Thermal Inactivation of Clostridium perfringens After Growth at Several Constant and Linearly Rising Temperatures

Inactivation kinetics of Clostridium perfringens strains NCTC 8238 and NCTC 8798 vegetative cells were evaluated in autoclaved ground beef after growth at constant (37, 41, 45, or 49°C) or linearly rising temperatures (4.0, 6.0, or 7.5 C°/hr) representative of long-time, low-temperature (LTLT) cooking. Inactivation temperatures of 55, 57, 59, 60, and 61°C were used. D values and z values were determined. For strain NCTC 8798 cells grown at 45°C, the average D₅₉°C was 7.2 min and the z_D was 3.8 C°. Both strains exhibited greater heat resistance after growth at higher constant temperatures. Also, NCTC 8798 was more heat resistant than NCTC 8238. With linearly rising temperature, terminal growth temperatures appeared dominant in resistance to inactivation. These data will permit predictions of growth and survival of C. perfringens during LTLT cooking of beef roasts.     

Thermal inactivation of Salmonella spp. in ground chicken breast or thigh meat

Thermal inactivation of a four‐strain mixture of Salmonella spp. was determined in chicken breast and thigh meat. Inoculated meat was packaged in bags and then completely immersed in a circulating water bath and held at 55, 57.5, 60 and 62.5 °C for pre‐determined lengths of time. When the surviving bacteria were enumerated on tryptic soya agar supplemented with 0.6% yeast extract and 1% pyruvate (non‐selective medium), D‐values (time to inactivate 90% of bacteria) in chicken breast meat were 6.08, 4.77, 3.00 and 0.66 min at 55, 57.5, 60 and 62.5 °C, respectively; the values in thigh meat ranged from 11.48 min at 55 °C to 0.84 min at 62.5 °C. As expected, the measured heat resistance was lower when the recovery medium was selective (xylose lysine deoxycholate agar). Thermal death time values from this study will assist food processors in designing the HACCP plans to effectively eliminate Salmonella spp. in cooked chicken breast and thigh meat.     

Lethality of heat to Escherichia coli O157:H7: D- and z-value determinations in turkey,lamb and pork

Thermal inactivation of a four-strain mixture of E. coli O157:H7 was determined in lean ground turkey, lamb and pork. Inoculated meat was packaged in bags completely immersed in a circulating water bath and held at 55, 57.5, 60, 62.5, and 65°C for predetermined lengths of time. The surviving cell population was enumerated by spiral plating meat samples on tryptic soy agar overlaid with Sorbitol MacConkey agar. D-values, determined by linear regression, in turkey were 11.51, 3.59, 1.89, 0.81 and 0.29 min at 55, 57.5, 60, 62.5 and 65°C, respectively (z=6.5°C). When a survival model was fitted to the non-linear survival curves, D-values in turkey ranged from 11.26 min at 55°C to 0.23 min at 65°C (z=6°C). When the E. coli O157:H7 four-strain cocktail was heated in ground pork or lamb, D-values calculated by both approaches were similar at all temperatures. Thermal-death-times from this study will assist the retail food industry to design cooking regimes that ensure safety of ground muscle foods contaminated with E. coli O157:H7.     

Thermal Inactivation of Escherichia coli O157:H7 in Cooked Turkey Products

Survival of Escherichia coli O157:H7 when heated in commercial-type turkey products was determined. Thermal death times (TDT) were determined at 52–60°C in ground turkey with no additives, 3% fat; ground turkey with no additives, 11% fat; turkey ham batter, 11% fat; turkey frank batter, 17% fat; and turkey sausage batter, 31% fat. Mean D₅₂-values ranged from 44.9 to 116 min; D₅₅-values from 6.63 to 39.4 min; D₅₇-values from 2.20 to 11.7 min; D₆₀-values from 0.68 to 5.86 min. At all temperatures, survival of E. coli O157:H7 was greater in formulated products than in turkey meat with no additives. Greatest survival occurred in the turkey frank batter. Using our z-value data, times to provide a 5 D kill of E. coli O157:H7 in turkey franks cooked at 60°C, 65.6°C, or 71°C would be 26, 3.1, or 0.37 min, respectively.     

Influence of sodium pyrophosphate on thermal inactivation of Listeria monocytogenes in pork slurry and ground pork

The thermal inactivation (55–62·5°C) of Listeria monocytogenes in pork slurry and ground pork that contained 0, 0·25 or 0·5% sodium pyrophosphate (SPP) was evaluated. Surviving cells were enumerated on Modified Oxford Medium. Decimal reduction (D)-values in pork slurry control (0% SPP) were 8·15, 2·57, 0·99, and 0·18 min, at 55, 57·5, 60 and 62·5°C, respectively; D-values in ground pork ranged from 15·72 min at 55°C to 0·83 min at 62·5°C. D-values in pork slurry that contained 0·25% SPP (w/v) were 4·75, 1·72, and 0·4 min, at 55, 57·5, and 60°C respectively; the values in ground pork ranged from 16·97 at 55°C to 0·80 min at 62·5°C. At 62·5°C,L. monocytogenes in slurry that contained SPP were killed too rapidly to allow determination of the D-value. Addition of 0·5% SPP further decreased (P<0·05) the heat resistance of L. monocytogenes in pork slurry but not in ground pork. The z-values in slurry ranged from 4·63 to 5·47°C whereas higher z-values (5·25 to 5·77°C) were obtained in ground pork. Degradation of SPP to orthophosphates in ground pork was two or three times greater than in pork slurry. Possible reasons for failure of SPP to reduce the thermal resistance of L. monocytogenes in ground pork are discussed.     

Heat resistance of Escherichia coli O157:H7 in cook‐in‐bag ground beef as affected by pH and acidulant†

Summary The heat resistance of a four‐strain mixture of Escherichia coli O157:H7 was tested. The temperature range was 55–62.5 °C and the substrate was beef at pH 4.5 or 5.5, adjusted with either acetic or lactic acid. Inoculated meat, packaged in bags, was completely immersed in a circulating water bath and cooked to an internal temperature of 55, 58, 60, or 62.5 °C in 1 h, and then held for pre‐determined lengths of time. The surviving cell population was enumerated by spiral plating meat samples on tryptic soy agar overlaid with Sorbitol MacConkey agar. Regardless of the acidulant used to modify the pH, the D ‐values at all temperatures were significantly lower (P < 0.05) in ground beef at pH 4.5 as compared with the beef at pH 5.5. At the same pH levels, acetic acid rendered E. coli O157:H7 more sensitive to the lethal effect of heat. The analysis of covariance showed evidence of a significant acidulant and pH interaction on the slopes of the survivor curves at 55 °C. Based on the thermal‐death–time values, contaminated ground beef (pH 5.5/lactic acid) should be heated to an internal temperature of 55 °C for at least 116.3 min and beef (pH 4.5/acetic acid) for 64.8 min to achieve a 4‐log reduction of the pathogen. The heating time at 62.5 °C, to achieve the same level of reduction, was 4.4 and 2.6 min, respectively. Thermal‐death–time values from this study will assist the retail food processors in designing acceptance limits on critical control points that ensure safety of beef originally contaminated with E. coli O157:H7.     

Heat resistance in Escherichia coli and its implications on ground beef cooking recommendations in Canada

This study assessed the adequacy of the current cooking recommendations in relation to heat resistant Escherichia coli by evaluating eight potentially heat resistant E. coli strains (four generic and four E. coli O157:H7) along with AW1.7. The D_60°C-values for these strains varied from 1.3 to 9.0 min, with J3 and AW1.7 being the least and most heat resistant strains, respectively. The D_60°C-values for E. coli 62 and 68 were similar and were not affected by growth medium, while the heat resistance of C37, J3, and AW1.7 varied with the growth medium. When heated in extra lean ground beef (100 g) in vacuum pouches, the mean D_54°C, D_57°C, and D_60°C-values were 44.8, 18.6, and 2.9 min for C37, 13.8, 6.9, and 0.9 min for J3, and 40.5, 9.1, and 6.1 min for AW1.7. Burger temperatures continued to rise after being removed from heat when the target temperature was reached, by 3–5°C, and resting of 1 min would result in a destruction of 133, 374 and 14 log C37, J3 and AW1.7. These findings along with the very low occurrence of heat resistant E. coli expected in ground beef show that cooking ground beef to 71°C should be adequate.     

Differences in thermotolerance of variousEscherichia coliO157:H7 strains in a salami matrix

Three strains ofEscherichia coliO157:H7 (ATCC 43895, Ent C9490 and 380–94) were inoculated into salami and heated in water baths at 50, 55 or 60°C. At intervals between 1 and 360 min, salami samples were removed from the water bath and examined for the presence of survivingE. coliO157:H7. Samples were directly plated onto sorbitol MacConkey (SMAC) agar, and onto tryptone soya agar (TSA) with SMAC overlay. The number of sub-lethally damaged cells in each sample was estimated from the differences between the resultant direct (uninjured cells only) and overlay (total recovery) counts. In samples heated at 50°C, the percentage of cell injury ranged from 71·8–88% for all strains. In samples heated at 55°C the percentage of sub-lethally damaged cells in strains ATCC 43895 and Ent C9490 was significantly higher (P< 0·001) at 97% than that observed in strain 380–94 (64%). Cell injury was not measured at 60°C. There were significant differences between the derived decimal reduction times (D-values) related to the different strains ofE. coliO157:H7, the heat treatment applied and the recovery/enumeration agars used. Significant interstrain differences (P< 0·05) in thermotolerance were noted. Strain Ent C9490 was significantly more heat resistant at 50°C and 60°C (D-values of 116·9 and 2·2 min, respectively), while at 55°C strain 380–94 was more thermotolerant (D-value of 21·9 min). The implications of these findings for the design of studies investigating the heat resistance ofE. coliO157:H7 in fermented meat environments are discussed.     

Modelling the effect of pH,sodium chloride and sodium pyrophosphate on the thermal resistance of Escherichia coli O157:H7 in ground beef

The objective of this study was to assess the combined effects of temperature, pH, sodium chloride (NaCl), and sodium pyrophosphate (SPP) on the heat resistance of Escherichia coli O157:H7 in minced beef meat. A fractional factorial design consisted of four internal temperatures (55.0, 57.5, 60.0 and 62.5 °C), five concentrations of NaCl (0.0, 1.5, 3.0, 4.5 and 6.0 wt/wt.%) and SPP (0.0, 0.1, 0.15, 0.2 and 0.3 wt/wt.%), and five levels of pH (4.0, 5.0, 6.0, 7.0 and 8.0). The 38 variable combinations were replicated twice to provide a total of 76 survivor curves, which were modelled by a modified three-parameter Weibull function as primary model. The polynomial secondary models, developed to estimate the time to achieve a 3-log and a 5-log reduction, enabled the estimation of critical pH, NaCl and SPP concentrations, which are values at which the thermo-tolerance of E. coli O157:H7 reaches it maximum. The addition up to a certain critical concentration of NaCl (~ 2.7–4.7%) or SPP (~ 0.16%) acts independently to increase the heat resistance of E. coli O157:H7. Beyond such critical concentrations, the thermo-resistance of E. coli O157:H7 will progressively diminish. A similar pattern was found for pH with a critical value between 6.0 and 6.7, depending upon temperature and NaCl concentration. A mixed-effects omnibus regression model further revealed that the acidity of the matrix and NaCl concentration had a greater impact on the inactivation kinetics of E. coli O157:H7 in minced beef than SPP, and both are responsible for the concavity/convexity of the curves. When pH, SPP or NaCl concentration is far above or below from its critical value, the temperatures needed to reduce E. coli O157:H7 up to a certain log level are much lower than those required when any other environmental condition is at its critical value. Meat processors can use the model to design lethality treatments in order to achieve specific log reductions of E. coli O157:H7 in ready-to-eat beef products.     

Heat-Resistance of Escherichia Coli O157:H7 in Meat and Poultry as Affected by Product Composition

The effects of fat level and low fat formulation on survival of Escherichia coli O157:H7 isolate 204P heated in ground beef [7%, 10% and 20% fat], pork sausage [7%, 10%, and 30% fat], chicken (3% and 11% fat), and turkey (3% and 11% fat) were determined by D- and z-values. D-values for E. coli 0157:H7 in lowest fat products were lower than in traditional beef and pork products (P < 0.05). Overall, higher fat levels in all products resulted in higher D-values. D₆₀ values (min) ranged from 0.45–0.47 in beef, 0.37–0.55 in pork sausage, 0.38–0.55 in chicken and 0.55–0.58 in turkey. D₅₅ and D₅₀ values were respectively longer. Z-values ranged from 4.4–4.8°C. Product composition affected lethality of heat to E. coli O157:H7.     

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.     

Effect of Gamma Radiation on Campylobacter jejuni

Radiation resistance of Campylobacter jejuni in broth, ground beef, and ground turkey meat was determined using dose levels from 0 - 200 Krad at −30 ± 10°C, at 0 - 5°C, and at 30 ± 10°C. Irradiation at −30°C increased radiation resistance of cultures in ground meats; broth cultures were not greatly influenced by temperature. The effect of culture age on radiation resistance was also evaluated using cells in various physiological phases. Age did not have a pronounced effect on radiation resistance. The largest D₁₀ value for C. jejuni was 32 Krad, which was less than D₁₀ values commonly reported for salmonellae.     

High pressure thermal processing for the inactivation of Clostridium perfringens spores in beef slurry

The spores of Clostridium perfringens can survive and grow in cooked/pasteurized meat, especially during the cooling of large portions. In this study, 600 MPa high pressure thermal processing (HPTP) at 75 °C for the inactivation of C. perfringens spores was compared with 75 °C thermal processing alone. The HPTP enhanced the inactivation of C. perfringens spores in beef slurry, resulting in 2.2 log reductions for HPTP vs. no reductions for thermal processing after 20 min. Then, the HPTP resistance of two C. perfringens spore strains in beef slurry at 600 MPa was compared and modeled, and the effect of temperature investigated. The NZRM 898 and NZRM 2621 exhibited similar resistance, and Weibull modeled well the log spore survivor curves. The spore inactivation increased when HPTP temperature was raised from 38 to 75 °C. The results confirm the advantage of high pressure technology to increase the thermal inactivation of C. perfringens spores in beef slurry.Industrial relevanceC. perfringens spores may cause food/meat poisoning as a result of improperly handled and prepared foods in industrial kitchens. Thermal processes at 100 °C or higher are generally carried out to ensure the elimination of these pathogenic spores. High pressure processing (HPP) is a food pasteurization technique which would help to maintain the sensorial and nutritional properties of food. Preservation of foods with HPP in conjunction with mild heat (HPTP) would enhance the spore inactivation compared to thermal processing alone at the same temperature, due to a known germination–inactivation mechanism. This technology, together with the application of Good Manufacturing Practices, including rapid cooling, is a good alternative to the traditional methods for producing safe processed meat and poultry products with enhanced sensory and nutritional quality.     

Resistance ofListeria monocytogenesto ultrasonic waves under pressure at sublethal (manosonication) and lethal (manothermosonication) temperatures

The influence of amplitude of ultrasonic waves, static pressure and temperature on the inactivation rate ofListeria monocytogenesby ultrasonic waves under pressure (manosonication; MS) has been investigated. Also the influence of growth temperature, pH and composition of treatment medium, and the presence of sodium chloride in the recovery medium on the MS resistance ofL. monocytogeneshas been investigated and compared with its heat resistance. The inactivation ofL. monocytogenesby high power ultrasonic waves (20 kHz, 117 μm) at ambient temperature and pressure was low (D_UW=4.3 min). The increase of relative pressure (MS) to 200 kPa decreasedD_MSto 1.5 min and the increase to 400 kPa to 1.0 min. Inactivation rate by MS increased exponentially with the amplitude of ultrasonic waves in the range of 62-150 μm. An amplitude increase of 100 μm decreased the MS resistance of approximately six times. The inactivation rate by MS was not influenced by treatment temperature up to 50°C. However at higher temperatures the lethality of this combined process (MTS) increased considerably. Inactivation by MTS was the result of the inactivation by heat in addition to that by ultrasound under pressure. Cells grown at 37°C were twice more heat resistant than those grown at 4°C but no differences were found between decimal reduction time values to MS (117 μm, 200 kPa, 40°C) of both suspensions. While the inactivation rate by heat in pH 4 buffer was two- and five-fold higher than in pH 7 buffer and skimmed milk respectively, the differences amongD_MS-value in these media were lower than 60%. The addition of 57% of sucrose to the treatment medium increasedD₆₁from 0.22 to 5.7 min andD_MSfrom 1.5 to 3.1 min. The addition of 3% sodium chloride to the recovery medium decreasedD₆₀by 1/3 but did not modifyD_MS-values.     

Predicting process lethality of Escherichia coli O157:H7, Salmonella,and Listeria monocytogenes in ground,formulated, and formed beef/turkey links cooked in an air impingement oven

The pathogen thermal lethality in ground and formulated beef/turkey was evaluated for a cocktail of E. coli O157:H7, Salmonella, and Listeria monocytogenes, respectively. At a temperature range of 55–70°C, the heat resistance of L. monocytogenes was not significantly (at α=0.05) different from those of Salmonella. The heat resistance of L. monocytogenes at 55–70°C was 45–81% higher than that of E. coli O157:H7. In this study, a practical approach was developed to predict log₁₀(CFU/g) reduction of E. coli O157:H7, Salmonella, or L. monocytogenes in ground, formulated, and formed beef/turkey links that were cooked in an air impingement oven. The predictions of pathogen thermal kills in the links were verified via the inoculation studies for at least a 7 log₁₀(CFU/g) reduction of E. coli O157:H7, Salmonella, and L. monocytogenes.     

Thermosonication versus thermal processing of skim milk and beef slurry: Modeling the inactivation kinetics of psychrotrophic Bacillus cereus spores

Non-thermal processed foods are generally cold stored and distributed. The use of ultrasound for food preservation has attracted the interest of many research groups. In the current study, the thermosonication (TS, simultaneous ultrasound and thermal process) inactivation of psychrotrophic Bacillus cereus spores was investigated (24 kHz, 210 μm, 0.33 W/mL or W/g). First, the effectiveness of a 1.5 min TS process at 70 °C in skim milk, beef slurry, cheese slurry, and rice porridge was investigated. The TS was more effective than sole thermal treatment in reducing B. cereus spores in rice porridge, beef slurry and cheese slurry by 7, 6, and 4 fold, respectively. Then, the first-order D- and z-values for TS and thermal processing in skim milk and beef slurry, and the best model to fit TS inactivation of B. cereus spores in beef slurry were determined. The D_70 °C-values in skim milk were 2.9 min for TS and 8.6 min for the thermal treatment. And in beef slurry, values of 0.4 min for TS and 2.3 min for thermal were estimated. It was found that the Log-logistic model better described the TS spore inactivation in beef slurry. The ultrasound technology required 20–30 °C lower temperatures for the same spore inactivation, which resulted in better food quality and energy saving gains.     

Determination of the effect of sodium lactate on the survival and heat resistance of Escherichia coli O157:H7 in two commercial beef patty formulations

The effect of 4% sodium lactate (NaL) in beefburger patty formulations on the survival and heat resistance of Escherichia coli O157:H7 was investigated. Fresh beef trimmings were inoculated with E. coli O157:H7 to a concentration of 6·0–7·0 log₁₀ cfu g⁻¹ and subjected to the processing stages of beefburger patty production. Two commercial beefburger patty formulations were produced: a ‘quality’ patty (100% beef) and an ‘economy’ patty (70% beef, 30% other ingredients, including onion, water, salt, seasoning, rusk and soya concentrate). Sodium lactate (4% w/v) was added to the beefburger patties during mincing and the formed patties were frozen and stored for 1 month. Beefburger patties without added NaL were used as controls. After frozen storage for 1 month, patties were examined for E. coli O157:H7 counts. There was a synergistic effect between freezing and NaL, which resulted in a small but significant reduction (P<0·05) (approximately 0·5 log₁₀ cfu g⁻¹) in E. coli O157:H7 numbers. The frozen beefburger patties were also heat-treated at 50, 55 and 60°C and the data analysed to derive D -values for E. coli O157:H7 cells. At each temperature treatment, theD -values of the quality and economy beefburger patties with 4% NaL were significantly lower (P<0·001) than the D -values of the patty formulations without NaL. The study demonstrates that the presence of 4% NaL in beefburger patty formulations can reduce the overall risks posed to consumers by the presence ofE. coli O157:H7 by, first; reducing pathogen survival during freezing and frozen storage of the uncooked product; and, second, by increasing the susceptibility of the pathogen to heat during normal cooking processes.     

Relationship between membrane fatty acid composition and heat resistance of acid and cold stressed Salmonella senftenberg CECT 4384

This study evaluates the adaptative response to heat (63 °C) and the modifications in membrane fatty acid composition of Salmonella senftenberg after its growth in an acidified medium and after its exposure to combinations of acid and cold stresses. Cells were grown in Brain Heart Infusion (BHI) buffered at pH 7.0 and acidified up to pH 4.5 (fresh cultures) and kept at refrigeration temperature (4 °C) for 7 days (refrigerated cultures). The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, but combinations of sublethal stresses reduced S. senftenberg heat tolerance, specially when the growth medium pH was decreased. Acid-adapted cells showed D₆₃-values ranging from 3.10 to 6.27 min, while non-acid-adapted cells showed D₆₃-values of 1.07 min. As pH decreased, over the pH range studied (7.4–4.5), D₆₃-values of the resulting cells increased. However, refrigerated acid-adapted cells showed lower D₆₃-values, which ranged from 0.95 to 0.49 min. A linear relationship between the thermotolerance of S. senftenberg cells and the previous growth medium pH was found in both fresh and refrigerated cultures, which allowed us to predict changes in heat resistance of S. senftenberg that occur at any pH value within the range used in the present study in which most foodstuffs are included.     

Resistance of Yeast to Dry Heat

The dry heat resistance of 10 strains of yeast was investigated to develop data useful for the evaluation of aseptic systems for packaging acid products and which sterilize containers with hot air. Although three of the strains tested showed little survival at 110°C, four other strains had Duo-c values between 1 and 4 min. Torulopsis glabrata had a D_126.7o.C of 0.78 min. Saccharomyces strains showed the highest dry heat resistance, with the most heat resistant strain tested having a D_126.7o of 5 min. The z values for these strains ranged from 9.1° to 13.3°C.     

Application of a biochemical time–temperature integrator to estimate pasteurisation values in continuous food processes

The microbiological safety of commercial fruit processes was evaluated using α-amylase time–temperature integrators (TTI) from either a Bacillus amyloliquefaciens or Bacillus licheniformis source. The TTIs were incorporated into silicone particles that were added to batches of fruit preparations to estimate the pasteurisation achieved during two different methods of continuous processing: a tubular heat exchanger and an ohmic column. Pasteurisation values (P-values) estimated with the TTIs represented the integrated thermal process at the core of 12-mm pear cubes for the tubular process of 12-mm strawberries, 12-mm pineapple and 10-mm blackcurrants for the ohmic process. The decimal reduction time at 85.0 °C (D₈₅) for the Bacillus amyloliquefaciens amylase was 6.8 min, with a kinetic factor (z-value) of 9.4±0.3 °C, and for the Bacillus licheniformis amylase the D₉₃ was 8.8 min with a z value of 9.1±0.3 °C. For the high-acid fruit products, the target P-value was equivalent to 5 min at 85 °C (T_ref=85 °C, z=10 °C). Amylase activity before and after processing was converted to P-values, with all of the processes showing a substantial safety margin, despite operating conditions that were deliberately set to represent the ‘worst case’ conditions. This method allowed P-value data to be collected under conditions that prevented the use of thermocouples.