Effect of high pressure combined with mild heat or nisin on inoculated bacteria and mesophiles of goat's milk fresh cheese

The effect of pressures ranging from 400 to 500 MPa combined with mild heat on Staphylococcus carnosus inoculated in fresh cheese and the concurrent use of 500 MPa and nisin to inactivate cheese indigenous populations has been studied. Staphylococcus carnosus counts could not be substantially decreased with treatments at 500 MPa at 10 or 25°C for 30 min, whereas treatments at 50°C for 5 min caused a reduction of 7-log₁₀cfu g⁻¹. Multiple-cycle treatments of 500 MPa and times between 15 and 30 min also improved the inactivation rate. Combination of 500 MPa and nisin was the most effective treatment to inactivate cheese indigenous microbiota. Inactivation of Bacillus subtilis spores inoculated in fresh cheese has also been studied. Germination treatments of 60 MPa at 40°C for 210 min followed by vegetative cells inactivation treatments of 500 MPa at 40°C for 15 min caused a lethality of 4·9-log₁₀ofB. subtilis , whereas the same combination of treatments applied at 25°C only caused a 2·7-log₁₀reduction.     

Combined high pressure and heat treatment effectively disintegrates spore membranes and inactivates Alicyclobacillus acidoterrestris spores in acidic fruit juice beverage

The commercial potential of high pressure and thermal processing (HPTP) was investigated against Alicyclobacillus acidoterrestris spores in commercial acidic apple juice beverage and in acidified and neutral potassium buffers. With starting spore counts prior to treatments being 6.5 and 7.2 log₁₀ respectively for strains AJA 66 (D_90°C 15.4 min) and ATCC 49025 (D_90°C 8.5 min), HPTP at 600 MPa at 80 °C for 3 min provided an optimal treatment with spore viability reduced below the detection limit for both strains. HPTP at 80 °C for 1 min and HPTP at 70 °C for 3 min achieved 4.1–4.5 log₁₀ CFU/mL reduction. HPTP at 70 °C for 1 min reduced the number of viable spores by 2.0–2.5-log₁₀ CFU/mL. Flow cytometry revealed the presence of membrane-compromised spores among culturable spores following HPTP and heat alone treatments at different temperatures. Electron microscopy clearly showed the efficiency of HPTP with crushed or hollow spores predominating after treatments. No correlation between HPTP susceptibility and genetic diversity was observed for two genotypes of A. acidoterrestris spores. The treatment combination provides a promising option for industrial utility since it requires lower heat and processing time.     

Mechanisms of Bacillus subtilis spore inactivation by single- and multi-pulse high hydrostatic pressure (MP-HHP)

Herein we investigate the effect of multi-pulse high hydrostatic pressure (MP-HHP) on the inactivation of Bacillus subtilis spores. B. subtilis spores were subjected to MP-HHP under pressures at 200–500 MPa at temperatures of 40 and 60 °C with 3 pulses (holding time of 3 min) with a total processing time of 10 min and compared it with a single pressurization (S-HHP).Mechanism of spore inactivation by S- or MP-HHP was explored by assessing germination by heat shock treatment, spore susceptibility to lysozyme and hydrogen peroxide (H₂O₂), release of dipicolinic acid (DPA), and the permeability of inner membrane and cortex. Our results presented the highest spore inactivation (5.8 log reduction), when MP-HHP was applied under the highest temperature and pressure. The increased inactivation appears to be largely due to mechanical disruption of spore coat and inner and outer membranes, as evidenced by DPA release, increased susceptibility to lysozyme and H₂O₂ (indicative of breakage of disulfide bonds in the spore coat), and membrane permeability as assessed by spore staining and fluorescence microscopy. No differences were seen in germination between MP-HHP and S-HHP. There was no evidence of any loss of cortex lytic enzymes or degradation of small acid-soluble proteins (SASPs) during both MP-HHP and S-HHP treatments.     

Oscillatory Compared with Continuous High Pressure Sterilization on Bacillus stearothermophilus Spores

TO ESTABLISH a sterilization method with minimal heating, the effect of high pressure on bacteriostasis was studied using thermoduric spores of Bacillus stearothermophilus. After exposure to 800 MPa for 60 min at 60°C, the spore count decreased from 10⁶ to 10²/mL. However, exposure to the same pressure at room temperature did not cause significant change in spore numbers. The synergistic effect of high pressurization on the bacteriostatic action of sucrose fatty acid ester at low concentration (< 10 ppm) was pronounced with sucrose palmitic acid ester but not with sucrose stearic acid ester. Oscillatory pressurization was more effective for spore sterilization. Six cycles oscillation of 5-min pressurization with 400 MPa at 70°C decreased the spore count from 10⁶ to 10²/mL, and with 600 MPa, complete sterilization was achieved.     

Kinetics of Clostridium sporogenes PA3679 Spore Destruction Using Computer-Controlled Thermoresistometer

A modified version of a computer-controlled thermoresistometer was used, with and without micropurge, to study the inactivation kinetics of Clostridium sporogenes PA 3679 spore destruction between 121–143°C in phosphate buffer (pH 7) and in mushroom extract acidified with citric acid. A shorter temperature come up time was observed with micropurge. The thermal death time (TDT) curve for spores in phosphate buffer with micropurge followed a straight line (z = 9.5°C). Without micropurge the curve could be described by two lines with z = 10.0 °C for temperatures up to 132.5 °C and z = 18.3 °C for higher temperatures. The spore heat resistance in mushroom extract was lower than in phosphate buffer. D_T. values decreased exponentially as temperature increased, but acidification did not reduce thermal resistance at high temperatures.     

Elucidation of the Mechanism of the Acid-blanch and EDTA Process Inhibition of Clostridium sporogenes PA 3679 Spores

Acid-blanching followed by addition of ethylenediamine tetraacetic acid to the can brine had been shown to control germination of mesophilic spores following sub-lethal thermal processes. The mechanism of this control was investigated using C. sporogenes PA 3679 spores in buffered solutions. Spore suspensions in buffered 0.05M citric acid solutions, pH 3.5 and 5.8, and Sorensons phosphate buffer, pH 7.0, were heat-activated at 80 °C and then heated for 5 min at 121 °C. The mechanism of inhibition appeared to involve destruction of the spore germination system by heat at reduced pH as a result of collapse of the cortex peptidoglycan layers, possible hydration of the core and concommitant increase in the core volume.     

Fate of Clostridium sporogenes PA 3679 in Fish Sausage Formulations with Adjusted aw using Food Binders

Fish sausage from minced Spanish mackerel was formulated to a_w of 097–0.92 using egg white solids alone and in combination with nonfat dry milk, alginate and soy protein isolate, without and with nitrite added at 150 ppm. The batter was inoculated with PA 3679 spores at ca. 10⁵/g, chopped, stuffed in casing, cooked to a core temperature of 85°C, vacuum-packaged and stored at 25°C for 60 days. The test organism did not reproduce in formulations having a_w < 0.95. Total counts decreased rapidly and spore counts were nil at the end of storage. Spores of PA 3679 germinated at a_w as low as 0.924 but the cells failed to grow. Products with a_w > 0.95 spoiled. Product stability was influenced by a_w regardless of binder type and presence of nitrite.     

Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.     

Elevation of heat resistance of Clostridium sporogenes following heat shock

Heat shock of strains of spores of Clostridium sporogenes at 80 or 100°C increased their apparent resistance to heating at 121·1°C, with the exception of NCIB 10696. The presence of glucose (0·5 – 1·0%) in the sporulation medium reduced the percentage of spores which could be activated by heat. Within individual spore populations, some spores required more heat than others to induce activation. Spores of PA 3679 remained activated for 18 months at 4°C and heat shock did not alter the z value. Experiments using dimethylglutaric acid/NaOH buffer suggest that heat shock in acid conditions has maximum effect on heat resistance.     

Temperature, pH, and Spore Load Effects on the Ability of Nisin to Prevent the Outgrowth of Clostridium botulinum Spores

The effectiveness of nisin in preventing the outgrowth of spores of Clostridium botulinum types A, B, and E in TPYG broth was profoundly affected by pH, temperature of heat-shocking, length of the heat-shocking period, and spore load. Nisin was considerably more effective at pH 6 than at either pH 7 or pH 8 in limiting the outgrowth of all six tested strains. Heat-damaged spores were also more sensitive to nisin. Both higher heat-shocking temperatures in the range 20-30°C higher than the optimal heat-shocking temperatures for the particular strain and longer heat-shocking periods served to lower the levels of nisin required to inhibit spore outgrowth. Nisin was more effective against spore loads of 10²spores/ml. than higher spore loads of 10³ or 10⁴ spores/ml with all of these variables taken into consideration, the order of sensitivity of the spores of the various strains of C. botulinum was strain 56A < strain 69A < strain 113 B = strain 213 B < strain Beluga E < strain Minnesota E     

HIGH PRESSURE–HIGH TEMPERATURE STERILIZATION: FROM KINETIC ANALYSIS TO PROCESS VERIFICATION†

The inactivation of Clostridium sporogenes PA 3679 spores by high pressure at high temperatures (HP–HT) in phosphate buffer was investigated in a lab‐scale temperature‐controlled HP system (QFP‐6) with an internal heater to maintain the sample temperature. Some inactivation of spores occurred during the pressurization come‐up time (CUT) and depressurization time. The inactivation of PA 3679 was found to be exponential during the adiabatic holding period of the HP cycle at constant pressures and temperatures. The inactivation rate increased with both pressure and temperature. The kinetic parameters – such as D‐values at tested temperatures and pressures that are necessary for the design of process parameters of HP sterilization process – were determined. Within the pressure range of 600–800 MPa, the calculated D‐values ranged from 270.3 to 357.4 and 49.0 to 67.6 s at 91 and 108C, respectively. These studies provided basic data on the effects of pressure and temperature on the inactivation of PA 3679 spores under conditions applicable to the development of preservation specifications for commercial HP–HT processing of low acid foods. The spore strips of C. sporogenes were used as indicators for microbiological verification of delivered lethality of HP–HT sterilization process at different processing conditions in a pilot scale HP vessel.     

Application of High Pressure for Spore Inactivation and Protein Denaturation

Our objective was to develop a method of sterilizing Bucillur stearothermophilus spores with minimal heating. Inactivation was achieved by spore destruction through six cycles of oscillatory pressurization at 70°C and 600 MPa, but inactivation was not complete within 60-min with continuous pressurization. Four pressurization cycles at 600 MPa and 5 min/cycle decreased the spore count from 10⁶ to 10²/mL, and six cycles decreased the count from 10⁶ to <10⁰. Spore inactivation was dependent on the adiabatic expansion velocity. On the other hand, protein denaturation by high pressures was due to phase changes of water under high pressure, and was not affected by adiabatic expansion velocity.     

Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage

High hydrostatic pressure represents an attractive non-thermal process for meat products to avoid post-processing contamination. When combined with antimicrobials, like bacteriocins, the death rate may be increased because of sub-lethal injuries to living cells. The behaviour of several foodborne bacteria inoculated in a meat model system with added bacteriocins (enterocins A and B, sakacin K, pediocin AcH or nisin) after pressurization (400 MPa, 10 min, 17°C) and during chilled storage was investigated. Although Staphylococcus was the genus least sensitive to pressurization, the samples including nisin displayed lower and significantly different counts during the 4°C storage than the rest of the treatments. A greater inactivation of Escherichia coli (>6 log₁₀) in the presence of nisin was recorded, the number of survivors remained unchanged during storage at 4°C for 61 days. Nisin was also the bacteriocin capable of maintaining slime-producing lactic acid bacteria below the detection limit (<10² cfu g⁻¹ ). Listeria monocytogenes in treatments with sakacin, enterocins or pediocin was kept <10² cfu g⁻¹ till the end of storage (61 days). Salmonella enterica subsp. enterica ser London and Salmonella enterica subsp.enterica ser Schwarzengrund counts in every treatment were kept at the level obtained after pressurization, with no significant differences between treatments during the chilled storage.     

Synergistic Processing of Skim Milk with High Pressure Nitrous Oxide,Heat, Nisin,and Lysozyme to Inactivate Vegetative and Spore-Forming Bacteria

Individual and combined effects of high pressure nitrous oxide (HPN₂O), heat, and antimicrobials on the inactivation of Escherichia coli, Listeria innocua, and Bacillus atrophaeus endospores in milk were all evaluated after 20-min treatments. Stand-alone milk treatments with HPN₂O (15.2 MPa), heat (45 and 65 °C), or nisin (50 and 150 IU mL^?1) resulted in log₁₀ reductions ranging only from 0.1 to 2.1 for E. coli and L. innocua. Combining HPN₂O (15.2 MPa) with heat (65 °C) inactivated 6.0 and 5.1 log₁₀ in the vegetative bacteria, respectively. Similarly, reductions of 5.9 and ≥ 6.0 log₁₀ of respective E. coli and L. innocua cells in milk were achieved through a combination of HPN₂O (15.2 MPa), heat (65 °C), and nisin (150 IU mL^?1). A 2.5 log₁₀ cycle inactivation of spores was obtained by HPN₂O, nisin (at both 50 and 150 IU mL^?1), and lysozyme (50 μg mL^?1) at 85 °C. Combining these processing techniques resulted in significantly greater microbial inactivation (p < 0.05) than the sum of individual reductions from each treatment alone, indicating synergistic effects. HPN₂O irrespective of processing temperatures did not cause any occurrence of sub-lethally injured cells or disruption in colloidal stability of milk at 65 and 85 °C (p ≥ 0.05). Color and pH changes in milk following the most demanding treatment conditions were minimal.     

Application of electro-activated potassium acetate and potassium citrate solutions combined with moderate heat treatment on the inactivation of Clostridium sporogenes PA 3679 spores

Combined effects of moderate temperatures and the electro-activated aqueous solutions of potassium acetate and potassium citrate on the inactivation of C. sporogenes PA 3679 spores (D_121°C = 1.18 min) were studied. Four types of solutions (potassium acetate with/without KCl and potassium citrate with/without KCl) were activated at 400 mA for 60 min. The oxidation reduction potential (ORP) and pH values ranged from + 417.50 to + 1043.33 mV and 3.18 to 3.47, respectively. The combination of these solutions with a moderate heat treatment (95 °C, 105 °C, and 115 °C) for different time (5, 10, 20, and 30 min) was sufficient to reach a 100% of spore destruction (inactivation) in a medium with an initial contamination level comprised between 7.0 and 7.8 log CFU/mL. The sporicidal effect of solutions was also present even if activated solutions were applied alone against spores without being combined with heat treatment. Spore morphology was determined under transmission electron microscopy and showed that there were important damages, such as rupture of spores and release of spore components in all of the treated spores. Thus, the sporicidal effect detected was the result of inactivation mechanisms of electro-activated solutions on spores. In almost all of observed micrographs, there were coreless spores, deformed spores, or debris of spores. The current investigation can be used for achieving further studies in order to better understand the mechanisms of inactivation of C. sporogenes spores by electro-activated solutions.Industrial relevanceThis research article aims to study the combined effect of electro-activated potassium acetate and citrate solutions and moderate heat treatment on the viability of Clostridium sporogenes in model solutions as a non-pathogenic surrogate of Clostridium botulinum. The objective was to use hurdle technology to produce nutritious, minimally processed foods while ensuring food safety. Moreover, this approach allowed for a reduced level of sodium in canned foods since the solutions were sodium-free.     

Ozone-high hydrostatic pressure synergy for the stabilization of refrigerated pitaya (Stenocereus pruinosus) juice

The synergistic effect of ozone (24 mg/O₃/L/min) and high hydrostatic pressure (HHP, 179–321 MPa) combination was evaluated with regard to individual effects of these over the microbial population in pitaya juice by using a response surface methodology and a repeated measures design with Tukey test. Treatment with 300 MPa/5 min reduced 6.89-log₁₀ and 0.89-log₁₀ CFU/mL of Saccharomyces cerevisiae and Listeria innocua population, respectively; and treatment with 9.6 min of ozonation time reduced 0.47-log₁₀ and 2.51-log₁₀ CFU/mL of S. cerevisiae and L. innocua population respectively; however, L. innocua was reduced 5.1-log₁₀ CFU/mL with exposure to ozone for 7 min followed by 316 MPa/5 min. Likewise, these treatment conditions kept native microbiota of the juice at non-detectable levels and achieved the highest sensory preference (79%) as compared to untreated juice at 30 d (5 ± 2 °C). Ozone-HHP reduced bacterial counts suggesting a microbiologically safe pitaya juice, contrary to what was obtained with individual application of these technologies.     

INCREASED INACTIVATION OF BACTERIAL SPORES AT HIGH TEMPERATURES IN THE PRESENCE OF MONOGLYCERIDES12

The presence of monolauryl-glyceride (monolaurin) in the heating menstruum (phosphate buffer, reconstituted non-fat dry milk or cream style corn) of Bacillus stearothermophilus 1518 spores increased the rates of spore inactivation at 113–121°C by 2–3 fold. The ZD-values for the spores heated with and without monolaurin were 8.7 and 6.9°C, respectively. Rates of inactivation of B. subtilis A spores also were enhanced, but spores of Clostridium perfringens NCTC 879B, C. sporogenes PA 3679, C. botulinum 62A, or C. botulinum 213B were unaffected. Increasing the concentration of monolaurin from 0.4 mM to 3.6 mM increased the rate of inactivation, but concentrations higher than 3.6 mM did not appear to influence the effectiveness of monolaurin. All monoglyerides containing C8-C16 saturated fatty acids appeared to increase the inactivation of B. stearothermophilus spores, but the enhancement of spore inactivation declined as the fatty acid chain length increased beyond 10 carbon atoms. The influence of monolaurin on B. stearothermophilus spores did not appear to be pH dependent over the range of pH 6 to 8. The spore-monolaurin interaction was not due to heat-injury of the spores, although the interaction was dependent on the heat treatment. The increased inactivation appeared to be due to a heat enhanced chemical inactivation of the spores.     

Interacting Effects of pH and NaCl on Heat Resistance of Bacterial Spores

Spores of PA3679 (C. sporogenes) and Clostridium botulinum 213B were less heat resistant when heated in a menstruum of decreasing pH. The D value of the spore population decreased by 50% when heated in pH 5.0 buffer compared to pH 7.0 buffer. Addition of NaCl to the recovery medium reduced the number of colony forming units in a population of heated spores. Presence of NaCl at 2.0% in the recovery medium decreased the spore D-value by 20-40% irrespective of pH of the heating menstruum. Combined effects of pH and NaCl could be illustrated by three-dimensional histograms.     

Microbiological quality of mechanically recovered poultry meat treated with high hydrostatic pressure and nisin

The combined effect of high hydrostatic pressure processing, addition of nisin and acidification on aerobic mesophilic and psychrotrophic bacterial populations of mechanically recovered poultry meat (MRPM) kept under refrigeration (2°C) was evaluated 1, 15 and 30 days after pressurization. Nisin (0, 12.055, 100 and 200 ppm) and glucono-delta-lactone (GdL; 0 and 1%) were added to MRPM. Vacuum-packaged samples were treated at 350 or 450 MPa and 2°C for 15 min using both continuous pressurization and three-cycle oscillatory pressurization for 5 min per cycle. In some samples a reduction of mesophile counts between 3.0544 and 5.0538 log cfu g⁻¹was found. Psychrotrophes seemed to be more sensitive; in samples with 100 ppm of nisin and GdL treated at 450 MPa with cycles they were reduced to undetectable levels (a lethality of approximately 7.055 log units). Cycle pressurization showed slightly better results than continuous pressurization. The combination of 350 MPa, 100 ppm of nisin and 1% of GdL was enough to extend the shelf life of MRPM during the 30 day chilled storage.     

Growth inhibition and inactivation of Alicyclobacillus acidoterrestris endospores in apple juice by hyperbaric storage at ambient temperature

Control of endospores of Alicyclobacillus acidoterrestris in pasteurized apple juice using hyperbaric storage at 18 to 23 °C was compared to storage at atmospheric pressure and 18 to 23 °C, as well as refrigeration at ~4 °C for up to 30 days. The juice samples were inoculated with approximately 1 × 10⁵ CFU/mL spores. The juice spoiled quickly at atmospheric pressure and ambient temperature, while under refrigeration spore levels remained unchanged for 30 days. Hyperbaric storage of inoculated apple juice at 25, 50 and 100 MPa at 18 to 23 °C resulted in spore inactivation at more rapid rates as pressure magnitudes increased, reaching levels below the detection limit of 10 CFU/mL at 50 and 100 MPa. In highly acid foods such as apple juice, hyperbaric storage at pressures ≤100 MPa and ambient temperature was effective in inactivating spores of A. acidoterrestris for periods up to 30 days.These results indicate hyperbaric storage at ambient temperature as a clearly more efficient preservation procedure to control the development of A. acidoterrestris endospores, compared to ambient temperature and refrigerated storage, in highly acidic foods as apple juice.