Application of bacteriophages during depuration reduces the load of Salmonella Typhimurium in cockles

As bivalve molluscs are filter feeder, often consumed raw or lightly cooked and are frequently cultivated in contaminated waters, they are implicated in food-borne disease transmission to human. The present study investigated the potential application of bacteriophage (or phage) phSE-2, phage phSE-5 and phage cocktail phSE-2/phSE-5 to decrease the concentration of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) during the depuration of natural and artificially contaminated cockles (Cerastoderma edule). Cockles were artificially infected with 10⁵ and 10⁶ colony-forming units (CFU)/mL of S. Typhimurium in static seawater and infected group were treated with phages at four different MOI values: 0.1, 1, 10 and 100. Depuration in static seawater at multiplicity of infection (MOI) of 0.1 with single phage suspensions of phSE-2 and phSE-5 provided the best results, as it decreased by ~ 1.3 and 1.7 log CFU/g, respectively, the concentration of Salmonella spp. after a 4 h treatment. At a MOI of 0.1, the rate of inactivation with single phage suspensions was higher when compared with the results obtained using the phage cocktail. However, in naturally contaminated cockles treated in static seawater with single phage suspensions and phage cocktail phSE-2/phSE-5, similar decreases in cultivable bacteria concentration (~ 0.7–0.9 log CFU/g) were achieved after 6 h of treatment. When artificially contaminated cockles were depurated with phage phSE-5 in a recirculated seawater system (mimicking industrial depuration conditions), a 0.9 and 2.0 log CFU/g reduction of Salmonella spp. was reached after 4 and 6 h treatment. Once the depuration process was performed without phage, a 6 h treatment was needed to obtain a 1.1 log CFU/g reduction of Salmonella spp. Results indicated that combining phage biocontrol with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency, proving that this technology can be transposed to the bivalves industry. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs.     

Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages

The ability of specific bacteriophages to inhibit Staphylococcus aureus growth in curd manufacturing processes was determined. Two lytic bacteriophages specific against S. aureus were obtained by DNA random deletion from the milk-isolated temperate phages, ΦH5 and ΦA72. A cocktail of these lytic phages, Φ88 and Φ35, at multiplicity of infection (MOI) of 100, produced a complete elimination of 3×10⁶ cfu mL⁻¹ of the pathogen in ultra-high-temperature (UHT) whole milk at 37 °C. Furthermore, the frequency of emergence of bacteriophage-insensitive mutants was reduced up to 200-fold in the presence of the two lytic phages compared with that detected with the combination of the temperate counterparts. The lytic phage derivatives, added to milk, were able to decrease rapidly the viable counts of S. aureus during curd manufacture. In acid curd, the pathogen was not detected after 4 h of incubation at 25 °C, whereas pathogen clearance was achieved within 1 h of incubation at 30 °C in renneted curd. These results indicate that lytic bacteriophages could be used as biopreservatives in the manufacture of particular dairy products.     

Hard surfaces decontamination of enteropathogenic and Shiga toxin-producing Escherichia coli using bacteriophages

Three Myoviridae phages (DT1, DT5 and DT6) specific for pathogenic Escherichia coli were studied, either individually or as cocktails, for their lytic activity on in vitro challenge tests. Also, cocktail ability to reduce artificial contamination on hard surfaces (glass coverslips and stainless steel coupons) by three pathogenic Escherichia coli strains (EPEC920, non-O157 STEC ARG4827 and O157:H7 STEC464) was tested. Assays of phage stability during refrigerated storage showed that the three phages evaluated retained a high viability after two months at 4 °C. Challenge tests showed high reductions in viable cells, of up to 6.4 log CFU ml^− 1, for all tested strains at 37 °C. Efficiency was somewhat lower at 4 °C, though biocontrol levels were still good, reaching values of up to 3.8 log CFU ml^− 1. Considering only results obtained at 37 °C, phage cocktails produced the highest reduction in most cases. Treatments with phage cocktails produced complete inactivation (ca. 5–6 log CFU ml^− 1) of EPEC920 and O157:H7 STEC464 on glass coverslips, and of EPEC920, non-O157 STEC ARG4827 and O157:H7 STEC464 on stainless steel coupons, at both temperatures (4 °C and 37 °C) and multiplicity of infection (ca. 10³ and 10⁷) tested. However, some strains not detected at 3 h were sometimes detected at 24 h, and inactivation of non-O157 STEC ARG4827 on glass coverslips was never accomplished; viable cell reductions in all these cases ranged from 1.2 to 5.4 log CFU ml^− 1. Our results suggest that lytic phages, either individually or as a cocktail, may be useful for reducing contamination on hard materials used in food processing surfaces. To our knowledge, this is the first study focused on the use of bacteriophages to reduce contamination of food processing surfaces by EPEC and non-O157 STEC strains.     

Effect of high pressure homogenization on lactic acid bacteria phages and probiotic bacteria phages

To investigate the effect of high pressure homogenization on virus inactivation, phages specific for Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus paracasei and Lactobacillus plantarum were studied. The influence of pressure, number of passes, suspension medium and phage concentration were studied at 25 °C. Reductions in viability were proportional to pressure and number of passes, though the inactivation extent was phage-dependent. At 100 MPa, some bacteriophages were completely inactivated (6 log₁₀ reduction) after 3 or 5 passes, while others remained infective after 8 passes. For all phages, treatment at 60 MPa was insufficient for complete inactivation, even after 8 passes. No clear influence of suspending medium was observed. Inactivation seems to depend on phage concentration; the higher the initial load, the bigger the reduction achieved. Although these results showed that several phages studied are resistant to high-pressure homogenization, this strategy could be combined with others to control their presence in raw milk.     

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.     

Inactivation of bacteriophages by thermal and high-pressure treatment

Dairy companies commonly experience fermentation failures due to bacteriophages that are spread mainly by milk, whey or air. Heat or high-pressure treatment may potentially reduce the phage titre, but further knowledge about the inactivation kinetics is desirable. Inactivation experiments were carried out with the commonly occurring lactococcal phages P001 and P008. Phage suspensions in calcium-enriched M17-broth were heated at 55–80 °C, or high-pressure treated at up to 600 MPa. Kinetic analysis showed that the order of inactivation reaction was above 1; thus, inactivation kinetics were approximated by a non-linear regression model. The Arrhenius parameters, rate constant, k_p,T, and activation energy, E_A (for heat treatments), and the volume of activation, ΔV^# (for pressure treatments) were calculated. Both measured and calculated results indicate that phage P008 was the more heat- and pressure-resistant of the two. By combining the results from heat and pressure inactivations, a pressure–temperature diagram for phage P008 was established.     

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.     

Phage adsorption on Enteropathogenic and Shiga Toxin-Producing Escherichia coli strains: Influence of physicochemical and physiological factors

Bacteriophages have proved to be useful tools against pathogenic Escherichia coli strains. The first step in the phage life cycle is the adsorption to the host cell surface. In the present work, three Myoviridae phages (DT1, DT5 and DT6) were used to characterize the adsorption process on three pathogenic E. coli strains, namely two Shiga-toxin producing E. coli (STEC) and one enteropathogenic E. coli (EPEC), in several conditions found on food. The influence of Na⁺, Mg^2 +, temperature, pH, periodate, proteinase K and physiological cell state on phage adsorption was investigated. The three phages evaluated showed high adsorption rates at pH 7.5 and 5.7 while they were moderate at the lowest pH evaluated (4.5). Sodium or magnesium ions were not indispensable for the adsorption of the three phages evaluated. Specifically, phage particles were adsorbed either in the presence or absence of Mg^2 +, while increasing Na⁺ concentration resulted in lower adsorption values for all the systems evaluated. Regarding temperature, phage adsorption was slightly affected at 4 °C and 50 °C, while it reached its maximum at 37 °C. Adsorption rates decreased after the thermal inactivation of cells, though, when chloramphenicol (as protein-synthesis inhibitor) was used, adsorption values on treated and untreated cells were similar. In addition, periodate was able to decrease phage adsorption, thus suggesting the receptors were carbohydrates in nature. All these results showed that the adsorption process was only partially affected and most conditions are suitable for the completion of the first step in the phage life cycle. Therefore, phages evaluated in this study can be used to prevent foodborne diseases on several food matrices since they are active in a wide range of environmental conditions.     

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.     

Examination of the internalization of Salmonella serovar Typhimurium in peanut,Arachis hypogaea,using immunocytochemical techniques

A variety of products of plant origin, such as tomatoes, melons, peppers, and peanuts, have been implicated in Salmonella spp. associated outbreaks in recent years. Although these bacteria have been found to internalize within some plants associated with foodborne-related outbreaks, the internalization in peanut plants has not been examined to date. To investigate internalization and where the bacteria localize within the plant, intact peanut seeds were contaminated with Salmonella serovar Typhimurium expressing green fluorescent protein (GFP) for 30 min and immunocytochemical techniques were used to localize the bacterium within the stem tissue of 16 day-old peanut plants. An average of 13.6 bacteria/mm³ were localized within the sampled tissue. The bacteria were found to be associated with every major tissue (cortical, vascular, epidermal, and pith) and corresponding cell type. The cortical cells located to the outside of the vascular bundles contained the majority of the Salmonella cells (72.4%). Additional growth experiments demonstrated peanut seedlings could support the reproduction of Salmonella to high levels (10⁹ CFU/plant) after 2 days following seed contamination. Together these results show that Salmonella Typhimurium can internalize within many different plant tissue types after a brief seed contamination event and that the bacteria are able to grow and persist within the plant.     

Effect of high hydrostatic pressure on the viability of Streptococcus thermophilus bacteriophages isolated from cheese

The effect of high hydrostatic pressure (HHP) on the inactivation of Streptococcus thermophilus bacteriophages ϕAbc2, ALQ13.2 and DT1 was investigated. Reductions in viability were proportional to pressure (400–600 MPa) and holding time (0–30 min), though the inactivation extent was phage-dependent. HHP treatment at 600 MPa for less than 5 min was sufficient to completely inactivate phages ϕAbc2 and DT1. However, ALQ13.2 only suffered a 3.7-log reduction which showed markedly higher resistance than ϕAbc2 and DT1. Weibull model was applied to describe the inactivation behavior of phages. Regression coefficients (R²), root mean square error (RMSE) and residual plots strongly suggested that Weibull model had a good fit to the data. Additionally, the Weibull models were simplified with a binary model since b values had a linear relationship at 400–600 MPa. The simplified Weibull model provided reasonable predictions of survival data at different pressures for ϕAbc2 and DT1.Industrial relevancePhage infection remains today the most serious and common problem in the manufacture of fermented cheese products. In particular, some S. thermophilus bacteriophages exhibited high thermal resistance, high burst size values and remarkably short latent periods. The use of alternative preservation techniques such as high hydrostatic pressure processing is an efficient method to improve cheese quality and achieve bacteriophages safety. The results showed HHP treatment was capable for complete inactivation of S. thermophilus bacteriophages. The lethal kinetics of S. thermophilus bacteriophages using the Weibull model could help the application of HHP in cheese-making industry.     

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.     

Salmonella bacteriophage diversity reflects host diversity on dairy farms

Salmonella is an animal and human pathogen of worldwide concern. Surveillance programs indicate that the incidence of Salmonella serovars fluctuates over time. While bacteriophages are likely to play a role in driving microbial diversity, our understanding of the ecology and diversity of Salmonella phages is limited. Here we report the isolation of Salmonella phages from manure samples from 13 dairy farms with a history of Salmonella presence. Salmonella phages were isolated from 10 of the 13 farms; overall 108 phage isolates were obtained on serovar Newport, Typhimurium, Dublin, Kentucky, Anatum, Mbandaka, and Cerro hosts. Host range characterization found that 51% of phage isolates had a narrow host range, while 49% showed a broad host range. The phage isolates represented 65 lysis profiles; genome size profiling of 94 phage isolates allowed for classification of phage isolates into 11 groups with subsequent restriction fragment length polymorphism analysis showing considerable variation within a given group. Our data not only show an abundance of diverse Salmonella phage isolates in dairy farms, but also show that phage isolates that lyse the most common serovars causing salmonellosis in cattle are frequently obtained, suggesting that phages may play an important role in the ecology of Salmonella on dairy farms.     

Comparison of antimicrobial resistance patterns and phage types of Salmonella Typhimurium isolated from pigs,pork and humans in Belgium between 2001 and 2006

Infections with non-typhoid Salmonella represent a major problem in industrialized countries.The emergence and spread of antimicrobial-resistant pathogens, among them Salmonella, has become a serious health hazard worldwide. One of the most commonly isolated non-typhoid Salmonella serovars in pigs, pork and humans is Salmonella Typhimurium. In this study the comparison of the incidences of resistance to nine antimicrobials, resistance patterns and phage types between S. Typhimurium isolated from pigs (n = 581), pork (n = 255) and humans (n = 1870) in Belgium in the period 2001 to 2006 was performed.Resistance to the antimicrobials ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline was frequently observed and varied between 23.5% and 83.1%. Resistance ranged from 15.6% to 20.7% for the combination trimethoprim–sulfonamides and from 3.4% to 5.8% for nalidixic acid. Resistance to the critical important antimicrobials cephalosporins and fluoroquinolones was found sporadically (≤ 1.2%). Resistance to the different antimicrobials was observed to be similar in S. Typhimurium isolates from the various origins. Twenty-seven antimicrobial resistance patterns representing in total 75.2%, 89.0% and 89.6% of the isolates from pigs, pork and humans respectively were found to be common among the three groups and 73 combinations antimicrobial resistance pattern/phage type were found to be common among pork and human isolates, representing 70.1% of the pork isolates and 51.0% of the human isolates. The high percentage of isolates that have a common resistance pattern, and in a less pronounced way a common combination phage type/resistance pattern, are in agreement with the hypothesis of transfer of antimicrobial resistant Salmonella from pigs via the consumption of pork to humans as one of the possible pathways. The most prevalent combination in Belgium within both the pork isolates (7.4%) and the human isolates (13.2%) was S. Typhimurium DT104 resistant to ampicillin, chloramphenicol, streptomycine, sulfonamides and tetracycline.     

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.     

Characterization of native microbial populations on Swiss chard (Beta vulgaris,type cicla) cultivated by organic methods

The native microflora of fresh Swiss chard (Beta vulgaris, type cicla) cultivated by organic methods was quantified and characterised. The concentration of the different microorganism groups presented less variability among different lots than results found on chard leaves produced by conventional methods. These results would indicate a greater degree of homogeneity among the different samples of chard produced by organic procedures.The microbial populations on Swiss chard leaves produced by organic methods were 6.8×10⁵ CFU/g for mesophilic microorganisms, 3.9×10³ CFU/g for psycrotrophic microorganisms, 1.4×10⁴ CFU/g for lactic acid microorganisms, 5.1×10³ CFU/g for total coliforms and 1.66×10³ CFU/g for Staphylococcus spp. Neither fecal coliforms nor human pathogens were identified. Immersion of organic Swiss chard leaves in tap water at 18°C for 4 min reduced microbial populations but did not accomplish more than 70% removal in any of the populations under study.     

Inactivation kinetics of Listeria monocytogenes,Salmonella enterica serovar Typhimurium,and Campylobacter jejuni in ready-to-eat sliced ham using UV-C irradiation

To investigate the applicability of UV-C irradiation on the inactivation of foodborne pathogenic bacteria in ready-to-eat sliced ham, UV-C treatment was evaluated. Irradiation dose required for 90% reduction of the populations of Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Campylobacter jejuni were determined to be 2.48, 2.39, and 2.18 J/m². Ready-to-eat sliced hams were inoculated with the pathogens and irradiated with UV-C light of 1000, 2000, 4000, 6000, and 8000 J/m². Microbiological data indicated that foodborne pathogen populations significantly (p < 0.05) decreased with increasing UV-C irradiation. In particular, UV-C irradiation at 8000 J/m² reduced the populations of L. monocytogenes, S. Typhimurium, and C. jejuni in the ham by 2.74, 2.02, and 1.72 log CFU/g. The results indicate that UV-C irradiation can be used as a microbial inactivation method for ready-to-eat sliced ham, and inactivation kinetics of the foodborne pathogens fit the Weibull model better than the first-order kinetics model.     

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.     

Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation

Lactobacillus fermentum was substituted for nitrite to produce cured pink color in a Chinese-style sausage. Treatments included inoculations (10⁴, 10⁶, and 10⁸ CFU/g meat) followed by fermentation at 30 °C for 8 h and then at 4 °C for 16 h. Control sausage (with sodium nitrite, 60 mg/kg meat) was cured at 4 °C for 24 h without L. fermentum. The UV–Vis spectra of pigment extract from L. fermentum-treated sausage were identical to that of nitrosylmyoglobin (NO-Mb) formed in nitrite-treated control. The NO-Mb concentration and the colorimetric a¹ value of sausage treated with 10⁸ CFU/g meat of L. fermentum essentially replicated those in nitrite-cured meat. Free amino acid content in sausage treated with L. fermentum was greater and the pH slightly lower compared with the nitrite-cured control sample. This study showed that L. fermentum has the potential to substitute for nitrite in the sausage production.     

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.