The effectiveness of treating irrigation water using ultraviolet radiation or sulphuric acid fertilizer for reducing generic Escherichia coli on fresh produce-a controlled intervention trial.

AIMS: The aims of this study were to: (i) estimate the effectiveness of ultraviolet radiation (UV) and sulphuric acid-based fertilizer (SA), at reducing levels of generic Escherichia coli in surface irrigation water and on produce and surface soil in open produce fields; and (ii) describe the population dynamics of generic E. coli in produce fields. METHODS AND RESULTS: Spinach and cantaloupe plots were randomly assigned to control, UV or SA treatment groups. Irrigation water was inoculated with Rifampicin-resistant E. coli prior to treatment. More than 75% of UV- and SA-treated tank water samples had counts below the detection limit, compared to a mean count of 3·3 Log10 CFU per ml before treatment. Levels of Rifampicin-resistant E. coli in soil and produce both increased and decreased over 10-15 days after irrigation, depending on the plot and time-period. CONCLUSIONS: UV and SA treatments effectively reduce the levels of E. coli in surface irrigation water. Their effectiveness at reducing contamination on produce was dependent on environmental conditions. Applying wait-times after irrigation and prior to harvest is not a reliable means of mitigating against contaminated produce. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are of timely importance for the agricultural industry as new FSMA guidelines require producers to demonstrate a low microbial load in irrigation water or allow producers to apply a wait-time to mitigate the risk of contaminated produce.

Nanoporous anodic alumina reduces Staphylococcus biofilm formation.

Staphylococcus epidermidis and Staphylococcus aureus, two bacterial strains commonly associated with biofilm-related medical infections and food poisoning, can rapidly colonize biotic and abiotic surfaces. The present study investigates the ability of anodic alumina surfaces with nanoporous surface topography to minimize the attachment and biofilm formation mediated by these pathogenic bacterial strains. Early attachment and subsequent biofilm development were retarded on surfaces with nanopores of 15-25 nm in diameter compared to surfaces with 50-100 nm pore diameter and nanosmooth surfaces. After 30 min of incubation in nutritive media, the biomass accumulation per unit surface area was 2·93 ± 1·72 µm3  µm-2 for the 15 nm, 3·49 ± 1·97 µm3  µm-2 for the 25 nm, as compared to 14·04 ± 6·39 µm3  µm-2 for the nanosmooth, 11·88 ± 9·72 µm3  µm-2 for the 50 nm and 12·09 ± 11·84 µm3  µm-2 for the 100 nm surfaces respectively. These findings suggest that anodic alumina with small size nanoscale pores could reduce the incidence of staphylococcal biofilms and infections, and shows promise as a material for a variety of medical applications and food contact surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on a simple, robust and scientifically sound method to reduce attachment and biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis to abiotic surfaces using a carefully designed nanoscale topography. This approach can help to reduce the incidence of staphylococcal biofilms and infections without imposing selective stresses on bacteria, thus preventing the creation of resistant strains.

Stability of alternariol and alternariol monomethyl ether during food processing of tomato products.

The stability of two Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), has been investigated during the food processing of tomato products simulating commercial processing conditions. The production stages assessed were the storage of raw fruits, fruit washing, and thermal processing. It was observed that time of storage significantly reduced the initial concentration of AOH, but only if tomatoes were stored at 35 °C. For AME, 12 h were sufficient to reduce the initial concentration, regardless of the temperature at which samples were stored (25, 30 and 35 °C). The washing step achieved the highest reduction of AOH and AME. This reduction was even more efficient when using sodium hypochlorite solutions. Finally, during the heat treatment (80-110 °C), results showed that heating tomato samples at 100 and 110 °C, significantly affected AOH stability, though AME seemed to not be affected by these thermal processes.

Short communication: Homologous expression of recombinant and native thurincin H in an engineered natural producer.

The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

Shelf-life evaluation of natural antimicrobials for Concord and Niagara grape juices.

This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10(2) and 10(4) CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21°C by yeast enumeration and measurement of turbidity, pH, and °Brix. Juices were deemed spoiled when yeast counts exceeded 10(6) CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, pH, and °Brix. Spoiled juices had lower pH and °Brix values and higher turbidity due to microbial activity and increased in microbial levels.

Inactivation of different strains of Escherichia coli O157:H7 in various apple ciders treated with dimethyl dicarbonate (DMDC) and sulfur dioxide (SO2) as an alternative method.

Escherichia coli has been identified as the causative agent in numerous foodborne illness outbreaks associated with the consumption of fresh apple cider. Apple cider has a pH which is normally below 4.0 and would not be considered a medium capable of supporting the growth of foodborne pathogens. The association of unpasteurized apple cider with foodborne illness due to E. coli O157:H7 has however, led to increased interest in potential alternative methods to produce pathogen free cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6)-10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895) and tested to determine the effectiveness of sulfur dioxide (SO(2)) and dimethyl dicarbonate (DMDC). Bacterial populations for treated and untreated samples were then enumerated by using non-selective media. Eight different ciders were treated with DMDC (125 and 250 ppm) and SO(2) (25, 50, 75, 100 ppm). Greater than a 5-log reduction was achieved at room temperature with 250 ppm of DMDC and 50 ppm of SO(2) after the incubation time of 6h and 24h, respectively. Addition of DMDC and/or SO(2) may offer an inexpensive alternative to thermal pasteurization for the production of safe apple cider for small apple cider producers.

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25.

AIMS: Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages. METHODS AND RESULTS: A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16. CONCLUSIONS: The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F. SIGNIFICANCE AND IMPACT OF THE STUDY: The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100 degrees C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.

Effects of ultraviolet irradiation on chemical and sensory properties of goat milk.

Sensory and chemical consequences of treating goat milk using an UV fluid processor were assessed. Milk was exposed to UV for a cumulative exposure time of 18 s and targeted UV dose of 15.8 +/- 1.6 mJ/cm2. A triangle test revealed differences between the odor of raw milk and UV irradiated milk. Oxidation and hydrolytic rancidity was measured by thiobarbituric acid reactive substances and acid degree values (ADV). As UV dose increased, there was an increase in thiobarbituric acid reactive substance values and ADV of the milk samples. A separate set of samples were processed using the fluid processor but with no UV exposure to see if lipase activity and agitation from pumping contributed to the differences in odor. The ADV increased at the same rate as samples exposed to UV; however, sensory studies indicated that the increase of free fatty acids was not enough to cause detectable differences in the odor of milk. Solid phase microextraction and gas chromatography were utilized for the analysis of volatile compounds as a result of UV exposure. There was an increase in the concentration of pentanal, hexanal, and heptanal (relative to raw goat milk) after as little as 1.3 mJ/cm2 UV dose. Ultraviolet irradiation at the wavelength 254 nm produced changes in the sensory and chemical properties of fluid goat milk.

Efficacy of UV light for the reduction of Listeria monocytogenes in goat's milk.

Certain types of goat's cheeses are produced using unpasteurized milk, which increases the food safety concerns for these types of products. Popularity and consumption of goat's milk products have increased, and the niche market includes gourmet goat's cheeses. The U.S. Code of Federal Regulations and the Pasteurized Milk Ordinance both address the possibility for processing alternatives to heat treatment, and the use of UV light treatment may be a viable alternative that still ensures the safety of the product. Fresh goat's milk was inoculated with Listeria monocytogenes (L-2289) at 10(7) CFU/ml and exposed to UV light using the CiderSure 3500 apparatus (FPE Inc., Macedon, NY). Inoculated milk was exposed to a UV dose range between 0 and 20 mJ/cm2 to determine the optimal UV dose. A greater than 5-log reduction was achieved (P < 0.0001) when the milk received a cumulative UV dose of 15.8 +/- 1.6 mJ/cm2. The results of this study indicate that UV irradiation could be used for the reduction of L. monocytogenes in goat's milk.

Influence of apple cultivars on inactivation of different strains of Escherichia coli O157:H7 in apple cider by UV irradiation.

This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6) to 10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm(2). Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (alpha < or = 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (alpha < or = 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of (o)Brix, pH, and malic acid content failed to show any statistically significant relationship (R(2) > or = 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested.

Modeling of Escherichia coli inactivation by UV irradiation at different pH values in apple cider.

This study examined the effects and interactions of UV light dose (1,800 to 20,331 microJ/cm2) and apple cider pH (2.99 to 4.41) on the inactivation of Escherichia coli ATCC 25922, a surrogate for E. coli O157:H7. A predictive model was developed to relate the log reduction factor of E. coli ATCC 25922 to the UV dose. Bacterial populations for treated and untreated samples were enumerated with the use of nonselective media. The results revealed that UV dose was highly significant in the inactivation of E. coli, whereas pH showed no significant effect at higher UV doses. Doses of 6,500 microJ/cm2 or more were sufficient to achieve a greater than 5-log reduction of E. coli. Experimental inactivation data were fitted adequately by a logistic regression model. UV irradiation is an attractive alternative to conventional methods for reducing bacteria in unpasteurized apple cider.

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

An alternative approach for enumeration of Escherichia coli in foods.

An assay to screen Escherichia coli in foods using MUG supplemented lauryl sulfate tryptose (LST) broth instead of tryptone water (TW) medium was evaluated. The results presented in this paper suggest that this method is more sensitive for lower levels of E. coli, faster (16-18 h vs. 6-10 days) and less expensive (2.454 vs. 2.887 EURO/sample) than the standard ISO procedure. Thus, this method may be beneficial for use when both fecal coliforms and E. coli analyses are required in food systems.

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli.

AIMS: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543. METHODS AND RESULTS: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively. CONCLUSION: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to develop better xylanase-utilizing yeast strains.

Analysis and modeling of the variability associated with UV inactivation of Escherichia coli in apple cider.

Raw data from validation studies of UV tubes used for nonthermal pathogen reduction in apple cider underwent comprehensive statistical analysis. Data from each tube that demonstrated at least a 5-log reduction of Escherichia coli ATCC 25922, a surrogate for E. coli O157:H7, in each of three trials were used in the analysis. The within- and between-tube variability was calculated for 70 tubes. The mean log reductions of the tubes fit a Beta distribution (Kolmogorov-Smirnov test, 0.0246), and the between-replicate variability followed a logistic distribution (Kolmogorov-Smirnov test, 0.0305). These two distributions can be used together to model UV cider treatment as part of an overall E. coli O157:H7 in cider risk assessment. Examples of codes from @RISK and Analyticato describe these distributions, such as one would find in a quantitative risk assessment, are included.

Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology.

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.

Characterization and purification of a bacteriocin produced by a potential probiotic culture, Lactobacillus acidophilus 30SC.

Lactobacillus acidophilus 30SC was tested for its potential as a probiotic culture. The strain exhibited good acid tolerance in an artificial gastric solution as well as bile resistance in media containing 0.3% bile acids. The strain produced a heat-stable antimicrobial compound that was shown to be proteinaceous in nature and, therefore, referred to as a bacteriocin. The bacteriocin was active over a wide pH range and inhibited a number of gram-positive bacteria including Listeria ivanovii and pathogenic strains. The bacteriocin was purified by 50% ammonium sulfate precipitation followed by hydrophobic interaction column chromatography. The SDS-PAGE of the active fractions resulted in a single band with estimated molecular mass of 3.5 kDa. These results demonstrate the potential of L. acidophilus 30SC as a probiotic culture that can be utilized in the manufacturing of dairy foods and dietary supplements.

Detection of the Cholera Toxin-binding Activity of κ-Casein Macropeptide and Optimization of Its Production by the Response Surface Methodology.

The cholera toxin (CT)-binding activity of purified κ-casein macropeptide (CMP) from bovine κ-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of κ-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5°C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.