Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food

In this study, we developed a rapid and sensitive fimY-based loop-mediated isothermal amplification (LAMP) assay on a real-time turbidimeter platform for the detection of Salmonella in food. Since turbidity of the reaction mixture would increase in correlation with the DNA yield, real-time monitoring of the LAMP reaction was achieved by real-time turbidimeter. Time threshold values which indicate positive results for 81 Salmonella strains of different serotypes ranged from 36 to 40 min. For the 20 non-Salmonella strains, turbidity did not increase in the reaction mixture. When testing 10-fold serial dilutions of Salmonella Typhimurium-ATCC 14128 DNA by LAMP, the time threshold ranged from 36 to 52 min on the real-time turbidimeter. The detection limit was 13 cells per reaction in pure culture, up to 10-fold more sensitive than that of PCR. When applied in deli food samples, the LAMP assay was able to detect Salmonella even though the sample was contaminated with very low concentration after 3 h enrichment culture. Increase in turbidity was observed on real-time turbidimeter. Additionally, the LAMP results detected by naked-eye or turbidity consistently matched with each other. Results from this study showed that the fimY-based LAMP assay is an effective method for the rapid detection of Salmonella.     

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein,rimM, for detection and enumeration of Streptococcus thermophilus in dairy products

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein, rimM, for specific detection of Streptococcus thermophilus was developed. The designed real-time PCR primers and probe were specific for S. thermophilus JCM20026, LMG6896, LMG18311, OJT101, OJT102 but not Enteroccocus spp., Lactococcus lactis subsp. lactis, and Streptococcus salivarius which are phylogenetically closely related to S. thermophilus and are difficult to identify using culture-based methods. The linear range of the developed real-time PCR method was from 2.7 to 8.6 log CFU ml^?1 with an amplification efficiency of 96%. Minor differences (about 0.4 log CFU ml^?1) were observed between counts of S. thermophilus obtained by culture and real-time PCR method in plain yoghurt and yoghurt containing fruits. Therefore, the developed real-time PCR method could be of potential application in specific detection and accurate enumeration of S. thermophilus in a wide range of dairy products.     

A meta-analytical estimation of the detection limits of methods for Salmonella in food

The validation of microbial detection methods for foods does not typically state a limit of detection value. Therefore performances of rapid and reference methods for Salmonella detection in foods were compared retrospectively using data from 49 published studies. A total of 576 values for the limit of detection (LOD₅₀) were calculated. The major scientific variables in these independent validation studies were food matrices, Salmonella serovars, and method types (reference, cultural and immunological and nucleic acid based). The basic design feature of the original studies was comparison of the performances of new methods with those of cultural reference methods. A major experimental design variable was the number of laboratories per study. There were 29 single laboratory studies with 20 replicates per level of Salmonella spiked into the food matrix. The 20 multi-laboratory (N ≥ 10) studies had 5–6 replicates per level of Salmonella. The LOD₅₀ values of the dataset had a mean value of 0.021 MPN g^? 1 (standard deviation range (0.013–0.036) and the distribution of their logarithms was symmetrical about the logarithm of the mean if the outlier values were discounted. Eighty-nine percent of the values ranged from 0.01 to 0.04 MPN g^? 1. On this basis about 11% of the values were deemed outliers. About three-quarters of them were > 0.04 MPN g^? 1. The distribution derived in this study can be used as a bench mark against which to evaluate LOD₅₀ data generated in future studies of detection methods for Salmonella in foods and potentially offers a possible way to streamline method validation study experimental design.     

Survival of Salmonella Enteritidis PT 30 on inoculated almond kernels in hot water treatments

Almonds are blanched by exposure to hot water or steam-injected water to remove the pellicle (skin) from the kernel. This study evaluated the survival of Salmonella Enteritidis PT 30, Salmonella Senftenberg 775W and Enterococcus faecalis on whole raw almond kernels exposed to hot water. Whole, inoculated (7 to 9 log CFU/g) Nonpareil almonds (40 g) were submerged in 25 L of water maintained at 60, 70, 80 and 88 °C. Almonds were heated for up to 12 min, drained for 2 s, and transferred to 80 mL of cold (4 °C) tryptic soy broth. Almonds in broth were stomached at high speed for 2 min, serially diluted, plated onto tryptic soy and bismuth sulfite agars (Salmonella) or bile esculin agar (Enterococcus) and incubated at 37 °C for 24 and 48 h, respectively. D values of 2.6, 1.2, 0.75 and 0.39 min were calculated for exposure of S. Enteritidis PT 30 to water at 60, 70, 80 and 88 °C, respectively; the calculated z value was 35 C°. D values determined for Salmonella Senftenberg 775W and E. faecalis at 88 °C were 0.37 and 0.36 min, respectively. Neither Salmonella serovar could be recovered by enrichment of 1-g samples after almonds inoculated at 5 log CFU/g were heated at 88 °C for 2 min. These data will be useful to validate almond industry blanching processes.     

Synergistic effect of sonication and high osmotic pressure enhances membrane damage and viability loss of Salmonella in orange juice

The efficacy of using sonication (50 ± 0.2 W, 20 kHz), combined with subsequent concentration and storage at high osmotic pressure, has been evaluated to reduce levels of Salmonella bacteria in different solutions (PBS, sucrose and orange juice) at varying concentrations. To visualize the impact on cell membranes, we used a staining protocol (propidium iodide [PI] and 4′,6′-diamidino-2-phenylindole [DAPI]). Sonication alone did not cause significant membrane damage. Storage alone, for 48 h and at high osmotic pressure (10.9 MPa), affected membrane permeability in 20% of cells. However, sonication, combined with storage, considerably increased loss of membrane integrity, resulting in a significant logarithmic reduction of microorganisms. When the combination was applied to contaminated orange juice, a 5 log₁₀ cfu ml^?1 reduction of Salmonella spp. was obtained. “Osmosonication”—the synergistic combination of sonication and subsequent storage at high osmotic pressure—is an innovative alternative for the non-thermal decontamination of liquid foods.     

Electrostatic spraying of organic acids on biofilms formed by E. coli O157:H7 and Salmonella Typhimurium on fresh produce

Electrostatic spraying which has an even and retained surface coverage could be an effective novel technique to completely cover the surface of fresh produce to disrupt biofilm formation by pathogenic bacteria. Spinach leaves and cantaloupe rind were spot-inoculated with a bacterial culture and stored at 8 °C for 72 h to allow biofilm formation. Among various green fluorescent protein-labeled strains, ED 14 strain of E. coli O157:H7 and SD 10 strain of Salmonella Typhimurium had the best attachment based on colony counts. The produce samples were electrostatically sprayed with malic (MA) and lactic (LA) acid solutions alone (1.0/2.0/3.0/4.0% w/v) or in combination (0.5 + 0.5/1.0 + 1.0/1.5 + 1.5/2.0 + 2.0% w/v) to test for a reduction in the attached bacteria. A combined treatment of LA 2.0% w/v + MA 2.0% w/v had the highest log reduction (CFU/disk) of 4.14 and 3.6 on the attached E. coli strain ED 14 (spinach) and Salmonella strain SD 10 (cantaloupe), respectively. Crystal violet assay demonstrated the disruptive effect of organic acids on biofilms formed by the pathogenic bacteria. Application of electrostatic spray with a combination of malic and lactic acids resulting in a log reduction (CFU/disk) of 3.6 or higher can improve the microbial safety of spinach and cantaloupe by preventing the pathogenic biofilm formation and bacterial growth.     

Salmonella contamination of laying-hen flocks in two regions of Algeria

A preliminary epidemiological study of Salmonella contamination in laying-hen flocks was carried out in the regions of Annaba and Eltarf, Algeria, from March to October 2008 and March to November 2009. Our objectives were (i) to estimate the prevalence of infection by Salmonella spp. in seven pooled samples during the hens' laying period (ii) to identify the serotypes and antimicrobial resistance phenotypes of isolates, and (iii) to characterize the factors that may be related to Salmonella contamination in Algerian henhouses. For this purpose, 18 out of 22 operational laying-hen houses were sampled one to three times during these periods: once at the start of laying (pullets aged 22–31 weeks), once in the middle of laying (47–60 week) and once at the end of laying prior to depopulation (70–86 week). The flocks'Salmonella status was assessed by collecting 2754 environmental samples that were analyzed according to the ISO 6579 method. The antibiotic resistance of Salmonella strains was tested as per the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The relationship between each potential risk factor and the Salmonella status of laying-hen flocks was evaluated by calculating the relative risk with 95% confidence intervals. Eight flocks tested positive for Salmonella spp., with a higher prevalence at the end of laying than at either the beginning or middle. Only 19 isolates were recovered from the 2754 samples analyzed and nine different serotypes identified. S. enteritidis (n = 4) was the most prevalent serovar, along with S. Kentucky and S. Hadar (n = 3), followed by S. Heidelberg, S. Manhattan and S. Virchow (n = 2), whereas S. Dublin, S. Typhimurium and S. Albany were found only once. Thirteen isolates were resistant to at least one antimicrobial agent. Of these, six were resistant to at least three different antimicrobial classes. Salmonella serovar Kentucky isolates were resistant to fluoroquinolones with ciprofloxacin MIC ≥ 8 mg/L. Six risk indicators were identified as potentially related to the Salmonella status of layer houses.     

Radio-sensitivity of pathogens in inoculated prepared foods of animal origin

The effectiveness of irradiation for inactivating Staphylococcus aureus, Listeria ivanovii, Salmonella Typhimurium, and Escherichia coli in the prepared foods of animal origin was investigated. Commercially available seasoned and cooked beef, fried egg, and ham were purchased, radiation-sterilized, and inoculated at 10⁶–10⁷ CFU/g with each of the four pathogens and stored at three storage conditions at 10 °C, 20 °C, and 30 °C. D₁₀-values of S. aureus, L. ivanovii, Salmonella Typhimurium, and E. coli were 0.34±0.01, 0.24±0.02, 0.24±0.01, and 0.27±0.01 kGy, respectively. No viable cells were detected at 3 kGy of irradiation. Salmonella mutagenicity assay (Ames test) indicated that the 10 kGy irradiated samples were statistically similar to non-irradiated control samples in the Salmonella mutagenicity assay (Ames test). These studies demonstrate that irradiation can be used as an additional safety tool to produce microbiologically safe and wholesome prepared foods of animal origin.     

A systematic review and meta-analysis of the effectiveness of biosecurity and vaccination in reducing Salmonella spp. in broiler chickens

Our objective was to estimate the effectiveness of vaccination and biosecurity on the prevalence of Salmonella spp. in broiler chickens using systematic review and meta-analysis. A comprehensive search of the global primary literature was conducted in: Current Contents (1999–2009), Agricola (1924–2009), MEDLINE (1860–2009), Scopus (1960–2009), CAB (1913–2009), and Centre for Agricultural Bioscience Global Health (1971–2009). The search algorithm was (Salmonell*) AND (chicken* OR chick* OR poultry* OR broiler* OR gallus*). Additional studies were identified by contacting five topic experts and hand-scanning bibliographies of recent review articles and a recently published textbook. Studies were included if they were English language and investigated the effects of vaccination and biosecurity on the prevalence of Salmonella spp. in broiler chickens. All study design types were included. Data extraction and methodological assessment were conducted by two reviewers independently. All meta-analyses were based on random-effects models. For biosecurity, sixteen challenge studies (n = 137 treatment-control comparisons) and one controlled study (n = 2) met the inclusion criteria. Significant heterogeneity (Cochran's Q-statistic, p < 0.001) was observed among biosecurity challenge studies examining hydrogen peroxide or polyhexamethylenebiguanide hydrochloride applied to hatching eggs, making it inappropriate to present a summary effects measure. For vaccination, 19 challenge studies (n = 226) and three controlled studies (n = 10) met the inclusion criteria. Among live Salmonella Typhimurium vaccine challenge studies heterogeneity was not significant (p = 0.138). Vaccination with a live Salmonella Typhimurium reduced the risk of Salmonella cecal colonization in the treated broiler group by 35 out of 1000 broilers when compared to the control group (OR = 0.21; 95% CI = 0.06–0.77) and this effect was significant (p = 0.018). One biosecurity study (n = 2 treatment-control comparisons) and three vaccination studies (n = 10) were conducted in a commercial setting. The two included studies in the vaccination meta-analysis were both conducted at research facilities. The live Salmonella Typhimurium vaccine showed the most promise in reducing the prevalence of Salmonella in broiler ceca. However, the meta-analysis included few studies, and these studies challenged the birds with different serotypes. We recommend that more large-scale randomized, blinded trials be conducted with a live Salmonella Typhimurium vaccine on commercial farms.     

Decontamination of Salmonella enterica Typhimurium on green onions using a new formula of sanitizer washing and pulsed UV light (PL)

Pathogenic bacteria such as Salmonella and Shigella flexneri have been linked to green onion contamination. This study was conducted to evaluate decontamination of Salmonella Typhimurium using a new formula of sanitizer washing (0.4 mg/mL thymol and five new formula sanitizers including 300 ppm H₂O₂ + 4% SDS, 2 mg/mL citric acid + 4% SDS, 0.2 mg/mL thymol + 4% SDS, 0.2 mg/mL thymol + 2 mg/mL citric acid and 0.2 mg/mL thymol + 2 mg/mL acetic acid), pulsed UV light (PL) as well as synergy between the sanitizer wash and PL. New formula sanitizers based on decontamination efficacy of single washing solutions (organic acids, hydrogen peroxide (H₂O₂), essential oil or surfactant) were applied to decontaminate spot inoculated green onions. PL, the novel technique, alone has been applied to inactivate Salmonella on both dip and spot inoculated green onions. Salmonella inactivation of PL–new formula sanitizer combinations on dip inoculated green onions was investigated for their potential synergy. As a result, for spot inoculated green onions, 0.4 mg/mL thymol individually and the five new formula sanitizers all achieved higher log reduction of Salmonella (4.5–5.3 log 10 CFU/g reduction) than the 200 ppm chlorine washing. These new formulas of sanitizer would be potential alternatives to chlorine. The 5 s dry PL (4.6 log 10 CFU/g) or 60 s wet PL treatment (3.6 log 10 CFU/g) was better or comparable as chlorine washing. The sanitizer combinations did not provide significantly higher log reduction than PL, and PL has the potential of being used in the green onion industry for decontamination purpose. For dip inoculated green onions, none of our treatment provided > 0.8 log 10 CFU/g (0.6–0.8 log 10 CFU/g) reduction of Salmonella. As a result, the PL–new formula sanitizer combinations had no or minimal synergy to inactivate Salmonella dip inoculated on green onions.     

Comparison of multiple chemical sanitizers for reducing Salmonella and Escherichia coli O157:H7 on spinach (Spinacia oleracea) leaves

Effectiveness of multiple chemical sanitizers on the reduction of Salmonella spp. and Escherichia coli O157:H7 on spinach was compared. Fresh spinach (Spinacia oleracea) was inoculated with a bacterial suspension containing multiple strains of rifampin-resistant Salmonella and E. coli O157:H7. Inoculated spinach leaves were treated with a water wash or water wash followed by 2% L-lactic acid at 55 °C, peroxyacetic acid (80 mg/L), calcium hypochlorite (200 mg/L), ozonated water (mg/L) or ClO₂ gas (1.2 or 2.1 mg/L). The l-lactic acid produced a 2.7 log CFU/g reduction for E. coli O157:H7 and a 2.3 log CFU/g reduction for Salmonella, statistically significant compared to water wash alone (P < 0.05), which resulted in a reduction of 0.7 log CFU/g for both pathogens. These findings indicate that 2% l-lactic acid at 55 °C may be an effective treatment for reducing pathogens on spinach leaves.     

Fat contributes to the buffering capacity of chicken skin and meat but enhances the vulnerability of attached Salmonella cells to acetic acid treatment

Chicken skin and chicken meat display different buffering effects which may impact the survival of Salmonella attached to them when treated with acids. This study investigated the role that differences in fat composition of chicken skin and meat play in their buffering capacity. The survival of Salmonella attached to chicken skin and meat in the presence of fat, and treated with acetic acid was also investigated. Fat was extracted from chicken skin and meat and the buffering capacities of chicken skin, meat, extracted fat and their respective remnants were determined. Two strains of Salmonella Typhimurium and two strains of S. Enteritidis were attached independently to each of the chicken component listed above and enumerated before and after treatment with 0.3 M acetic acid. Chicken skin has a higher fat content as compared to chicken meat. Skin (13 mmol H⁺/(pH1 kg)) had a stronger buffering capacity (p < 0.05) than the extracted fat alone and skin remnants alone (7.0 mmol H⁺/(pH 1 kg) and 6.9 mmol H⁺/(pH 1 kg) respectively). From an initial inoculum (~ 9 log CFU/g), Salmonella cells attached better (p < 0.05) to chicken meat (~ 8 log CFU/g) and chicken skin (~ 7 log CFU/g) than extracted fat (~ 1.5 log CFU/g). Skin remnants without fat were better (p < 0.05) at protecting attached Salmonella than other chicken components. For example S. Typhimurium ATCC 33062 decreased ~ 1 log CFU/g (p < 0.05) on skin remnants after acetic acid treatment while its viable counts on other components decreased from ~ 1.5 to 7 log CFU/g (p < 0.05). We suggest that the fat content present in the skin may enhance the vulnerability of attached cells to acetic acid.     

Rapid detection of Salmonella from vegetable rinse-water using real-time PCR

A PCR-based method for the detection of Salmonella spp. from fresh vegetable rinse-water was developed. The method is a modification of an existing Association of Official Analytical Chemist (AOAC)-approved protocol and is compatible for high throughput analysis. The protocol is sensitive enough to detect contamination in mixed-salad (made up of approximately 80% leaf lettuce, 10% red cabbage and 10% carrots by weight) that was washed with water, which was artificially contaminated with Salmonella spp. at the estimated level of 1–10 cells ml⁻¹. The modified protocol, which includes a confirmatory melt–curve analysis of PCR products, requires 8–10 h. The method should help implementation of the HACCAP program for fresh produce.     

Salmonella in food surveillance: PCR,immunoassays, and other rapid detection and quantification methods

Foodborne diseases caused by Salmonella have always been a significant health burden to many countries. Recent epidemiological data have indicated that Salmonella was the most common bacterial etiologic agent in food poisoning outbreaks both in the United States and in Asian regions like Hong Kong. In the past, labor-intensive traditional standard culture methods with long turnaround time have always been employed by many laboratories of public health services for the detection of Salmonella in Food Surveillance Programmes. To cope with the enormous volume of sample received for Salmonella detection, recent advances in nucleic acid- and immunoassay-based methods, and the subsequent commercialization and automation of the technologies have provided more rapid, specific, and productive alternatives for routine applications in testing laboratories. Fluorogenic or real-time PCR methods are able to generate results in a day, whereas immunoassay-based methods can produce negative results in 1–3 days. Some of these rapid methods have already been validated and accepted by international authorities as standard methods and have become increasingly popular among testing laboratories.     

A modified methylene blue assay for accurate cell counting

Cell counting is a common technique in cellular and molecular biology research applications, such as cell culture maintenance, cell plating, cell growth and cell doubling time determinations, as well as cell proliferation and cytotoxicity measurements. Many commonly employed cell counting methods exhibit limitations that influence resulting accuracy or versatility. For example, the trypan blue method typically underestimates cell numbers in culture, and the Lowry protein assay can be influenced by cell cycle. An urgent need exists for a method of cell counting that is both accurate and versatile. This work intended to explore an adaptation of the methylene blue assay to overcome the existing limitations of the procedure, enabling application to a broader range of cell densities and various cell culture plates. This new methylene blue assay was found to be more efficient, accurate and sensitive. A linear relationship (r² > 0.99) was established between cell number and absorbance at 570 nm wavelength when the new methylene blue assay was applied to three cell lines (HepG2, Caco-2, and MCF-7) plated in a broad range of cell densities (5 × 10⁴ to 2.5 × 10⁶) in four different types of culture plates (6-, 12-, 24-, and 96-well plates). Growth curves were determined using both the trypan blue and methylene blue methods. At each time point in the HepG2 growth curve, the cell count obtained using the trypan blue assay was statistically significantly lower than that obtained using the methylene blue assay (p < 0.05). The same was true for the Caco-2 growth curve at all time points (p < 0.05) except at the 0 h. The methylene blue method proposed in this paper may serve as a direct, automated counting method for cells grown in any type of culture plates. This assay has clear advantages over traditional methods and is a powerful tool for any application requiring a versatile, efficient, and accurate method of cell counting, such as bioavailability and cytotoxicity assays, and more basic experiments such as cell growth curve or doubling time determination, especially in the research of natural products, bioactive compounds, phytochemicals, functional foods and nutraceuticals.     

Risk of salmonellosis from consumption of almonds in the North American market

Salmonellosis outbreaks from consumption of raw almonds in 2001 and 2004 led to regulations that require mandatory treatment of almonds sold in North America to give a minimum 4-log reduction of Salmonella. This study aims to: 1) assess the risk of salmonellosis associated with almond consumption in North America, with current treatments in effect; 2) determine the resilience of the current production system to increases in prevalence or concentration of Salmonella on almonds; 3) assess the impact of treating less than 100% of the crop; and 4) investigate conditions that could explain the number of cases associated with the 2001 outbreak. Risk was assessed using a Monte Carlo simulation, based on an established dose–response relationship. Data for almond amounts sold, Salmonella prevalence and concentration on almonds, storage time and temperature at different handling steps, population reductions during storage at various temperatures and with different treatments, and consumer handling were based on data from published sources and almond industry or academic expert opinion. What-if scenarios were evaluated for Salmonella prevalence varying from 1 to 65%, concentrations of Salmonella varying from 1 to 120 MPN/100 g, and portions of untreated crop varying from 0 to 10%. The estimated incidence of salmonellosis in North America from almonds as currently treated is on average 0.008 cases per billion servings (with an estimated 6.6 billion servings consumed annually). Increases in Salmonella prevalence to 25%, mean concentrations above 25 MPN/100 g, or leaving 0.05% of the crop untreated all resulted in an arithmetic mean risk greater than 1 case/year (with geometric means remaining below 1 case/year for all variables). Assuming 4000 kg at a prevalence of 65% (observed in recalled lots) and an average concentration of 120 MPN/100 g in raw almonds (back calculated from levels in recalled almonds) predicted over the 2800 cases estimated for the 2001 outbreak. Applying a 4-log reduction to these almonds reduced the average number of predicted cases to less than a single case. The current regulation is effective in maintaining the risk of salmonellosis from consumption of almonds below an arithmetic mean of 1 case/year, although significant increases in either prevalence or concentration, or small increases in proportion of untreated almonds would frequently lead to exceeding this threshold.     

Water activity change at elevated temperatures and thermal resistance of Salmonella in all purpose wheat flour and peanut butter

Water activity (a_w) is a major factor affecting pathogen heat resistance in low-moisture foods. However, there is a lack of data for a_w at elevated temperatures that occur during actual thermal processing conditions, and its influence on thermal tolerance of pathogens. The objective of this study was to gain an in-depth understanding of the relationship between temperature-induced changes in a_w and thermal resistance of Salmonella in all purpose flour and peanut butter at elevated temperatures (80 ^oC). Equilibrium water sorption isotherms (water content vs. water activity) for all purpose wheat flour and peanut butter over the range of 20 to 80 °C were generated using a vapor sorption analyzer and a newly developed thermal cell. The thermal resistance (D₈₀-values) of Salmonella in all purpose wheat flour and peanut butter with initial a_w of 0.45 (measured at room temperature, ~ 20 °C) was determined via isothermal treatment of small (< 1 g) samples. When increasing sample temperature from 20 to 80 °C in sealed cells, the a_w of all purpose flour increased from 0.45 to 0.80, but the a_w of peanut butter decreased from 0.45 to 0.04. The corresponding estimated D₈₀-values of Salmonella in all purpose flour and peanut butter with 20 ^oC a_w of 0.45 were 6.9 ± 0.7 min and 17.0 ± 0.9 min, respectively. The significantly (P < 0.05) higher D₈₀-value of Salmonella in peanut butter than in all purpose flour may be partially attributed to the reduced a_w in peanut butter in comparison to the increased a_w in all purpose flour at 80 °C. The improved understanding of temperature-induced changes in a_w of low-moisture products of different composition provides a new insight into seemly unpredictable results, when using heat treatments to control Salmonella in such food systems.     

Short communication: The effect of centrifugation and resuspension on the recovery of Mycoplasma species from milk

Low sensitivity of a single bulk tank milk culture is a major limitation for detection of mycoplasma organisms. We hypothesized that sedimentation of Mycoplasma spp. in a milk sample by centrifugation followed by resuspension in a small volume of fluid before agar plating would increase the ability to detect Mycoplasma spp. compared with direct conventional culture. The experiment was conducted to determine recovery of Mycoplasma spp. from milk as affected by 1) treatment (centrifugation vs. conventional method); 2) 2 species (Mycoplasma bovis and Mycoplasma californicum and 4 strains for each species); and 3) 4 different concentrations of Mycoplasma spp. (1,000, 100, 10, and 1 cfu/mL). A 5-mL portion of mycoplasma suspension from each strain was inoculated into 45 mL of fresh bulk tank milk to achieve concentrations of 1,000, 100, 10, and 1 cfu/mL. Treatment samples were vigorously mixed and centrifuged at 5,000 × g for 30 min. Control samples were vigorously mixed. All samples were plated on modified Hayflick agar. Plates were incubated at 37°C and 5% CO₂ for 5 d. Mean (±SE) log₁₀ mycoplasma counts (cfu/mL) in the treatment groups (1.91 ± 0.15) were higher than those in the control groups (1.70 ± 0.16). Recovery of at least 1 mycoplasma colony on agar culture was 100% in both treatment and control groups at high, medium, and low concentrations. At the lowest concentration, recovery of at least 1 mycoplasma colony on agar culture in treatment and control groups was 75% (n = 12/16) and 18.75% (n = 3/16), respectively. Centrifugation of milk followed by suspension in a smaller volume of saline before conventional culture increased the ability to detect mycoplasma microorganisms in the milk sample compared with controls. Recovery by centrifugation appeared best at the lowest concentration where detection of a positive sample was 4 times more likely than when conventional methods were used.     

Effect of reducing nitrate and nitrite added to dry fermented sausages on the survival of Salmonella Typhimurium

Salmonella is one of the pathogens that most frequently contaminate pork processing lines. Several hurdles can control this organism in dry fermented sausages, among them is nitrite. However, the traditional use of nitrate/nitrite in the meat industry is being questioned due to their involvement in nitrosamine formation. In this study, minced pork and sausages inoculated with Salmonella Typhimurium were prepared with 150 ppm NaNO₃ and 150 ppm NaNO₂ (maximum amounts allowed by EU), and with a reduction of 25% and 50%. The absence of nitrate/nitrite favored Salmonella growth, with 2–2.5 log cfu/g higher counts at the end of ripening, compared to nitrate/nitrite added batches. The 50% reduction showed the same inhibitory effect as the maximum amounts. Nitrate/nitrite represented an essential hurdle to control Salmonella even when pH and a_w were below the values considered as minimum for its growth. The effect of this reduction on other pathogens should be considered.     

Routine and sensitive SPE-HPLC method for quantitative determination of pheophytin a and pyropheophytin a in olive oils

A sensitive and specific HPLC method with tandem diode array-fluorescence detection (DAD-FL) has been developed and validated for the simultaneous determination of pheophytin a (phy a) and pyropheophytin a (pyrophy a) in olive oils. Pigments were extracted with reverse phase solid phase extraction (RP-SPE) and subsequently analysed by HPLC-DAD-FL. The chromatographic analysis was carried out isocratically on ODS2 RP column using methanol–acetone (1:1 v/v) at flow-rate of 2.0 ml min⁻¹. Specificity of the method was assured by the simultaneous detection by UV–visible (410 nm) and FL (λ_Ex: 410 nm; λ_Em: 672 nm). Both compounds could be baseline separated within 7 min. The method was validated and applied in olive oil samples recently extracted as well as stored during 12 months. The limit of detection (LOD) defined at a signal-to-noise ratio of about 3 was ∼21.6 ng g⁻¹ for pyrophy a and ∼24.6 ng g⁻¹ for phy a under FL detection, and ∼148.0 ng g⁻¹ for both analytes under UV–visible detection. The calibration graphs were linear (r² > 0.9999; p < 0.01) between 0.25–14.00 ng μl⁻¹ for pyrophy a and 0.25–19.00 ng μl⁻¹ for phy a, under both fluorescence and UV–visible detection conditions. Recoveries of phy a and pyrophy a were over 94% as estimated by the standard addition method. Relative standard deviation for the intra-day and inter-day determination of phy a and pyrophy a were lower than 3.7% and 8.0%, respectively.