. The inclusion of the heterofermenter provided the required acid balance for correct product flavour and aroma by enhancing production of acetic acid. Acetate is also a better antimicrobial than lactate. A shorter fermentation time was also obtained, reducing the time from 3-4 weeks in the natural fermentation to only 7 days with the use of the mixed starter. When reduced salt concentrations were tried, 1% NaCl (w/w) resembled the spontaneous fermentation more closely, in regard to microbial sequence, pH and total acidity. Different ratios of the two lactic acid bacteria in combination were tried, the best being that in which L, mesenteroides and L. pentosus were initially present in the same proportions. Survival of Listeria monocytogenes in fermenting sauerkraut was shorter when starter cultures were used, but no difference was detectable between mixed and single cultures.