Comparison of Capillary and Test Tube Procedures for Analysis of Thermal Inactivation Kinetics of Mold Spores

Transcription

1 1404 Journal of Food Protection, Vol. 63, No. 10, 2000, Pages Copyright, International Association for Food Protection Comparison of Capillary and Test Tube Procedures for Analysis of Thermal Inactivation Kinetics of Mold Spores HIROSHI FUJIKAWA, 1 * SATOSHI MOROZUMI, 1 GLEN H. SMERAGE, 2 AND ARTHUR A. TEIXEIRA 2 1 Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakunin-cho, Shinjuku, Tokyo Japan; and 2 Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida, USA MS : Received 17 November 1999/Accepted 3 March 2000 ABSTRACT Characteristics of capillary and test tube procedures for thermal inactivation kinetic analysis of microbial cells were studied for mold spores. During heating, capillaries were submerged in a water bath and test tubes were held with their caps positioned above the level of the heating medium. Thermal inactivation curves of Aspergillus niger spores in capillaries at around 60 C consisted of a shoulder and a fast linear decline, whereas curves in test tubes consisted of a shoulder, a fast linear decline, and a horizontal tail. There were no significant differences in values of the rate and the delay of fast declines in curves between the procedures. Some experiments were done to clarify the cause for tailing with test tubes. There were no tails with test tubes whose inner walls were not contaminated by A. niger spores, suggesting that tails arise from A. niger spores contaminating the inner walls of test tubes. Temperature of the inner wall at the level of a heating medium was lower than that of the medium. Further, there were no tails for test tubes submerged in the heating medium. These results showed that the reason for survival of contaminants on the upper wall of test tubes was that cells were not subjected to sufficient inactivation temperature. Finally, thermal inactivation curves of A. niger spores in capillaries at various constant temperatures were studied. Curves consisted of a shoulder and a fast linear decline at 57 C and above, whereas curves at below 57 C consisted of a shoulder, a fast linear decline, and a sloping tail. Knowledge of kinetic data on thermal inactivation of food-contaminating microorganisms is considerably important for evaluating thermal processing conditions of foods from a food safety aspect. Experimental procedures to obtain those data for mesophiles, such as Salmonella, are often done in test tubes in water baths, but for thermoresistant cells, such as bacterial spores, are done in capillaries in oil baths (6, 17). Donnelly et al. (6) and Schuman et al. (17) described thermal inactivation curves of bacteria, such as Listeria monocytogenes in milk and Aeromonas hydrophila in liquid whole egg, that differed according to the heating procedures: inactivation curves of bacterial cells in capillaries consisted of fast linear declines only, while those in test tubes consisted of fast linear declines followed by tails. In those studies (6, 17), capillaries were submerged in a heating medium, but test tubes stood vertically with their tops above the surface of the heating medium. Neither group clarified the reason(s) for differences in inactivation curves from the two procedures. Compared with bacteria, thermal inactivation kinetics of mold spores have not been well studied, except thermoresistant spores (3, 7, 11, 16). Recently, Fujikawa and Itoh (8) experimentally obtained thermal inactivation curves of Aspergillus niger spores at constant temperatures in test tubes that had three segments: a shoulder, a fast linear decline, and a horizontal tail. However, potential differences in inactivation curves obtained via the above two * Author for correspondence. Tel: ; Fax: ; fujik@tokyo-eiken.go.jp. heating procedures have not been studied for mold spores. The purpose of this study was, therefore, to obtain and compare inactivation curves for mold spores via the two heating procedures and to identify and validate reasons for differences, especially tailing in the test tube procedure. Further, thermal inactivation kinetics of the spores in capillaries at various temperatures were analyzed. MATERIALS AND METHODS All experiments were done under sterile conditions. Preparation of mold spore suspension. Spores of A. niger ATCC (8) strain were cultured on potato dextrose agar (PDA; Eiken Chemicals, Tokyo, Japan) plates at 25 C for 7 days. After a portion (about 15 ml) of 0.1 M dibasic sodium phosphate adjusted with 0.05 M citric acid monohydrate to ph 7.0 with 0.005% Tween 80 was poured on the plate, spores were suspended in the buffer by rubbing with a glass rod. The suspension was filtered twice through a glass wool column to remove hyphae. After the spore concentration of the suspension was estimated with a hemocytometer, the suspension was diluted with buffer to make and spore/ml for capillaries and test tubes, respectively (8). Procedures for thermal inactivation: (i) capillary tube procedure. Portions of 91 l of each spore suspension were put into glass capillaries (1 mm in inner diameter) with a syringe, and the capillaries were flame sealed. The capillaries were submerged in a circulating water bath unit (DH-12; Taitec Corporation, Koshigaya, Japan) at constant temperatures. Subsequently, they were removed at various time intervals and cooled in ice water. Outer surfaces of the capillaries were sterilized by submergence in eth-

2 J. Food Prot., Vol. 63, No. 10 COMPARISON OF MOLD SPORE ANALYSIS PROCEDURES 1405 in the saline were estimated with PDA plates. After through vortexing, survivors in suspensions in the tubes were enumerated with PDA plates. Temperature measurement of inner wall surface. Temperatures at points A, B, and C on the inner wall surface of a test tube (Fig. 1) were measured separately with the thermometer during heating. Point B was at the level of the circulating water. Points A and C were 1.5 cm higher and 2 cm lower than point B, respectively. A probe of the thermometer was attached to each point on the inner wall surface by cellophane tape. The temperature at each point was measured until it became constant. FIGURE 1. Schematic diagram of a test tube in the water bath unit. Temperatures at points A, B, and C on the inner wall surface of a test tube were measured separately with the thermometer during heating. Point B was at the level of the circulating water. Points A and C were 1.5 cm higher and 2 cm lower than point B, respectively. A gray region in a tube is a region swabbed. anol for 10 min. After removal of residual ethanol with sterile water, each capillary was crushed into small pieces with a glass rod in a plastic tube containing 9 ml of saline (0.85% NaCl solution). This made the spore concentration of an unheated sample to be spore/ml. The resultant cell suspension was fully vortexed and sampled (9). Concentrations of survivors in the samples were enumerated by the dilution method with PDA plates (2). (ii) Test tube procedure. Pyrex test tubes, 10 mm in inner diameter and 100 mm long, with tight screw caps, were used in this study. Tubes were injected with a 2.5-ml portion of the cell suspension ( spores/ml) with a glass pipette. Racks of test tubes standing vertically were placed in the circulating water bath unit at specific constant temperatures. Cap portions of the tubes were above the surface of the circulating water. The surface of the cell suspension in each tube was 4 cm below than that of the circulating water. Tubes were subsequently removed from the bath after various time intervals and cooled in ice water (8). Concentrations of survivors in thoroughly vortexed samples were enumerated by the dilution method with PDA plates (2). Temperature measurement of spore suspension. Temperature histories of the spore suspensions in capillaries and test tubes during heating were recorded with a digital thermometer (AM- 7002; Anritsu Meter Co., Ltd., Tokyo). The probe of the thermometer was positioned at the geometrical center of each suspension. From temperature history curves, come-up times, t c, for suspensions in the capillaries and test tubes at specified constant temperatures were estimated. Inner wall contamination of test tube. Portions of 2.5 ml spore suspension were placed into test tubes without spore contamination of the inner walls of the tubes above the levels of suspensions. To accomplish this, the suspension was injected from the tip of a capillary positioned near the bottom of a tube with special care. The suspensions were heated in the water bath and cooled. Throughout the procedures, a test tube was treated very carefully in order not to wet its upper inner wall by swinging it or splashing the suspension. Control samples were made with the test tube procedure described above. A 2.5-cm-deep region of the inner wall below the level of the circulating water in Figure 1 was then swabbed with wet, sterile cotton to collect spores contaminating that region. The cotton was fully washed in 10 ml sterile saline, and viable spore cells Submergence of test tubes. Spore suspensions in test tubes standing in racks were heated while completely submerged in the water bath unit for various periods at a specified temperature and then cooled in ice water. Control samples whose cap portions were above the surface of the circulating water were made with the test tube procedure described above. The 2.5-cm-deep region of the inner wall below the level of the circulating water (Fig. 1) was swabbed with the procedure described above, and survivors of that region were estimated with PDA plates. After through vortexing, survivors in suspensions in the tubes were then enumerated with PDA plates. Analysis of data. The total heating interval, t, at a constant temperature consisted of come-up time, t c, and temperature-holding period, t h ; thus t t c t h. To compensate for the decrease in survivors during the come-up time, the experimental data were analyzed in terms of the survivor ratio during the heating period immediately after the come-up time, that is, N h /N c during t h (8). Here, N h and N c are the numbers of survivors heated for t h and t c, respectively. Thermal inactivation curves were described with the survivor ratio of N h /N c during t h. A fast linear decline in an inactivation curve was analyzed for its slope, k, and delay, t d. The value of k, i.e., rate constant of the linear decline, was obtained by a linear regression of the sloping segment. The duration of a shoulder, t d, was calculated at the intersection of the regression line with the horizontal axis in the graph. An inactivation curve with a sloping tail was analyzed with a thermotolerant subpopulation (TTSP) model, which is described below (8). N h /N c p exp( k 1 t h ) (1 p)exp[ k 2 (t h t d )] In this model we postulated that during heating a thermotolerant and thermosensitive subpopulation of a total population would be thermally inactivated following the first-order kinetics with rate constants k 1 and k 2. Here, k 1 k 2. Initial ratios of the thermotolerant and thermosensitive subpopulation would be p and 1 p, respectively. Here, 0 p 1. A shoulder of the inactivation curve was treated as a time delay, t d, of the thermosensitive population inactivation. A single sample was used for each experimental point. Experiments in this study were replicated three times and gave similar results. Thus, representative results were shown here. Values of k and t d were presented as average standard deviation of three individual experiments. Statistical analyses were done with Student s t-test at a significant level of 5%. RESULTS Thermal inactivation curves in capillaries and test tubes. Thermal inactivation curves of A. niger spores in capillaries at 60 C consisted of a shoulder and a linear decline. Those in test tubes consisted of a shoulder, a linear

3 1406 FUJIKAWA ET AL. J. Food Prot., Vol. 63, No. 10 FIGURE 2. Thermal inactivation curves of A. niger spores at 60 C in capillaries and test tubes. Symbols:, test tube;, capillary. Linear regression lines are generated from data of the fast decline segments in the inactivation curves. The graph shows the logarithm of survival ratios of spores heated during temperatureholding periods. Here, N h and N c are the numbers of survivors heated for the temperature-holding period, t h and come-up time, t c, respectively. decline, and an essentially horizontal tail (Fig. 2). The concentration of survivors in the capillary procedure finally reduced to zero. Curves from the two procedures differed only in the presence of a tail in the data for test tubes. There were no significant differences of values of rate constant, k, of the linear decline and duration of the shoulder, t d between the procedures. Values of k were min 1 for capillaries and min 1 for test tubes. Values of t d were min for capillaries and min for test tubes. Here t c was 2 s for capillaries and 1.8 min for test tubes. Cell contamination of the inner wall of a test tube. When A. niger spore suspensions were injected into test tubes from a glass pipette, or test tubes containing suspensions were jiggled during an experiment, or both, it was possible for the inner wall of the tube above the level of the suspension to become contaminated with a wet layer of A. niger spores from the suspension. Whether those contaminating spores could cause tails in inactivation curves was examined. Thermal inactivation curves of A. niger spores in test tubes with uncontaminated upper inner walls did not exhibit tails: the concentration of survivors finally reduced to zero. (Fig. 3). However, control test tubes by the test tube procedure exhibited horizontal tails (Fig. 3). There were no FIGURE 3. Thermal inactivation curve of A. niger spores in test tubes with uncontaminated inner walls. Spores were heated at 59 C. Symbols:, test tube with uncontaminated inner wall;, the control. Linear regression lines are generated from data of the fast decline segments in the inactivation curves. The graph shows the logarithm of survival ratios of spores heated during temperature-holding periods. significant differences in values of k and t d between uncontaminated tubes and contaminated control tubes. Values of k were min 1 for uncontaminated tubes and min 1 for control tubes. Values of t d were min for uncontaminated tubes and min for control tubes. Inner walls of uncontaminated tubes were negative for A. niger spores above the level of the spore suspension throughout heating (Table 1). In contrast, A. niger spores that contaminated upper inner walls of the control tubes survived the heating (Table 1). These results supported the proposition that tails in inactivation curves obtained via test tubes could arise from cells that contaminate and survive on the inner walls of test tubes above the level of the cell suspension. Inner wall temperature variation of test tube. Vertical temperature variation along the inner wall of a test tube in the water bath was measured. When the temperature of the heating medium was 59.0 C, temperatures of the inner wall at points A, B, and C in Figure 1 were 40.6, 53.2, and 58.9 C, respectively. Temperatures of points A and B at and above the level of the medium were considerably lower than that of the medium. The temperature at C, 2 cm below the level, was the same as that of the medium. These results showed the temperature of a swabbed region ranged from that of the heating medium (point C) to a considerably lower value (point B). TABLE 1. Survival of A. niger spore contaminants on the inner walls of test tubes during heating a Heating time (t h, min) Test tube Non-contaminated inner wall Control a After heating at 59 C for given periods, the regions of the inner walls of test tubes illustrated in Figure 1 were swabbed and washed in 10 ml of saline. Viable spore counts in the saline were then measured. Symbols:, 100 CFU/ml;, 50 to 100 CFU/ml;, 1 to 50 CFU/ml;, 0 CFU/ml.

4 J. Food Prot., Vol. 63, No. 10 COMPARISON OF MOLD SPORE ANALYSIS PROCEDURES 1407 FIGURE 4. Thermal inactivation curve of A. niger spores in submerged test tubes. Spores were heated at 59 C. Symbols:, submerged test tube;, the control. Linear regression lines are generated from data of the fast decline segments in the inactivation curves. The graph shows the logarithm of survival ratios of spores heated during temperature-holding periods. Submergence of test tube. In the test tube procedure, temperatures of inner walls of test tubes at and near the level of the circulating water did not reach the inactivation temperature set for the bath, and inner wall contaminants survived. On the other hand, the equilibrated temperature at any point in a submerged test tube in a bath should equal that of the bath. This leads to a hypothesis that when test tubes are completely submerged into a bath, inner wall contaminants of the test tubes would be killed by the inactivation temperature. Therefore, the effect of submergence of test tubes on tail generation was studied. Test samples were processed in the same manner as control tubes by the test tube procedure except for submergence in the bath. There were no tails in inactivation curves from submerged test tubes: the concentration of survivors finally reduced to zero (Fig. 4). There were no significant differences in values of k and t d between submerged tubes and control tubes by the test tube procedure. Values of k were min 1 for submerged tubes and min 1 for control tubes. Values of t d were min for submerged tubes and min for control tubes. During heating, spores that contaminated inner walls of submerged tubes above the levels of their spore suspensions were completely inactivated, while the contaminating spores for the controls survived (Table 2). These results and the inner wall temperature variations described above clarified that inner wall contaminants of A. niger spores in the test tube procedure survived because they were not exposed to inactivation temperature. Thermal inactivation curves in capillaries at various temperatures. Thermal inactivation curves of A. niger spores in capillaries at various constant temperatures of 55 to 66 C were studied (Fig. 5). The values of t c in this temperature range were all 2 s. At temperatures of 57 C and above, inactivation curves consisted of a shoulder and a linear decline (Figs. 2 and 5A). Interestingly, at temperatures below 57 C, tails were observed in the thermal inactivation curves (Fig. 5B and 5C). The tails were slopes: the number of tail spores was finally reduced to zero, essentially different from the horizontal tails by the test tube procedure (Fig. 2). The TTSP model well described the inactivation curves at the lower temperatures (Fig. 5B and 5C). DISCUSSION Essentially horizontal tails in thermal inactivation curves of mold spores were obtained in this study with the test tube procedure but not with the capillary procedure. The horizontal tail with the test tube procedure was observed for periods longer than 60 min at 58 C in our previous study (8). Donnelly et al. (6) and Schuman et al. (17) reported similar results for bacterial cells. Further, this study clarified the reason for tailing in the test tube procedure. Survivors in tails were located on the inner wall of the test tube above the level of the spore suspension (Fig. 3 and Table 1); they were not in the suspensions. Different thermal inactivation curves by the two procedures (Fig. 2) and tails generated by microbial contamination of the inner walls of test tubes (Fig. 3) were also observed for other microorganisms of mold spores (Penicillium frequentans), bacterial cells (Escherichia coli), and yeast cells (Saccharomyces cerevisiae) in our preliminary studies. These findings led to the conclusion that tailing is to be anticipated whenever employing the test tube procedure for microorganisms and that it is caused by contamination of inner test tube walls by a test organism. Therefore, now we have to say that our previous results on the horizontal tail with the test tube procedure (8) were artificial by such contamination by the test organism. Actually, it is quite usual to wet and contaminate the inner walls of test tubes by injecting cell suspensions and normal handing of tubes. On the other hand, as easily imagined, special and laborious care is required to avoid contamination of inner walls by the test organism throughout the experiment of Figure 3. TABLE 2. Survival of A. niger spore contaminants on the inner walls of submerged test tubes during heating a Heating time (t h, min) Procedure Submergence Control a After heating at 59 C for given periods, the regions of the inner walls of test tubes illustrated in Figure 1 were swabbed and washed in 10 ml of saline. Viable spore counts in saline were then measured. Symbols:, 100 CFU/ml;, 50 to 100 CFU/ml;, 1 to 50 CFU/ml;, 0 CFU/ml.

5 1408 FUJIKAWA ET AL. J. Food Prot., Vol. 63, No. 10 FIGURE 5. Thermal inactivation curves of A. niger spores in capillaries at constant temperatures. (A) 66 C, (B) 56 C, and (C) 55 C. Lines are those fitted with the TTSP model. The model is explained in the Materials and Methods section. The graph shows the logarithm of survival ratios of spores heated during temperature-holding periods. The temperature at any point on the inner wall of a submerged tube at equilibrium should equal that of the heating medium. On the other hand, lower temperature equilibrium was made on upper inner walls of unsubmerged test tubes. Consequently, it was believed that spores contaminating the inner wall in the submerged tubes were exposed to and inactivated by the inactivation temperature used (Table 2). Further, there were no tails in submerged test tubes. These results showed that the reason for survival of contaminants on the upper wall of test tubes was that cells were not subjected to sufficient inactivation temperature. It was usually observed in experiments with lengthy heating of spore suspension that colony growth, especially the sporulation, of the survivors in suspension incubated on PDA plates was inhibited. That may have been due to thermal stress against the cells. However, colonies of tail cells grew well, indicating further that those cells were insufficiently heated for inactivation. Donnelly et al. (6) presented some possible explanations for tailing: (i) refluxing of condensate and splashed cells could collect in the cap of a test tube above the level of the heating medium, or (ii) cells coating the wall of a test tube above the level of the heating medium could survive. For the former, results in this study (Fig. 3 and Table 1) indicated no refluxing of condensate. For the latter, it was found that cells coating the inner wall at and slightly below the level of the heating medium, that is, the swabbed region in Figure 1, survived the inactivation temperature (Table 1). A preliminary study indicated that cells contaminating above the level of the heating medium also survived. Some investigators have thought that tailing with the test tube procedure might arise from aggregation or chaining of microbial cells during heating (5, 18). However, microscopic observation of heated spores in this study demonstrated no aggregation or chaining of tail cells. Also, some may think that the tailing might arise from creeping up the inner wall of cells. This is also thought to be incorrect. Because, in this study, heated spore suspensions in test tubes were thoroughly vortexed just prior to sampling for survivor estimation, to make the suspensions homogenous. If some of the spores in a suspension might creep up the inner test tube wall, those cells would go back into the whole cell suspension by vortexing. Actually, when a suspension in a test tube was vortexed, the surface of the suspension climbed up the inner wall and even point B in Figure 1 was fully washed by the suspension. Moreover, the spores studied in this study were suspended in a buffer containing a surfactant (Tween 80) to avoid such creeping up and aggregation. Therefore, no possible reasons other than the inner wall contamination of a test tube by the test organism during an experiment could be considered so far. This study gave some useful information on capillary and test tube procedures for analyzing thermal inactivation curves of microorganisms. The capillary procedure was believed to be superior for obtaining thermal inactivation curves of microorganisms due to homogeneity of temperature and considerably short come-up time of temperature in microbial suspensions. However, the capillary procedure is laborious and time-consuming. It requires several special processes for injecting cell suspensions into a capillary, sealing it with flame, sterilizing the outer surface in alcohol, and crushing it into small pieces in sterile saline. The test tube procedure is easy and requires no special attention. Values of k and t d obtained with the test tube procedure essentially equal those obtained with the capillary procedure, as shown in Figure 2. However, come-up times are relatively long, and more importantly, invalid tails can occur in inactivation curves. Therefore, when just values of k and t d are sought, the test tube procedure should suffice. The submerged test tube procedure, which does not generate invalid horizontal tails, would be better. The effort

6 J. Food Prot., Vol. 63, No. 10 COMPARISON OF MOLD SPORE ANALYSIS PROCEDURES 1409 required to prepare test tubes without microbial contamination of upper inner walls (Fig. 3) is impractical. As described above, horizontal tails in the inactivation curves by the test tube procedure were artificial. However, in this study, we found other tails by the capillary procedure. For tails by the capillary procedure, slopes were dependent on the temperatures, and the number of tail cells was finally reduced to zero (Fig. 5). These findings showed that sloping tails by the capillary procedure were completely different from horizontal tails by the test tube procedure (8). In our previous study (8), the TTSP model was applied for the thermal inactivation curves with horizontal tails by the test tube procedure. In this study, however, we found that the TTSP model also successfully described the curves with sloping tails by the capillary procedure (Fig. 5). Several investigators reported that there would be two subpopulations in a single microbial population; one is thermosensitive and the other thermoresistant (5, 10, 14, 18). This is identical to the TTSP model. A two-component model also has been applied for the thermal inactivation kinetics of biochemical substances such as enzymes (4, 12, 15). Recently, Humpheson et al. (10) reported that there were sloping tails in thermal inactivation curves of Salmonella Enteritidis PT4 and that generation of heat shock proteins in bacterial cells during heating might be related to tailing, which is possibly by a thermoresistant subpopulation (1, 13). However, the reason for generation of sloping tails or the unhomogeneity of the thermal sensitivity of a microbial population has not been clarified. For mold spores, it should be further studied why the thermoresistant subpopulation appears at low temperatures by the capillary procedure (Fig. 5). REFERENCES 1. Allan, B., M. Linseman, L. A. MacDonald, J. S. Lam, and A. M. Kropinski Heat shock response of Pseudomonas aeruginosa. J. Bacteriol. 170: Anonymous Standard methods of analysis in food safety regulation. Japan Food Hygiene Association, Tokyo, Japan. 3. Bayne, H. G., and H. D. Michener Heat resistance of Byssochlamys ascospores. Appl. Environ. Microbiol. 37: Borhan, M., and H. E. Snyder Lipogenase destruction in whole soybeans by combinations of heating and soaking in ethanol. J. Food Sci. 44: Cerf, O A review: tailing of survival curves of bacterial spores. J. Appl. Bacteriol. 42: Donnelly, C. W., E. H. Briggs, and L. S. Donnelly Comparison of heat resistance of Listeria monocytogenes in milk as determined by two methods. J. Food Prot. 50: Engel, G., and M. Teuber Heat resistance of ascospores of Byssochlamys nivea in milk and cream. Int. J. Food Microbiol. 12: Fujikawa, H., and T. Itoh Tailing of thermal inactivation curve of Aspergillus niger spores. Appl. Environ. Microbiol. 62: Fujikawa, H., and T. Itoh Thermal inactivation analysis of mesophiles using Arrhenius and z-value models. J. Food Prot. 61: Humpheson, L., M. R. Adams, W. A. Anderson, and M. B. Cole Biphasic thermal inactivation kinetics in Salmonella enteritidis PT4. Appl. Environ. Microbiol. 64: King, A. D., H. G. Bayne, and G. Alderton Nonlogarithmic death rate calculations for Byssochlamys fulva and other microorganisms. Appl. Environ. Microbiol. 37: Ling, A. C., and D. B. Lund Determining kinetic parameters for thermal inactivation of heat-resistant and heat-labile isozymes from thermal destruction curves. J. Food Sci. 43: Neidhardt, F. C., and R. A. Van Bogelen Heat shock response, p In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 14. Palop, A., F. J. Sala, and S. Condon Occurrence of a highly heat-sensitive spore subpopulation of Bacillus coagulans STCC 4522 and its conversion to a more heat-stable form. Appl. Environ. Microbiol. 63: Park, K.-H., Y.-M. Kim, and C.-W. Lee Thermal inactivation kinetics of potato tuber lipoxygenase. J. Agric. Food Chem. 36: Rajashekhara, E., E. R. Suresh, and S. Ethiraj Influence of different heating media on thermal resistance of Neosartorya fischeri isolated from papaya fruit. J. Appl. Bacteriol. 81: Schuman, J. D., B. W. Sheldon, and P. M. Foegeding Thermal resistance of Aeromonas hydrophila in liquid whole egg. J. Food Prot. 60: Stumbo, C. R Themobacteriology in food processing. Academic Press, Orlando, Fla.