Influence of recovery conditions on apparent heat resistance of Bacillus stearothermophilus spores

The effects of post-treatment environmental factors on the heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Nutrient Agar (NA), Antibiotic Assay Medium (AAM), Dextrose Tryptone Agar (DTA) and Tryptic Soy Agar (TSA) with Ca^{2+ added to a final concentration of 100 p.p.m. were used as recovery media. No significant differences were seen between D-values obtained except in the case of strain 12980 when comparing TSA with the other media and for strain 7953 comparing AAM and DTA. The optimum incubation temperature was slightly lower for heated than for unheated spores of} each strain, although, in general, 50 °C was adequate. Higher D-values were obtained at 50–55 °C. The effects of the pH of the medium in the range 5.0–7.0 and the addition of starch and phosphate on heat resistance have also been investigated. Maximum colony counts of heated spores were obtained at pH 7.0 and decreased as pH fell. D-values were significantly lower at pH ≤ 5.5. Increasing the concentration of phosphate in the recovery medium from 0 to 0.2% resulted in a progressive decrease in spore recovery and D-values. The addition of starch improved recoverability. The z-values obtained for the four strains studied under the different recovery conditions were similar with a mean value of 7.58 °C ± 0.28.     

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry.     

Modelling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores.

A simple overall model is proposed to describe the effect of both the pH of the heating menstruum and the pH of the recovery medium on the apparent spore heat resistance of Bacillus cereus. Applied to foods making up both heating and recovery media, the model can be reduced to only two parameters. Its goodness of fit and its robustness enable it to be applied to the optimisation of heat treatments. However. further experiments should be undertaken to validate the model for other species and to determine the parameters related to reference species such as Clostridium botulinum.     

Influence of nisin on the resistance of Bacillus anthracis sterne spores to heat and hydrostatic pressure

The influence of nisin on the heat and pressure resistance of Bacillus anthracis Sterne spores was examined. The decimal reduction times (D-value) of spores in milk (2% fat) at 80, 85, and 90 degrees C were determined. In the absence of nisin, the D-values were 30.09, 9.30, and 3.86 min, respectively. The D-values of spores heated in the presence of nisin (1 mg/ml) were not significantly different (P = 0.05). However, spores heated in the presence of nisin had a 1- to 2-log reduction in viability, after which the death kinetics became similar to those of spores in the absence of nisin. The z-values all were 11.2 degrees C regardless of the presence or absence of nisin. The pressure sensitivity of B. anthracis Sterne spores in the presence and absence of nisin also was determined. Spores treated with nisin were 10 times more pressure sensitive than were spores subjected to pressure in the absence of nisin under the conditions used in this study.     

Factors affecting the heat resistance of Bacillus stearothermophilus spores

Summary. Maximum colony counts of unheated and heated spores of Bacillus stearothermophilus were obtained after incubation at 50-65°C and 45-50°C, respectively. the composition of the plating medium was found to have a marked effect upon recovery of unheated and heated spores. the effects of diluent and incubation time on recovery have also been investigated.     

Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce

Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60 °C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10 min at different pressures (100-800 MPa) at 40 °C. None of these treatments caused any significant inactivation, except perhaps at 800 MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300 MPa) for A. acidoterrestris and only in a high pressure window (600-800 MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800 MPa at 25, 40 and 60 °C for 10 min. At 40 °C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60 °C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria.     

Modeling the combined effect of pH and temperature on the heat resistance of Bacillus stearothermophilus spores heated in a multicomponent food extract

The combined effect of pH and temperature on the heat resistance of Bacillus stearothermophilus spores heated in an extract of complex food was studied. The results showed that, in general, reducing the pH reduced the heat resistance of the spores. Similarly, the value for the D parameter in the nonacidified extract was between 30 and 70% lower than the one obtained with double-distilled water. This result once again shows the importance of the substrate in inactivation studies of microorganisms. The experimental data were used to carry out a comparison of two predictive mathematical models of inactivation, one based on a multiparametric regression obtained in this study and the other obtained from the bibliography and based on a linear-Bigelow equation. Both models predict reasonably well, although the multiparametric model presented a slightly better accuracy factor (1.11) than the one obtained with the linear-Bigelow equation (1.13).     

Relationship between the apparent heat resistance of Bacillus cereus spores and the pH and NaCl concentration of the recovery medium

Conventional heat resistance data, D values, were previously established by other workers at optimal condition for spores outgrowth. However, in canned food conditions of outgrowth are generally suboptimal in term of pH, salt concentration, water activity. The combined effects of pH and NaCl level of the recovery medium for the D value and z(pH) value were studied. Spores of Bacillus cereus were heated at 95 degrees C in phosphate-citrate buffer media at pH 7. Cells were recovered at 25 degrees C in Nutrient Agar with pH ranging from 5 to 7 and 1% to 4% (w/w) NaCl concentration. For each condition D' values (decimal reduction time associated with the recovery media characteristics) were determined. The results show a major influence of the recovery pH on the D' values. This effect is characterised by the z'(pH) values, distance of recovery medium pH from optimum recovery pH* medium (6.7) which leads to a tenfold reduction time of D value. The increase of the salt concentration leads to a slight decrease of D' value. However z'(pH) values are not significantly affected by the salt concentration. A simple three parameter model describing the effects of pH and NaCl concentration of the recovery medium upon the heat resistance of spores is proposed. The interaction between pH and salt concentration is sufficiently low to be neglected by the model.     

A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH

Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8. Inactivation of spores under these conditions was maximally 80% and was not increased at low pH. However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH. This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5). Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed. Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B. subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons. Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed.     

Growth and Corresponding Elevation of Tomato Juice pH by Bacillus coagulans

B. coagulans was inoculated into sterile, tubed tomato juice and growth and pH were observed for 21 days. Juice samples initially at pH 4.5 supported good growth; pH was elevated to 5.0 after 6 days incubation at 35°C. Other tomato juice samples (initial pH 5.0 or 5.5) also supported good growth of these flat-sour bacteria and with corresponding increases in pH. The results indicate that flat-sour spoilage of tomatoes and similar foods may cause decreased acidity, thus possibly allowing outgrowth of Clostridium spores in these water bath-processed foods.     

Effect of heat activation and inactivation conditions on germination and thermal resistance parameters of Bacillus cereus spores

The effect of isothermal and non-isothermal heat activation on germination and thermoresistance of two strains of Bacillus cereus spores was studied. Results indicated that the germination after isothermal activation was lower than after non-isothermal heating. The activation rate affected the z value, which increased with faster heating rates. For each temperature and inactivation rate, the non-isothermal activation at rate of 2 degrees C/min resulted in larger D values (D90 = 4.70 min) than isothermal activation (D90 = 4.04 min). The two mathematical equations used to analyse non-isothermal data produced similar predicted D and z values, nevertheless the Hayakawa equation modified in this work for non-linear regression analysis, requires less computational effort.     

Heat treatment for the control of Bacillus cereus spores in foods

The effect of heat treatments on food products related to soybean curd contaminated with Bacillus cereus spores was investigated for the purpose of preventing food-poisoning outbreaks by B. cereus. In the case of B. cereus strains isolated from foods, heating foods inoculated with the spores for 20 min at 70 degrees C, 5 min at 75 degrees C, 2 min at 100 degrees C, or 10 sec with a microwave oven plus reheating after standing for 2 hr at 25 degrees C reduced the number of surviving cells within the foods to less than one-hundredth. In the case of a heat-resistant strain isolated from a food poisoning outbreak, heating for 20 min at 70 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, heating for 10 min at 75 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, or heating for 2 min at 100 degrees C plus reheating after standing for 4 hr at 25 degrees C was effective. There was not much difference between the flavor components in foods with and without heat treatment at 70 degrees C, as analyzed by gas chromatography-mass spectrometry. These results indicate that these heat treatments are available to control B. cereus spores, without affecting the sensory quality of the foods.     

Performance evaluation of aluminum test cell designed for determining the heat resistance of bacterial spores in foods

Thermal inactivation kinetic studies are necessary to determine heat resistances of spores in the development of new thermal processes for low-acid shelf-stable products. Most currently available sample holders used for solid and semi solid samples in the kinetic studies take long time to reach the target sample temperature, hence fail to provide isothermal condition. In this research, novel aluminum test cells were developed to facilitate easy loading and unloading samples in a hermetically sealed 1 ml cavity to evaluate the heat resistance of bacterial spores when heated at temperatures above 100 °C. Design of the test cell was governed by minimum come-up time. A finite element model based on the commercial software ‘FEMLAB’ was used to simulate transient heat transfer and finalize the test cell dimensions. Performance of the new test cell was evaluated against capillary and aluminum thermal death time tube methods in characterizing the heat resistance of Clostridium sporogenes PA 3679 spores in a phosphate buffer and mashed potato at 121 °C. D₁₂₁ values of PA 3679 spores in both the phosphate buffer and mashed potato using the new test cells were not significantly different (P>0.05) from those by the capillary tube method. The results indicated that the new test cell is appropriate for studying the inactivation kinetics of bacterial spores in microbial validation of conventional and novel thermal processes for low-acid shelf-stable foods.     

Effect of heat treatment of milk on activation of Bacillus spores

The quality and shelf life of fluid milk products are dependent on the amount and type of microorganisms present following pasteurization. This study evaluated the effects of different pasteurization processes on the microbial populations in fluid milk. The objective was to determine whether certain pasteurization processes lead to an increase in the amount of bacteria present in pasteurized milk by activating Bacillus spores. Samples of raw milk were collected on the day of arrival at the dairy plant. The samples were pasteurized at 63 degrees C for 30 min (low temperature, long time), 72 degrees C for 15 s (high temperature, short time), 76 degrees C for 15 s, and 82 degrees C for 30 min. The pasteurized samples were then stored at 6 and 10 degrees C for 14 days. The samples were analyzed for standard plate count and Bacillus count immediately after pasteurization and after 14 days of storage. Pasteurization of milk at 72 and 76 degrees C significantly (P < 0.05) increased the amount of Bacillus spore activation over that of 63 degrees C. There was no detection of Bacillus in initial samples pasteurized at 82 degrees C for 30 min, but Bacillus was present in samples after storage for 14 days, indicating that injury and recovery time preceded growth. The majority of isolates were characterized as Bacillus mycoides and not Bacillus cereus, suggesting that this organism might be more a cause of sweet curdling of fluid milk than previously reported.     

Comparative effect of different test methodologies on Bacillus coagulans spores inactivation kinetics in tomato pulp under isothermal conditions

Heat resistant micro‐organisms are an ongoing challenge to the food industry. Various factors may influence the heat resistance of micro‐organisms including type and strain; the environmental influences during cell and spore formations and during heat exposure; and the equipment and test tools used to perform the experimental process. In an attempt to analyse the influence of different test tools used on the heat inactivation processes, this study aimed to define the isothermal inactivation kinetics of Bacillus coagulans spores in tomato pulp at different temperatures and compare the inactivation of this bacterium when thermal death time (TDT) and capillary tube methods were used. Temperature ranges from 95 °C to 120 °C were studied, and inactivation kinetic parameters were estimated through the application of primary models. TDT inactivation curves consisted of shoulder and linear decline, while capillary method inactivation curves consisted of shoulder, linear decline and long tail. A secondary model was used to describe the influence of the temperature on spore inactivation parameters. The results showed test methods are at least as important in determining thermal processes as the micro‐organisms and media used.     

Modelling combined effects of temperature and pH on the heat resistance of spores ofBacillus cereus

The development of relationships between the pH of a heating medium and the thermal resistance of contaminant micro-organisms is important and of public health significance. A number of mathematical models have been presented in recent years, including that of Mafart and Leguérinel (1998). However, in this model the effect of possible interactions between temperature and pH onD-values was not assessed. The consequences of ignoring interaction terms in models have been assessed, and a comparison with Mafart's model that includes an interaction term showed that interaction terms can be neglected and that Mafart's model can be used in thermal process calculations. It appears possible to adopt a standard value ofZ_pH, for example the 3.6 value, and the conventional concept of biological destruction value L(T) (ratio of the sterilization value and the exposure time at a fixed heating temperature) may then be extended to L(T,pH) (the same ratio at a fixed temperature with a fixed pH of the heating menstruum).     

Evaluation of the stability of Bacillus coagulans MTCC 5856 during processing and storage of functional foods

The study was aimed to find out the stability of Bacillus coagulans MTCC 5856 in functional foods during processing and storage. B. coagulans MTCC 5856 was found to be stable during baking and storage at frozen conditions of banana muffins (92% viability) and waffles (86% viability) upto 12 months. After brewing coffee at 90 °C for 2 min, there was 87% viability and retained 66% viability even after maintaining temperature at 77 °C for 4.0 h. It showed over 95% viability in chocolate fudge frosting, hot fudge toppings, peanut butter, strawberry preserve and vegetable oil at room temperature upto 12 months. It was found to be stable in apple juice upto 6 months at refrigerated condition and concentrated glucose syrup at 4 ± 3 and 25 ± 2 °C upto 24 months. B. coagulans MTCC 5856 showed promising stability during processing and storage of functional foods and could be an excellent probiotic ingredient to use in various food applications.     

Bacillus coagulans spore inactivation through the application of oregano essential oil and heat

The present study evaluated the effect of thermal (temperature) and thermochemical (temperature + oregano essential oil (EO)) inactivation of Bacillus coagulans spores in Nutrient Broth (NB) adjusted at 4 °Brix and pH of 4.2. Thermal treatments included temperatures between 95 and 103 °C. For thermochemical treatment, first temperature was fixed at 100 °C and EO concentration varied between 250 and 1000 μg/g. Thermochemical treatment significantly reduced the time needed to reduce a 6 log level of spores compared to thermal treatment, for example around 1.4 min with 400 μg/g of EO. Then, EO concentration was fixed at 400 μg/g and temperature varied between 90 and 100 °C. Although the first results showed a faster spore reduction with 500 μg/g, the fixed EO concentration was 400 μg/g, since it represents a lower organoleptic impact and also a significant reduction in the spores’ resistance. For instance, at 97 °C and 400 μg/g, about 4.3 min was needed to reduce the spores in 6 log, without the EO this time was 5.0 min. These findings indicate that oregano EO may be used to render B. coagulans spores more susceptible to the lethal effect of heat.     

A quantitative approach for studying the effect of heat treatment conditions on resistance and recovery of Bacillus cereus spores.

When a bacterial population undergoes an unfavourable transient increase in temperature, a death phase followed by a lag and growth phase are observed for the surviving and cultivable population. The lag phase is of great interest in regard to food safety, but for bacterial spores, very few studies have been carried out on the evolution of lag time versus heat treatment duration. The experiments monitored on spores of two strains of Bacillus cereus showed a biphasic evolution of the lag time for germination of stressed spores with increase in heat treatment duration, at 90 degrees C, 95 degrees C and 100 degrees C and for different recovery conditions in laboratory medium. Initially, the lag time increased with the duration of the thermal stress up to an optimum. Subsequently, the lag time decreased, with increasing stress duration, to a threshold. The evolution of the lag time to germination of surviving spores, under the range of temperature tested, was similar to the evolution of the lag time to growth after mild-temperature treatments observed with vegetative cells of Escherichia coli and Listeria monocytogenes by Breand et al. (1997) [Breand, S., Fardel, G., Flandrois, J.-P., Rosso, L., Tomassone, R., 1997. A model describing the relationship between lag time and mild temperature increase duration. Int. J. Food Microbiol. 38, 157-167]. The mathematical model established from these investigations could also be applied in the case of heat-stressed spores. Moreover, it was observed that there was a significant linear relationship between the D-value and the treatment duration that gave the maximum lag time.