Mehmet Calicioglu

Viability of Escherichia coli O157:H7 in Fermented Semidry Low-Temperature-Cooked Beef Summer Sausage

The population of inoculated Escherichia coli O157:H7 was monitored during the manufacture and storage of a semidry beef summer sausage processed by fermentation and cooking at a low temperature by heating to an internal temperature of 130°F (54°C). The all-beef batter (11% fat and nonmeat ingredients) was inoculated with the commercial starter culture Pediococcus acidilactici HP (≥8.6 log CFU/g of batter) and a five-strain mixture of E. coli O157:H7 (≥7 log CFU/g) and then hand stuffed into 2.5-inch (64-mm) diameter fibrous casings. The sausages were fermented at an initial temperature of 85°F (29°C) to a final temperature of 105°F (41°C) over ca. 13 h at 80% relative humidity (RH) to pH 4.6 or pH 5.0. After fermentation to pH 4.6, the internal temperature of the chubs was raised to 130°F (54°C) instantaneous over 3.6 h at 60% RH. After fermentation to pH 5.0, the internal temperature of the chubs was raised to 130°F (54°C) over 3.6 h at 60% RH and the chubs were maintained under these conditions for 0, 30, or 60 min. he chubs were cold water showered for 15 min and then chilled at 39°F (4°C) for 6 h before being vacuum packaged and stored at 39°F (4°C) or 77°F (25°C) for 7 days. Regardless of the target pH, fermentation alone resulted in only a 1.39-log CFU/g decrease in pathogen numbers. However, fermentation to pH 4.6 and heating to an internal temperature of 130°F (54°C) instantaneous reduced counts of E. coli O157:H7 by ≥7.0 log units to below detection levels (<10 CFU/g). Pathogen numbers remained below levels detectable by direct plating, but viable E. coli O157:H7 cells were recovered by enrichment of samples during sausage storage at either refrigeration or abuse temperatures. In contrast, fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous resulted in a 3.2-log-unit decrease in counts of E. coli O157:H7. No appreciable reductions in pathogen numbers were observed thereafter following storage at either 39°F (4°C) or 77°F (25°C) for 7 days. Fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous followed by holding for 30 or 60 min resulted in about a 5- or 7-log reduction, respectively, in pathogen numbers. For chubs held for 30 min at 130°F (54°C), pathogen numbers decreased to 2.02 and <1.0 log CFU/g at 39°F (4°C) and 77°F (25°C), respectively, after 7 days; viable cells were only observed by enrichment after storage at 77°F (25°C). For chubs held for 60 min at 130°F (54°C), pathogen numbers remained below levels detectable by direct plating, but viable cells were recoverable by enrichment after 7 days at both storage temperatures. These data will be useful guidelines to manufacturers for developing processing conditions to further ensure the safety of this category of fermented sausages relative to food-borne pathogens such as serotype O157:H7 strains of E. coli .

Viability of Escherichia coli O157:H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63, or 68°C in a home-style dehydrator

Beef jerky batter was prepared to fat contents of about 5 and 20% and inoculated with about 10(8) cfu g(-1) of a five-strain inoculum of Escherichia coli O157:H7. Pathogen numbers were determined in the raw batter and in the strips formed from it after drying at 52, 57, 63, and 68 degrees C for times that ranged from 2 to 20 h. For both the high and low fat products, pathogen numbers were reduced by about 5 log10 cfu g(-1) within 4 h drying at 68 degrees C and within 8 h drying at 63 degrees C. At 57 degrees C, a 5-log10-unit reduction was achieved within 10h drying for the 5% fat product and within 16 h drying for the 20% fat product. At 52 degrees C, a 5-log10-unit reduction was achieved within 10 h drying for the 5% fat product and within 20 h drying for the 20% fat product. In at least one of the three trials for all four drying temperatures tested, the pathogen was present following enrichment of the samples in synthetic media. The calculated D values decreased from 2.59, 2.48, 1.23, and 1.17 as the temperature increased from 52, 57, 63, and 68 degrees C and as the fat content decreased from 20 to 5%. However, there was no direct correlation between the moisture-to-protein ratio and either the doneness of the strips or the viability of the pathogen. These data indicate that the fat content and the time and temperature at which strips are dried directly impact the viability of E. coli O157:H7 in ground and formed beef jerky.

Effectiveness of spraying with tween 20 and lactic acid in decontaminating inoculated Escherichia coli O157:H7 and indigenous Escherichia coli biotype I on beef.

Beef carcass quarters and fat-covered subprimal cuts were suspended vertically and inoculated with a bovine manure slurry containing a five-strain mixture of Escherichia coli O157:H7 to deliver about 4 to 5 log10 CFU/cm2. To identify treatments that would improve the effectiveness of spraying with lactic acid (LA), the inoculated quarters and cuts were treated as follows: experiment A, (i) not treated (control), (ii) sprayed with 2% (vol/vol) LA, (iii) tempered at 21 degrees C for 4 h, and (iv) tempered and then sprayed with LA; experiment B, (v) sprayed with water, (vi) sprayed with LA, (vii) sprayed with LA containing 0.5% (vol/vol) sodium benzoate (SB), and (viii) sprayed with LA containing SB and 5% (vol/vol) Tween 20 (TW20); and experiment C, (ix) sprayed with water (no prespray), (x) presprayed with TW20 and then sprayed with LA, and (xi) presprayed with TW20 and then sprayed with LA containing SB. In experiment A, spraying carcasses with LA significantly (P < 0.05) reduced the numbers of E. coli Biotype I and serotype O157:H7 after 1 and 3 days of storage, respectively. The tempering process employed did not affect the effectiveness of the LA spray on either type of E. coli. In experiment B, there was no significant difference in the reduction of E. coli O157:H7 on subprimal cuts sprayed with water and that on cuts sprayed with LA alone or with LA in combination with SB and TW20 after 1 or 3 days of storage (total reductions ranged from about 1.6 to 2.8 log10 CFU/cm2). In experiment C, prespraying subprimal cuts with TW20 significantly (P < 0.05) increased the effectiveness of LA (reductions of 2.8 and 3.2 log10 CFU/cm2, respectively) and that of LA with SB (reductions of 2.6 and 3.3 log10 CFU/cm2, respectively) compared with spraying with water alone (reductions of ca. 1.0 and 2.0 log10 CFU/cm2, respectively) after I and 3 days of storage, respectively. In a separate experiment, the incorporation of TW20 (0.1 or 0.25%) into buffered peptone water prior to the maceration of excised carcass surface samples resulted in the recovery of significantly larger numbers (ca. 5.1 to 5.2 log10 CFU/cm2) of E. coli O157:H7 cells than did the control treatment without added TW20 (ca. 3.8 to 4.6 log10 CFU/cm2). These results demonstrate that the treatment of beef carcasses with LA reduces the number of viable E. coli O157:H7 cells and that this inactivation or removal by LA is enhanced by prespraying of the carcass with a 5% solution of TW20.

Inactivation of acid-adapted and nonadapted Escherichia coli O157:H7 during drying and storage of beef jerky treated with different marinades

The inactivation of both acid-adapted and unadapted Escherichia coli O157:H7 during the processing of beef jerky was studied. Following inoculation with the pathogen, beef slices were subjected to different predrying marinade treatments, dried at 60 degrees C for 10 h, and stored at 25 degrees C for 60 d. The predrying treatments evaluated were as follows: (i) no treatment (C), (ii) traditional marinade (TM), (iii) double-strength TM modified with added 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (iv) dipping into 5% acetic acid for 10 min followed by application of TM (AATM), and (v) dipping into 1% Tween 20 for 15 min and then into 5% acetic acid for 10 min followed by TM (TWTM). Bacterial survivors were determined during drying and storage using tryptic soy agar with 0.1% pyruvate, modified eosin methylene blue agar, and sorbitol MacConkey agar. Results indicated that bacterial populations decreased during drying in the order of TWTM (4.9 to 6.7 log) > AATM > MM > C > or = TM (2.8 to 4.9 log) predrying treatments. Populations of acid-adapted E. coli O157:H7 decreased faster (P < 0.05) in AATM and TWTM than nonadapted cells during drying, whereas no significant difference was found in inactivation of acid-adapted and nonadapted inocula in C and TM samples. MM was more effective in inactivating the nonadapted than the adapted inoculum. Bacterial populations continued to decline during storage and dropped below the detection limit (-0.4 log10 CFU/cm2) as early as day 0 (after drying) or as late as day 60, depending on acid adaptation, predrying treatment, and agar medium. The results indicated that acid adaptation may not increase resistance to the hurdles involved in jerky processing and that use of additional antimicrobial chemicals or preservatives in jerky marination may improve the effectiveness of drying in inactivating E. coli O157:H7.

Validation of a manufacturing process for fermented, semidry Turkish soudjouk to control Escherichia coli O157:H7.

Two soudjouk batters were prepared from ground beef (20% fat) and nonmeat ingredients and inoculated with a five-strain mixture of Escherichia coli O157:H7 to yield an initial inoculum of 7.65 log10 CFU/g. One batter contained a commercial-starter culture mixture (approximately 8.0 log10 CFU/g) and dextrose (1.5%), while the other batter relied upon a natural fermentation with no added carbohydrate. Following mixing, sausage batters were held at 4 degrees C for 24 h prior to stuffing into natural beef round casings. Stuffed soudjouk sticks were fermented and dried at 24 degrees C with 90 to 95% relative humidity (RH) for 3 days and then at 22 degrees C with 80 to 85% RH until achieving a product moisture level of approximately 40%. After fermentation and drying with an airflow of 1 to 1.5 m/s, the sticks were either not cooked or cooked to an instantaneous internal temperature of 54.4 degrees C (130 degrees F) and held for 0, 30, or 60 min. The sticks were then vacuum packaged and stored at either 4 or 21 degrees C. For each of three trials, three sticks for each treatment/batter were analyzed for numbers of E. coli O157:H7 after inoculation, after fermentation, after cooking, and after storage for 7, 14, 21, and 28 days. Reductions in numbers of E. coli O157:H7 after fermentation and drying for sticks fermented by the starter culture (pH 4.6) and for sticks naturally fermented (pH 5.5) were 1.96 and 0.28 log10 CFU/g, respectively. However, cooking soudjouk sticks produced with a starter culture and holding at 54.4 degrees C for 0, 30, or 60 min reduced pathogen numbers from an initial level after fermentation and drying of 5.69 log10 CFU/g to below a detectable level by either direct plating (<1.0 log10 CFU/g) or by enrichment. In contrast, cooking soudjouk sticks produced without an added starter culture decreased pathogen numbers from an initial level after fermentation and drying of 7.37 to 5.65 log10 CFU/g (54.4 degrees C, no hold), 5.04 log10 CFU/g (54.4 degrees C, 30 min hold), and 4.67 log10 CFU/g (54.4 degrees C, 60 min hold). In general, numbers of E. coli O157:H7 within both groups of soudjouk sticks decreased faster during storage at 21 degrees C compared to 4 degrees C. After 28 days of storage, total reductions in pathogen numbers in soudjouk sticks produced using a starter culture but that were not subsequently cooked were 7.65 and 3.93 log10 CFU/g at 21 and 4 degrees C, respectively. For naturally fermented soudjouk, total reductions varied from 4.47 to 0.45 log10 CFU/g, depending on the cooking time and storage temperature. These data provide guidelines for manufacturers of dry sausage of ethnic origin, including soudjouk, to assess the safety of their processes for control of E. coli O157:H7.

Inactivation of Salmonella during Drying and Storage of Apple Slices Treated with Acidic or Sodium Metabisulfite Solutions

This study was undertaken to determine whether pretreating inoculated Gala apple slices with metabisulfite or acidic solutions enhanced the inactivation of Salmonella during dehydration and storage. Apple slices inoculated with a five-strain mixture of Salmonella (7.6 log CFU/g) were pretreated, dried for 6 h at 60 degrees C, and stored aerobically at 25 degrees C for 28 days. Predrying treatments included (i) no treatment, (ii) 10 min of immersion in sterile water, (iii) 10 min of immersion in a 4.18% sodium metabisulfite solution, (iv) 10 min of immersion in a 3.40% ascorbic acid solution, and (v) 10 min of immersion in a 0.21% citric acid solution. Samples were plated on tryptic soy agar with 0.1% pyruvate (TSAP), brilliant green sulfa (BGS) agar, and xylose lysine tergitol 4 (XLT4) agar for the enumeration of bacteria. Populations were not significantly (P > 0.05) reduced by immersion in water but were reduced by 0.7 to 1.1 log CFU/g by immersion in acidic solutions. Immersion in the sodium metabisulfite solution reduced populations by 0.4, 1.3, and 5.4 log CFU/g on TSAP, BGS agar, and XLT4 agar, respectively. After 6 h of dehydration at 60 degrees C, populations on untreated and water-treated slices were reduced by 2.7 to 2.8, 2.7 to 2.9, and 4.0 to 4.2 log CFU/g as determined with TSAP, BGS agar, and XLT4 agar, respectively. In contrast, populations on slices treated with sodium metabisulfite, ascorbic acid, and citric acid were reduced after 6 h of dehydration by 4.3, 5.2, and 3.8 log CFU/g, respectively, as determined with TSAP; by 4.7, 5.5, and 3.9 log CFU/g, respectively, as determined with BGS agar; and by 5.5, 5.7, and 5.6 log CFU/g, respectively, as determined with XLT4 agar. Bacteria were still detectable by direct plating after 28 days except on slices treated with ascorbic acid. Immersion in metabisulfite or acidic solutions prior to dehydration should enhance the inactivation of Salmonella during the dehydration and storage of Gala apple slices.

Viability of Escherichia coli O157:H7 during Manufacturing and Storage of a Fermented, Semidry Soudjouk-Style Sausage

Soudjouk-style batter was inoculated with a five-strain mixture of Escherichia coli O157:H7 at about 7.6 log10 CFU/g in each of two trials. The sticks were fermented and dried at 22 degrees C and 50% relative humidity (RH) for 3 days and then at 9 degrees C and 40% RH for 18 h. After being flattened to about 1.25 cm, the sticks were conditioned at 38 degrees C and 70% RH or at 22 degrees C and 50% RH for about 3 days. After the latter conditioning treatment, sticks either were cooked to an internal temperature of 63 degrees C or received no heat treatment. Final mean pH values after conditioning at 22 degrees C and 50% RH for soudjouk manufactured with a starter culture and dextrose (1.0%) and for soudjouk manufactured without a starter culture were about 4.9 and 6.0, respectively. For soudjouk produced with a starter culture, pathogen numbers were reduced by 4.53 and 0.88 log10 CFU/g after conditioning at 38 degrees C and 70% RH and at 22 degrees C and 50% RH, respectively. For soudjouk produced via natural fermentation, pathogen numbers were reduced by 1.39 and 0.09 log10 CFU/g after conditioning at 38 degrees C and 70% RH and at 22 degrees C and 50% RH, respectively. Cooking reduced pathogen numbers to below the levels detectable by direct plating (<1.0 log10 CFU/g) and by enrichment for soudjouk produced with a starter culture and also reduced pathogen numbers by 6.28 log10 CFU/g for soudjouk produced via natural fermentation. However, cooking also resulted in an unacceptable product. In general, the reduction in pathogen numbers achieved by storage at ambient temperature (25 degrees C) was greater than that achieved by storage at cooler temperatures (4 and 15 degrees C), particularly for soudjouk prepared with a starter culture (for which a final pH value of 4.8 and a 6.4-log10 reduction were obtained after 21 days at 25 degrees C) rather than for that prepared without a starter culture (for which a final pH value of 6.1 and a 2.6-log10 reduction were obtained after 21 days at 25 degrees C). These results indicate that naturally fermented old-country-type sausage may allow the survival of E. coli O157:H7 in the absence of controlled fermentation, postfermentation cooking, and/or an ambient-storage processing step.

Effects of Acid Adaptation and Modified Marinades on Survival of Postdrying Salmonella Contamination on Beef Jerky during Storage

This study was undertaken to evaluate the survival of acid-adapted and nonadapted Salmonella cultures inoculated after drying on beef jerky that had been treated with marinades before drying at 60 degrees C for 10 h. Beef slices were (i) not treated prior to refrigeration at 4 degrees C for 24 h (control [C]); (ii) marinated with traditional marinade (TM), (iii) marinated with TM modified with 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce containing 5% ethanol (MM) at twice the amount used in the TM treatment; (iv) dipped into 5% acetic acid and then marinated with TM (AATM); and (v) dipped into 1% Tween 20, then dipped into 5% acetic acid, and then marinated with TM (TWTM); after each treatment, meat slices were refrigerated at 4 degrees C for 24 h prior to drying. Dried slices were inoculated with acid-adapted or nonadapted Salmonella (ca. 5.7 log CFU/cm2) prior to aerobic storage at 25 degrees C for 60 days. Tryptic soy agar with 0.1% pyruvate, as well as xylose-lysine-tergitol 4 (XLT4) agar, was used to determine survivor counts. Bacterial decreases achieved with the different treatments were found to be in the following order: TWTM (5.4 to 6.3 log units) > or = AATM > or = MM > C > or = TM (2.9 to 5.1 log units). Acid-adapted Salmonella decreased faster than nonadapted Salmonella for all treatments. Bacterial populations decreased to below the detection limit (-0.4 log CFU/cm2) in as few as 14 days or remained detectable by direct plating after 60 days of storage, depending on acid adaptation, treatment, and agar media. The results of this study indicate that the modified marinades used in jerky processing and the low water activity of the dried product provide antimicrobial effects against possible postprocessing contamination with Salmonella, while the preparation of cultures under acid-adaptation conditions did not increase Salmonella survival during storage and may have reduced it.

Recovery of Escherichia coli Biotype I and Enterococcus spp. during refrigerated storage of beef carcasses inoculated with a fecal slurry.

Three beef front quarters/carcasses were inoculated with a slurry of cattle manure. During storage at 4 degrees C, two sponge samples from each of three sites (i.e., 100 cm2 from each of two fat surfaces and 100 cm2 from a lean surface) were taken from each of the three carcasses on days 0, 1, 3, 7, and 10 after inoculation. The initial numbers of Escherichia coli averaged 2.0 log10 CFU/cm2 (1.21 to 2.47 log10 CFU/cm2) using the Petrifilm method and 2.09 log10 most probable number (MPN)/cm2 (0.88 to 2.96 log10 MPN/cm2) using the MPN method. The initial numbers of enterococci averaged 3.34 log10 CFU/cm2 (3.07 to 3.79 log10 CFU/cm2) using kanamycin esculin azide agar. In general, an appreciable reduction in the numbers of E. coli occurred during the first 24 h of storage; for the Petrifilm method an average reduction of 1.37 log10 CFU/cm2 (0.69 to 1.71 log10 CFU/cm2) was observed, and for the MPN method an average reduction of 1.52 log10 MPN/cm2 (0.47 to 2.08 log10 MPN/cm2) was observed. E. coli were not detected (<-0.12 log10 CFU/cm2) using Petrifilm on day 7 of the storage period on two (initial counts of 1.21 and 2.29 log10 CFU/cm2) of the three carcasses. However, viable E. coli cells were recovered from these two carcasses after a 24-h enrichment at 37 degrees C in EC broth. Viable E. coli cells were detected at levels of -0.10 log10 CFU/cm2 on the third carcass (initial count of 2.47 log10 CFU/cm2) after 7 days at 4 degrees C. No significant difference in recovery of viable cells was observed between the MPN and Petrifilm methods on days 0, 1, and 3 (P > 0.05). However, viable E. coli cells were recovered from all three carcasses by the MPN method on day 7 at an average of -0.29 log10 MPN/ cm2 (-0.6 to -0.1 log10 MPN/cm2). On day 10, viable cells were recovered by the MPN method from two of the three carcasses at -0.63 and -0.48 log10 MPN/cm2 but were not recovered from the remaining carcass (<-0.8 log10 MPN/cm2). Similar to E. coli, the greatest reduction (average of 1.26 log10 CFU/cm2, range = 1.06 to 1.45 log10 CFU/cm2) in the numbers of enterococci occurred during the first 24 h of storage. Because of higher initial numbers and a slightly slower rate of decrease, the numbers of Enterococcus spp. were significantly higher (P < 0.017) than the numbers of E. coli Biotype I after 3, 7, and 10 days of storage. These results suggest that enterococci may be useful as an indicator of fecal contamination of beef carcasses.

Survival of Listeria monocytogenes during Production and ripening of traditional Turkish Savak tulum cheese and in synthetic gastric fluid.

This study investigated the survival and acid tolerance of Listeria monocytogenes during the 2-day processing stage and 90-day ripening of Savak tulum cheese, a traditional cheese in Turkey. Experimental Savak tulum cheese was produced from raw sheeps milk that was inoculated with a L. monocytogenes mixture consisting of five strains (average 7.0 log CFU/ml) and was ripened at 6°C for 90 days. Microbiological and chemical analyses and acid exposure experiments in synthetic gastric fluid (SGF) (pH 1.5 to 2.5) were carried out on days 1 and 2 during processing and on days 0, 15, 30, 45, 60, and 90 during ripening. The numbers of L. monocytogenes did not decrease during processing, but a total of 4.1 log CFU/g reduction was observed during ripening. Throughout the ripening period, L. monocytogenes cells survived direct 90-min exposures of the cheese samples to SGF. These results suggest that, although the pathogen numbers decreased in Savak tulum cheese ripened at 6°C for 90 days, a sublethal environment may have occurred in the cheese during the production stage, activating the acid-tolerance mechanisms of the pathogen and allowing L. monocytogenes to maintain its viability in the SGF for 90 min.