Heat‐resistance characteristics of ascospores of Eurotium chevalieri isolated from apricot juice

A heat‐resistant fungus was isolated from aseptically packaged apricot pulp. The fungus was identified asEurotium chevalieri. Heat resistance of the fungus was studied at four different temperatures (70, 75, 80 and 83°C) after activation of its ascospores for 30 min at 70°C. D₇₀, D₇₅, D₈₀ and D₈₃ values of ascospores ofEurotium chevalieri were estimated by linear regression (log‐survival vs. heating time) as 118.58, 34.15, 5.50 and 3.77 min, respectively. The z‐value was determined in the same way (regression of log‐D values vs. heating temp.) and was found as 8.23°C.     

Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation kinetics of beta-lactoglobulin in raw milk under isothermal and dynamic temperature conditions

A detailed kinetic study of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin was carried out in the context of identifying intrinsic time-temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5.3 deg C (D60 degrees C = 24.6 min) and 4.3 deg C (D71 degrees C = 38.6 min) respectively. For the denaturation of beta-lactoglobulin we found z values of 7.9 deg C (D7.5 degrees C = 49.9 min) in the temperature range 70-80 degrees C and 24.2 deg C (D85 degrees C = 3.53 min) in the range 83-95 degrees C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.     

Modulation of thermal resistance of ascospores of Neosartorya fischeri by acidulants and preservatives in mango and grape juice

Minimal thermal processing is desirable for near natural organoleptic and nutritional qualities of fruit based products. In the present investigation, the effect of heat (85°C) in combination with acidulants or common preservatives on inactivation of ascospores of Neosartorya fischeri, a heat resistant mould isolated from grapes, has been studied in mango and grape juice. The ascospores were found to survive for >300 min of heating at 70, 75 and 80°C in these fruit juices and complete inactivation required 120 min of heating at 85°C. The synergistic effect of heat and organic acids or preservatives in fruit juices was noticed. The thermal death rate (1/k_85°C) values did not vary much in the presence of lactic (20), malic (20) and citric (19) acids, but tartaric acid showed least inactivation effect (1/k_85°C=54 min) in mango juice. The 1/k_85°Cvalues for ascospores of N. fischeri in mango juice containing 0·1% of potassium sorbate or sodium benzoate or combination of both at 0·05% were found to be 44, 35 and 29 min respectively. These values were respectively, 32, 13 and 14 min in grape juice. Nearly 50 and 67% of the heating time was reduced by the use of potassium sorbate and sodium benzoate (0·05% each) in mango and grape juice to inactivate 3 log number of ascospores of N. fischeri. These results may be useful in thermal processing of fruit juices.     

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.     

Efficacy of media for promoting ascospore formation by Neosartorya fischeri,and the influence of age and culture temperature on heat resistance of ascospores

Gorodkowa agar (GA), Fowell's acetate agar (FAA), Kleyn's acetate agar (KAA), McClary's acetate agar (MAA), grape juice agar (GJA), apple juice agar (AJA) and vegetable juice agar (VJA) were evaluated for their efficacy to promote ascospore formation by three strains of Neosartorya fischeri. All three strains produced abundant mature ascospores when grown on FAA, GJA and AJA, but not on the other test media. Ascospores produced on GJA developed greater heat resistance with time when suspended in commercial apple juice (AJ), grape juice (GJ) and 0·1 M potassium phosphate buffer (PB) during heat treatment. From 10 to 13 days of incubation at 30°C, decimal reduction times at 80°C (D_80°C values) for ascospores increased from 27·0 to 66·7 min in AJ, 28·5 to 33·3 min in GJ and 18·2 to 50·0 min in PB. The rate of development of heat resistance was dependent upon the incubation temperature at which ascospores were produced. After treatment at 70°C for 60 min, survival percentages for 21-day-old ascospores were 0·32, 8·13, 54·95 and 53·70% of ascospores produced at 18, 21, 25 and 30°C, respectively. At 42 days, 0·23 and 38·02% of ascospores produced at 18 and 21°C, respectively survived 85°C for 30 min; this treatment did not inactivate 42-day-old ascospores produced at 25 and 30°C. During a 114-day test period, ascospores produced at all incubation temperatures became dormant to some extent and required heat activation to facilitate germination.     

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.     

Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.     

Influence of pH on heat resistance of spores of Bacillus coagulans in buffer and homogenized foods

The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus coagulans spores has been investigated and compared with the heat resistance in homogenized tomato and asparagus at pH 7 and 4 at a wide range of temperatures. Spores were less heat resistant in all menstrua at acid pH. The magnitude of this effect was greatest at the lowest heating temperatures tested. z values in buffer increased from 8.9 degrees C at pH 7 to 10.5 degrees C at pH 4. pH of menstrua was the main influencing factor, but media composition also influenced heat resistance: at pH 7 heat resistance was similar in all menstrua (D111 degrees C = 1.6 min) but at pH 4 the heat resistance in homogenized foods (D111 degrees C = 0.26 min in tomato and D111 degrees C = 0.28 min in asparagus) was lower than in buffer (D111 degrees C = 0.49 min). The reduced influence of the acidification of media on the heat resistance of B. coagulans at higher temperatures should be taken into account when a rise in the temperature of treatment for canned vegetables is considered to shorten duration of heat processes.     

Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing.

Resistance of 19 mold and 6 yeast species to 15 commercial disinfectants was investigated by using a suspension method in which the fungicidal effect and germination time were determined at 20 degrees C. Disinfectants containing 0.5% dodecyldiethylentriaminacetic acid, 10 g of chloramine-T per 1, 2.0% formaldehyde, 0.1% potassium hydroxide, 3.0% hydrogen peroxide, or 0.3% peracetic acid were ineffective as fungicides. The fungicidal effect of quaternary ammonium compounds and chlorine compounds showed great variability between species and among the six isolates of Penicillium roqueforti var. roqueforti tested. The isolates of P roqueforti var. carneum, P. discolor, Aspergillus versicolor, and Eurotium repens examined were resistant to different quaternary ammonium compounds. Conidia and vegetative cells were killed by alcohols, whereas ascospores were resistant. Resistance of ascospores to 70% ethanol increased with age. Both P. roqueforti var. roqueforti and E. repens showed great variability of resistance within isolates of each species.     

THERMAL KINETICS of STREPTOCOCCUS FAECIUM IN NUTRIENT BROTH/SOUS VIDE PRODUCTS UNDER PASTEURIZATION CONDITIONS

There is current major interest in the microbiological aspects and safety of chilled foods such as sous vide. Thermal kinetics for a target microorganism, Streptococcus faecium, was performed and is described in this paper. an E-20 S. faecium was tested for heat resistance in cooked meat nutrient broth. Known cell concentrations were inoculated into ampoules containing the heating medium and exposed to various heating temperatures (60-85C). Swival curves for the recovered S. faecium cells were constructed. the D-values calculated from the logarithm of number of survivors versus time plot were 22.53, 4.47, 1.11, 0.61, 0.29, 0.20 min for 60, 65, 70, 75, 80, 85C, respectively. the z-value obtained from 60–85C was 12.4C; however when only 60–75C was considered, the z-value obtained was 9.4C. Pasteurization values (P_v) calculated for a 13D reduction of S. faecium at 65 and 75C based on the above kinetics and heat penetration data under sous vide processing conditions compared favorably with experimental values in both nutrient broth and in sous vide spaghetti with meat sauce and mackerel with rice products. Using D-values of a heat resistant representative, S. faecium E-20 as a basis, parameters for pasteurization that effectively eliminate vegetative nonspore forming bacteria may be developed. However, the variation in z-values suggest that their usefulness in determining pasteurization process parameters should be carefully considered.     

Validation of radio-frequency dielectric heating system for destruction of Cronobacter sakazakii and Salmonella species in nonfat dry milk

Cronobacter sakazakii and Salmonella species have been associated with human illnesses from consumption of contaminated nonfat dry milk (NDM), a key ingredient in powdered infant formula and many other foods. Cronobacter sakazakii and Salmonella spp. can survive the spray-drying process if milk is contaminated after pasteurization, and the dried product can be contaminated from environmental sources. Compared with conventional heating, radio-frequency dielectric heating (RFDH) is a faster and more uniform process for heating low-moisture foods. The objective of this study was to design an RFDH process to achieve target destruction (log reductions) of C. sakazakii and Salmonella spp. The thermal destruction (decimal reduction time; D-value) of C. sakazakii and Salmonella spp. in NDM (high-heat, HH; and low-heat, LH) was determined at 75, 80, 85, or 90°C using a thermal-death-time (TDT) disk method, and the z-values (the temperature increase required to obtain a decimal reduction of the D-value) were calculated. Time and temperature requirements to achieve specific destruction of the pathogens were calculated from the thermal destruction parameters, and the efficacy of the RFDH process was validated by heating NDM using RFDH to achieve the target temperatures and holding the product in a convection oven for the required period. Linear regression was used to determine the D-values and z-values. The D-values of C. sakazakii in HH- and LH-NDM were 24.86 and 23.0 min at 75°C, 13.75 and 7.52 min at 80°C, 8.0 and 6.03 min at 85°C, and 5.57 and 5.37 min at 90°C, respectively. The D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75°C, 10.45 and 12.54 min at 80°C, 8.63 and 8.68 min at 85°C, and 5.82 and 4.55 min at 90°C, respectively. The predicted and observed destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the behavior of the organisms was similar regardless of the heating system (conventional vs. RFDH). Radio-frequency dielectric heating can be used as a faster and more uniform heating method for NDM to achieve target temperatures for a postprocess lethality treatment of NDM before packaging.     

Thermal inactivation kinetics of Bacillus stearothermophilus spores using a linear temperature program.

A systematic study of the inactivation kinetics of Bacillus stearothermophilus spores was carried out in nonisothermic heating conditions using a linear temperature increase program and analyzing the experimental data by means of a one-step nonlinear regression. The D and z values estimated are close to those obtained in isothermic conditions and estimated by using a two-step model, first D values are calculated, and then in the second step a z value is deduced (D(121 degrees C) = 3.08 and 4.38 min, respectively, and z = 7 and 7.9 degrees C, respectively). No convergence problems were observed when using the one-step nonlinear regression proposed. The results indicated that the methodology applied in this study can be used to obtain kinetic data for bacterial spores, which could mean a significant reduction in the amount of experimental work employed to generate these data.     

Buffalo-milk enzyme levels, their sensitivity to heat inactivation, and their possible use as markers for pasteurization

The activities and rates of inactivation of four enzymes in raw buffalo milk were measured in relation to the process of heating to determine the value of these enzymes as markers for the evaluation of milk pasteurization. The activities of the enzymes alkaline phosphatase (ALP), lactic dehydrogenase (LDH), gamma-glutamyltransferase (GGT), and aspartate aminotransferase (AST) were measured before and after heating at 50, 60, 70, and 80 degrees C for 1, 3, 5, 10, 20, and 30 min. The enzyme GGT showed the highest activity (712 +/- 601 IU/liter), followed by LDH (386 +/- 183 IU/liter), ALP (295 +/- 164 IU/liter), and AST (18 +/- 4 IU/liter). Heating the milk at 50 degrees C for 1 to 30 min resulted in no effect on the activity of any of the enzymes. At 60 degrees C, ALP showed the highest sensitivity to heat inactivation, whereas all other enzymes showed resistance. At 70 degrees C, ALP activity was abolished completely after 1 min, whereas GGT and LDH lost most activity after 10 min, and AST still maintained 50% activity even after 30 min. At 80 degrees C, the activities of LDH and GGT were lost, whereas AST still retained some of its activity. The results suggest that in addition to ALP, LDH and GGT, but not AST, are potential markers for heat denaturation in buffalo milk, with GGT having the advantage that its concentration is the highest.     

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Heat Resistance of Sporolactobacillus inulinus

Sporolactobacillus inulinus was grown on 24 media varying in carbohydrate source and concentration. A heat-resistant total count (80°C–5 min) and the thermal resistance of the spores isolated from each cell crop were determined. The highest heat-resistant couunts were obtained when S. inulinus was grown on Sporolactobacillus agar with 2%α-methyl glucoside. Cells grown in Sporolactobacillus broth with 1%α-methyl glucoside yielded the most heat resistant spores. D values (decimal reduction times) and z values were determined for each spore crop. Average D values were 53.2 min at 75°C, 19.5 min at 80°C, 6.8 min at 85°C and 5.1 min at 90°C. The average z value was 13.0°C.     

Heat Resistance of Eurotium herbariorum, a Xerophilic Mold

Eurotium haerbariorum, isolated from a spoilage outbreak involving grape preserves, was a true osmophile in that optimal growth was obtained in media containing 40-60% sucrose. Survival curves showed logarithmic death of heated spores followed by a tailing. In 5° Brix grape juice D-values at 70°C and z-values were 2.5 min and 9.1°C compared to 5.2 min and 7.1°C in 65°C Brix juice. Low concentrations of sorbic acid and fumaric acid in the heating menstruum had little effect on heat resistance.     

A cardinal model to describe the effect of water activity on the growth of moulds

A simple model was proposed to describe the effect of water activity (Aw) on the radial growth rate of moulds. This model is deduced from the cardinal model family proposed by Rosso in 1995, which is only defined from cardinal values of environmental factors (minimum, optimum and maximum values), the growth rate observed at the optimal value of the environmental factors, and n, a shape parameter. For Aw, a simple form of cardinal model is proposed. This form is obtained for n = 2 and Aw(max) = 1.0 (pure water). The final model is so defined from only three parameters: Aw(min), Aw(opt), and optimal radial growth rate (RGR(opt)). This model was successfully fitted on a data set of Aspergillus flavus, Aspergillus nomius, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus candidus, Aspergillus sydowii, Eurotium amstelodami, Eurotium chevalieri, and Xeromyces bisporus. The same quality of fit was obtained for different solutes used to control the Aw (NaCl, glucose/fructose mixture, glycerol), and at different pH values. From this model and using cardinal values extracted from the literature, theoretical evolutions of the RGR of enicillium roqueforti, and Paecilomyces variotii, were proposed and superimposed on data published in the literature. The results showed a good concordance between the predicted and the observed values for these species. The use of this model in Predictive Microbiology is discussed.     

SOYMILK VISCOSITY AS INFLUENCED BY HEATING METHODS AND SOYBEAN VARIETIES

In the two‐step heating experiments, soymilk with 5% protein was first heated at 55, 60, 65, 70, 75 or 80C for 10 min, and then heated at 97C for another 10 min. As a control, the soymilk was heated at 97C for 10 to 60 min by continuous one‐step heating. The viscosity of the heated soymilk was measured after cooling to room temperature. Results showed that the two‐step heating increased the soymilk viscosity by up to six times the viscosity of the control. The maximal viscosity was reached when the first‐step heating temperature was 70C. Differential scanning calorimetry showed separate denaturation of soy 7S and 11S proteins was related to the viscosity increase. Soymilk protein content and soybean variety also influenced the viscosity. Three heat transfer methods, including ohmic heating, steam injection and water bath heating, produced different viscosities of soymilk.     

Heat Stability of Bovine Milk Immunoglobulins and Their Ability to Bind Lactococci as Determined by an ELISA

Immunoglobulins obtained from bovine colostrum were heat treated at 70, 75, 80 and 85°C for 0-90 min. An ELISA was developed to determine the susceptibility of lactococci to bind heat treated IgG, IgM and IgA. IgA was the most heat sensitive of the immunoglobulins. Treatment at 80°C for 25 min completely inactivated IgA as evidenced by the inability of lactococci to bind IgA. IgM was completely inactivated upon heating to 85°C for 20 min whereas IgG was completely inactivated at 85°C after 30 min. Laser Scanning Microscopy verified results with ELISA. Severe thermal treatment is needed to reduce immunogenicity of bovine milk to prevent lactococcal agglutination.     

Effects of heating procedures on deoxynivalenol, nivalenol and zearalenone levels in naturally contaminated barley and wheat.

The influence of heating temperature and time on deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) contents in naturally co-contaminated barley and wheat was investigated intending to establish the basis for a decontamination model of Fusarium mycotoxins in cereals. The standard toxins and whole barley powder samples were heated in a convection oven at 140, 160, 180, 200, or 220 degrees C, and kernel subsamples (200 g each) were roasted in an experimental rotary gas-fired roaster at 150, 180 or 220 degrees C. All treatments resulted in a time-temperature-dependent decomposition of the toxins; the logarithm of the toxin remaining % presented a linear relationship with heating time. The lines equations were used to estimate the half (H) and decimal (D) decomposition times (time required to destroy 50 or 90% of the toxin, respectively). DON and NIV H and D decomposition times were similar and 50% shorter for heated standards than for whole barley powder. ZEA standard values were considerably longer, while whole barley powder values were comparable with those of DON and NIV. At 220 degrees C, D decomposition times of DON, NIV and ZEA heated standards were 11, 10 and 85 min, respectively, while the values obtained in whole barley powder were the same for the three toxins (25 min). The determination of H and D decomposition values constitutes a basis to understand the heating stability nature of each toxin.