Inactivation of Listeria innocua and Escherichia coli in carrot juice by combining high pressure processing,nisin, and mild thermal treatments

This study explored the effectiveness of high pressure (200–500 MPa) alone or in combination with mild thermal treatments (35 and 50 °C) and nisin (25 and 50-ppm) on the inactivation of L. innocua and E. coli in carrot juice. Processing at 500 MPa at 20 °C for 2 min without nisin resulted in 4- and 5-log CFU/mL reduction of L. innocua and E. coli, respectively while incorporating 25-ppm nisin at same pressure and temperature rendered 7-log CFU/mL reduction. There was synergism between high pressure, nisin, and heat in all treatments to inactivate both microorganisms. After a 28-d of refrigerated storage, total plate counts were <2-log CFU/mL in carrot juice treated with combination of 300 MPa and 25-ppm nisin at 35 °C. All combinations resulted in less intense use of pressure, i.e. more energy efficient, cost effective processes while attaining high quality juices. The results of this study suggest that by using selected combinations of high pressure, nisin and mild temperatures, safe, clean-label, high-quality juices can be produced.Industrial relevanceThe results from this study show a synergistic effect on the inactivation of L. innocua and E. coli in carrot juice from the combined application of HPP, nisin, and mild temperatures. By replacing the use of HPP alone by these combinations will allow the use of reduced pressures over shorter period of times to process low-acid juices, lowering energy requirements and increasing throughput. This study will aid the beverage processing industry in the development of clean label juice products with fresh-like quality attributes and using considerable less energy to conventional processing.     

Inactivation of Escherichia coli by high hydrostatic pressure at different temperatures in buffer and carrot juice

The inactivation of Escherichia coli MG1655 was studied at 256 different pressure (150-600 MPa)-temperature (5-45 degrees C) combinations under isobaric and isothermal conditions in Hepes-KOH buffer (10 mM, pH 7.0) and in fresh carrot juice. A linear relationship was found between the log10 of inactivation and holding time for all pressure-temperature combinations in carrot juice, with R2-values>or=0.91. Decimal reduction times (D-values), calculated for each pressure-temperature combination, decreased with pressure at constant temperature and with temperature at constant pressure. Further, a linear relationship was found between log10D and pressure and temperature. A first order kinetic model, describing log10D in carrot juice as a function of pressure and temperature was formulated that allows to identify process conditions (pressure, temperature, holding time) resulting in a desired level of inactivation of E. coli. For Hepes-KOH buffer, the Weibull model more accurately described the entire set of inactivation curves of E. coli MG1655 compared to the log-linear or the biphasic model. Several secondary models (first and second order polynomial and Weibull) were evaluated, but all had poor fitting capacities. When the Hepes-KOH dataset was limited to 22 of the 34 pressure-temperature combinations, a first order model was appropriate and enabled us to use the same model structure as for carrot juice, for comparative purposes. The major difference in kinetic behaviour of E. coli in buffer and in carrot juice was that inactivation rate as a function of temperature showed a minimum around 20-30 degrees C in buffer, whereas it increased with temperature over the entire studied temperature range in carrot juice.     

Modeling survival of high hydrostatic pressure treated stationary- and exponential-phase Listeria innocua cells

High Hydrostatic Pressure (HHP) inactivation (325–400 MPa; 0–20 min; maximum temperature 30 °C) of cells of Listeria innocua CECT 910 was studied in two different growth phases (exponential and stationary), and the corresponding survival curves were obtained for each case. The curves were fitted to two nonlinear models, the modified Gompertz equation and the Baranyi model. The kinetic constants calculated for both models, µ_max and k_max, indicated that cells in exponential growth phase were more sensitive to pressure than those in stationary phase. Both mathematical models were suitable for describing L. innocua HHP survival curves, rendering kinetic constants that increased with increasing pressure. When considering the experimental models validation, both Gompertz and Baranyi predicted in a similar way, however Baranyi had slightly lower A_f (Accuracy factor) and B_f (Bias factor) values, which indicated better prediction values. In summary, both mathematical models were perfectly valid for describing L. innocua inactivation kinetics under HHP treatment.Industrial relevanceThe mathematical models for inactivation and growth of microorganisms are the foundation of predictive microbiology and are used in risk assessments procedures as part of the food safety management system. Besides, these models together with those applied to inactivation of enzymes and destruction of quality factors are essential to optimize processes and thus to lay the foundations for industrial processing. It is therefore necessary to identify generally applicable kinetic models that will produce primary and secondary kinetic parameters and are statistically reliable as a key tool to predict the behaviour of microorganisms, enzymes and quality factors after processing.     

Inactivation kinetics of three Listeria monocytogenes strains under high hydrostatic pressure

High hydrostatic pressure (HHP) inactivation of three Listeria monocytogenes strains (EGDe, LO28, and Scott A) subjected to 350 MPa at 20 degrees C in ACES buffer resulted in survival curves with significant tailing for all three strains. A biphasic linear model could be fitted to the inactivation data, indicating the presence of an HHP-sensitive and an HHP-resistant fraction, which both showed inactivation according to first-order kinetics. Inactivation parameters of these subpopulations of the three strains were quantified in detail. EGDe showed the highest D-values for the sensitive and resistant fraction, whereas LO28 and Scott A showed lower HHP resistance for both fractions. Survivors isolated from the tail of LO28 and EGDe were analyzed, and it was revealed that the higher resistance of LO28 was a stable feature for 24% (24 of 102) of the resistant fraction. These HHP-resistant variants were 10 to 600,000 times more resistant than wild type when exposed to 350 MPa at 20 degrees C for 20 min. Contrary to these results, no stable HHP-resistant isolates were found for EGDe (0 of 102). The possible effect of HHP survival capacity of stress-resistant genotypic and phenotypic variants of L. monocytogenes on the safety of HHP-processed foods is discussed.     

Listeria innocua Multi-target Inactivation by Thermo-sonication and Vanillin

Hurdle technology combining an emerging preservation technique such as low-frequency ultrasound is an alternative for processing juices that are susceptible to suffer a loss of quality due to traditional heat treatments. Predictive microbiology allows evaluation of the effectiveness of preservation techniques and its combinations in order to enhance both food quality and safety. Listeria innocua inactivation by thermo-sonication along with vanillin was investigated. Fermi model (R ² _adj= 0.970 ± 0.02) and surface response methodology (p < 0.05) were utilized in order to evaluate the survival of L. innocua to a multi-target treatment and to predict the interactions of studied techniques, high-intensity/low-frequency ultrasound (20 kHz/400 W) at selected wave amplitudes (60, 75, or 90 μm), temperature (40, 50, or 60 °C), and vanillin (200, 350, or 500 mg/kg). A combination of ultrasound, vanillin, and temperature enhanced L. innocua inactivation as described by Fermi parameters a and t _c, which decreased as the studied effects increased. A multi-target inactivation effect was observed for a temperature range of 45–55 °C.     

Inactivation of high pressure resistant Escherichia coli by lysozyme and nisin under high pressure

We studied the inactivation (by high hydrostatic pressure at 20°C) of Escherichia coli MG1655 and the selected pressure-resistant mutants that were derived previously from this strain, and which are the most pressure-resistant vegetative cells described to date. The natural antimicrobial peptides, lysozyme (50 μg/ml) and nisin (100 IU/ml), enhanced considerably the inactivation of the target bacteria under pressure. However, kinetic inactivation experiments in the presence of these compounds revealed pronounced tailing, which limited the level of inactivation that could be achieved under mild conditions of pressure and temperature. Interrupted pressure treatments enhanced the effectiveness of lysozyme and nisin, allowing a reduction by at least 6 logs of all strains at 400 MPa. A hypothetical mechanism of ‘pressure-promoted uptake’ is proposed to explain E. coli outer membrane permeabilization for lipophilic and cationic peptides like lysozyme and nisin under pressure.     

Antimicrobial activity of essential oils on Salmonella enteritidis, Escherichia coli, and Listeria innocua in fruit juices

The antimicrobial properties of essential oils (EOs) and their derivatives have been known for years. However, the information published about the minimal effective concentration of EOs against microorganisms in fruit juices is scarce. In this study, both MIC and MBC of six EOs (lemongrass, cinnamon, geraniol, palmarosa, or benzaldehyde) against Salmonella Enteritidis, Escherichia coli, and Listeria innocua were determined by the agar and broth dilution methods, respectively. All of the six EOs inhibited the microbial (Salmonella Enteritidis, E. coli, and L. innocua) growth at a concentration from 1 microl/ ml (MIC). These studies led to choosing the three most effective EOs. Lemongrass, cinnamon, and geraniol were found to be most effective in inhibiting the growth of the microorganisms and thus were used for the MBC analysis. On this last point, significant differences (P < 0.05) among EOs, their concentrations, and culture media (apple, pear, and melon juices, or tryptone soy broth medium) were found after comparing the results on MBC for each microorganism. A concentration of 2 microl/ml from lemongrass, cinnamon, or geraniol was enough to inactivate Salmonella Enteritidis, E. coli, and L. innocua in apple and pear juices. However, in melon juice and tryptone soy broth medium, concentrations of 8 and 10 microl/ml from cinnamon, respectively, or 6 microl/ml from geraniol were necessary to eliminate the three microorganisms, whereas lemongrass required only 5 micro/ml to inactivate them. These results suggest that EOs represent a good alternative to eliminate microorganisms that can be a hazard for the consumer in unpasteurized fruit juices. The present study contributes to the knowledge of MBC of EOs against pathogenic bacteria on fruit juices.     

Inactivation of pathogenic Escherichia coli in skimmed milk using hhigh hydrostatic pressure

The pressure resistance of a range of pathogenic Escherichia coli strains was determined in skimmed milk. Pressure resistances varied widely among the 12 strains tested, with the two most resistant being an enterotoxigenic E. coli (NCTC 11601) and an enteroinvasive E. coli (NCTC 9706). A pressure treatment of 500 MPa for 40 min gave only a 4-log reduction of these two most resistant strains, but after a treatment of 600 MPa for 30 min, no survivors of either strain could be detected (>7 log reduction). The effect of the length of time in the stationary phase on the pressure resistances of these two strains was determined. Pressure resistance (600 MPa for 10 min at 20°C) was determined after 12 h incubation at 37°C in tryptone soya broth with 0.6% yeast extract added (TSBYE) (early stationary phase) and at 24-h intervals after this. The pressure resistances of these strains did not change significantly for the first 5 days after the onset of the stationary phase, after which time their resistances decreased. When cells were pressure-treated 7 days after they went into stationary phase, no survivors could be detected.     

Effect of high hydrostatic pressure on Listeria innocua 910 CECT inoculated into Ewe's milk.

Ewe's milk standardized to 6% fat was inoculated with Listeria innocua 910 CECT at a concentration of 10(7)CFU/ml and treated by high hydrostatic pressure. Treatments consisted of combinations of pressure (200, 300, 350, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). To determine numbers of L. innocua, listeria selective agar base with listeria selective supplement and plate count agar was used. Low-temperature (2 degrees C) pressurizations produced higher L. innocua inactivation than treatments at room temperatures (25 degrees C). Pressures between 450 and 500 MPa for 10 to 15 min were needed to achieve reductions of 7 to 8 log units. The kinetics of destruction of L. innocua were first order with D-values of 3.12 min at 2 degrees C and 400 MPa and 4 min at 25 degrees C and 400 MPa. A baroprotective effect of ewe's milk (6% fat) on L. innocua was observed in comparison with other studies using different media and similar pressurization conditions.     

Process Parameters Affecting Listeria innocua Inactivation by Pulsed Light

The impact of several processing parameters on the effectiveness of pulsed light (PL) technology was investigated. The distribution of the light on the surface of the PL treatment chamber and the fluence striking on the sample under different treatment conditions were evaluated. Higher pulse fluences were registered when input voltage increased and the distance from the sample to the light source decreased. Distance from the sample to the xenon lamp also played a major role in the light distribution onto the surface of the treatment zone. Whereas quartz shelf placed close to the xenon lamp resulted in a non-uniform distribution of fluence, a homogeneous light distribution was found when increasing the distance to the flash lamp. Moreover, the impact of several PL processing parameters on microbial inactivation was investigated. Neither pulse voltage nor total voltage determined the antimicrobial effectiveness of PL technology in the operating range tested. Reduction in Listeria innocua counts increased with the number of pulses and the pulse fluence, being total fluence reaching the samples the most relevant process factor affecting microbial inactivation by PL. Considering the importance of this processing parameter, an accurate quantification of the fluence striking on the sample will be needed in order to design effective PL treatments.     

Inactivation of Listeria innocua in milk and orange juice by ultrahigh-pressure homogenization

The aim of this work was to evaluate the bactericidal efficacy of ultrahigh-pressure homogenization (UHPH) against Listeria innocua ATCC 33090 inoculated into milk and orange juice. We also intended to study the effect of inlet temperature on the lethality and production of sublethal injuries in this microorganism and its ability to survive, repair, and grow in refrigerated storage after UHPH treatment. Samples of ultrahigh-temperature whole milk and ultrahigh-temperature orange juice inoculated at a concentration of approximately 7.0 log (CFU per milliliter) were immediately pressurized at 300 MPa on the primary homogenizing valve and at 30 MPa on the secondary valve, with inlet temperatures of 6.0 +/- 1.0 degrees C and 20 +/- 1.0 degrees C. L. innocua viable counts and injured cells were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4 degrees C for milk and after 3, 6, 9, 12, 15, 18, and 21 days of storage at 4 degrees C for orange juice. Both the inlet temperature and the food matrix influenced significantly (P < 0.05) the inactivation of L. innocua, which was higher in whole milk at the 20 degrees C inlet temperature. The UHPH treatment caused few or no sublethal injuries in L. innocua. During storage at 4 degrees C after treatments, counts increased by approximately 2 logarithmic units from day 0 to 9 in whole milk, whereas in orange juice counts diminished by approximately 2.5 logarithmic units from day 0 to 18.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.     

Inactivation of avian influenza virus by heat and high hydrostatic pressure

Avian influenza viruses threaten the life of domestic terrestrial poultry and contaminate poultry meat and eggs. Recently, these viruses rarely infected humans but had a high mortality rate in Southeast Asia, the Middle East, and Egypt. Thereby, these viruses caused high economic costs for production of poultry and health protection. We inactivated a highly pathogenic avian influenza A virus of subtype H7N7 in cell culture medium and chicken meat by heat and high hydrostatic pressure. Because heat and pressure inactivation curves of the H7N7 virus showed deviations from first-order kinetics, a reaction order of 1.1 had to be selected. A mathematical inactivation model has been developed that is valid between 10 and 60 degrees C and up to 500 MPa, allowing the prediction of the reduction in virus titer in response to pressure, temperature, and treatment time. Incubation at 63 degrees C for 2 min and 500 MPa at 15 degrees C for 15 s inactivated more than 10(5) PFU/ml, respectively. Thus, we suggest high-pressure treatment of poultry and its products to avoid the possible health threat by highly pathogenic avian influenza viruses.     

Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices

Listeria monocytogenes was subjected to ultra high hydrostatic pressure (UHHP) treatments from 200 to 700 MPa at 25 °C in broth, raw milk, peach juice and orange juice. Survivor curves showed that cell death increased as pressure increased. After 10 min pressure treatment at 400 MPa reductions of about 2.09 and 2.76 log CFU mL^?1 in aerobic bacteria and L. monocytogenes, respectively, were produced in raw milk, this increased to 5.09 and 6.47 log CFU mL^?1, respectively, at 600 MPa. Death of bacteria at UHHP treatment was greater in orange juice than peach juice, and in peach juice than milk. Listeria monocytogenes was more sensitive to increased pressure than increased pressurization time. Injury of L. monocytogenes occurred from 0 to 100%. Factors effecting the rate of microbial inactivation are: pressure, age of cell, composition of medium, and pressurization time. UHHP inactivation can be used to extend shelf life and increase food quality during storage, and may also contribute to inactivation of L. monocytogenes.     

Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure

A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), which had been developed previously for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeasts. Fluorescence intensities of the yeast population were measured by fluorescence spectrophotometry upon exposure to antifungal agents after staining with 2-NBDG and were compared to the number of colony forming units (CFU). A good correlation was obtained between the yeast viability, determined by the CFU, and the accumulation of 2-NBDG by yeast cells (correlation constant: r=0.98). Susceptibility testing of amphotericin B and miconazole against yeast strains by plate count and 2-NBDG fluorescence method yielded corresponding results. In conclusion, we found that staining with 2-NBDG is a rapid and sensitive method for the assessment of yeast cell viability.     

Inactivation of Listeria monocytogenes by high hydrostatic pressure: effects and interactions of treatment variables studied by analysis of variance

High hydrostatic pressure is regarded as possible alternative method for food preservation. One of the primary considerations for industrial applications is the ability of this method to eradicate pathogenic micro-organisms. This study subjected L. monocytogenes suspensions, in a phosphate buffer (pH 7·0) or in a citrate phosphate buffer (pH 5·6), to high hydrostatic pressure treatments relative to the following variables: pressure (200–600 MPa), treatment time (3, 10 and 20 min), temperature (4, 20 and 40°C) and the pH of the suspension medium (5·6 and 7·0). An optimal design of 40 runs was obtained using the Fedorov algorithm, and responses were studied by analysis of variance in terms of cell survival on plate count agar. Efficiency was determined by log₁₀comparisons of the numbers of live cells before and after treatment. A statistically significant relationship was found between the four variables considered (pressure, pH, treatment time and temperature), their interactions (treatment time vs pressure, pH vs treatment time, pH vs pressure, pressure vs temperature, treatment time vs temperature) and the inactivation of L. monocytogenes. R -squared statistical analysis indicated that the linear model used accounted for more than 98·5% of the variability in the inactivation ofL. monocytogenes .     

Multi-pulsed high hydrostatic pressure treatment for inactivation and injury of Escherichia coli

Escherichia coli cells in peptone water were pressurized at 300?MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300?s for single- (300?s?×?1 pulse) and multi-pulsed (150?s?×?2 pulses, 100?s?×?3 pulses, 75?s?×?4 pulses, 60?s?×?5 pulses, 50?s?×?6 pulses and 30?s?×?10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log₁₀ more reduction on selective medium than the non-selective medium indicating that more than 95?% of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4?°C revealed that there was less recovery from injury for multi-pulsed HHP treatment.     

Inactivation ofEscherichia coliinoculated in liquid whole egg by high hydrostatic pressure

The resistance ofEscherichia coliin liquid whole egg was studied at several pressures (300, 350, 400 and 450MPa), temperatures (50, 20, 2 and –15°C) and times (5, 5+5, 10, 5+5+5, 15min). The highest reduction was obtained at 50°C (about 7log₈units). At 20 and –15°CE. coliwas more resistant to pressure than at 50 and 2°C. The intermittent treatments were more effective than continuous treatments at lower pressures (350MPa). The destruction increases upon increasing the pressure and the time treatment. Survivor curves were studied at 400MPa for two temperatures (20 and 2°C) and different times (0–60min), obtaining a decimal reduction time of 14.1min at 20°C and 9.5min at 2°X.     

Effect of hydrostatic pressure and antimicrobials on survival of Listeria monocytogenes and enterohaemorrhagic Escherichia coli in beef

The effects of marinades on Listeria monocytogenes and enterohaemorrhagic Escherichia coli were investigated in pressure treated beef steaks. Meat was treated with 600 MPa or 450 MPa. Marinades did not enhance pressure inactivation of E. coli in beef steaks and marinades prevented pressure-induced sublethal injury in L. monocytogenes. Membrane-active essential oils carvacrol and thymol, and thiol-reactive allyl-isothiocyanate (AITC) and cinnamaldehyde were selected to investigate potential synergistic activity of clean label antimicrobials with pressure. Carvacrol accelerated pressure inactivation of E. coli in beef steaks; however, carvacrol increased pressure resistance of E. coli in buffer, and had no effect on survival of E. coli in ground beef. Thymol had no effect in either buffer or meat. AITC and cinnamaldehyde exhibited synergistic activity with pressure on E. coli in buffer; however, cinnamaldehyde did not affect survival of E. coli after pressure treatment of meat. Synergistic inactivation of AITC with pressure was observed only at concentrations that are negatively affect meat quality. AITC and carvacrol may be practically applied for enhancing the bacterial inactivation and extending the shelf life of beef steaks.