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.     

Inactivation of a calicivirus and enterovirus in shellfish by high pressure

This study demonstrates the potential of high pressure (HP) processing to reduce viral contamination in shellfish. Bovine enterovirus, which is structurally similar to hepatitis A virus, was more pressure-resistant than feline calicivirus, a surrogate for norovirus. Both viruses were more pressure-resistant when treated in “naturally” contaminated mussels and oysters, compared to seawater and culture medium, suggesting that the medium can have a significant protective effect against HP treatment. Treatment at pressures of 250 MPa showed only a limited inactivation of either virus in shellfish, suggesting that relatively mild HP treatments (approximately 260 MPa) currently used for the commercial processing of oysters, principally to assist the shucking process, may be insufficient to ensure the safety of shellfish for human consumption, particularly in relation to human pathogenic viruses.Industrial relevanceHigh pressure (HP) processing is increasingly being used in the commercial processing of oysters, principally to assist the opening or shucking of oysters. Little is known about the effect of HP treatment on pathogens in shellfish, particularly human enteric viruses, which are the predominant cause of shellfish-borne disease. This article demonstrates the inactivation of surrogate animal viruses, as potential models for noroviruses and hepatitis A virus, by HP processing and compares the efficacy of inactivation when viruses are treated in culture medium, seawater and shellfish. The potential of HP processing to reduce viral contamination in shellfish is discussed in light of these findings.     

Inactivation of hepatitis A virus,poliovirus and a norovirus surrogate by high pressure processing

Hepatitis A virus (HAV), feline calicivirus (FCV, a surrogate for non-culturable norovirus), and poliovirus (PV), suspended in buffered cell culture media, were treated with pressures ranging from 200 to 600 MPa at ambient temperature for between 30 and 600 s. HAV was inactivated by > 1-log₁₀ tissue culture infectious dose 50% mL^− 1 (TCID₅₀ mL^− 1) and > 2-log₁₀ TCID₅₀ mL^− 1 after 600 s treatment with 300 and 400 MPa, respectively, and was undetectable (> 3.5-log₁₀ TCID₅₀ mL^− 1 reduction) within 300 s treatment with 500 MPa. FCV was inactivated by 3.6-log₁₀ TCID₅₀ mL^− 1 after 120 s treatment with 300 MPa, and was undetectable after 180 s treatment with 300 MPa. PV was the most resistant with little or no substantial reduction in titre after 300 s treatment with 600 MPa. The studies were designed to determine the efficacy of using high pressure to inactivate enteric viruses and generate inactivation data to assist in determining appropriate process criteria for safe shellfish production.Industrial relevanceThe high pressure treatment of raw oysters has proved commercially successful, due in part to a marked increase in the product’s shelf life, yet little alteration of its organoleptic properties. Illnesses from human enteric viruses such as hepatitis A virus and norovirus have traditionally been associated with shellfish consumption, and for this reason, studies have examined the stability of enteric viruses under high pressure. However, kinetic data on enteric virus stability under pressure is needed by processors to better understand the response of viruses throughout the entire treatment time. The kinetic data obtained in this study may be useful for processors wishing to alter high pressure processing conditions to ensure a high quality product in terms of organoleptic and microbiological properties.     

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.     

Inactivation of Bacillus cereus spores by high hydrostatic pressure at different temperatures

The effect of pH on the initiation of germination and on the inactivation of Bacillus cereus (KCTC 1012) spores during high hydrostatic pressure processing (HPP) with pressures of 0.1 to 600 MPa at different temperatures was investigated. Two different high-pressure treatments were adopted to evaluate the effect of pH on the inactivation of B. cereus on sporulation medium and in suspension medium. Inactivation of B. cereus spores with HPP treatment was affected more by sporulation medium pH than by suspension medium pH. B. cereus spores obtained through sporulation at pH 6.0 showed more resistance to inactivation by HPP at 20, 40, and 60 degrees C than did those obtained through sporulation at pHs of 7.0 and 8.0. Constituents of B. cereus spores obtained through sporulation at pH 6.0 may undergo electrochemical charge changes comparable to those for spores obtained through sporulation at pH 7.0. The initiation of B. cereus spore germination was more sensitive to pressure around 300 MPa at 20 degrees C. Increasing processing temperatures during HPP enhanced the effect of sporulation medium pH (i.e., environmental pH) on the inactivation of B. cereus spores.     

Temperature and treatment time influence high hydrostatic pressure inactivation of feline calicivirus, a norovirus surrogate

Interest in high hydrostatic pressure processing as a nonthermal pasteurization process for foods continues to increase. Feline calicivirus (FCV), a propagable virus that is genetically related to the nonpropagable human noroviruses, was used for detailed evaluation of the high pressure processing parameters necessary for virus inactivation. Pressure inactivation curves of FCV strain KCD in Dulbecco's modified Eagle medium with 10% fetal bovine serum were obtained at 200 and 250 MPa as a function of time at room temperature. Pressure inactivation curves at 200 and 250 MPa also were determined as a function of temperature ranging from --10 to 50 degrees C at treatment times of 4 and 2 min, respectively. Tailing was observed for inactivation as a function of treatment time, indicating that the linear model was not adequate for describing these curves. The two nonlinear models, the log logistic and Weibull functions, consistently produced better fit to inactivation curves than did the linear model. The mean square errors were 0.381 for the log logistic model, 0.425 for the Weibull model, and 1.546 for the linear model. For inactivation as a function of temperature, FCV was most resistant to pressure at 20 degrees C. Temperatures above and below 20 degrees C significantly increased pressure inactivation of FCV. A 4-min treatment of 200 MPa at --10 and 50 degrees C reduced the titer of FCV by 5.0 and 4.0 log units, respectively; whereas at 20 degrees C the same treatment only reduced the titer by 0.3 log units. These novel results point to the potential for using temperatures above and particularly below room temperature to lower the pressure needed to cause the desired level of virus inactivation.     

Inactivation of Mucor plumbeus by the combined actions of chitinase and high hydrostatic pressure

Sporangiospores were treated with high hydrostatic pressure and/or fungal chitinase in order to study the inhibition of germination and growth of the food spoiling mold Mucor plumbeus. Total fungal inhibition was obtained either at 4.0 kbar or by 10 U/ml of chitinase from Penicillium janthinellum. A pretreatment with 1 U/ml of the same chitinase reduced the pressure necessary to obtain complete spore inhibition to 3 kbar.     

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.     

Inactivation of Salmonella typhimurium DT 104 in UHT whole milk by high hydrostatic pressure

Cell suspensions of Salmonella typhimurium DT 104 in ultra-high temperature (UHT) whole milk were exposed to high hydrostatic pressure at 350, 400, 450, 500, 550, and 600 MPa at ambient temperature (ca. 21 degrees C). Tailing was observed in all survival curves, and sigmoidal survival curves were observed at relatively high pressure (500-600 MPa). Four modeling methods (linear and nonlinear including Weibull, modified Gompertz, and log-logistic models) were fitted to these data at 500, 550, and 600 MPa. Performances of the modeling methods were compared using mean square error (MSE). The linear regression model at these three pressure levels had a mean square error (MSE) of 1.260-2.263. Nonlinear regressions using Weibull, modified Gompertz, and log-logistic models had MSE values in the range of 0.334-0.764, 0.601-1.479, and 0.359-0.523, respectively. Modeling results indicated that first-order kinetics could not accurately describe pressure inactivation of S. typhimurium DT 104 in UHT milk; the log-logistic model produced the best fit to data.     

Inactivation of Escherichia coli and Listeria innocua in kiwifruit and pineapple juices by high hydrostatic pressure

Escherichia coli and Listeria innocua in kiwifruit and pineapple juices were exposed to high hydrostatic pressure (HHP) at 300 MPa for 5 min. Both bacteria showed equal resistance to HHP. Using low (0 degrees C) or sub-zero (-10 degrees C) temperatures instead of room temperature (20 degrees C) during pressurization did not change the effectiveness of HHP treatment on both bacteria in studied juices. Pulse pressure treatment (multiple pulses for a total holding time of 5 min at 300 MPa) instead of continuous (single pulse) treatment had no significant (p>0.05) effect on the microbial inactivation in kiwifruit juice; however, in pineapple juice pulse treatment, especially after 5 pulses, increased the inactivation significantly (p<0.05) for both bacteria. Following storage of pressure-treated (350 MPa, 20 degrees C for 60 s x 5 pulses) juices at 4, 20 and 37 degrees C up to 3 weeks, the level of microbial inactivation further increased and no injury recovery of the bacteria were detected. This work has shown that HHP treatment can be used to inactivate E. coli and L. innocua in kiwifruit and pineapple juices at lower pressure values at room temperature than the conditions used in commercial applications (>400 MPa). However, storage period and temperature should carefully be optimized to increase the safety of HHP treated fruit juices.     

Inactivation of hepatitis A virus by high-pressure processing: the role of temperature and pressure oscillation

Inactivation of hepatitis A virus (HAV) in Dulbecco's modified Eagle medium with 10% fetal bovine serum was studied at pressures of 300, 350, and 400 MPa and initial sample temperatures of -10, 0, 5, 10, 20, 30, 40, and 50 degrees C. Sample temperature during pressure application strongly influenced the efficiency of HAV inactivation. Elevated temperature (> 30 degrees C) enhanced pressure inactivation of HAV, while lower temperatures resulted in less inactivation. For example, 1-min treatments of 400 MPa at -10, 20, and 50 degrees C reduced titers of HAV by 1.0, 2.5, and 4.7 log PFU/ml, respectively. Pressure inactivation curves of HAV were obtained at 400 MPa and three temperatures (-10, 20, and 50 degrees C). With increasing treatment time, all three temperatures showed a rapid initial drop in virus titer with a diminishing inactivation rate (or tailing effect). Analysis of inactivation data indicated that the Weibull model more adequately fitted the inactivation curves than the linear model. Oscillatory high-pressure processing for 2, 4, 6, and 8 cycles at 400 MPa and temperatures of 20 and 50 degrees C did not considerably enhance pressure inactivation of HAV as compared with continuous high-pressure application. These results indicate that HAV exhibits, unlike other viruses examined to date, a reduced sensitivity to high pressure observed at cooler treatment temperatures. This work suggested that slightly elevated temperatures are advantageous for pressure inactivation of HAV within foods.     

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.     

Food processing by high hydrostatic pressure

The use of high hydrostatic pressures (HHP) for food processing is finding increased application within the food industry. One of the advantages of this technology is that because it does not use heat, sensory, and nutritional attributes of the product remain virtually unaffected, thus yielding products with better quality than those processed traditional methods. HHP have the ability to inactivate microorganisms as well as enzymes responsible for shortening the life of a product. In addition to lengthening the shelf-life of food products, HHP can modify functional properties of components such as proteins, which in turn can lead to the development of new products. Equipment for large-scale production of HHP processed products are commercially available nowadays. Guacamole, sliced ham, oysters, and fruit juices are some of the products currently available on the market. HHP technology is one of the most promising nonthermal processes.     

Inactivation of Colletotrichum gloeosporioides spores by high hydrostatic pressure combined with citral or lemongrass essential oil

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is the main post-harvest disease of the papaya. Inactivation of the spores of C. gloeosporioides in saline solution by the use of high hydrostatic pressure, citral oil and lemongrass oil, alone and in combination, was studied. C. gloeosporioides spores were efficiently inhibited after a pressure treatment of 350 MPa for 30 min. When C. gloeosporioides was treated with 0.75 mg ml(-1) of citral or lemongrass oil, the pressure needed to achieve the same spore inhibition was 150 MPa. This work suggests the use of high hydrostatic pressure and plant essential oils as an alternative control for fruit diseases.     

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO₂ photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm² spore reduction (initial spore count was 6.6 log CFU/cm²), including an approximate 0.8-log CFU/cm² reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products.Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.     

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 .     

Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure

The resistance of five gram-positive bacteria, Enterococcus faecalis, Staphylococcus aureus, Lactobacillus plantarum, Listeria innocua and Leuconostoc dextranicum, and six gram-negative bacteria, Salmonella enterica serovar typhimurium, Shigella flexneri, Yersinia enterocolitica, Pseudomonas fluorescens and two strains of Escherichia coli, to high-pressure homogenisation (100-300 MPa) and to high hydrostatic pressure (200-400 MPa) was compared in this study. Within the group of gram-positive bacteria and within the group of gram-negative bacteria, large differences were observed in resistance to high hydrostatic pressure, but not to high-pressure homogenisation. All gram-positive bacteria were more resistant than any of the gram-negative bacteria to high-pressure homogenisation, while in relative to high hydrostatic pressure resistance both groups overlapped. Within the group of gram-negative bacteria, there also existed another order in resistance to high-pressure homogenisation than to high hydrostatic pressure. Further it appears that the mutant E. coli LMM1010, which is resistant to high hydrostatic pressure is not more resistant to high-pressure homogenisation than its parental strain MG1655. The preceding observations indicate a different response of the test bacteria to high-pressure homogenisation compared to high hydrostatic pressure treatment, which suggests that the underlying inactivation mechanisms for both techniques are different. Further, no sublethal injury could be observed upon high-pressure homogenisation of Y. enterocolitica and S. aureus cell population by using low pH (5.5 7), NaCl (0 6%) or SDS (0-100 mg/l) as selective components in the plating medium. Finally, it was observed that successive rounds of high-pressure homogenisation have an additive effect on viability reduction of Y. enterocolitica and S. aureus.     

Inactivation of gram-negative bacteria in milk and banana juice by hen egg white and lambda lysozyme under high hydrostatic pressure

The effect of hen egg white lysozyme (HEWL) and bacteriophage lambda lysozyme (LaL) in combination with high pressure (HP) treatment on the inactivation of four gram-negative bacteria (Escherichia coli O157:H7, Shigella flexneri, Yersinia enterocolitica and Salmonella typhimurium), was studied in skim milk (pH 6.8; a(w) 0.997) and in banana juice (pH 3.8; a(w) 0.971). In the absence of lysozymes, S. flexneri was more sensitive to HP in milk than in banana juice, while the opposite was observed for the other three bacteria. In combination with HP treatment, LaL was more effective than HEWL on all bacteria in both milk and banana juice. Depending on the bacteria, inactivation levels in banana juice were increased from 0.4-2.7 log units by HP treatment alone to 3.6-6.5 log units in the presence of 224 U/ml LaL. Bacterial inactivation in milk was also enhanced by LaL but only by 0.5-2.1 log units. Under the experimental conditions used, LaL was more effective in banana juice than in milk, while the effectiveness of HEWL under the same conditions was not significantly affected by the food matrix. This effect could be ascribed to the low pH of the banana juice since LaL was also more effective on E. coli in buffer at pH 3.8 than at pH 6.8. Since neither LaL nor HEWL are enzymatically active at pH 3.8, we analysed bacterial lysis after HP treatment in the presence of these enzymes, and found that inactivation proceeds through a non-lytic mechanism at pH 3.8 and a lytic mechanism at pH 6.8. Based on these results, LaL may offer interesting perspectives for use as an extra hurdle in high pressure food preservation.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

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.