Inducing the production of the bacteriocin paenibacillin by Paenibacillus polymyxa through application of environmental stresses with relevance to milk bio-preservation.

Food processors are keenly interested in natural alternatives to currently used chemical preservatives. Although bacteriocins of beneficial bacteria are one of the promising alternatives, low productivity of bacteriocin producers has been limiting the application of these agents in food processing. The present study was designed to examine the possibility of inducing the production of paenibacillin, a bacteriocin produced by Paenibacillus polymyxa OSY-DF, by exposing this organism to environmental stresses. Cold stress (15 °C for 105 min) caused 2- to 4-fold increase in the antibacterial and specific antibacterial activities of bioreaction's cell-free filtrate against Listeria innocua and Bacillus subtilis. Paenibacillin production increased when the organism was exposed during incubation to pH 6.0. Presence of 1% NaCl in the growth medium, application of oxidative stress by exposure to 3 ppm H2O2, and adjusting aeration to a relatively low rate were also found to induce the expression of paenibacillin but to a limited degree, when compared to the effect of cold stress. Exposure to pH 6 increased the transcription of the paenibacillin structural gene, paeA, whereas no significant change was noticed for the same gene under cold stress. Paenibacillin, produced in sufficient amounts under cold stress, was tested as a potential preservative to control the sporeforming bacterium, B. subtilis, in milk. The initial population of B. subtilis of ~7 log10 CFU/ml decreased to an undetectable level (≤ 1 log10 CFU/ml) within 24 h of incubation of treated milk held at 37 °C. Similarly, paenibacillin was active against L. innocua in milk; the treatment caused 5-log decrease in the organism's population after 7 days of incubation. Taken together, these results demonstrated increased expression of paenibacillin by exposure to selected environmental stresses, particularly cold stress, and that paenibacillin seems promising in the biopreservation of milk and potentially milk products.

Enhancing titre and production stability of paenibacillin from Paenibacillus polymyxa by sequential drug resistance screening.

AIMS: Paenibacillin is a naturally biosynthesized antimicrobial lantibiotic peptide which is produced by wild-type Paenibacillus polymyxa OSY-DF in low but detectable levels. The aim was to increase paenibacillin titre and production consistency through sequential drug resistance screening. METHODS AND RESULTS: Spontaneous mutants of P. polymyxa OSY-DF were isolated by subjecting the bacterium to two rounds of screening for resistance to rifampicin, which targets RNA polymerase, and gentamicin, which targets ribosomes. Changes in antimicrobial production of the mutants were monitored using a bioassay method. A spontaneous mutant, P. polymyxa OSY-EC, capable of producing high paenibacillin titre, was selected and compared phenotypically to the wild-type strain. The mutant was found to produce paenibacillin at five-fold higher titre than the wild type. The mutant constantly produced paenibacillin while the wild type produced the antimicrobial agent variably. Fourier transformation mid-infrared spectroscopy revealed an interclass distance of 6·4 between the wild type and the mutant strain, suggesting significant phenotypic change during the mutation. CONCLUSIONS: P. polymyxa OSY-EC, a spontaneous mutant capable of consistent production of high paenibacillin titre, was isolated from the wild type after sequential screening on rationally selected antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The study will help make paenibacillin available for large-scale testing by interested researchers and industries seeking applications that improve food safety and quality.

Modified microassay for the isolation of antimicrobial-producing, spore-forming and nonspore-forming bacteria.

AIMS: To develop a microassay method to detect antimicrobials produced by spore-forming bacteria, thus speeding up the discovery of new antimicrobials. METHODS AND RESULTS: Environmental isolates were grown in 96-well plates, to allow production of antimicrobial agents, then treated with lysozyme and heated sequentially. Lysozyme heat treatment inhibited or prevented spore-to-cell transformation, thus eliminating interference from spore outgrowth while detecting antimicrobials by indicator bacteria. Supplementation of the indicator strain medium with 2,3,5-triphenyltetrazolium chloride, as a vital stain, made it easy to rapidly differentiate between antimicrobial-deficient (indicator growth) and antimicrobial-containing (no growth) wells. The method was used to rapidly screen 657 bacteria isolated from eight soil samples. Results revealed 46 Bacillus sp. producing antimicrobials against Listeria sp., and a Bacillus sp. producing antimicrobial(s) against Escherichia coli. CONCLUSIONS: A microassay method was successfully developed and implemented to screen and detect antimicrobial agents from spore-forming, in addition to nonspore-forming, bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobials are needed to combat antibiotic- and preservative-resistant bacteria. Spore-forming bacteria are prolific producers of antimicrobials. This assay will speed the discovery of antimicrobials from spore-forming bacteria; these new antimicrobials are urgently needed in food and medicinal applications.

Lactobacillus acidophilus and Lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus.

Probiotic lactic acid bacteria (LAB) have been shown to alleviate inflammation, enhance the immunogenicity of rotavirus vaccines, or reduce the severity of rotavirus diarrhoea. Although the mechanisms are not clear, the differential Th1/Th2/Th3-driving capacities and modulating effects on cytokine production of different LAB strains may be the key. Our goal was to delineate the influence of combining two probiotic strains of Lactobacillus acidophilus and Lactobacillus reuteri on the development of cytokine responses in neonatal gnotobiotic pigs infected with human rotavirus (HRV). We demonstrated that HRV alone, or HRV plus LAB, but not LAB alone, initiated serum cytokine responses, as indicated by significantly higher concentrations of IFN-α, IFN-γ, IL-12, and IL-10 at postinoculation day (PID) 2 in the HRV only and LAB+HRV+ pigs compared to LAB only and LAB-HRV- pigs. Peak cytokine responses coincided with the peak of HRV replication. LAB further enhanced the Th1 and Th2 cytokine responses to HRV infection as indicated by significantly higher concentrations of IL-12, IFN-γ, IL-4 and IL-10 in the LAB+HRV+ pigs compared to the LAB-HRV+ pigs. The LAB+HRV+ pigs maintained relatively constant concentrations of TGF-ß compared to the HRV only group which had a significant increase at PID 2 and decrease at PID 7, suggesting a regulatory role of LAB in maintaining gut homeostasis. At PID 28, cytokine secreting cell (CSC) responses, measured by ELISpot, showed increased Th1 (IL-12, IFN-γ) CSC numbers in the LAB+HRV+ and LAB-HRV+ groups compared to LAB only and LAB-HRV- pigs, with significantly increased IL-12 CSCs in spleen and PBMCs and IFN-γ CSCs in spleen of the LAB+HRV+ group. Thus, HRV infection alone, but not LAB alone was effective in inducing cytokine responses but LAB significantly enhanced both Th1 and Th2 cytokines in HRV-infected pigs. LAB may also help to maintain immunological homeostasis during HRV infection by regulating TGF-ß production.

Quality of shell eggs pasteurized with heat or heat-ozone combination during extended storage.

The physical quality and functionality of shell eggs, pasteurized with heat or a combination of heat and ozone, were assessed during eight weeks of storage at 4 or 25 °C. Shell eggs were treated as follows: (1) immersion heating that mimics commercial pasteurization processes (egg internal temperature of 56 ± 0.1 °C for 32 min), or (2) a newly developed combination process comprised of heating (56 ± 0.1 °C, internal, for 10 min) followed by gaseous ozone treatment. Eggs were tested for yolk index, Haugh units, albumen pH, albumen turbidity, and percent overrun. Additionally, albumen samples were assayed for lysozyme activity and free sulfhydryl group content, and were analyzed using differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopy. Both processed and unprocessed eggs maintained superior quality when stored at 4 °C, as opposed to 25 °C. Pasteurization, regardless of method, led to superior maintenance of Haugh units during storage but also increased albumen opacity and decreased albumen overrun. Detrimental effects on quality markers were more severe in heat-pasteurized eggs than those treated with the ozone-based process. Pasteurization of shell eggs by either process did not affect lysozyme activity or sulfhydryl group content. Changes in protein secondary structure, as indicated by FTIR analysis, suggest that the ozone-based process is less damaging to albumen proteins than is the heat-alone process. In conclusion, heat-ozone pasteurization, by virtue of its less severe heat treatment, yields a safe final product that more closely resembles untreated shell eggs.

Inactivation of Bacillus amyloliquefaciens spores by a combination of sucrose laurate and pressure-assisted thermal processing.

The aim of this research was to study the effect of sucrose laurate ester (SL) on enhancing pressure-assisted thermal processing (PATP) inactivation of Bacillus amyloliquefaciens Fad 82 spores. B. amyloliquefaciens spores (∼108 CFU/ml) were suspended in deionized water, solutions of 0.1, 0.5, and 1.0% SL, and mashed carrots without or with 1% SL. Samples were treated at 700 MPa and 105°C for 0 (come-up time), 1, 2, and 5 min and analyzed by pour-plating and most-probable-number techniques. Heat shock (80°C, 10 min) was applied to untreated and treated samples to study the germination rates. Results were also compared against samples treated by high pressure processing (700 MPa, 35°C) and thermal processing (105°C, 0.1 MPa). Among the combinations tested, SL at concentrations of 1.0% showed the best synergistic effect against spores of B. amyloliquefaciens when combined with PATP treatments. In the case of high pressure and thermal processing treatments, SL did not enhance spore inactivation at the conditions tested. These results suggest that SL is a promising antimicrobial compound that can help reduce the severity of PATP treatments.

Production of shelf-stable ranch dressing using high-pressure processing.

High-pressure processing (HPP) can reduce or eliminate microorganisms of concern in food without deteriorating product quality; however, quality benefits must justify the substantial capital investment for the utilization of this technology. HPP is particularly a beneficial preservation technology for products damaged by thermal treatments or when product quality could be improved by reformulation to raise pH or eliminate chemical preservatives. The primary objectives of this study were to determine the efficacy of HPP to protect premium ranch dressing (pH 4.4) from microbial spoilage and to assess changes in physical, chemical, and sensory attributes throughout the product's shelf life. In inoculated-packages studies, the efficacy of HPP was measured against ranch dressing spoilage organisms: Pediococcus acidilactici, Lactobacillus brevis, and Torulaspora delbrueckii. HPP treatment (600 MPa, 3 min) decreased population of P. acidilactici, the most pressure-resistant spoilage organism tested, by >or= 6.4 log CFU/g. During a shelf-life study of edible product, treating ranch dressing at 600 MPa for 5 min effectively prevented microbial spoilage throughout the storage period (26 wk at 4 and 26 degrees C). The pH and emulsion stability of ranch dressing were not adversely influenced by HPP. Extended storage of HPP product for 16 to 26 wk at 26 degrees C resulted in a decrease in consumer acceptance and significant changes in color and organic acid profile (specifically, increased pyroglutamic acid). These changes were consistent with those expected during extended storage of commercially available products. HPP may be used to produce premium ranch dressing, with defined shelf-life and storage conditions, without significantly changing product attributes.

Image analysis based quantification of bacterial volume change with high hydrostatic pressure.

Scanning electron microscopy (SEM) images of Staphylococcus aureus 485 and Escherichia coli O157:H7 933 were taken after pressure treatments at 200 to 400 MPa. Software developed for this purpose was used to analyze SEM images and to calculate the change in view area and volume of cells. Significant increase in average cell view area and volume for S. aureus 485 was observed in response to pressure treatment at 400 MPa. Cell view area for E. coli O157:H7 933 significantly increased at 325 MPa, the maximum pressure treatment tested against this pathogen. In contrast to S. aureus, cells of E. coli O157:H7 exhibited significant increase in average view area and volume at 200 MPa. The pressure-induced increase in these parameters may be attributed to modifications in membrane properties, for example, denaturation of membrane-bound proteins and pressure-induced phase transition of membrane lipid bilayer.

Inactivation of Salmonella enterica serovar enteritidis in shell eggs by sequential application of heat and ozone.

AIMS: To assess the contribution of ozone to lethality of Salmonella enterica serovar Enteritidis in experimentally inoculated whole shell eggs that are sequentially treated with heat and gaseous ozone in pilot-scale equipment. METHODS AND RESULTS: Whole shell eggs were inoculated with small populations of Salmonella Enteritidis (8.5 x 10(4)-2.4 x 10(5) CFU per egg) near the egg vitelline membrane. Eggs were subjected to immersion heating (57 degrees C for 21 min), ozone treatment (vacuum at 67.5 kPa, followed by ozonation at a maximum concentration of approx. 140 g ozone m(-3) and 184-198 kPa for 40 min) or a combination of both treatments. Survivors were detected after an enrichment process or enumerated using modified most probable number technique. Ozone, heat and combination treatments inactivated 0.11, 3.1 and 4.2 log Salmonella Enteritidis per egg, respectively. CONCLUSIONS: Sequential application of heat and gaseous ozone was significantly more effective than either heat or ozone alone. The demonstrated synergy between these treatment steps should produce safer shell eggs than the heat treatment alone. SIGNIFICANCE AND IMPACT OF THE STUDY: Shell eggs are the most common vehicle for human infection by Salmonella Enteritidis. Many cases of egg-related salmonellosis are reported annually despite efforts to reduce contamination, including thermal pasteurization of shell eggs and egg products. Treatment with ozone-based combination should produce shell eggs safer than those treated with heat alone.

Proposed mechanism of inactivating Escherichia coli O157:H7 by ultra-high pressure in combination with tert-butylhydroquinone.

AIMS: Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert-butylhydroquinone (TBHQ) during ultra-high pressure (UHP) treatment. METHODS AND RESULTS: Escherichia coli O157:H7 EDL-933, and 14 E. coli K12 strains with mutations in selected genes, were treated with dimethyl sulfoxide solution of TBHQ (15-30 ppm), and processed with UHP (400 MPa, 23 +/- 2 degrees C for 5 min). Treatment of wild-type E. coli strains with UHP alone inactivated 2.4-3.7 log CFU ml(-1), whereas presence of TBHQ increased UHP lethality by 1.1-6.2 log CFU ml(-1); TBHQ without pressure was minimally lethal (0-0.6 log reduction). Response of E. coli K12 mutants to these treatments suggests that iron-sulfur cluster-containing proteins ([Fe-S]-proteins), particularly those related to the sulfur mobilization (SUF system), nitrate metabolism, and intracellular redox potential, are critical to the UHP-TBHQ synergy against E. coli. Mutations in genes maintaining redox homeostasis and anaerobic metabolism were associated with UHP-TBHQ resistance. CONCLUSIONS: The redox cycling activity of cellular [Fe-S]-proteins may oxidize TBHQ, potentially leading to the generation of bactericidal reactive oxygen species. SIGNIFICANCE AND IMPACT OF THE STUDY: A mechanism is proposed for the enhanced lethality of UHP by TBHQ against E. coli O157:H7. The results may benefit food processors using UHP-based preservation, and biologists interested in piezophilic micro-organisms.

Effect of moderate electric field on the metabolic activity and growth kinetics of Lactobacillus acidophilus.

Moderate electric fields (MEF), applied across microbial growth media may potentially affect the permeability of cell membranes. We investigated the effects of MEF on bacteriocin (lacidin A) production during fermentation and on microbial growth kinetics of Lactobacillus acidophilus OSU 133. We comparatively investigated the following treatments: conventional, MEF (1 V/cm, 60 Hz, for 40 h), combinations of MEF (1 V/cm, 60 Hz, for the first 5 h) and conventional fermentation (for 35 h), and discrete MEF (1 V/cm, 2 min on and off, for 40 h). In all treatments, except as noted below, temperature was set at 30 degrees C. The two exceptions were control (conventional) and discrete MEF treatment, which were conducted both at 30 and 37 degrees C. MEF treatments at the early stage of fermentation at 30 degrees C showed the maximum bacteriocin activity. Minimum bacteriocin production was observed under conventional fermentation at 37 degrees C. A mathematical model based on Monod growth kinetics was used to predict bacteriocin production and showed results consistent with conventional treatment data. MEF did not have a significant effect on the lag time, maximum specific growth rate, biomass production and pH change under the different experimental conditions at each specific temperature. Based on the observations, bacteriocin activity under the presence of MEF at the early stage of fermentation increased without significant change in the final biomass.

Inactivation kinetics of selected aerobic and anaerobic bacterial spores by pressure-assisted thermal processing.

The combined pressure-thermal inactivation kinetics of spores from three strains of anaerobic (Clostridium sporogenes, C. tyrobutylicum, and Thermoanaerobacterium thermosaccharolyticum), and six strains of aerobic (Bacillus amyloliquefaciens and B. sphaericus) bacteria were studied. Spores of these bacteria were prepared in deionized water and treated in a custom-made kinetic tester over various pressure (0.1 and 700 MPa) and thermal (105 and 121 degrees C) combinations. Survivor data were modeled using log-linear and Weibull models to obtain relevant kinetic parameters. In comparison to thermal treatment alone, the combined pressure-thermal conditions accelerated the inactivation of the spores tested. A measurable fraction of spore populations was inactivated during the pressure come-up time. Pressure-assisted thermal processing (PATP) at 700 MPa and 121 degrees C for 1 min inactivated up to 7-8 log for some of spores tested. Among bacteria evaluated, based on survivor curve data T. thermosaccharolyticum, B. amyloliquefaciens Fad 82, and Fad 11/2 were found to produce the most PATP-resistant spores. PATP inactivation plots showed characteristic upward curvature, which is indicative of the tailing behavior. Since both log-linear and Weibull kinetic models did not consider microbial reduction during process come-up time, our results demonstrated that the estimated model parameters were not adequate to compare combined pressure-thermal resistance of various bacterial spores tested.

Combined pressure-thermal inactivation kinetics of Bacillus amyloliquefaciens spores in egg patty mince.

Bacillus amyloliquefaciens is a potential surrogate for Clostridium botulinum in validation studies involving bacterial spore inactivation by pressure-assisted thermal processing. Spores of B. amyloliquefaciens Fad 82 were inoculated into egg patty mince (approximately 1.4 x 10(8) spores per g), and the product was treated with combinations of pressure (0.1 to 700 MPa) and heat (95 to 121 degrees C) in a custom-made high-pressure kinetic tester. The values for the inactivation kinetic parameter (D), temperature coefficient (zT), and pressure coefficient (zP) were determined with a linear model. Inactivation parameters from the nonlinear Weibull model also were estimated. An increase in process pressure decreased the D-value at 95, 105, and 110 degrees C; however, at 121 degrees C the contribution of pressure to spore lethality was less pronounced. The zP-value increased from 170 MPa at 95 degrees C to 332 MPa at 121 degrees C, suggesting that B. amyloliquefaciens spores became less sensitive to pressure changes at higher temperatures. Similarly, the zT-value increased from 8.2 degrees C at 0.1 MPa to 26.8 degrees C at 700 MPa, indicating that at elevated pressures, the spores were less sensitive to changes in temperature. The nonlinear Weibull model parameter b increased with increasing pressure or temperature and was inversely related to the D-value. Pressure-assisted thermal processing is a potential alternative to thermal processing for producing shelf-stable egg products.

A real-time polymerase chain reaction-based method for rapid and specific detection of spoilage Alicyclobacillus spp. in apple juice.

AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. METHODS AND RESULTS: The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development. Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison. Selected bacteria were examined for cross-reactivity by the new method. Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity. Using the newly developed Taqman real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A. acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms. Detection of <10 cells per PCR reaction from juice samples was accomplished within 3-5 h. CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: As a favourable alternative for the laborious and time-consuming culture- or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control.

Susceptibility of human rotavirus to ozone, high pressure, and pulsed electric field.

The rotavirus causes a food-transmitted gastroenteritis that affects mainly children. Currently, the food industry is interested in alternative food-processing technologies, but research on the control of food-transmitted viruses by these technologies is limited. In this study, the human rotavirus was cultured on MA104 cells, and suspensions of the virus were prepared and treated with ozone, high pressure, and pulsed electric field (PEF). Virus viability was quantified as 50% tissue culture infectious doses (TCID50) per milliliter. Ozone at 25 microg/ml decreased rotavirus infectivity by 8 to 9 log10 TCID50/ml. High pressure was extremely effective against the rotavirus; treatment with 300 MPa for 2 min at 25 degrees C inactivated approximately 8 log10 TCID50/ml. A small fraction of the virus population, however, remained resistant to pressure treatments of up to 800 MPa for 10 min. Viruses surviving these extreme pressures showed a cytopathic effect different from that of the untreated viruses. The rotavirus was found to be resistant to PEF treatment at 20 to 29 kV/cm, for which no appreciable reductions in virus titer were observed.

Immunoassay method for quantitative determination of nisin in solution and on polymeric films.

AIMS: To develop a non-competitive direct immunoassay method for quantifying nisin in solution or adsorbed to surfaces. METHODS AND RESULTS: The developed method differs from traditional ELISA in pre-forming a complex of the nisin antibodies and the secondary antibody conjugate before addition to the nisin-coated solid phase. The modifications may help decrease interference from the nisin degradation molecules and reduce non-specific adsorption of secondary antibody conjugate. The new method has a detection limit of 65 ng nisin and correlates well (r=0.982) with the agar diffusion bioassay. CONCLUSIONS: A new sensitivity method was developed to determine the amounts of nisin adsorbed to a polymeric surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The new technique can provide a reproducible and accurate method to quantitate nisin molecules in solutions and those bound on polymeric surfaces, and it is free of the limitations of the traditional agar diffusion bioassay.

Inactivation of Escherichia coli O1 57:H7, Listeria monocytogenes, and Lactobacillus leichmannii by combinations of ozone and pulsed electric field.

Pulsed electric field (PEF) and ozone technologies are nonthermal processing methods with potential applications in the food industry. This research was performed to explore the potential synergy between ozone and PEF treatments against selected foodborne bacteria. Cells of Lactobacillus leichmannii ATCC 4797, Escherichia coli O157:H7 ATCC 35150, and Listeria monocytogenes Scott A were suspended in 0.1% NaCl and treated with ozone, PEF, and ozone plus PEE Cells were treated with 0.25 to 1.00 microg of ozone per ml of cell suspension, PEF at 10 to 30 kV/cm, and selected combinations of ozone and PEF. Synergy between ozone and PEF varied with the treatment level and the bacterium treated. L. leichmannii treated with PEF (20 kV/cm) after exposure to 0.75 and 1.00 microg/ml of ozone was inactivated by 7.1 and 7.2 log10 CFU/ml, respectively; however, ozone at 0.75 and 1.00 microg/ml and PEF at 20 kV/cm inactivated 2.2, 3.6, and 1.3 log10 CFU/ml, respectively. Similarly, ozone at 0.5 and 0.75 microg/ml inactivated 0.5 and 1.8 log10 CFU/ml of E. coli, PEF at 15 kV/cm inactivated 1.8 log10 CFU/ml, and ozone at 0.5 and 0.75 microg/ml followed by PEF (15 kV/cm) inactivated 2.9 and 3.6 log10 CFU/ml, respectively. Populations of L. monocytogenes decreased 0.1, 0.5, 3.0, 3.9, and 0.8 log10 CFU/ml when treated with 0.25, 0.5, 0.75, and 1.0 microg/ml of ozone and PEF (15 kV/cm), respectively; however, when the bacterium was treated with 15 kV/cm, after exposure to 0.25, 0.5, and 0.75 microg/ml of ozone, 1.7, 2.0, and 3.9 log10 CFU/ml were killed, respectively. In conclusion, exposure of L. leichmannii, E. coli, and L. monocytogenes to ozone followed by the PEF treatment showed a synergistic bactericidal effect. This synergy was most apparent with mild doses of ozone against L. leichmannii.

Sporicidal action of ozone and hydrogen peroxide: a comparative study.

Elimination of contaminating spores on packaging materials and food-contact surfaces remains a challenge to the food industry. Hydrogen peroxide and chlorine are the most commonly used sanitizers to eliminate these contaminants, and ozone was recommended recently as an alternative. Hence, we compared the sporicidal action of ozone and hydrogen peroxide against selected foodborne spores of Bacillus spp. Under identical treatment conditions, 11 microg/ml aqueous ozone decreased spore counts by 1.3 to 6.1 log10 cfu/ml depending upon the bacterial species tested. Hydrogen peroxide (10%, w/w), produced only 0.32 to 1.6 log10 cfu/ml reductions in spore counts. Thus, hydrogen peroxide, at approximately 10,000-fold higher concentration, was less effective than ozone against Bacillus spores. Resistance of spores to ozone was highest for Bacillus stearothermophilus and lowest for B. cereus. Therefore, spores of B. stearothermophilus are suitable for testing the efficacy of sanitization by ozone. Electron microscopic study of ozone-treated B. subtilis spores suggests the outer spore coat layers as a probable site of action of ozone.

Solvent extraction of bacteriocins from liquid cultures.

A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133. The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium. Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations. Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively. The new extraction procedure saves time and is easy to perform. This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B. subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively.

Inactivation of Listeria monocytogenes Scott A on artificially contaminated frankfurters by high-pressure processing.

Vacuum-packaged frankfurters, inoculated with 24-h cultures of Listeria monocytogenes Scott A (approximately 10(9) CFU/ml) by injection into the packages, were held at pressures of 300, 500, and 700 MPa for up to 9 min. L. monocytogenes were washed from the surface of the frankfurter and plated onto brain heart infusion agar. During the time to achieve 300, 500, and 700 MPa (come-up time), L. monocytogenes populations decreased by 1, >3, and >5 logs, respectively. Additional inactivation of L. monocytogenes occurred while the samples were held at 300 and 500 MPa. A 5-log reduction in bacterial population was possible at all pressure treatments; however, pressurization at 700 MPa showed the fastest inactivation with L. monocytogenes reduced from 10(8) to 10(2) CFU/package during the come-up time. These results show that high-pressure processing may be a viable method for controlling foodborne pathogens in postprocessed, packaged frankfurters.