Evaluation of high pressure processing (HPP) inactivation of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in acid and acidified juices and beverages.

Increasing consumer demand for high-quality foods has driven adoption by the food industry of non-thermal technologies such as high pressure processing (HPP). The technology is employed as a post-packaging treatment step for inactivation of vegetative microorganisms. In order to evaluate HPP inactivation of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in acid and acidified juices and beverages, pressure tolerance parameters were determined using log-linear and Weibull models in pH-adjusted apple juice (pH 4.5) at 5 °C. A commercial processing HPP unit was used. The Weibull model better described the inactivation kinetics of the three tested pathogens. According to estimates from the Weibull model, 1.5, 0.9, and 1.5 min are required at 600 MPa to produce 5-log reductions of E. coli, Salmonella, and L. monocytogenes, respectively, whereas according to the log-linear model, 3.2, 1.8, and 2.1 min are required. The effects of process conditions were verified using commercial products (pH between 3.02 and 4.21). In all tested commercial juices or beverages, greater than 5-log reductions were achieved for all tested pathogens using HPP process conditions of 550 MPa for 1 min. These findings demonstrate that the HPP conditions of 600 MPa for 3 min, typically used by the food industry provide an adequate safety margin for control of relevant vegetative pathogens in acid and acidified juices and beverages (pH < 4.5). Results from this study can be used by food processors to support validation studies and may be useful for the future establishment of safe harbors for the HPP industry.

The combined effect of high pressure processing and dimethyl dicarbonate to inactivate foodborne pathogens in apple juice.

Novel processing technologies can be used to improve both the microbiological safety and quality of food products. The application of high pressure processing (HPP) in combination with dimethyl dicarbonate (DMDC) represents a promising alternative to classical thermal technologies. This research work was undertaken to investigate the combined effect of HPP and DMDC, which was aimed at reaching over 5-log reduction in the reference pathogens Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes inoculated in apple juice. Different strains of each species were tested. The pressure (ranging from 100 to 600 MPa), dwell time (from 26 to 194 s), and DMDC (from 116 to 250 mg/L) were tested based on a central composite rotatable design. The dwell time, in the studied range, did not have a significant effect (p > 0.1) on the pathogens´ reduction. All treatments achieved a greater than 5-log reduction for E. coli O157:H7 and L. monocytogenes. The reductions for S. enterica were also greater than 5-log for almost all tested combinations. The results for S. enterica suggested that it is more resistant to HPP and DMDC compared with E. coli O157:H7 and L. monocytogenes. The findings of this study showed that DMDC at low concentrations can be added to apple juice to reduce the parameters conventionally applied in HPP. The combined use of HPP and DMDC was highly effective under the conditions of this study.

Association of fungal genera from spoiled processed foods with physicochemical food properties and processing conditions.

The processing conditions and physiochemical properties used in food manufacturing create niches which support the growth of a limited number of spoilage fungi. This study was designed to evaluate the influence of intrinsic and extrinsic food product variables on the identity of spoilage fungi genera isolated from commercially produced foods. The spoilage etiology was identified in 127 products through ITS region sequencing. The prevalence and diversity of the identified spoilage fungi were evaluated in relationship to product-specific attributes using various descriptive statistics and a bipartite network analysis. Additionally, recursive partitioning was used to generate a classification tree with the outcomes, genera of the spoilage isolates, divided into increasingly homogenous subgroups. All of the isolated fungi belonged to the Ascomycete phylum, except four mucoralian isolates and the basidiomycete Rhodotorula. The occurrence of filamentous fungi repeatedly isolated ranged from 2% (Phoma spp.) to 18% (Penicillium spp.). In order of decreasing contribution to subgroup homogeneity, the split rules for the classification tree were based on process, water activity, food matrix category, and pH. Fungal genera representation in the terminal nodes indicated that production failures, in addition to product-specific attributes, were responsible for determination of the most probable spoilage organism.

Inactivation of Salmonella enterica and spoilage microorganisms in orange juice treated with dimethyl dicarbonate (DMDC).

Salmonella enterica is the pertinent pathogen associated with orange juice products that have resulted in numerous foodborne outbreaks. Although fresh orange juice typically has a pH below 4.0, which inhibits most pathogen growth, S. enterica can survive at low pH for extended periods. Additionally, fresh juice contains spoilage microorganisms such as natural yeasts and molds, which can grow at low pH, and may cause fermentation and product spoilage if left untreated. Numerous Salmonella outbreaks linked to fresh orange juice, as well as the burden of product spoilage, have generated increased demand for alternative, non-thermal treatments that can ensure pathogen- and spoilage-free products. In this study, the effect of dimethyl dicarbonate (DMDC) on pathogen and spoilage microorganism inactivation in orange juice has been investigated with two experiments. First, pasteurized orange juice was inoculated with approximately 106-107 CFU/ml of five serotypes of S. enterica per ml and treated with DMDC to test the effectiveness of inactivation against Salmonella. For the fungal spoilage microorganism study, fresh orange juice was held at room temperature to increase natural yeast and mold count to roughly 105-106 CFU/ml, followed with treatment with DMDC. DMDC at two concentrations (172 and 200 ppm) was used, and the tests were carried out at ambient (21 °C ±â€¯3 °C) and refrigeration (4 °C) temperatures. There was a >5-log reduction of Salmonella at 4 °C after 24 h at both 172 and 200 ppm of DMDC. For the treatment of fungal spoilage microorganisms, a nearly 5 and 4 log reduction of yeasts and molds was observed at ambient temperature and 4 °C, respectively. These results suggest that DMDC is most effective for use under the 4 °C holding conditions to inactivate S. enterica, and should be coupled with an additional preservative system for fungal spoilage control to produce safe orange juice that retains fresh quality.

Isolation of Bacteriocin-producing Staphylococcus spp. Strains from Human Skin Wounds, Soft Tissue Infections and Bovine Mastitis.

A collection of 206 Staphylococcus spp. isolates was investigated for their ability to produce compounds exhibiting antistaphylococcal activity. This group included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus xylosus strains recovered from bovine mastitis (n = 158) and human skin wounds and soft tissues infections (n = 48). Production of substances with antimicrobial activity was observed in six strains. Five of them were recovered from bovine mastitis, and one was isolated from the infected human skin wound. Three of the six antimicrobials produced by the different strains showed substantial loss of antimicrobial activity upon treatment with proteolytic enzymes, which suggests their peptidic structure. Additional studies have shown that one of the putative bacteriocins was efficiently secreted to the liquid medium, facilitating its large-scale production and isolation. The peptide produced by the M2B strain exhibited promising activity; however, against narrow spectrum of Staphylococcus spp. clinical and animal isolates. Growth inhibition was observed only in the case of 13 (including nine S. aureus, three S. xylosus and one S. epidermidis strains) out of 206 strains tested. Important advantage of the produced agent was its high thermal stability. Fifteen minutes of incubation at 90°C did not affect its antimicrobial potential. The highest efficiency of production of the agent was demonstrated in TSB medium after 24 hours at 37°C. The researches revealed that ability to production of bacteriocin among staphylococci is not very common. Only one (S. xylosus strain assigned as M2B) out of 206 strains tested produced satisfactory amounts of antistaphylococcal bacteriocin. In spite of that, we would encourage other researchers for investigation of their collections of Staphylococcus spp. isolates towards selection strains producing antimicrobial agents.

Effect of Water Activity on the Thermal Tolerance and Survival of Salmonella enterica Serovars Tennessee and Senftenberg in Goat's Milk Caramel.

The low thermal tolerance of Salmonella enterica in foods with intermediate moisture levels, such as caramel sauces, ensures that mild heat treatment is sufficient to achieve 5-log reductions of this pathogen. This treatment mitigates the risk posed by salmonellae in raw materials; however, recontamination might occur because of survival of the pathogen in products that are not heated before consumption. This study was conducted to evaluate the effect of water activity (aw) on the thermal tolerance and survival of S. enterica serovars Tennessee and Senftenberg. The D-values at 76, 78, and 80°C, z-values, and survival at 20.0 ± 0.5°C for 32 weeks of these two serovars were determined in goat's milk caramel at three aw values (0.85, 0.90, and 0.93). The highest thermal tolerance was observed at aw = 0.85 for Salmonella Senftenberg (D76°C = 2.9 ± 0.3 min), and the lowest was at aw = 0.93 for Salmonella Tennessee (D80°C = 0.131 ± 0.007 min). After a logarithmic transformation of the z-values, a significant interaction between serovar and aw was found (P < 0.0001), but no consistent trends were observed at the three evaluated aw levels for either serovar. Survival response was modeled using two sigmoidal three-parameter models. A significant interaction was found between nominal variables aw and serovar when comparing inflection points of the resulting curves: P < 0.0016 for the logistic model (R2 = 0.91) and P < 0.0014 for the Gompertz model (R2 = 0.92). Although a >8-log reduction was observed at week 20 of storage, regardless of the product's aw and the serovar, low levels of salmonellae were found in the product up to week 32 of storage. Our findings may assist the food industry with the establishment of critical limits for the safe thermal treatment of milk- and sugar-based foods with intermediate moisture levels. The survival data presented here highlight the relevance of implementing and effectively maintaining good sanitation and hygiene practices during the production of goat's milk caramel and similar food products.

Microbial dynamics of indicator microorganisms on fresh tomatoes in the supply chain from Mexico to the USA.

Quality and safety of fresh produce are important to public health and maintaining commerce between Mexico and USA. While preventive practices can reduce risks of contamination and are generally successful, the variable environment of the supply chain of fresh produce can be suitable for introduction or proliferation of pathogenic microorganisms. As routine surveillance of these pathogens is not practical, indicator microorganisms are used to assess the sanitary conditions of production and handling environments. An opportunity exists to use indicators on fresh produce to measure how handling and transport from field to market may affect microbial populations that contribute to their quality or safety. The objective was to quantify indicator microorganisms on tomatoes sampled along the supply chain during the harvest year, in order to observe the levels and changes of populations at different locations. Roma tomatoes (n=475) were taken from the same lots (n=28) at four locations of the postharvest supply chain over five months: at arrival to and departure from the packinghouse in México, at the distribution center in Texas, and at retail in USA. Samples were analyzed individually for four microbial populations: aerobic plate count (APC), total coliforms (TC), generic Escherichia coli, and yeasts and molds (YM). APC population differed (p<0.05) from 1.9±1.1, 1.7±1.1, 2.3±1.1 and 3.5±1.4logCFU/g at postharvest, packing, distribution center and supermarket, respectively. TC populations were <1logCFU/g at postharvest, increased at packing (0.7±1.0logCFU/g), decreased in distribution (0.4±0.8logCFU/g) and increased in supermarkets (1.4±1.5logCFU/g). Generic E. coli was not identified from coliform populations in this supply chain. YM populations remained <1logCFU/g, with the exception of 1.1±1.3logCFU/g at supermarkets and tomatoes were not visibly spoiled. The levels reported from this pilot study demonstrated the dynamics within populations as influenced by time and conditions in one supply chain during a harvest year, while the large variances in some locations indicate opportunities for improvement. Overall, packinghouse and supermarket locations were identified as crucial points to control microbial safety risks.

Characterization and control of Mucor circinelloides spoilage in yogurt.

Consumer confidence in the food industry is severely affected by large-scale spoilage incidents. However, relatively little research exists on spoilage potential of members of the fungal subphylum Mucormycotina (e.g. Mucor), which includes dimorphic spoilage organisms that can switch between a yeast-like and hyphal phase depending on environmental conditions. The presence of Mucor circinelloides in yogurt may not cause spoilage, but growth and subsequent changes in quality (e.g. container bloating) can cause spoilage if not controlled. The purpose of this study was to evaluate the effects on M. circinelloides of pasteurization regimen, natamycin concentrations, and storage temperature in yogurt production, as measured by fungal proliferation and carbon dioxide production. A strain of M. circinelloides isolated from commercially spoiled yogurt showed greater yogurt-spoilage potential than clinical isolates and other industrial strains. D-values and z-values were determined for the spoilage isolate in milk as an evaluation of the fungus' ability to survive pasteurization. Natamycin was added to yogurt at 0, 5, 10, 15, and 20ppm (µg/ml) to determine its ability to inhibit M. circinelloides over the course of month-long challenge studies at 4°C, 15°C, and 25°C. Survivors were recovered on acidified PDA and carbon dioxide levels were recorded. The D-values at 54°C, 56°C, and 58°C for hyphae/sporangiospores were (in min) 38.31±0.02, 10.17±0.28, and 1.94±0.53, respectively, which yielded a z-value of 3.09°C. The D-values at 51°C, 53°C, and 55°C for yeast-like cells were (in min) 14.25±0.12, 6.87±1.19, and 2.44±0.35, respectively, which yielded a z-value of 0.34°C. These results indicated that M. circinelloides would not survive fluid milk pasteurization if contamination occurred prior to thermal treatment. CO2 production was only observed when M. circinelloides was incubated under low-oxygen conditions, and occurred only at temperatures above 4°C. Addition of 10ppm and greater of natamycin inhibited the growth and CO2 production of M. circinelloides under moderate temperature abuse when compared to the untreated control. These data suggest that yogurt spoilage (container bloating) caused by anaerobic growth of M. circinelloides is due to post-pasteurization contamination. Temperature abuse facilitated spoilage as CO2 production was observed in yogurt incubated at 15°C and 25°C, but not at 4°C. The addition of at least 10ppm of natamycin prevented M. circinelloides growth in both hyphal and yeast-like phases, as well as CO2 production in temperatures of up to 15°C for 30days.

Variable Efficacy of the Proteinaceous Antifungal YvgO in Select Fruit Juices and Teas as a Complement with UV Methods of Food Protection.

Heat-resistant fungal spores present a processing challenge for beverages and fruit juices, as thermal and UV strategies are often inadequate in reducing heat-resistant fungal burdens to acceptable levels. While effective against pathogenic or invasive bacteria, germicidal UV light treatments also fail to achieve an appreciable reduction of heat-resistant fungal spores. As an alternative, the efficacy of the antifungal protein YvgO was examined across a selection of fruit juices and teas, as well as solid model matrices. Compared with its efficacy in analogous liquid matrices, the apparent efficacy of YvgO was diminished on acidified solid matrices due to a reduction in YvgO diffusion. Using an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] tetrazolium dye cytotoxicity assay, the effective concentrations to reduce growth by 50% were elucidated in samples challenged with Byssochlamys fulva H25. The MICs were determined and ranged from 2 ppm in apple juice and acidified teas to approximately 3 to 12 ppm for lemonade and orange, white cranberry, blueberry, prune, cherry, and grape juices. Apple cider and nonacidified teas showed reduced efficacy, with MICs exceeding 100 ppm. Tannin-rich products readily removed YvgO from the product, impairing its efficacy. Adding bovine serum albumin as a competitive inhibitor effectively reversed the YvgO-tannin association and restored efficacy in black but not green tea matrices. When challenged with a 5-log CFU inoculum of B. fulva, the shelf lives of the products were extended for various times up to 28 days in a concentrationdependent manner. However, initial efficacy was not predictive of shelf life extension, as some products exhibited improved protection at just two- and fourfold concentrations above the MIC, while others only exhibited long-term stability when concentrations exceeded 20 times the MIC. As such, YvgO may be an attractive alternative to currently available protection strategies and will provide needed diversity for natural food protectants.

Efficient reduction of pathogenic and spoilage microorganisms from apple cider by combining microfiltration with UV treatment.

Thermal pasteurization can achieve the U. S. Food and Drug Administration-required 5-log reduction of pathogenic Escherichia coli O157:H7 and Cryptosporidium parvum in apple juice and cider, but it can also negatively affect the nutritional and organoleptic properties of the treated products. In addition, thermal pasteurization is only marginally effective against the acidophilic, thermophilic, and spore-forming bacteria Alicyclobacillus spp., which is known to cause off-flavors in juice products. In this study, the efficiency of a combined microfiltration (MF) and UV process as a nonthermal treatment for the reduction of pathogenic and nonpathogenic E. coli, C. parvum, and Alicyclobacillus acidoterrestris from apple cider was investigated. MF was used to physically remove suspended solids and microorganisms from apple cider, thus enhancing the effectiveness of UV and allowing a lower UV dose to be used. MF, with ceramic membranes (pore sizes, 0.8 and 1.4 µm), was performed at a temperature of 10 °C and a transmembrane pressure of 155 kPa. The subsequent UV treatment was conducted using at a low UV dose of 1.75 mJ/cm(2). The combined MF and UV achieved more than a 5-log reduction of E. coli, C. parvum, and A. acidoterrestris. MF with the 0.8-µm pore size performed better than the 1.4-µm pore size on removal of E. coli and A. acidoterrestris. The developed nonthermal hurdle treatment has the potential to significantly reduce pathogens, as well as spores, yeasts, molds, and protozoa in apple cider, and thus help juice processors improve the safety and quality of their products.

Determination of the validation frequency for commercial UV juice processing units.

The CiderSure 3500 is one of the most commonly used UV juice processing units in the United States for the nonthermal processing of apple cider and fulfills the 5-log performance standard established by the federal juice HACCP regulation. However, the appropriate validation frequency of this machine's quartz tubes is currently unknown by juice processors and regulatory agencies. Presently, an annual validation is recommended by the manufacturer. Historical validation data from 1998 to 2013 of commercially used quartz tubes underwent comprehensive statistical analysis. A total of 400 tubes were validated one time, and 212 of those units were revalidated at least once over the evaluated time frame. Validations were performed at 14 mJ·cm(-2) UV dose and under turbulent flow conditions. Every validation showed a greater than 5-log reduction of Escherichia coli ATCC 25922, a nonpathogenic surrogate for pathogenic E. coli O157:H7, in each of three replicates. For initial validations, a mixed-effect model with log reduction of E. coli as response was constructed (400 tubes analyzed in triplicate). The model showed that the year of analysis and the initial inoculum level significantly affected the log reduction of E. coli (P < 0.0001), which on average was 7.0 ± 0.7. A quadratic relationship between the year of analysis and the response was found. Likewise, for revalidations (212 tubes analyzed in triplicate), the constructed random coefficient model showed that the year of analysis, quadratic effect of year of analysis, and initial inoculum level significantly affected the log reduction of E. coli (P < 0.0001). For this model, the major source of variance was explained by the year of analysis. The models describe the UV reactor's performance over time and suggest that a validation frequency of every 3 years would be conservatively adequate during the first 8 years of quartz tube use. After that, due to the reported quadratic trend, yearly validation would be recommended.

Nutrient-Dependent Efficacy of the Antifungal Protein YvgO Correlates to Cellular Proliferation Rate in Candida albicans 3153A and Byssochlamys fulva H25.

YvgO is a recently characterized antifungal protein isolated from Bacillus thuringiensis SF361 that exhibits a broad spectrum of activity and pH stability. Customized colorimetric metabolic assays based on standard broth microdilution techniques were used to determine the variable tolerance of Byssochlamys fulva H25 and Candida albicans 3153A to YvgO exposure under select matrix conditions impacting cellular proliferation. Normalization of the solution pH after antifungal challenge expanded the available pH range under consideration allowing for a comprehensive in vitro assessment of YvgO efficacy. Indicator susceptibility was examined across an array of elementary growth-modifying conditions, including media pH, incubation temperature, ionic strength, and carbohydrate supplementation. Under suboptimal temperature and pH conditions, the indicator growth rate reduced, and YvgO-mediated susceptibility was attenuated. While YvgO association but not efficacy was somewhat influenced by solution ionic strength, carbohydrate supplementation was shown to be the most influential susceptibility factor, particularly for C. albicans. Although the specific choice of carbohydrate/nutrient supplement dictated the extent of enhanced YvgO efficacy, D-glucose additionally improved the association between antifungal and target. Indeed, when exposed to YvgO under conditions that lead to increased cellular proliferation, both indicators displayed a stronger association and susceptibility to YvgO when compared to carbohydrate-deprived media or suboptimal incubation environments. With further study, YvgO may have the capacity to function as a prophylaxis for food safety and preservation, as well as a pharmaceutical agent against opportunistic fungal pathogens either independently or in combination with other established treatments applied to both livestock and human health concerns.

Bactericidal thurincin H causes unique morphological changes in Bacillus cereus F4552 without affecting membrane permeability.

Thurincin H is an antilisterial bacteriocin produced by Bacillus thuringiensis SF361. It exhibits inhibitory activity against a wide range of Gram-positive foodborne pathogens and spoilage bacteria including Listeria monocytogenes, B. cereus, and B. subtilis. This hydrophobic, anionic bacteriocin folds into a hairpin structure maintained by four pairs of unique sulfur to α-carbon thioether bonds. As its hydrophobicity and structure are quite different from most archived bacteriocins, this study aimed to elucidate its mode of action and compare it with the mechanisms of other well-characterized bacteriocins. The results indicated that, although bactericidal to B. cereus F4552, thurincin H did not lead to optical density reduction or detectable changes in cell membrane permeability. B. cereus F4552 imaged by scanning electron microscopy after treatment with thurincin H at 32 × MIC showed regular rod-shaped cells, while only cells treated with thurincin H at the elevated levels of 256 × MIC showed loss of cell integrity and rigidity. Both concentrations caused greater than 99% of cell viability reduction. In contrast, nisin caused significant cell membrane permeability at concentration as low as 2 × MIC. These results indicated a difference in the mode of action for thurincin H compared with the generalized pore-forming mechanism of many lantibiotics, such as nisin.

Large-Scale Purification, Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361.

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.

Development of a homologous expression system for and systematic site-directed mutagenesis analysis of thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361.

Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.

Pickled egg production: inactivation rate of Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus during acidification step.

Based on current U.S. Food and Drug Administration acidified foods guidelines, regulatory approval of commercial pickled egg production without a final heat treatment requires challenge studies. We conducted challenge studies to verify common pickled egg processing parameters. Hard-boiled eggs were acidified in ambient temperature brine at a 60:40 egg/brine ratio. Four acidification treatments were studied in triplicate: 5% acetic acid (AA) or 2.5% AA brine with and without 0.05% sodium benzoate. These treatments resulted in 2% or 1% AA with or without 0.02% sodium benzoate, respectively, in the total system. Samples were stored at 7 °C until pH at the yolk center was ≤ 4.6; subsequently, samples were held at ambient temperature. Egg pH was measured at 24- to 48-h intervals until equilibrium pH was reached (4.0 and 4.4). Eggs and jar lids were challenged with separate pathogen cocktails (six strains and/or serovars) of Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus. After 5 and 9 days, the pH fell below 4.6 in 2% AA and 1% AA eggs, respectively. Sodium benzoate did not affect acidification rate for these brine treatments (P ≥ 0.05), nor did sodium benzoate affect pathogen die-off. E. coli O157:H7, Salmonella, and L. monocytogenes were undetectable (<1 CFU/g) in pickled eggs in 2% AA at 72 h; S. aureus was undetectable after 7 days. In 1% AA eggs, Salmonella was undetectable after 10 days. No pathogens were detectable after 14 days. No pathogens were detectable on lids within 72 h for the 2% AA treatment. Only S. aureus was detectable on lids after 72 h in the 1% AA treatment and died off rapidly at ambient temperature. Although pathogens began die-off under refrigeration, heat treatment (ambient temperature storage) was required to reach undetectable levels. Minimal inversion was adequate treatment for lids. Pickled eggs should be held under refrigeration for the length of time needed to acidify them to ≤ 4.6 and then held at ambient temperatures to ensure pathogen inactivation.

Efficacy of UV, acidified sodium hypochlorite, and mild heat for decontamination of surface and infiltrated Escherichia coli O157:H7 on green onions and baby spinach.

Produce-associated foodborne illnesses outbreaks have highlighted the need for more effective decontamination methods to ensure the safety of fresh produce. The main objective of this study was to evaluate the individual and combined efficacies of germicidal UV light (12.5 to 500 mJ/cm(2)), acidified sodium hypochlorite (ASC 10 to 200 ppm), and mild heat (40 to 50°C) for decontaminating green onions and baby spinach infected with Escherichia coli O157:H7. Samples were inoculated by spot and dip inoculation methods to mimic surface and infiltrated E. coli O157:H7 contamination, respectively. In green onions and baby spinach, the individual efficacies of UV, ASC, and mild-heat treatments varied based on the produce type and contamination method. Following analysis of the efficacies of the single treatments, a combined treatment with 125 mJ/cm(2) UV and 200 ppm of ASC at 50°C was selected for spot-inoculated green onions, and a combined treatment with 125 mJ/cm(2) UV and 200 ppm of ASC at 20°C was selected for spot- and dip-inoculated baby spinach. While a >5-log reduction was achieved with the combination treatment for spot-inoculated green onions with an initial contamination level of 7.2 log CFU per spot, the same treatment reduced E. coli O157:H7 populations below the detection limit (<1 log) on green onions spot inoculated at a lower contamination level (4.3 log CFU per spot). On spot- and dip-inoculated baby spinach, the combined treatment reduced E. coli O157:H7 populations by 2.8 log CFU per spot and 2.6 log CFU/g, respectively. The combined treatment of 500 mJ/cm(2) UV and 200 ppm of ASC at 50°C selected for the decontamination of dip-inoculated green onions resulted in a 2.2-log CFU/g reduction. These findings suggest that when foodborne pathogens contaminate produce and subsequently infiltrate, attach to, or become localized into protected areas, the individual or combined applications of UV, ASC, and mild-heat treatments have limited decontamination efficacies on both green onions and baby spinach (<3 log). However, treatments combining UV, ASC, and mild heat could be a promising application for reducing pathogen populations (>5 log) on E. coli O157:H7 surface-contaminated green onions. This study also highlights the importance of developing and optimizing produce-specific decontamination methods to ensure the safety of fresh produce commodities.

Decontamination of green onions and baby spinach by vaporized ethyl pyruvate.

Foodborne illnesses associated with fresh produce continue to be a major concern as consumer demand for healthier and nonthermally processed food increases. The objective of this study was to evaluate vaporized ethyl pyruvate (EP; CAS 617-35-6) as a safe alternative antimicrobial agent for the decontamination of Escherichia coli O157:H7 on green onions and spinach. Baby spinach leaves and green onions were inoculated with a five-strain cocktail of E. coli O157:H7 (pGFP) by the dipping method. Samples were treated with concentrations of 0, 42, 105, and 420 mg/liter vaporized EP in a 2.6-liter enclosed container. The efficacy of EP vapors for reducing E. coli O157:H7((GFP)) populations on green onions and baby spinach at 4 and 10°C was monitored for 7 and 5 days, respectively. The lowest EP concentration (42 mg/liter) resulted in a 1.7-log reduction of E. coli O157:H7((GFP)) on green onions after 7 days at 4°C and a 1.9-log reduction after 5 days at 10°C (P < 0.05). In baby spinach, the same concentration resulted in 0.9-log and 1.4-log reductions (P < 0.05) of E. coli O157:H7((GFP)) after 7 days at 4°C and 5 days at 10°C, respectively. On green onions, the highest concentration of EP (420 mg/liter) reduced the population of E. coli O157:H7((GFP)) by >4.7 log CFU/g after 7 days at 4°C and 5 days at 10°C. The same concentration was also effective for reducing E. coli O157:H7((GFP)) populations in baby spinach by 4.3 log CFU/g after 7 days at 4°C and by >6.5 log CFU/g after 3 days at 10°C. Although the successful EP treatments minimally affected the sensory attributes of green onions, the treatments resulted in significant changes in the sensory attributes of baby spinach samples stored at 4 and 10°C. These results indicate that EP is an effective antimicrobial that could be used to enhance the safety of fresh produce depending on the sensory characteristics of the product.

Patulin reduction in apple juice from concentrate by UV radiation and comparison of kinetic degradation models between apple juice and apple cider.

Patulin, a mycotoxin produced by several genera of fungi, including Byssochlamys, Aspergillus, and Penicillium, has been an important concern in apple cider and apple juice due to its toxicity and health consequences. In this study, the effects of UV on the patulin level, physical and chemical properties, and sensory attributes in apple juice from concentrate were investigated. Kinetic modeling of patulin reduction by UV radiation in apple juice from concentrate was calculated and compared with the degradation rate observed previously in apple cider. From an initial patulin contamination of approximately 1,000 ppb (µg/liter), the UV exposure, ranging from 14.2 mJ/cm(2) (one pass) to 99.4 mJ/cm(2) (seven passes), was successful in reducing patulin levels by 72.57% ± 2.76% to 5.14% ± 0.70%, respectively. Patulin reduction by UV radiation followed first-order kinetic modeling in a fashion similar to first-order microbial inactivation. An exponential correlation between UV exposure and the percentage of patulin remaining was observed, giving an r(2) value of 0.9950. Apple juice was repeatedly exposed to 14.2 mJ/cm(2) for each treatment, and patulin levels were significantly decreased when compared with the level obtained with the previous UV exposure treatment. While there were no significant differences in the percentages of titratable acidity and ascorbic acid (P > 0.05), there were minor yet random sampling differences in pH and degrees Brix (1 °Brix is 1 g of sucrose in 100 g of solution; the °Brix represents the soluble solids content of the solution as percentage by weight [%, wt/wt]) (P ≤ 0.05). A significant difference (P ≤ 0.05) in sensory perception for the finished apple juice was detected between the control and the full seven-pass UV radiation treatment using an experienced consumer panel and a triangle test. Patulin reduction by UV radiation from both the current study and a previous study involving apple cider was compared, which showed that both matrices strongly fit a first-order kinetic degradation model. However, the kinetic constant for degradation in apple juice was approximately 5.5 times greater than that observed in an apple cider matrix.

Functional assignment of YvgO, a novel set of purified and chemically characterized proteinaceous antifungal variants produced by Bacillus thuringiensis SF361.

This study reports a novel class of antifungal protein derived from bacterial origin. Bacillus thuringiensis SF361, the strain also responsible for producing the novel bacteriocin thurincin H, exhibits broad antifungal activity against select members of several fungal genera, including Aspergillus, Byssochlamys, and Penicillium, as well as the pathogenic yeast Candida albicans. Optimal antifungal production and secretion were observed after-log phase growth when incubated at 37°C in a carbohydrate-free growth medium. High-performance liquid chromatography purification was performed after pH-selective ammonium sulfate precipitation and size-exclusion chromatography. Intact mass analysis and peptide mass fingerprinting identified the 13,484-Da protein to be a mass homolog to the YvgO protein construct sequenced from Bacillus cereus AH 1134. Further analysis via amino-terminal sequencing also revealed the existence of four distinct yet equally efficacious YvgO variants differing only within the first four N-terminal residues. YvgO was found to be remarkably stable, maintaining its antifungal activity under a wide pH and temperature range. When assayed against the toxigenic species Byssochlamys fulva H25, the selected primary filamentous fungal indicator, the MIC was estimated to be 1.5 ppm. Candida albicans 3153 was more resistant, exhibiting MICs between 25 and 800 ppm, depending on growth conditions. YvgO is unique among antifungals, showing no known sequential or functional homology to the typical classes of antifungal proteins, including common membrane-acting agents such as cellulases and glucanases. Due to its activity against an array of pathogenic and spoilage fungi, the potentials for clinical, agricultural, and food-processing applications are encouraging.