Statistical Modeling of D- and z-value of E. coli O157:H7 and pH in Apple Cider Containing Preservatives

ABSTRACT: The objective of this study was to model the heat resistance of E. coli O157:H7 in apple cider as a function of added sorbate, benzoate, and malic acid. Inactivation experiments were done at 47, 50, and 53 °C, and D and z values were calculated. Models for D₄₇, D₅₀, and D₅₃ showed that all 3 additives significantly decreased heat resistance. Furthermore, z-values increased with addition of preservatives and low pH. Model predictions showed that at 50 °C, 5-log reduction is attainable in a few minutes when the cider contains additives, while preliminary test results showed that at 20 °C, a 5-log reduction could be obtained in about 90 min in cider with 0.1% sorbate, 0.1% benzoate, and 1% malic acid.     

Addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of Escherichia coli O157:H7 populations in apple cider

A study was conducted to develop a preservative treatment capable of the Food and Drug Administration-mandated 5-log reduction of Escherichia coli O157:H7 populations in apple cider. Unpreserved apple cider was treated with generally recognized as safe acidulants and preservatives before inoculation with E. coli O157:H7 in test tubes and subjected to mild heat treatments (25, 35, and 45 degrees C) followed by refrigerated storage (4 degrees C). Fumaric acid had significant (P < 0.05) bactericidal effect when added to cider at 0.10% (wt/vol) and adjusted to pH 3.3, but citric and malic acid had no effect. Strong linear correlation (R2 = 0.96) between increasing undissociated fumaric acid concentrations and increasing log reductions of E. coli O157:H7 in apple cider indicated the undissociated acid to be the bactericidal form. The treatment that achieved the 5-log reduction in three commercial ciders was the addition of fumaric acid (0.15%, wt/vol) and sodium benzoate (0.05%, wt/vol) followed by holding at 25 degrees C for 6 h before 24 h of refrigeration at 4 degrees C. Subsequent experiments revealed that the same preservatives added to cider in flasks resulted in a more than 5-log reduction in less than 5 and 2 h when held at 25 and 35 degrees C, respectively. The treatment also significantly (P < 0.05) reduced total aerobic counts in commercial ciders to populations less than those of pasteurized and raw ciders from the same source (after 5 and 21 days of refrigerated storage at 4 degrees C, respectively). Sensory evaluation of the same ciders revealed that consumers found the preservative-treated cider to be acceptable.     

Modeling the survival of Escherichia coli O157:H7 in apple cider using probability distribution functions for quantitative risk assessment

Outbreaks of foodborne illness from apple cider have prompted research on the survival of Escherichia coli O157:H7 in this food. Published results vary widely, potentially due to differences in E. coli O157:H7 strains, enumeration media, and other experimental considerations. We developed probability distribution functions for the change in concentration of E. coli O157:H7 (log CFU/day) in cider using data from scientific publications for use in a quantitative risk assessment. Six storage conditions (refrigeration [4 to 5 degrees C]; temperature abuse [6 to 10 degrees C]; room temperature [20 to 25 degrees C]; refrigerated with 0.1% sodium benzoate, 0.1% potassium sorbate, or both) were modeled. E. coli survival rate data for all three unpreserved cider storage conditions were highly peaked, and these data were fit to logistic distributions: ideal refrigeration, logistic (-0.061, 0.13); temperature abuse, logistic (-0.0982, 0.23); room temperature, logistic (-0.1, 0.29) and uniform (-4.3, -1.8), to model the very small chance of extremely high log CFU reductions. There were fewer published studies on refrigerated, preserved cider, and these smaller data sets were modeled with beta (4.27, 2.37) x 2.2 - 1.6, normal (-0.2, 0.13), and gamma (1.45, 0.6) distributions, respectively. Simulations were run to show the effect of storage on E. coli O157:H7 during the shelf life of apple cider. Under every storage condition, with and without preservatives, there was an overall decline in E. coli O157:H7 populations in cider, although a small fraction of the time a slight increase was seen.     

Heat resistance of Escherichia coli O157:H7 in apple juice.

The objective was to determine the effect of cider composition on the heat resistance of Escherichia coli O157:H7. The average D52 value in a model Empire apple juice was 18 min with a z value of 4.8 degrees C. Increasing the Brix from 11.8 to 16.5 degrees had no effect on thermal resistance, while increasing L-malic acid from 0.2 to 0.8%, or reducing the pH from 4.4 to 3.6 sensitized the cells to heat. The greatest effect on heat resistance was afforded by the preservatives benzoic and sorbic acids: D50 values in ciders containing 1,000 mg/l were 5.2 min in the presence of sorbic acid and only 0.64 min in the presence of benzoic acid. Commercial apple juice concentrates yielded lower numbers of survivors than single-strength juices even though their higher sugar concentrations of about 46 degrees Brix increased heat resistance.     

Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature

Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone was evaluated. A five-strain mixture of E. coli O157:H7 or a five-serovar mixture of Salmonella was inoculated (7 log CFU/ml) into apple cider and orange juice. Ozone (0.9 g/h) was pumped into juices maintained at 4 degrees C, ambient temperature (approximately 20 degrees C), and 50 degrees C for up to 240 min, depending on organism, juice, and treatment temperature. Samples were withdrawn, diluted in 0.1% peptone water, and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on tryptic soy agar (TSA), sorbitol MacConkey agar, hemorrhagic coli agar, and modified eosin methylene blue agar; recovery of Salmonella was compared on TSA, bismuth sulfite agar, and xylose lysine tergitol 4 (XLT4) agar. After treatment at 50 degrees C, E. coli O157:H7 populations were undetectable (limit of 1.0 log CFU/ml; a minimum 6.0-log CFU/ml reduction) after 45 min in apple cider and 75 min in orange juice. At 50 degrees C, Salmonella was reduced by 4.8 log CFU/ml (apple cider) and was undetectable in orange juice after 15 min. E. coli O157:H7 at 4 degrees C was reduced by 4.8 log CFU/ml in apple cider and by 5.4 log CFU/ml in orange juice. Salmonella was reduced by 4.5 log CFU/ml (apple cider) and 4.2 log CFU/ml (orange juice) at 4 degrees C. Treatment at ambient temperature resulted in population reductions of less than 5.0 log CFU/ml. Recovery of E. coli O157:H7 and Salmonella on selective media was substantially lower than recovery on TSA, indicating development of sublethal injury. Ozone treatment of apple cider and orange juice at 4 degrees C or in combination with mild heating (50 degrees C) may provide an alternative to thermal pasteurization for reduction of E. coli O157:H7 and Salmonella in apple cider and orange juice.     

Critical Factors Affecting the Destruction of Escherichia coli O157:H7 in Apple Cider Treated with Fumaric Acid and Sodium Benzoate

Destruction of Escherichia coli O157:H7 in apple cider treated with fumaric acid and sodium benzoate (0.15% and 0.05% w/v, respectively) was determined under pH and storage temperatures that commonly occur in apple cider. At 5°C storage, while destruction of E. coli O157:H7 in the presence of preservatives increased with time, there was little decline in E. coli O157:H7 populations in the absence of the preservatives. Increasing storage temperatures to 15°C and 25°C significantly increased the rate of destruction of E. coli O157:H7 in cider with the preservatives (P < 0.05). Increasing the pH of cider (from 3.2 to 4.7) decreased the rate of destruction of E. coli O157:H7.     

Survival of Escherichia coli O157:H7 in Apple Cider as Affected by Dimethyl Dicarbonate, Sodium Bisulfite, and Sodium Benzoate

Survival of Escherichia coli O157:H7 in apple cider containing no preservatives, 0.025% dimethyl dicarbonate (DMDC), 0.045% sodium benzoate (SB), 0.0046% sodium bisulfite (NaS; 65.5% sulfur dioxide), or a combination of NaS and SB (NaS/SB) and stored at 4, 10, and 25°C was evaluated. E. coli O157:H7 survived for up to 18 days at 4,10, and 25°C in unpreserved apple cider. At 4 and 10°C, DMDC was most efficient at inactivating E. coli O157:H7, generally followed by NaS/SB SB, and NaS (p<0.05). E coli O157:H7 was more resistant to preservatives at 4°C than at 25°C (P < 0.05). E. coli O157:H7 was sublethally injured in cider containing preservatives, and to a lesser extent, in unpreserved cider. Generally, injury was more pronounced in cider containing DMDC, followed by NaS/SB, SB, and NaS (p<0.05).     

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.     

Controlling Listeria monocytogenes on sliced ham and turkey products using benzoate, propionate, and sorbate

The objective of this study was to identify concentrations of sorbate, benzoate, and propionate that prevent the growth of Listeria monocytogenes on sliced, cooked, uncured turkey breast and cured ham. Sixteen test formulations plus a control formulation for each product type were manufactured to include potassium sorbate, sodium benzoate, or sodium propionate, used alone and combined (up to 0.3% [wt/wt]), or with sodium lactate-sodium diacetate combinations. Products were inoculated with L. monocytogenes (5 log CFU/100-g package) and stored at 4, 7, or 10 degrees C for up to 12 weeks, and triplicate samples per treatment were assayed biweekly by plating on modified Oxford agar. Data showed that 0.1% benzoate, 0.2% propionate, 0.3% sorbate, or a combination of 1.6% lactate with 0.1% diacetate prevented the growth of L. monocytogenes on ham stored at 4 degrees C for 12 weeks, compared with greater than a 1-log increase at 4 weeks for the control ham without antimicrobials. When no nitrite was included in the formulation, 0.2% propionate used alone, a combination of 0.1% propionate with 0.1% sorbate, or a combination of 3.2% lactate with 0.2% diacetate was required to prevent listerial growth on the product stored at 4 degrees C for 12 weeks. Inhibition was less pronounced when formulations were stored at abuse temperatures. When stored at 7 degrees C, select treatments delayed listerial growth for 4 weeks but supported significant growth at 8 weeks. All treatments supported more than a 1-log increase in listerial populations when stored at 10 degrees C for 4 weeks. These results verify that antimycotic agents inhibit the growth of L. monocytogenes on ready-to-eat meats but aremore effective when used in combination with nitrite.     

Acceptability of a multi-step intervention system to improve apple cider safety

A multi-step intervention system against Escherichia coli O157:H7 in apple cider was studied. The system consisted of adding 0.05% (w/v) each of sodium benzoate and potassium sorbate, holding cider at 35 °C for 6 h, followed by freezing and thawing the cider. This system was tested in triplicate 10 ml volumes of eight lots of cider throughout the 2000 season. For all ciders, E. coli O157:H7 numbers were reduced by ⩾3.0 log units, with ⩾5.0 log unit reductions for 6 of 8 ciders. For blends of early- and late-season (2000) ciders treated and then thawed in 3.5 l volumes (jugs), no survivors were detected (⩾3.3–4.3 log unit reductions). In consumer acceptance panels (one cider tasted per panel) each treated cider received a mean (n of 191–201) score of 5.5–6.1 (5=“like slightly”; 6=“like moderately”). In a direct comparison, the treated cider had a significantly (P<0.05) lower mean score (n=189) of 5.6 compared to heat-pasteurized cider (mean score of 6.1). Although the multi-step intervention system may provide sufficient lethality under certain conditions, its inconsistent lethality and lower consumer acceptability reduce its usefulness as an alternative to high-temperature short-time pasteurization.     

Antimicrobial Activity and Synergistic Effect of Cinnamon with Sodium Benzoate or Potassium Sorbate in Controlling Escherichia coli O157:H7 in Apple Juice

ABSTRACT: Antimicrobial effects of cinnamon, sodium benzoate, potassium sorbate, and combinations were examined against Escherichia coli O157:H7 in apple juice at 8°C and 25°C. E. coli O157:H7 was reduced by 1.6 log colony-forming units (CFU)/mL at 8°C and 2.0 log CFU/mL at 25°C by 0.3% cinnamon. At 8°C, 5.2 log CFU/mL of E. coli O157:H7 was eliminated in 11 d by 0.3% cinnamon with 0.1% sodium benzoate, and in 14 d by 0.3% cinnamon with 0.1% potassium sorbate. At 25°C, 5.3 log CFU/mL E. coli O157:H7 was eliminated in 3 d by the same combinations. A synergistic effect was observed between cinnamon and preservatives against E. coli O157:H7 at 8°C and 25°C.     

Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties

Thermal inactivation studies were used to determine the D- and z-values of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. Inoculated meat was packaged in sterile bags, which were immersed in a circulated water bath and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different lengths of time. D- and z-values were determined with a linear regression model. Average D-values at temperatures 55 to 70 degrees C were 27.62 to 0.04 min for E. coli 0157:H7, 67.68 to 0.22 min for Salmonella, and 81.37 to 0.31 min for L. monocytogenes. The z-values were 5.2 degrees C for E. coli O157:H7, 6.0 degrees C for Salmonella, and 6.1 degrees C for L. monocytogenes. The results of this study can be used by food processors to validate their processes and help eliminate pathogenic bacteria associated with chicken-fried beef products.     

Thermal inactivation of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in fruit juices

The heat resistance of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella enterica (serotypes Typhimurium, Enteritidis, Gaminara, Rubislaw, and Hartford), and Listeria monocytogenes was evaluated in single-strength apple. orange, and white grape juices adjusted to pH 3.9. The heat resistance increased significantly (P < 0.05) after acid adaptation. Salmonella had an overall lower heat resistance than the other pathogens. Acid-adapted E. coli O157:H7 presented the highest heat resistance in all juices at the temperatures tested, with lower z-values than Salmonella and L. monocytogenes. The heat resistance (D(60 degrees C)-values) of all three pathogens, assessed in tryptic soy broth adjusted to different pH values, increased above pH 4.0. From the results obtained in this study, one example of a treatment that will inactivate 5 logs of vegetative pathogens was calculated as 3 s at 71.1 degrees C (z-value of 5.3 degrees C). Normal processing conditions calculated for hot-filled, shelf-stable juices achieve a lethality in excess of 50,000 D for all three pathogens.     

INACTIVATION OF E. COLI 0157:H7 IN APPLE CIDER BY OZONE AT VARIOUS TEMPERATURES AND CONCENTRATIONS

The effect of temperature (5–20C) at 860 ppm (v/v) ozone and different gaseous ozone concentrations above 1,000 ppm on inactivation of E. coli O157:H7 in apple cider was studied. Lag times ranged from 3.5 min at 20C to 6.7 min at 10C before the on-set of E. coli O157:H7 inactivation. D-values ranged from 0.6 to 1.5 min at 20C and 5C, respectively. After ozone treatment of cider for 14 min, dissipation of ozone from cider was slow, decreasing to about 5 mg/L after 2 h at 5C. At high gaseous ozone concentration, log time was shortest and D-value lowest. There was a critical concentration of dissolved ozone of about 5–6 mg/L at 20C, before the on-set of E. coli O157:H7 inactivation in the cider. Total processing times, based on lag time plus 5D, ranged from about 4 to 14 min depending on temperature and ozone concentration. Overall, inactivation of E. coli O157:H7 by ozone was fast enough to allow practical applications in cider production, and it should be considered as an alternative to thermal pasteurization.     

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.     

Inactivation of microorganisms in milk and apple cider treated with ultrasound

Nonthermal technologies are emerging as promising alternatives to heat treatment for food processing. Ultrasound, defined as sound waves with a frequency greater than 20 kHz, has proven bactericidal effects, especially when combined with other microbial-reduction strategies such as mild heating. In this study, ultrasound treatment (sonifier probe at 20 kHz, 100% power level, 150 W acoustic power, 118 W/cm2 acoustic intensity) with or without the effect of mild heat (57 degrees C) was effective at reducing microbial levels in raw milk, Listeria monocytogenes levels inoculated in ultrahigh-temperature milk, and Escherichia coli O157:H7 in apple cider. Continuous flow ultrasound treatment combined with mild heat (57 degrees C) for 18 min resulted in a 5-log reduction of L. monocytogenes in ultrahigh-temperature milk, a 5-log reduction in total aerobic bacteria in raw milk, and a 6-log reduction in E. coli O157:H7 in pasteurized apple cider. Inactivation regressions were second-order polynomials, showing an initial period of rapid inactivation, eventually tailing off. Results indicate that ultrasound technology is a promising processing alternative for the reduction of microorganisms in liquid foods.     

Power ultrasound treatment of Listeria monocytogenes in apple cider

Inactivation experiments with Listeria monocytogenes 10403S, an ultrasound-resistant strain, were conducted at sublethal (20, 30, and 40 degrees C) and lethal (50, 55, and 60 degrees C) temperatures in saline solution (pH 7.0), acidified saline solution (pH 3.4), and apple cider (pH 3.4) with and without application of ultrasound (20 kHz, 457 mW.ml(-l)). The survival of recoverable L. monocytogenes 10403S in apple cider was evaluated, and the effects of temperature, ultrasound, pH, and food matrix on inactivation were studied. Application of ultrasound increased the inactivation rate at both sublethal and lethal temperatures. Additional death of L. monocytogenes 10403S was due to low acidity at the lethal temperatures. The reduction in surviving L. monocytogenes 10403S followed first order kinetics at sublethal temperatures, but at lethal temperatures, a two-section linear model described the inactivation behavior. The bactericidal effect of thermosonication was additive in apple cider. The survival tests of L. monocytogenes 10403S in apple cider indicated the possibility of using a mild treatment condition in combination with ultrasound to achieve a 5-log reduction in number of listerial cells.     

Polyphenol oxidase activity as a potential intrinsic index of adequate thermal pasteurization of apple cider

In response to increasing concerns about microbial safety of apple cider, the U.S. Food and Drug Administration has mandated treatment of cider sufficient for a 5-log reduction of the target pathogen. Pasteurization has been suggested as the treatment most likely to achieve a 5-log reduction, with Escherichia coli O157:H7 as the target pathogen. Regulators and processors need a reliable method for verifying pasteurization, and apple cider polyphenol oxidase (PPO) activity was studied as a potential intrinsic index for thermal pasteurization. The effect of pasteurization conditions and apple cider properties on PPO activity and survival of three pathogens (E. coli O157:H7, Salmonella, and Listeria monocytogenes) was studied using a Box-Behnken response surface design. Factors considered in the design were pasteurization conditions, i.e., hold temperature (60, 68, and 76 degrees C), preheat time (10, 20, 30 s), and hold time (0, 15, 30 s), pH, and sugar content ((o)Brix) of apple cider. Response surface contour plots were constructed to illustrate the effect of these factors on PPO activity and pathogen survival. Reduction in PPO activity of at least 50% was equivalent to a 5-log reduction in E. coli O157:H7 or L. monocytogenes for cider at pH 3.7 and 12.5 (o)Brix. Further studies, however, are needed to verify the relationship between PPO activity and pathogen reduction in cider with various pH and (o)Brix values.     

Influence of cinnamon and clove essential oils on the D- and z-values of Escherichia coli O157:H7 in apple cider

Escherichia coli O157:H7 has become a concern within the apple cider industry. The purpose of this study was to screen several essential oils and isolated components for antimicrobial activity against E. coli O157:H7 in tryptic soy broth at neutral and acidic pH and to assess the effect of these additives on the D-value of E. coli O157:H7 in apple cider in combination with mild heat treatments. Cinnamon oil and clove oil strongly inhibited the growth of E. coli O157:H7 at neutral and acidic pH, (R)-(-)-carvone and (S)-(-)-perillaldehyde were moderately inhibitory at both pH 7.2 and pH 4.5, and citral and geraniol displayed moderate activity at pH 4.5 only. Lemon oil, methyl jasmonate, and p-anisaldehyde displayed little or no antibacterial activity. A synergistic effect between the essential oils and the lower pH of the growth medium was evident by consistently lower MICs at pH 4.5. Cinnamon and clove oils (0.01%, vol/vol) were further tested in apple cider in combination with mild heat treatments for the practical control of E. coli O157:H7 in apple cider. The addition of either essential oil resulted in lower D-values than those for cider alone, suggesting a synergistic effect and the potential efficacy of a mild heat treatment for apple cider.