Extreme acid tolerance of Escherichia coli O157:H7 has raised doubts about the safety of acidic foods. This study examined whether prior storage in acidic and/or cold conditions enhanced survival of E. coli O157:H7 in synthetic gastric fluid (SGF). Three E. coli O157:H7 strains were stored in trypticase soy broth (TSB; acidified with HCl, malic acid, citric acid, or lactic acid) or pH 3.5 and 6.5 (nonacidic control) apple juice at 4 and 21 degrees C for < or = 7 days and then were incubated in pH 2.5 SGF at 37 degrees C for 4 h. Cells survived better in apple juice than in TSB containing organic acids, suggesting that juice constituents other than organic acids protect E. coli O157:H7. Refrigeration combined with low pH best protected cells in apple juice and acidified TSB, but, compared to the nonacidic control, only acidified TSB enhanced subsequent survival in pH 2.5 SGF. Equal survival in SGF occurred after storage in pH 3.5 or 6.5 apple juice at 4 degrees C, suggesting that low temperature alone in apple juice enhanced acid tolerance. Two strains stored at 4 degrees C in TSB containing malic or citric acid subsequently survived better in SGF than cells stored in nonacidified TSB but poorer than cells stored in the presence of HCl. These differences reflect the higher pKa of these organic acids. However, subsequent survival of these strains in SGF was poorer after refrigerated storage in apple juice than in TSB containing citric or malic acids. Cells stored in lactic acid were most likely to be completely eliminated upon transfer to SGF. Differences in survival in storage media or SGF related to strain, storage conditions, or acidifier were consistent and often statistically significant (P < 0.05). Although the survival of E. coli O157:H7 in refrigerated acidic beverages may not be affected by the type of acidifier used, the subsequent survival in SGF of this pathogen may be critically dependent on this factor.

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimuriumDT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log10-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.

In apple beverage manufacture, cider and juice may be stored for a short time prior to pasteurization. Storage time and temperature may affect the subsequent thermotolerance of bacteria in these beverages. This study examined whether prior storage in pH 3.4 apple cider or apple juice affected the thermotolerance of two Escherichia coli O157:H7 strains in the same beverages at 61 degrees C. Both strains exhibited biphasic survivor curves. Strain ATCC 43894 was consistently more thermotolerant than strain ATCC 43889, with 33 to 153% greater D values derived from the linear portion of each survivor curve. Prior storage at 21 degrees C for 2 or 6 h hastened thermal destruction of both strains in apple cider, but not to a statistically significant extent. In apple juice, prior storage at 21 degrees C for 2 h significantly decreased thermotolerance of strain ATCC 43889, but not of strain ATCC 43894. During 6 h of storage in 21 degrees C apple juice, populations of strains ATCC 43889 and 43894 decreased by 2.1 and 0.5 log10 CFU/ml, respectively, and died rapidly during subsequent heating. Prior storage in apple juice at 4 degrees C for 24 h significantly decreased thermotolerance of both strains, but this effect was not seen after 2 h of storage at 4 degrees C. Experiments with filtered apple cider showed that presence of filterable pulp enhanced the thermotolerance of both strains. These results show that short-term (< or = 6h) room temperature storage of pH 3.4 apple cider and apple juice may enhance the lethality of subsequent pasteurization.

The behavior of Listeria monocytogenes was evaluated during storage of a queso blanco type of cheese produced with acidulants (citric, malic, or acetic acids) and a commercial lactic acid bacterium fermentation product, ALTA™2341 (ALTA). The cheese was prepared by direct acidification (final pH 5.2), with and without 0.6% ALTA, inoculated with 106 CFU/g of L. monocytogenes, and stored at 4 or 20°C for 42 and 7 days, respectively. Levels of L. monocytogenes increased in cheese coagulated with citric or malic acids and stored at 4°C, but decreased slightly in cheese coagulated with acetic acid. At 20°C, counts of L. monocytogenes increased in cheeses acidified with citric or malic acid, but counts did not increase appreciably in cheese acidified with acetic acid. When cheese was stored at 4°C, the presence of 0.6% ALTA resulted in lower counts of L. monocytogenes compared with counts in cheese that did not contain ALTA. However, at 20°C populations of L. monocytogenes increased in cheese containing ALTA regardless of acid type. Additional studies compared the effects of acetic acid, alone or in combination with 0.6 or 2.5% ALTA, against low (102 CFU/g) and high (106 CFU/g) inoculum levels. When inoculum levels were low, pathogen counts decreased by &gt; 1.1 log10 CFU/g in all formulations at 4°C. After 7 days at 20°C, pathogen counts increased in the queso blanco type of cheese prepared with acetic acid alone. In contrast, in the presence of 0.6 or 2.5% ALTA, 7-day counts were less than the initial inoculum. With high inoculum levels at 4°C, counts of L. monocytogenes were less than the initial inoculum in the acetic acid-coagulated queso blanco type of cheese with or without ALTA. At 20°C, counts increased in the queso blanco type of cheese prepared with 0.6% ALTA, but decreased appreciably in cheese prepared with 2.5% ALTA. These results demonstrate that acetic acid is significantly more effective than malic or citric acids for controlling L. monocytogenes in queso blanco, and that inclusion of ALTA can provide added protection against the pathogen.

Probabilistic models were used as a systematic approach to describe the response of Escherichia coli O157:H7 populations to combinations of commonly used preservation methods in unpasteurized apple cider. Using a complete factorial experimental design, the effect of pH (3.1 to 4.3), storage temperature and time (5 to 35°C for 0 to 6 h or 12 h), preservatives (0, 0.05, or 0.1% potassium sorbate or sodium benzoate), and freeze-thaw (F-T; ؊20°C, 48 h and 4°C, 4 h) treatment combinations (a total of 1,600 treatments) on the probability of achieving a 5-log 10 -unit reduction in a three-strain E. coli O157:H7 mixture in cider was determined. Using logistic regression techniques, pH, temperature, time, and concentration were modeled in separate segments of the data set, resulting in prediction equations for: (i) no preservatives, before F-T; (ii) no preservatives, after F-T; (iii) sorbate, before F-T; (iv) sorbate, after F-T; (v) benzoate, before F-T; and (vi) benzoate, after F-T. Statistical analysis revealed a highly significant (P < 0.0001) effect of all four variables, with cider pH being the most important, followed by temperature and time, and finally by preservative concentration. All models predicted 92 to 99% of the responses correctly. To ensure safety, use of the models is most appropriate at a 0.9 probability level, where the percentage of false positives, i.e., falsely predicting a 5-log 10 -unit reduction, is the lowest (0 to 4.4%). The present study demonstrates the applicability of logistic regression approaches to describing the effectiveness of multiple treatment combinations in pathogen control in cider making. The resulting models can serve as valuable tools in designing safe apple cider processes.