Food Protection with High Temperatures 429 was found by Stumbo et al.55 to have a Dr value of 2.47 in cream-style corn, whereas flat-sour (FS) spore strain 617 was found to have a Dr of 0.84 in whole milk The approximate heat resistance of spores of thermophilic and mesophilic spoilage organisms may be compared by use of Dr values Geobacillus stearothermophilus: Thermoanaerobacterium thermosaccharolyticum: Clostridium nigrificans: C botulinum (types A and B): C sporogenes (including PA 3679): B coagulans: 4.0–5.0 3.0–4.0 2.0–3.0 0.10–0.2 0.10–1.5 0.01–0.07 The effect of pH and suspending menstrum on D values of C botulinum spores is presented in Table 17–9 As noted above, microorganisms are more resistant at and around neutrality and show different degrees of heat resistance in different foods In order to determine the z value, D values are plotted on the log scale, and degrees Fahrenheit are plotted along the linear axis From the data presented in Figure 17–3, the z value is 17.5 Values of z for C botulinum range from 14.7 to 16.3, whereas for PA 3679, the range 6.6–20.5 has been reported Some spores have been reported to have z values as high as 22 Peroxidase has been reported to have a z value of 47; and 50 has been reported for riboflavin, and 56 for thiamine 12-D Concept The 12-D concept refers to the process lethality requirement long in effect in the canning industry and implies that the minimum heat process should reduce the probability of survival of the most resistant C botulinum spores to 10−12 Because C botulinum spores not germinate and produce toxin below pH 4.6, this concept is observed only for foods above this pH value An example from Stumbo54 illustrates this concept from the standpoint of canning technology If it is assumed that each container of food contains only one spore of C botulinum, F0 may be calculated by use of the general survivor curve equation with the other assumptions noted above in mind: F0 = Dr (log a − log b), F0 = 0.21(log − log 10−12 ), F0 = 0.21 × 12 = 2.52 Processing for 2.52 minutes at 250◦ F, then, should reduce the C botulinum spores to one spore in of billion (1012 ) containers When it is considered that some flat-sour spores have Dr values of about 4.0 and some canned foods receive F0 treatments of 6.0–8.0, the potential number of C botulinum spores is reduced to nondetectable levels SOME CHARACTERISTICS OF THERMOPHILES On the basis of growth temperatures, thermophiles may be characterized as organisms with a minimum of around 45◦ C, an optimum between 50◦ C and 60◦ C, and a maximum of 70◦ C or above By this definition, thermophilic species/strains are found among the cyanobacteria, archaebacteria, actinomycetes,58 the anaerobic photosynthetic bacteria, thiobacilli, algae, fungi, bacilli, clostridia, 430 Modern Food Microbiology Figure 17–4 Growth curves of a bacterial strain incubated at 20◦ C, 37◦ C, and 55◦ C Source: Reprinted with permission from Tanner and Wallace.57 lactic acid bacteria, and other groups Those of greatest importance in foods belong to the genera Bacillus, Clostridium, Alicyclobacillus, Geobacillus, and Thermoanaerobacterium In thermophilic growth, the lag phase is short and sometimes difficult to measure Spores germinate and grow rapidly The logarithmic phase of growth is of short duration Some thermophiles have been reported to have generation times as short as 10 minutes when growing at high temperatures The rate of death or “die off” is rapid Loss of viability or “autosterilization” below the thermophilic growth range is characteristic of organisms of this type The growth curves of a bacterium at 55◦ C, 37◦ C, and 20◦ C are compared in Figure 17–4 Why some organisms require temperatures of growth that are destructive to others is of concern not only from the standpoint of food preservation but also from that of the overall biology of thermophilism Some of the known features of thermophiles are summarized below Enzymes The enzymes of thermophiles can be divided into three groups: Some enzymes are stable at the temperature of production but require slightly higher temperatures for inactivation—for example, malic dehydrogenase, adenosinetriphosphatase (ATPase), inorganic pyrophosphatase, aldolase, and certain peptidases Some enzymes are inactivated at the temperature of production in the absence of specific substrates—for example, asparagine deamidase, catalase, pyruvic acid oxidase, isocitrate lyase, and certain membrane-bound enzymes Some enzymes and proteins are highly heat resistant—for example, α-amylase, some proteases, glyceraldehyde-3-phosphate dehydrogenase, certain amino-acid-activating enzymes, flagellar proteins, esterases, and thermolysin In general, the enzymes of thermophiles produced under thermophilic growth conditions are more heat resistant than those of mesophiles (Table 17–13) Of particular note is α-amylase produced by a strain of G stearothermophilus, which retained activity after being heated at 70◦ C for 24 hours In one 431 α-Amylase α-Amylase B subtilis G stearothermophilus 45 50 80 86 10 1.5 60 95 50 55 100 (%) (50◦ C, 30 minutes) (60◦ C, 15 minutes) (60◦ C, 10 minutes) (70◦ C, 10 minutes) (75◦ C, 10 minutes) (70◦ C, 30 minutes) (50◦ C, 20 minutes) (60◦ C, 10 minutes) (60◦ C, 120 minutes) (80◦ C, 60 minutes) (65◦ C, 20 minutes) (70◦ C, 24 hours) Heat Stability ∗ 50,000 15,500 42,700 0 32,000 90,000 28,000 44,700 48,400 39,500 Molecular Weight 0 0 4.6 Half-Cystine (mole/mole of Protein) Source: Matsubara,34 copyright c 1967 by The American Association for the Advancement of Science remaining after heat treatment shown in parentheses Streptococcal protease Collagenase Pronase Thermolysin Group A streptococci C histolyticum S griseus B thermoproteolyticus ∗ Activity Subtilisin BPN’ Neutral protease Alkaline protease Elastase Enzyme B subtilis B subtilis P aeruginosa P aeruginosa Species Yes Yes Yes Yes Yes Yes Yes Metal Required for Stability Table 17–13 Comparison of Thermostability and Other Properties of Enzymes from Mesophilic and Thermophilic Bacteria 432 Modern Food Microbiology study, the optimum temperature for the activity of amylase from G stearothermophilus was found to be 82◦ C with a pH optimum of 6.9.53 The enzyme required Ca2+ for thermostability The heat stability of cytoplasmic proteins isolated from four thermophiles was greater than that from four mesophiles.28 Several possibilities exist as to why the enzymes of thermophiles are thermostable Among these is the existence of higher levels of hydrophobic amino acids than exist in similar enzymes from mesophiles A more hydrophobic protein would be more heat resistant Regarding amino acids, it has been shown that lysine in place of glutamine decreased the thermostability of an enzyme, whereas replacements with other amino acids enhanced thermostability.30 Another factor has to with the binding of metal ions such as Mg2+ The structural integrity of the membrane of G stearothermophilus protoplasts was shown to be affected by divalent cations.63 Overall, the proteins of thermophiles are similar in molecular weight, amino acid composition, allosteric effectors, subunit composition, and primary sequences to their mesophilic counterparts Extremely thermophilic and obligately thermophilic organisms synthesize macromolecules that have sufficient intrinsic molecular stability to withstand thermal stress.1 Ribosomes In general, the thermal stability of ribosomes corresponds to the maximal growth temperature of a microorganism (Table 17–14) Heat-resistant ribosomes have been reported but not DNA In a study of the ribosomes of G stearothermophilus, no unusual chemical features of their proteins could be found that could explain their thermostability,2 and in another study, no significant differences in either the size or the arrangement of surface filaments of G stearothermophilus and Escherichia coli ribosomes could be found.4 The base composition of ribosomal RNA (rRNA) has been shown to affect thermal stability In a study of 19 organisms, the G–C content of rRNA molecules increased and the A–U content decreased with increasing maximal growth temperatures.43 The increased G–C content makes for a more stable structure through more extensive hydrogen bonding On the other hand, the thermal stability of soluble RNA from thermophiles and mesophiles appears to be similar Flagella The flagella of thermophiles are more heat stable than those of mesophiles, with the former remaining intact at temperatures as high as 70◦ C, whereas those of the latter disintegrate at 50◦ C.27,29 The thermophilic flagella are more resistant to urea and acetamide than those of mesophiles, suggesting that more effective hydrogen bonding occurs in thermophilic flagella OTHER CHARACTERISTICS OF THERMOPHILIC MICROORGANISMS Nutrient Requirements Thermophiles generally have a higher nutrient requirement than mesophiles when growing at thermophilic temperatures Although this aspect of thermophilism has not received much study, changes in nutrient requirements when incubation temperature is raised may be due to a general lack of efficiency on the part of the metabolic complex Certain enzyme systems might well be affected by the increased temperature of incubation, as well as the overall process of enzyme synthesis Food Protection with High Temperatures 433 Table 17–14 Maximal Growth Temperatures and Ribosome Melting Organism and Strain Number V marinus (15381) 7E-3 1–1 V marinus (15382) 2–1 D desulfuricans (cholinicus)∗ D vulgaris (8303)∗ E coli (B) E coli (Q13) 10 S itersonii (SI–1)† 11 B megaterium (Paris) 12 B subtilis (SB-19) 13 B coagulans (43P) 14 D nigrificans (8351)‡ 15 Thermophile 194 16 G stearothermophilus (T-107) 17 G stearothermophilus (1503R) 18 Thermophile (Tecce) 19 G stearothermophilus Maximum Growth Temperature (◦ C) Ribosome Tm(◦ C) 18 20 28 30 35 40 40 45 45 45 45 50 60 60 73 73 73 73 73 69 69 74 71 70 73 73 72 72 73 75 74 74 75 78 78 79 79 79 ∗ Desulfovibrio † Spirillum ‡ Desulfotomaculum Source: From Pace and Campbell.43 Oxygen Tension Thermophilic growth is affected by oxygen tension As the temperature of incubation is increased, the growth rate of microorganisms increases, thereby increasing the oxygen demand on the culture medium while reducing the solubility of oxygen This is thought by some investigators to be one of the most important limiting factors of thermophilic growth in culture media Downey15 has shown that thermophilic growth is optimal at or near the oxygen concentration normally available in the mesophilic range of temperatures—143 to 240 µM Although it is conceivable that thermophiles are capable of high-temperature growth due to their ability to consume and conserve oxygen at high temperatures, a capacity that mesophiles and psychrophiles lack, further data in support of this notion are wanting Cellular Lipids The state of cellular lipids affects thermophilic growth Because an increase in degree of unsaturation of cellular lipids is associated with psychrotrophic growth, it is reasonable to assume that a reverse effect occurs in the case of thermophilic growth This idea finds support in the investigations of many authors Gaughran19 found that mesophiles growing above their maximum range showed decreases in lipid ...430 Modern Food Microbiology Figure 17–4 Growth curves of a bacterial strain incubated at 20◦ C, 37◦ C, and 55◦ C Source: Reprinted with permission from Tanner and... Yes Yes Yes Metal Required for Stability Table 17–13 Comparison of Thermostability and Other Properties of Enzymes from Mesophilic and Thermophilic Bacteria 432 Modern Food Microbiology study, the... affected by oxygen tension As the temperature of incubation is increased, the growth rate of microorganisms increases, thereby increasing the oxygen demand on the culture medium while reducing