From hazard analysis to risk control using rapid methods in microbiology: A practical approach for the food industry.

The prevention of foodborne diseases is one of the main objectives of health authorities. To this effect, analytical techniques to detect and/or quantify the microbiological contamination of foods prior to their release onto the market are required. Management and control of foodborne pathogens have generally been based on conventional detection methodologies, which are not only time-consuming and labor-intensive but also involve high consumable materials costs. However, this management perspective has changed over time given that the food industry requires efficient analytical methods that obtain rapid results. This review covers the historical context of traditional methods and their passage in time through to the latest developments in rapid methods and their implementation in the food sector. Improvements and limitations in the detection of the most relevant pathogens are discussed from a perspective applicable to the current situation in the food industry. Considering efforts that are being done and recent developments, rapid and accurate methods already used in the food industry will be also affordable and portable and offer connectivity in near future, which improves decision-making and safety throughout the food chain.

Advanced oxidation technology with photohydroionization as a surface treatment for controlling Listeria monocytogenes on stainless steel surfaces and ready-to-eat cheese and turkey.

Listeria monocytogenes is difficult to control in food and processing environments due to its widespread nature and ability to survive in a range of adverse conditions, including low temperatures, pH, and high salt concentrations. The objective of this study was to evaluate the efficacy of Photohydroionization™ (PHI; RGF Environmental Group, Inc., Riviera, Beach, FL), a novel advanced oxidation technology, as a surface treatment to control L. monocytogenes on food-contact surfaces, sliced American cheese, and ready-to-eat (RTE) turkey. A five-strain cocktail of L. monocytogenes was used to inoculate sample surfaces. Food-contact surfaces were exposed to ultraviolet and other oxidative gases produced by the PHI system for 10, 20, 30, 45, 60, and 120 s and 5, 10, and 15 min; cheese and turkey samples were treated for 30, 60, and 120 s and 5 min. For each matrix at each time point, seven samples were treated and enumerated by plating appropriate dilutions onto modified oxford medium and thin-agar-layer modified oxford medium. Results showed reductions (p<0.05) in L. monocytogenes: 4.37 log colony-forming units (CFU)/coupon on stainless steel after 15-min treatment. A 1.39 and 1.63 log CFU/sample after 120 s and 2.16 and 2.52 log CFU/sample after 5 min were seen on American cheese and ready-to-eat turkey, respectively. Lipid oxidation analyses performed on cheese and turkey samples indicated that PHI treatment did not affect (p>0.05) thiobarbituric acid-reactive substances values. This study demonstrates the efficacy of PHI treatment to reduce L. monocytogenes on stainless steel and RTE foods and may serve as a processing intervention to ensure safe production of food.

Evaluation of two thermal processing schedules at low relative humidity for elimination of Escherichia coli O157:H7 and Salmonella serovars in chopped and formed beef jerky.

Foodborne outbreaks have been linked to jerky produced under insufficient thermal processing schedules. Reduction of Escherichia coli O157:H7 and Salmonella serovars during thermal processing of chopped and formed beef jerky was evaluated under two processing schedules representative of those used by large-scale (LS) and small-scale (SS) jerky production facilities. Fresh chopped and formed all-beef jerky batter was inoculated with 5.8 to 7.3 log CFU of E. coli O157:H7 or Salmonella per g, extruded into strips, and thermally processed by LS or SS schedules. A >or=5.0-log CFU/g reduction of both pathogens occurred with <10% relative humidity and a cumulative process of 44 min at 55.6 degrees C followed by 46 min at 77.8 degrees C into the LS schedule. Additional drying at 77.8 degrees C for 3.5 h was needed to achieve a water activity of 0.67 and a moisture-to-protein ratio (MPR) of 0.77. For the SS process, a >or=5.0-log CFU/g reduction of both pathogens occurred with 15 to 20% relative humidity and a cumulative process of 45 min at 52 degrees C, 60 min at 57 degrees C, 45 min at 60 degrees C, 45 min at 63 degrees C, 90 min at 68 degrees C, and finishing with 30 min at 77 degrees C. After processing for an additional 90 min at 77 degrees C, water activity was 0.60 while the MPR was 0.82. The LS and SS processes for producing chopped and formed jerky provided >or=5.0 log lethality to control E. coli O157:H7 and Salmonella. However, both processes would require additional drying to achieve an MPR of 0.75 to be labeled as jerky.

Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation.

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0-5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R (2) = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41-47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (mu(max)) were 0.42 and 0.32 h(-1) for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production.

Screening procedures for clenbuterol residue determination in raw swine livers using lateral-flow assay and enzyme-linked immunosorbent assay.

Clenbuterol, which may cause symptoms of increased heart rate, muscular tremors, headache, nausea, and muscular cramps in patients, has been prohibited for consumption in many countries including the European Union, the United States, and China. A rapid lateral-flow strip assay was developed in our laboratory, and results obtained with this assay were compared with those obtained with a commercial enzyme-linked immunosorbent assay (ELISA) kit for the screening of clenbuterol in raw swine liver. A total of 128 swine livers were acquired from five local markets and prepared for analysis by the lateral-flow strip assay and ELISA. Analysis was completed in 10 min with the lateral-flow strip assay and in 90 min with the ELISA. In parallel with the ELISA, the rapid detection strip produced no false-negative results but had a false-positive rate of 6.3%. Cross-reactivity of the strip was assessed and was negative after tests with clenbuterol analogues such as terbutaline, salbutamol, ractopamine, ritodrine, and fenoterol. These data suggest that a lateral-flow strip assay can be used safely as a screening method as part of a clenbuterol residue surveillance program and should be a valuable tool in the food safety field, especially in developing countries.

Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments.

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was developed. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochromatographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data indicated that clenbuterol rapid detection strip had no false negative. The false positive rate was 4.4%. Immunochromatographic strip has great applied value in the food safety field because it possesses benefits of sensitivity, stability, reproducibility, ease of use and inexpensive.

Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy.

Abundant literature information is available on sodium chloride, NaCl, as an antimicrobial and a preservative, however, information on NaCl effects on bacterial cell morphology is lacking. The effect of NaCl, on Escherichia coli O157:H7 and Staphylococcus aureus cells individually grown in a laboratory medium was examined using transmission electron microscopy (TEM). Cultures were grown in brain heart infusion (BHI) broth containing dissolved 0%, 5%, or 10% (w/v) commercially obtained fine (FN) and extra coarse (EC) grade granular NaCl. The pathogens were incubated at 35 degrees C for 12 and 24 h. Then, a mixture of five strains of each pathogen per treatment was prepared. Samples were centrifuged, pellets collected, fixed immediately with glutaraldehyde, and prepared for TEM examination. Cells morphology on TEM micrographs verified that the magnitude of morphological damage to E. coli O157:H7 cells was significantly greater than that of S. aureus cells. More cell injury occurred as NaCl concentration increased from 5% to 10%. Generally, S. aureus maintained its cellular structure and no severe cell wall or plasma membrane damage and/or shrinkage was observed. At 10% NaCl, the damage to E. coli O157:H7 cells was extensive, and the pathogen seemed to have lost its cellular integrity. Although NaCl affected the morphology of E. coli O157:H7 and S. aureus, the coarse grade of NaCl seemed to have a milder effect with respect to cell damage, especially on S. aureus. The 24 h-old cultures were more susceptible to NaCl treatment compared to the 12 h-old cells. Thus, the age of the cells has an impact on their resistance to salt--the environmental stressor.

Effects of chilling rate on outgrowth of Clostridium perfringens spores in vacuum-packaged cooked beef and pork.

Cooked, chilled beef and cooked, chilled pork were inoculated with three strains of Clostridium perfringens (NCTC 8238 [Hobbs serotype 2], NCTC 8239 [Hobbs serotype 3], and NCTC 10240). Inoculated products were heated to 75 degrees C, held for 10 min in a circulating water bath to heat activate the spores, and then chilled by circulating chilled brine through the water bath. Samples were chilled from 54.4 to 26.6 degrees C in 2 h and from 26.6 to 4.4 degrees C in 5 h. Differences in initial C. perfringens log counts and log counts after chilling were determined and compared with the U.S. Department of Agriculture (USDA) stabilization guidelines requiring that the chilling process allow no more than 1 log total growth of C. perfringens in the finished product. This chilling method resulted in average C. perfringens increases of 0.52 and 0.68 log units in cooked beef and cooked pork, respectively. These log increases were well within the maximum 1-log increase permitted by the USDA, thus meeting the USDA compliance guidelines for the cooling of heat-treated meat and poultry products.

Comparison of recovery of airborne microorganisms in a dairy cattle facility using selective agar and thin agar layer resuscitation media.

Thin agar layer (TAL) medium was developed at Kansas State University to improve the resuscitation of injured cells and has been shown to result in higher recovery than is obtained with selective media alone for cold-, heat-, salt-, and acid-injured cells. The experiment presented here was designed to determine the effectiveness of the TAL method for the recovery of possibly injured organisms from air. Eleven agar media were used for the experiment: tryptic soy agar (TSA), MacConkey sorbitol agar (MSA), TAL-MSA, Baird-Parker (BP) agar, TAL-BP agar, modified Oxford (MOX) agar, TAL-MOX agar, xylose lysine sodium desoxycholate (XLD) agar, TAL-XLD agar, Yersinia-selective (CIN) agar, and TAL-CIN agar. The TAL plates were prepared by pipetting 6 ml of selective agar into a BBL Rodac plate (65 by 15 mm). Selective agar was allowed to solidify, and then each plate was overlaid with 6 ml of TSA. Selective agar plates were prepared by pipetting 12 ml of agar into BBL Rodac plates and allowing the agar to solidify. Samples were taken at an indoor cattle facility at five separate locations with a BioScience SAS air-sampling instrument. For each plate, 60 liters of air was sampled. Three replications of the experiment were performed. The TAL method resulted in higher counts of microorganisms on all media tested. In addition, 175 isolates were selected randomly and identified in order to test the selectivity of TAL and the selective media for target organisms. The data obtained in this study show that the TAL resuscitation method is effective and necessary for the recovery of airborne organisms that may be injured.

Predictions for rapid methods and automation in food microbiology.

A discussion is presented on the present status of rapid methods and automation in microbiology. Predictions are also presented for development in the following areas: viable cell counts; real-time monitoring of hygiene; polymerase chain reaction, ribotyping, and genetic tests in food laboratories; automated enzyme-linked immunosorbent assay and immunotests; rapid dipstick technology; biosensors for Hazard Analysis Critical Control Point programs; instant detection of target pathogens by computer-generated matrix; effective separation and concentration for rapid identification of target cells; microbiological alert systems in food packages; and rapid alert kits for detecting pathogens at home.

Use of Nisin and Microwave Treatment Reduces Clostridium sporogenes Outgrowth in Precooked Vacuum-Packaged Beef.

Nisin (NS), microwave (MW), and nisin plus microwave (NS + MW) treatments were applied to precooked beef semitendinosus muscles inoculated with spores of Clostridium sporogenes . Samples were then vacuum packaged and stored at 4°C or 10°C for 21 or 70 days. After 21 days of storage at 10°C, NS and NS + MW reduced viable vegetative cell (VVC) counts by approximately 3 log cycles compared to the control. After 70 days storage, VVC counts were higher (P < 0.05) at 10°C than 4°C only for the control. However, at 4°C, VVC counts for each treatment were not different (P > 0.05) from those of the control. After 70 days of storage at 10°C, VVC counts were lower (P < 0.05) for NS and NS + MW treatments than for the control.

Effects of Mode of Sterilization on the Recovery of Phenolic Antioxidants in Laboratory Media Assessed by Nonderivatizing Gas Chromatography-Mass Spectrometry ‡.

The effects of autoclaving and filter sterilization on the recovery of butylated hydroxyanisole, butylated hydroxytoluene, tertiary butylhydroquinone, and propyl gallate in laboratory media were investigated by a nonderivatizing gas chromatography-mass spectrometry (GC-MS) method. Lauryl tryptose broth (LTB) or brain heart infusion (BHI) broth were treated with the combinations of the phenolic antioxidants at 200 ppm. The antioxidants were dissolved in 95% ethanol and either added directly to the media followed by autoclaving or filter-sterilized and then added to sterilized media in a flask. Results suggested that antioxidant recoveries were affected by the modes of sterilization as well as by the complexity of the medium. More antioxidants were recovered from the filter-sterilized than from the autoclave-sterilized samples for LTB medium, but no clear difference was seen for BHI broth. The recovery was lowest with butylated hydroxytoluene regardless of medium type or sterilization mode used. The extraction procedure applied as well as the nonderivatizing GC-MS method appeared to be adequate for the simultaneous determination of the four antioxidants.

Growth of Clostridium sporogenes PA 3679 in Home-Style Canned Quick Breads.

The safety of a home-style canned quick bread was investigated using spores of Clostridium sporogenes putrefactive anaerobe (PA) 3679. Baking was done at 177°C for 30, 40 and 50 min, at 191°C for 45, 50 and 55 min, and at 204°C for 40, 45 and 50 min. Products were analyzed for pH, water activity (aw) and vacuum level. The microbial quality of the products was determined before and after baking. Of the products baked at 177°C, some were stored for 90 days at room temperature (23 to 25°C) or in an incubator at 35°C to study their shelf-life. Inoculated and endogenous vegetative cells and their spores were counted before and after baking and after storage using Fung's Double Tube method. Results showed germination of endogenous spores in uninoculated products after baking at 177°C for 30 min and storage at 35°C for 90 days. Survival of inoculated C. sporogenes PA 3679 was detected for all baking and storage treatments. Further work is recommended to determine safe processing procedures for this type of product.

Comparisons of Selected Methods with the Fung-Yu Tube Procedure for Determining Listeria monocytogenes and Other Listeria spp. in Meats.

The ability of the motility enrichment Fung-Yu tube procedure with Oxyrase™ enzyme to detect the presence of Listeria monocytogenes inoculated into ground beef samples was compared to the USDA-FSIS method. Three strains of L. monocytogenes (LM 101M, LM 103M, and Scott A) were inoculated separately into sterilized ground beef or culture broth. The inoculum levels used were as low as 1 to 1000 Listeria cells per g of meat or per ml of broth. The Fung-Yu tube procedure produced results as sensitive as the USDA procedures and provided a shorter detection time of 26-48 h. A total of 215 retail-level meat and poultry products were analyzed comparatively by the Fung-Yu tube and the GENETRAK® DNA hybridization methods for Listeria detection. Six Listeria spp. ( L. denitrificans , L. grayi , L. innocua , L. ivanovii , L. monocytogenes , and L. murrayi ) were identified among the isolates. All 48 presumptively positive samples determined by the Fung-Yu tube method were further confirmed to harbor Listeria by biochemical tests. Eleven samples were missed by the GENETRAK® procedure, probably because of the enrichment procedure. L. monocytogenes was isolated from ground beef, pork sausage, smokie links, and cheese hot dogs. Among cooked samples examined, only cheese hot dogs and macaroni and cheese loaf showed substantial incidence of Listeria contamination.

Comparison of Redigel, Petrifilm, Spiral Plate System, Isogrid, and Aerobic Plate Count for Determining the Numbers of Aerobic Bacteria in Selected Foods.

The numbers of aerobic bacteria from chicken, ground beef, ground pork, shelled pecan, raw milk, thyme, and flour (20 samples from each food) were determined by four alternative viable cell count methods (Redigel, Petrifilm, Spiral Plate System, and Isogrid) to ascertain the effectiveness of these methods in providing viable cell counts compared with the widely used Aerobic Plate Count (APC) method. The results indicated that all five methods were highly comparable (r=0.97 and higher, with the exception of Petrifilm versus Spiral Plate System, which was 0.88) and exhibited a high degree of accuracy and agreement. Thus, the four alternative methods were found to provide accurate aerobic bacterial counts of foods compared with the APC method.

Microbiological Study of Tofu.

The microbiological qualities of tofu juice and cake were studied. Seven brands of tofu from four grocery stores were tested, at day 1 and after 30 d of storage in a refrigerator. The microbial load at day 1 was different from brand to brand, but cell counts in juice and cake of the same brand were correlated. The number of cells observed at day 30 was different from brand to brand but was not related to the initial cell count. The pH had a great effect on the type of contaminating microorganisms present. All brands spoiled after 30 d of storage at 7°C; 112 isolates from both the fresh juice and cake at day 1 and at day 30 were obtained. The most common gram-positive organisms isolated were Streptococcus sp., Pediococcus sp., and Lactobacillus sp., and the most common gram-negative bacteria were Pseudomonas putida , P. aeruginosa , Enterobacter agglomerans , and E. cloacae .

Rapid Methods and Automation: A Survey of Professional Microbiologists.

Participants of an international workshop on rapid methods and automation were surveyed concerning the numbers of total plate counts and coliform counts performed per year, the numbers and kinds of pathogen detection tests routinely performed, and the type of instruments and diagnostic kits routinely used in their laboratories. The candid opinions on what is needed in the near future and the general perceptions of the field of rapid methods and automation in microbiology and their wish list were also solicited. Responses from 55 professional practicing microbiologists were analyzed. The data should be of interest to educators and the developers of instruments and diagnostic kits as well as applied microbiologists concerned with the current status and future development of the field of rapid methods and automation in microbiology.

Microbiology and Water Activity Relationship in the Processing and Storage of Sudanese Dry Meat (Sharmoot).

The Sudanese dry meat SHARMOOT is a major food product in East Africa. No information is available on microbial profiles or improved methods of processing the product. We have developed a pre-cooking and grinding procedure that produces the meat efficiently. The product is chemically and microbiologically stable for at least 4 months without refrigeration. While staphylococci and Enterobacteriaceae were the most common major bacterial groups isolated from dried meat samples at the beginning, micrococci and bacilli predominated during the last stages of storage. Microbiological data (total, spore, yeast and mold, and Staphylococcus aureus counts and Clostridium perfringens detection) indicated that the product made by us is microbiologically more acceptable than a comparable product made traditionally in Sudan. The potential exists for large-scale production of SHARMOOT.

Destruction of Indigenous Bacillus Spores in Soymilk by Heat 1.

Boiling of soymilk in preparation for soy yogurt production does not destroy the indigenous Bacillus spores which may affect the fermentation process. Soymilk (2.2 × 103 CFU/ml) boiled for 1 min in a Microwave had 33 CFU/ml, steamed for 20 min at 110°C had 25 CFU/ml, and autoclaved at 121°C for 15 min had no detectable organisms (<1 × 10). Soy yogurt made from these heat-treated soymilks had 4.2 × 106 Bacillus spores/ml, 3.4 × 104 Bacillus spores ml, and no detectable spores, respectively. We recommend autoclaving (121°C, 15 min) soymilk before performing research work on microbiology and biochemistry of soy yogurt fermentation.