The heat sensitivity of various micro-organisms is illustrated by Table 4.3 which shows their D values. Generally psychrotrophs are less heat resistant than mesophiles, which are less heat resistant than thermophiles; and Gram-positives are more heat resistant than Gram-negatives. Most vegetative cells are killed almost instantaneously at 100 °C and their D values are measured and expressed at temperatures appropriate to pasteurization.
Bacterial spores are usually far more heat resistant than vegetative cells; thermophiles produce the most heat resistant spores while those of psychrotrophs and psychrophiles are most heat sensitive. Since spore inactivation is the principal concern in producing appertized foods, much higher temperatures are used in appertization processes and in the measurement of spore D values.
Yeast ascospores and the asexual spores of moulds are only slightly more heat resistant than the vegetative cells and will normally be killed by temperatures at or below 100 °C, e.g. in the baking of bread. Ascospores of the mould By ssochlamysfulva do show a more marked heat resistance and can be an occasional cause of problems in canned fruits which receive a relatively mild heat process.
The heat resistance exhibited by the bacterial endospore is due mainly to its ability to maintain a very low water content in the central DNA-containing protoplast; spores with a higher water content have a lower heat resistance. The relative dehydration of the protoplast is maintained by the spore cortex, a surrounding layer of electronegative peptidoglycan which is also responsible for the spore’s refractile nature.
The exact mechanism by which it does this is not known, although it may be some combination of physical compression of the protoplast by the cortex and osmotic extraction of the water. As the cortex is dissolved during germination and the protoplast rehydrates, so the spore’s heat resistance declines. Suspension of a germinated spore population in a strong solution of a non- permanent solute such as sucrose will reverse this process of rehydration and restore the spore’s heat resistance.
The total picture is probably more complex than this however, since other features of the spore such as its high content of divalent cations, particularly calcium, are thought to make some contribution to heat resistance.
Thermal sensitivity as measured by the D value can vary with factors other than the intrinsic heat sensitivity of the organism concerned. This is most pronounced with vegetative cells where the growth conditions and the stage of growth of the cells can have an important influence. For example, stationary phase cells are generally more heat resistant than log phase cells.
Heat sensitivity is also dependent on the composition of the heating menstruum; cells tend to show greater heat sensitivity as the pH is increased above 8 or decreased below 6. Fat enhances heat resistance as does decreasing aw through drying or the addition of solutes such as sucrose.
The practical implications of this can be seen in the more severe pasteurization conditions used for high sugar or high fat products such as ice cream mix and cream other than that used for milk. This effect is quite dramatic in the instance of milk chocolate where the D70 value of Salmonella senftenberg 775W has been measured as between 6 and 8 hours compared with only a few seconds in milk.
A more specialized example of medium effects on heat sensitivity occurs in brewing where the ethanol content of beer has been shown to have a profound effect on the heat sensitivity of a spoilage Lactobacillus’, an observation that has implications for the pasteurization of low-alcohol beers (Figure 4.3).
At present all thermal process calculations are based on the assumption that the death of micro-organisms follows the log-linear kinetics described by Equation 4.4. Though this is often the case, deviations from log-linear behaviour are also often observed (Figure 4.4). Sometimes these deviations can be rationalized on the basis of some special property of the organism.
For example, an apparent increase in viable numbers of organisms or a lag at the start of heating may be ascribed to heat activation of spores so that in the first moments of heating the number of spores being activated equals or exceeds the number being destroyed. Alternatively a lag phase may reflect the presence of clumps of cells, all of which require to be inactivated before that colony forming unit is destroyed.
The frequently observed tailing of the curves, which has greater practical significance, may be due to a sub- population of cells that are more heat resistant. These deviations from the accepted model tend to be observed more often when studying the thermal death of vegetative organisms and in some cases may reflect inadequacy of the logarithmic death concept in this situation.
The primary assumption which gives rise to log-linear kinetics is that at a constant temperature each cell has an equal chance of inactivation at any instant. This can be explained on theoretical grounds if there is a single target molecule in each cell whose inactivation causes death.
If this is not the case and there is, say, a large number of the same target molecules present or a large number of different targets, then several inactivation events would be required for cell death and a lag would be observed in the thermal death curve.
Damage to DNA has been identified as the probable key lethal event in both spores and vegetative cells. In spores, however, inactivation of germination mechanisms is also important. If this inactivation can be bypassed in some way, then apparently dead spores may be cultured. This has been demonstrated by the inclusion of lysozyme in recovery media where the enzyme hydrolyses the spore cortex, replacing the spore’s own inactivated germination system.
Deviations from log-linear kinetics in the thermal death of vegetative cells probably reflect a greater multiplicity of target sites for thermal inactivation such as the cytoplasmic membrane, key enzymes, RNA and the ribosomes. This type of damage can be cumulative rather than’ instantly lethal. Individual inactivation events may not kill the cell but will inflict sub-lethal injury making it more vulnerable to other stresses.
If however injured cells are allowed time in a non- inhibitory medium, they can repair and recover their full vigour. Examples of sub-lethal damage can be seen when cells do not grow aerobically but can be cultured anaerobically or in the presence of catalase, or when selective agents such as bile salts or antibiotics, which are normally tolerated by the organism, prove inhibitory.
Two other factors also contribute to deviations from log-linear behaviour in vegetative organisms. Individual cells within a population may exhibit a broader range of heat resistance than is seen with spores and, since vegetative cells are not metabolically inert, they may also respond and adapt to a heating regime modifying their sensitivity.