Molecular characterization of Prototheca strains isolated from Italian dairy herds

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.     

Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method.     

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35−68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.     

Short communication: Dairy bedding type affects survival of Prototheca in vitro

Protothecae are algal pathogens, capable of causing bovine mastitis, that are unresponsive to treatment; they are believed to have an environmental reservoir. The role of bedding management in control of protothecal mastitis has not been studied. The purpose of this study was to evaluate the growth of either environmental or mastitis-associated Prototheca genotypes in dairy bedding materials that are commonly used in Maine. Prototheca zopfii genotypes 1 and 2 (gt1 and gt2) were inoculated into sterile broth only (control ), kiln-dried spruce shavings, “green” hemlock sawdust, sand, or processed manure-pack beddings with broth, and incubated for 2 d. Fifty microliters of each isolate was then cultured onto plates and the resulting colonies counted at 24 and 48 h postinoculation. Shavings were associated with significantly less total Prototheca growth than other bedding types. Growth of P. zopfii gt1 was significantly higher than that of gt2 in the manure-pack bedding material. Spruce shavings, compared with manure, sand, or sawdust, may be a good bedding type to prevent growth of Prototheca. Based on these in vitro findings, bedding type may affect Prototheca infection of cattle in vivo.     

Heat resistance of Neosartorya fischeri in various juices

Heat resistance of Neosartorya fischeri was studied in three different juices (apple, pineapple and papaya). The optimum heat activation temperature and time for the ascospores of the N. fischeri (growth for 30 days at 30 °C) was 85 °C for 10 min. Of the three juices tested, apple juice exhibited maximum 1/k values at 80, 85 and 90 °C (208.3, 30.1 and 2.0 min, respectively). The 1/k values for papaya juice (129.9, 19.0 and 1.9 min) and pineapple juice (73.5, 13.2 and 1.5 min) decreased with acidity and °Brix/acidity (ratio) level. The Z^* values for apple, papaya and pineapple juices were 5, 5.5 and 5.9 °C, respectively. The sterilization F values (4-log reduction) for apple, pineapple and papaya juices were 56.3, 38.0 and 7.2 s, respectively. Considering the thermal treatments commercially applied to pineapple (96 °C/30 s) and apple juices (95 °C/30 s), it is concluded that such treatments will not guarantee that less than 1 ascospore in each set of 10³ packs survive. Only the treatment applied to papaya juice (100 °C/30 s) will be sufficient because the F value is less than 30 s.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd

The use of raw milk in the processing of buffalo Mozzarella cheese is permitted, but the heat treatment used for stretching the curd must ensure that the final product does not contain pathogens such as Shiga toxin-producing Escherichia coli (STEC) that may be present on buffalo dairy farms. This study carried out challenge tests at temperatures between 68°C and 80°C for 2 to 10 min to simulate curd temperatures during the stretching phase. Curd samples were inoculated with 2 STEC strains (serotypes O157 and O26), and their inactivation rates were assessed in the different challenge tests. The curd samples were digested with papain to ensure a homogeneous dispersion of bacteria. The STEC cells were counted after inoculation (range 7.1–8.7 log cfu/g) and after heat treatments using the most probable number (MPN) technique. A plot of log MPN/g versus time was created for each separate experiment. The log linear model with tail was used to provide a reasonable fit to observed data. Maximum inactivation rate (k_max, min⁻¹), residual population (log MPN/g), decimal reduction time (min), and time for a 4D (4-log₁₀) reduction (min) were estimated at each temperature tested. A 4D reduction of the O26 STEC strain was achieved when curd was heated at 68°C for 2.6 to 6.3 min or at 80°C for 2.1 to 2.3 min. Greater resistance was observed for the O157 strain at 68°C because k_max was 1.48 min⁻¹. The model estimates can support cheesemakers in defining appropriate process criteria needed to control possible STEC contamination in raw milk intended for the production of Mozzarella.     

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4 °C for 24 h), heat (55 °C for 5 min), and desiccation (cells were dried at 40 °C for 2 h and held at 21 °C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.     

Heat resistance of Listeria species to liquid whole egg ultrapasteurization treatment

The lethality of ultrapasteurization treatments (70 °C/1.5 min.) applied at constant temperature (isothermal condition) and at a constantly raising temperature of 2 °C/min (non-isothermal condition) in liquid whole egg (LWE) against two strains of Listeria monocytogenes (STCC 5672 and 4032) and one of Listeria innocua has been investigated. Isothermal survival curves up to 71 °C were obtained, which followed first-order inactivation kinetics. The obtained D_t values indicated that L. innocua was significantly (p < 0.05) more heat resistant than L. monocytogenes strains. Non-significant (p > 0.05) differences were observed among z values (12.4 ± 0.4 °C, 13.1 ± 0.4 °C and 12.2 ± 0.7 °C for L. innocua and L. monocytogenes 5672 and 4032, respectively). Based on obtained D_t and z values, isothermal ultrapasteurization treatment (70 °C/1.5 min.) would provide 3.5-, 5.0-, and 6.5-Log₁₀ cycles of L. innocua and L. monocytogenes 5672 and 4032, respectively. Non-isothermal heating lag phase increased the thermotolerance of Listeria species in LWE. The simulated industrial pasteurization treatment for LWE (heating-up phase from 25 to 70 °C followed by 1.5 min. at 70 °C) would attain 5-Log₁₀ reductions of L. monocytogenes 5672 and 4032, and 3.7-Log₁₀ reductions of L. innocua. Therefore, the safety level of industrial ultrapasteurization concerning L. monocytogenes could be lower than that estimated with data obtained under isothermal conditions.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

Effects of combining microbial transglutaminase and high pressure processing treatments on the mechanical properties of heat-induced gels prepared from arrowtooth flounder (Atheresthes stomias)

The objective of this study was to evaluate the effect of setting conditions (25 °C for 2 h or 40 °C for 30 min) and combining of microbial transglutaminase (MTGase) and high pressure processing (HPP) on the mechanical properties of heat induced gels obtained from paste from arrowtooth flounder (Atheresthes stomias). Treatments included fish paste control without added MTGase, fish paste incubated with MTGase but not pressurized (MTGase + cooking), fish paste incubated with MTGase and pressurized at 600 MPa for 5 min (MTGase + HPP + cooking) and fish paste pressurized at 600 MPa for 5 min and incubated with MTGase (HPP + MTGase + cooking). The controls and the treated samples were then subjected to one of two thermal treatments: 90 °C for 15 min or 60 °C for 30 min before cooking at 90 °C for 15 min. Samples of fish paste heated at 60 °C before cooking could not be used to prepare gels for texture profile analysis (TPA). TPA showed that pressurization improved the mechanical properties of gels made from paste treated with MTGase and set at 25 °C. The opposite was observed for samples set at 40 °C. Setting at 40 °C appeared to induce proteolytic degradation of myofibrillar proteins.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log₁₀ cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log₁₀ cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log₁₀ cfu/g; ON) or in the curds (ca. 7.0 log₁₀ cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.     

Survival of Vibrio parahaemolyticus under environmental stresses as influenced by growth phase and pre-adaptation treatment

In this study, the susceptibility of Vibrio parahaemolyticus in different growth phases after exposure to lethal stresses including 47 °C and 8% ethanol was first investigated. The effect of a culture's growth phase on both the heat and ethanol shock response of V. parahaemolyticus was then examined. It was found that cells of V. parahaemolyticus in the mid-exponential phase, regardless of adaptation, were most susceptible to environmental stresses, while cells in the stationary phase were least susceptible to the lethal stresses examined. Adaptation with heat shock at 42 °C for 45 min or ethanol shock with 5% ethanol for 60 min induced an increased resistance of V. parahaemolyticus to subsequent lethal stresses at 47 °C and 8% ethanol. While the adaptation treatments resulted in a reduced resistance of the test organism to pH 4.4 and 20% NaCl. Generally, the extent of changes in the resistance of V. parahaemolyticus to lethal stresses between the adapted and control cells was found to be growth phase dependent. Compared with the respective control cells, the adapted late-exponential phase cells exhibited the greatest extent of change, while the adapted stationary phase cells showed the least change in their resistance to the lethal stresses examined.     

Inactivation of Bacillus stearothermophilus spores in egg patties by pressure-assisted thermal processing

The use of pressure-assisted thermal processing (PATP) to inactivate bacterial spores in shelf-stable low-acid foods, without diminishing product quality, has received widespread industry interest. Egg patties were inoculated with Bacillus stearothermophilus spores (10⁶ spores/g) and the product was packaged in sterile pouches by heat sealing. Test samples were preheated and then PATP-treated at 105 °C at various pressures and pressure-holding times. Thermal inactivation of spores was studied at 121 °C using custom-fabricated aluminum tubes; this treatment served as a control. Application of PATP at 700 MPa and 105 °C inactivated B. stearothermophilus spores, suspended in egg matrix rapidly, (4 log reductions in 5 min) when compared to thermal treatment at 121 °C (1.5 log reduction in 15 min). Spore inactivation by PATP progressed rapidly (3 log reductions at 700 MPa and 105 °C) during pressure-hold for up to 100 s, but greater holding times (up to 5 min) had comparatively limited effect. When PATP was applied to spores in water suspension or egg patties, D values were not significantly different. While thermal inactivation of spores followed first-order kinetics, PATP inactivation exhibited nonlinear inactivation kinetics. Among the nonlinear models tested, the Weibull model best described PATP inactivation of B. stearothermophilus spores in the egg product.     

Resistances to benzalkonium chloride of bacteria dried with food elements on stainless steel surface

To confirm the importance of washing food sediments from the surface of food-related environments, we examined resistances against benzalkonium chloride of pathogenic bacterial (Escherichia coli O26, Pseudomonas aeruginosa and Staphylococcus aureus) cells dried and adhered on stainless steel dishes with milk, beef gravy or tuna gravy. Suspensions (0.1 ml) of these bacteria (8-9 log cfu/ml) were put on a 5 cm ? stainless steel dish and dried at room temperature (20-24 °C) for 90 min in a bio-clean bench with ventilation. Though these bacteria suspended with distilled water decreased 30-40 fold during the drying period, milk and the gravies protected the bacteria. Without the food elements, the adhered E. coli and Stap. aureus were decreased from 6 to<2 log cfu/dish by 0.5 mg/ml benzalkonium chloride (BKC) for 10 min treatment. Although Ps. aeruginosa showed resistance to BKC, the adhered cells were inactivated by 2.0 mg/ml BKC. However, the bactericidal effect disappeared by the food elements, particularly with milk, even at 1.0 and/or 2.0 mg/ml BKC levels. The protective efficiency of milk on bacteria disappeared if washed with water.     

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.     

Milk thermization by pulsed electric fields (PEF) and electrically induced heat

Whole milk was processed using selected combinations of pulsed electric fields (PEF) and thermal treatments to inactivate Listeria innocua. Electric field intensities of 30 and 40 kV/cm were applied at selected number of pulses (1–30) and temperatures (20–72 °C) for less than 10 s. A maximum microbial reduction of 4.3 log cycles was achieved using 10, 17.5, 20 and 25 pulses, when processing milk at 30 kV/cm and initial temperatures of 43, 33, 23 and 13 °C, respectively. Around 4.3 log cycles of L. innocua was observed when treating milk at 40 kV/cm using 3, 10, 12.5, 15, and 20 pulses and 53, 33, 23, 15, and 3 °C, respectively. Milk treated with 40 kV/cm of electric field intensity, few pulses, and initial temperature close to 55 °C showed the best balance between L. innocua inactivation and energy-consumption. An energy expenditure of around 244 J/mL was achieved, which can be further reduced to 44 J/mL using a thermal regeneration system.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

Strategy to inactivate Clostridium perfringens spores in meat products

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200 MPa, 7 min) and elevated temperature (80 °C, 10 min); spore germination at high temperatures (55, 60 or 65 °C); and inactivation of germinated spores with elevated temperatures (80 and 90 °C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 °C, 10 min). Low pressures (100–200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 °C, 10 min), and germinated at temperatures lethal for vegetative cells (≥55 °C) when incubated for 60 min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90 °C for 20 min) required a long germination period (55 °C for 60 min). However, similar inactivation level was reached with shorter germination period (55 °C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 °C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 °C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 °C in about 20 min and further incubation at 55 °C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 °C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.