Validation of the CompactDry "Nissui" BC for Enumeration of Bacillus cereus in a Variety of Foods: AOAC Performance Tested MethodSM 092201.

BACKGROUND: The CompactDry "Nissui" BC is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of Bacillus cereus in products after incubation at 30 ± 1°C for 24 ± 2 h. OBJECTIVE: The CompactDry "Nissui" BC method was validated to achieve AOAC Performance Tested MethodsSM certification. METHOD: The performance of the CompactDry "Nissui" BC was compared to that of ISO 7932:2004 for 10 matrixes, including panna cotta, double cream, dried baby food, dried vegetable soup mix, seafood sticks, salmon pâté, sliced ham, pork liver pâté, ham and cheese sandwich, and Caesar pasta salad with chicken and bacon. Performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. RESULTS: After log10 transformation of the data, the relative standard deviation of repeatability (RSDr) was ≤9.2% for 28 of the 30 materials (10 matrixes each at three contamination levels) analyzed by the CompactDry "Nissui" BC method and ≤13% for 27 of the 30 matrix/level combinations analyzed by the reference method. Method equivalence was demonstrated in 28 of the 30 matrix/level combinations based on the 90% confidence interval of the DOM being within (-0.5, 0.5). For inclusivity, 47 of 50 strains tested showed typical colonies and confirmed positive. For exclusivity, 28 of 33 strains tested resulted in no growth or were negative, and five were positive. Inclusivity and exclusivity results were similar on the reference method agar. The method was shown to be robust to changes in sample volume, incubation temperature, and incubation time, and data are presented supporting product consistency and 18-month shelf life. CONCLUSIONS: The CompactDry "Nissui" BC method is validated for the determination of Bacillus cereus in a variety of matrixes. HIGHLIGHTS: The CompactDry "Nissui" BC method is equivalent to the ISO 7932:2004 reference method and is suitable for Performance Tested MethodsSM certification for the matrixes tested.

Validation of the CompactDry "Nissui" YMR for Enumeration of Yeasts and Molds in a Variety of Foods: AOAC Performance Tested MethodSM 092002.

BACKGROUND: The CompactDry "Nissui" YMR is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of yeasts and molds in products after incubation at 25 ± 1°C for 3 days. OBJECTIVE: The CompactDry "Nissui" YMR method was validated in order to achieve AOAC Performance Tested MethodsSM certification. METHOD: The performance of the CompactDry "Nissui" YMR was compared to that of ISO 21527-1:2008 for 10 matrixes including cooked prawns, deli vegetable salad, tuna pâté, fermented yogurt drink, spinach and ricotta quiche, egg custard tarts, fruit and vegetable smoothie, cream cheese, egg salad sandwich, and deli pasta salad. Performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. RESULTS: After log10 transformation of the data, the relative standard deviation of repeatability (RSDr) was <10% for all 30 materials (10 matrixes each at 3 levels) analyzed by the CompactDry "Nissui" YMR method and for 29 of the 30 matrix/level combinations analyzed by the reference method. The DOM ranged from -0.284 (-0.310, -0.257) log10 CFU/g to 0.307 (-0.013, 0.627) log10 CFU/g. Method equivalence was demonstrated in 29 of the 30 matrix/level combinations based on the 90% confidence interval of the DOM being within (-0.5, 0.5). All 51 inclusivity strains showed expected or atypical results, and all 32 exclusivity organisms showed no growth on the medium. The method was shown to be robust to changes in sample volume, incubation temperature, and incubation time, and data are presented supporting product consistency and a 24-month shelf life. CONCLUSIONS: The CompactDry "Nissui" YMR method is validated for the determination of yeasts and molds in a variety of matrixes. HIGHLIGHTS: The CompactDry "Nissui" YMR method is equivalent to the ISO 21527-1:2008 reference method and is suitable for Performance Tested MethodsSM certification for the matrixes tested.

Soleris®Enterobacteriaceae for the Detection of Enterobacteriaceae in Select Foods: AOAC Performance Tested MethodSM 121901.

BACKGROUND: Soleris®Enterobacteriaceae is a growth-based, automated method for detection of Enterobacteriaceae in food. OBJECTIVE: A study was conducted to validate the Soleris method for detection of Enterobacteriaceae in select foods (pasteurized milk, yogurt, mozzarella cheese, ice cream, dried milk, pasteurized liquid egg, frozen cooked chicken, deli ham, lettuce, and dry dog food) at a threshold of ≥ 10 CFU/g of product. METHODS: Inclusivity and exclusivity of the Soleris method were assessed by testing 55 and 38 target and non-target bacterial strains, respectively. Matrix testing was performed with one naturally contaminated and nine inoculated foods. Efficacy of the Soleris method was compared to that of the ISO 21528-2:2017 direct plating reference method using probability of detection analysis. Independent laboratory testing was conducted to verify method performance in two matrixes (yogurt and deli ham). Method robustness, stability, and lot-to-lot consistency of the Soleris reagents were also assessed. RESULTS: Inclusivity of the Soleris test was 91% and exclusivity was 100%. In matrix testing, there were no significant differences in the number of positive results obtained with the Soleris and reference methods for any of the matrixes examined. Overall, of 370 test portions, there were 176 positive results by the Soleris method and 177 positive results by the reference procedure. CONCLUSIONS: Soleris Enterobacteriaceae is an effective method for detection of Enterobacteriaceae in the foods evaluated, with performance equivalent to that of the ISO 21528-2:2017 reference method. HIGHLIGHTS: The Soleris method offers the advantages of labor savings and results within 18 h.

MC-Media Pad CC for Enumeration of Total Coliforms in a Variety of Foods.

BACKGROUND: Standard coliform count methods require the preparation of agar, the use of the pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad CC for the enumeration of coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. OBJECTIVE: Using a paired study design, the MC-Media Pad CC was compared to standard method ISO 4832:2006 for 10 matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice. METHODS: Each matrix was tested at three levels of coliform contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad CC and ISO 4832:2006 methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: The candidate and reference methods demonstrated SDs ranging from 0.027 to 0.264 and 0.025 to 0.157, respectively. The difference of means ranged from -0.015 to 0.381, showing no practical difference between the methods. The MC-Media Pad CC detected 58/62 inclusivity strains and correctly excluded 26/31 exclusivity organisms, similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. CONCLUSIONS: The results support the conclusions that the MC-Media Pad CC is a suitable alternative to the ISO 4832:2006 reference method for the matrixes examined and the data support AOAC Performance Tested MethodSM certification.

MC-Media Pad EC for Enumeration of Escherichia coli and Coliforms in a Variety of Foods.

BACKGROUND: Standard coliform count methods require preparation of agar, the use of pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad EC for enumeration of Escherichia coli and coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. OBJECTIVE: Using a paired study design, the MC-Media Pad EC was compared with standard method ISO 4832:2006. Ten matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice were evaluated in the study. METHODS: Each matrix was tested at three levels of contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad EC, ISO 4832:2006, and ISO 16649-2:2001 (Part 2) methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: The candidate and reference methods demonstrated standard deviations ranging from 0.034 to 0.188 and 0.028 to 0.181, respectively, for E. coli counts and 0.047-0.188 and 0.025-0.157, respectively, for total coliforms. The difference of means ranged from -0.025 to 0.331 for E. coli and from -0.037 to 0.372 for total coliforms, showing no practical difference between the methods. The MC-Media Pad EC detected 49/50 E. coli and 60/63 coliform inclusivity strains and correctly excluded 30/32 exclusivity organisms for E. coli and 24/31 exclusivity organisms for total coliforms, which was similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. CONCLUSIONS: The results support the conclusions that the MC-Media Pad EC is a suitable alternative to the ISO 4832:2006 and ISO 16649-2:2001 reference methods for the matrixes examined and the data support AOAC Performance Tested MethodSM certification. HIGHLIGHTS: The MC-Media Pad EC was approved for Performance Tested Method certification No. 011901.

MC-Media Pad SA (Sanita-kun SA) for the Enumeration of Staphylococcus aureus in a Variety of Foods.

MC-Media Pad SA (formerly known as Sanita-kun SA) is a dry rehydratable film medium for the enumeration of Staphylococcus aureus. The performance of the method in a variety of foods was compared with that of ISO 6888-1:1999, Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Enumeration of Coagulase-Positive Staphylococci (Staphylococcus aureus and Other Species) - Part 1: Technique Using Baird-Parker Agar Medium. The validated matrixes included pastrami, a sliced cooked chicken roll, cooked prawns, cold-smoked salmon, pasta salad, sandwich spread, fresh uncooked pasta, infant cereal, custard, and raw-milk Brie cheese. In the matrix study, five replicates at each of three contamination levels were tested as paired test portions. Across all matrixes, the difference in mean log10 values ranged from -0.32 to 0.10, which was within the acceptable range of -0.50 to 0.50. Thus, all 10 matrixes met the acceptance criterion at all concentration levels. Further, only two matrixes, cooked prawns and raw-milk Brie cheese, had 95% confidence limits outside the -0.50 to 0.50 criterion, and these were at the lowest concentration level for each matrix. The candidate method sr varied from 0.03 to 0.22 log10 CFU/g. This compares favorably with the reference method SD, which ranged from 0.06 to 0.30 log10 CFU/g. The candidate and reference methods detected 51 of 53 inclusivity strains, with both methods not detecting the same two strains. The candidate method did not detect any of the 32 exclusivity strains, whereas the reference method did not detect 30 of the 32 exclusivity strains; the 2 strains detected by the reference method were S. delphini and S. hyicus, both developing atypical colonies on Baird-Parker plates. The product consistency study demonstrated no significant difference between lots of product and supported the 1 year shelf life. Robustness testing yielded no significant differences when small variations were made in sample volume, incubation temperature, and incubation time. Thus, the data show equivalent or better performance of the Sanita-kun SA/MC-Media Pad SA method compared with the International Organization for Standardization reference method, in support of AOAC Performance Tested MethodSM certification.

MC-Media Pad ACplus™ for Enumeration of Aerobic Counts in a Variety of Foods.

The MC-Media Pad ACplus™ is a dry, rehydratable film medium for the enumeration of aerobic bacterial colonies. The performance of the method in a variety of foods was compared to that of U.S. reference methods: U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 3.02 "Quantitative Analysis of Bacteria in Foods as Sanitary Indicators" (USDA/FSIS MLG 3.02); Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6 "Microbiological Count Methods, Standard Plate Count Method" (SMEDP 6); AOAC Official MethodSM 966.23 Microbiological Methods; and ISO 4833-1:2013 "Microbiology of the food chain-Horizontal method for the enumeration of microorganisms-Part 1: Colony count at 30 degrees C by the pour plate technique." The validated matrixes included raw chicken breast and raw ground pork for USDA/FSIS MLG 3.02; cream cheese and yogurt drink for SMEDP 6; parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for AOAC Method 966.23, and raw chicken breast, raw ground pork, cream cheese, yogurt drink, parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for ISO 4833-1:2013. In each matrix study, five replicates at each of three contamination levels were tested as paired test portions. All 10 matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus standard-usage conditions (35 ± 1°C for 48 ± 2 h). Across all matrixes, the difference of mean log10 values ranged from -0.43 to 0.44, within the acceptable range of -0.50 to 0.50. The candidate method repeatability SD (sr) varied from 0.03 to 0.23 log10 CFU/g, comparing favorably to the reference method SD, which ranged from 0.06 to 0.30 log10 CFU/g. Seven matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus rapid-usage conditions (35 ± 1°C for 24 ± 2 h). Of the 21 matrix/concentration combinations, only three instances of difference of mean >0.5 log were observed. The ranges of sr values of the rapid-usage candidate method (0.023-0.324) and the reference method (0.013-0.236) were similar for the seven matrixes tested. All 10 matrixes were compared to the International Organization for Standardization (ISO) reference method under MC-Media Pad ACplus alternate-method conditions (30 ± 1°C for 72 ± 3 h). All 10 matrixes yielded a mean difference between methods of <0.5 log, and the ranges of sr values were similar between the candidate alternate method (0.037-0.378) and the ISO reference method (0.037-0.437). The product consistency study demonstrated no significant difference between lots of product and supported the 2-year shelf life. Robustness testing yielded no significant differences when small variations were made in sample volume, incubation temperature, and incubation time. Thus, the data show equivalent or better performance of the MC-Media Pad ACplus method compared to the relevant reference methods in support of AOAC Performance Tested MethodSM certification.

Membrane toxicity of antimicrobial compounds from essential oils.

Natural antimicrobial compounds perform their action mainly against cell membranes. The aim of this work was to evaluate the interaction, meant as a mechanism of action, of essential oil antimicrobial compounds with the microbial cell envelope. The lipid profiles of Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas fluorescens, and Brochothrix thermosphacta cells treated with thymol, carvacrol, limonene, eugenol, and cinnamaldehyde have been analyzed by gas chromatography. In line with the fatty acids analysis, the treated cells were also observed by scanning electron microscopy (SEM) to evaluate structural alterations. The overall results showed a strong decrease of the unsaturated fatty acids (UFAs) for the treated cells; in particular, the C18:2trans and C18:3cis underwent a notable reduction contributing to the total UFA decreases, while the saturated fatty acid C17:0 raised the highest concentration in cinnamaldehyde-treated cells. SEM images showed that the used antimicrobial compounds quickly exerted their antimicrobial activities, determining structural alterations of the cell envelope.

Changes in membrane fatty acids composition of microbial cells induced by addiction of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol in the growing media.

Major active compounds from essential oils are well-known to possess antimicrobial activity against both pathogen and spoilage microorganisms. The aim of this work was to determine the alteration of the membrane fatty acid profile as an adaptive mechanism of the cells in the presence of a sublethal concentration of antimicrobial compound in response to a stress condition. Methanolic solutions of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol were added into growth media of Escherichia coli O157:H7, Salmonella enterica serovar typhimurium, Pseudomonas fluorescens, Brochothrix thermosphacta, and Staphylococcus aureus strains. Fatty acid extraction and gas chromatographic analysis were performed to assess changes in membrane fatty acid composition. Substantial changes were observed on the long chain unsaturated fatty acids when the E. coli and Salmonella strains grew in the presence of limonene and cinnamaldehyde and carvacrol and eugenol, respectively. All compounds influenced the fatty acid profile of B. thermosphacta, while Pseudomonas and S. aureus strains did not show substantial changes in their fatty acid compositions.

Use of survival analysis and Classification and Regression Trees to model the growth/no growth boundary of spoilage yeasts as affected by alcohol, pH, sucrose, sorbate and temperature.

This paper describes the application of a survival analysis model and a Classification and Regression Trees (CART) model to a data set comprising times to growth of a yeast cocktail inoculated into media simulating a fruit-based or alcoholic food or drink, and covering over 900 combinations of five environmental factors (alcohol, pH, sucrose, sorbate and temperature). Growth was determined as either the time to growth within a 150-day time period or as no-growth after 150 days. Models were developed which could either predict the likelihood of growth occurring within the 150 day period, or the time to grow, either in days or in one of three categories chosen to represent a rapid (1-14 days), medium (15-30 days) or slow (31-150 days) growth response. Growth was observed in 29% of the experimental conditions and demonstrated that the yeasts used were able to grow under extreme environmental conditions, for example at a pH value of 2.1, a temperature of 2 degrees C, a sucrose concentration of 55% (w/w) or an alcohol concentration of 12% (w/v). Generally, both models provided a reasonable fit to the data, and successfully predicted the growth class in 84% of cases. Direct comparisons of the models were made to determine the more suitable for predicting the growth of yeasts in food systems. The survival analysis model was preferred for this data set because it was more fail-safe than the CART model. In food validation studies, the survival model generally gave reliable predictions of time to growth in a range of 23 different food and drink products and is considered to be a reliable model to predict the likelihood and speed of yeast spoilage for a range of fruit-based or alcoholic food or drinks.