[Show abstract][Hide abstract]ABSTRACT: Sanitization of fresh fruits and vegetables, particularly leafy greens, is limited by penetration of sanitizers to the location of internalized pathogens. It is necessary, therefore, to adapt sanitization operations to practices in the existing produce chain. In this work, we investigated experimentally in a pilot scale, various potential sanitization options using gaseous ozone during and post vacuum cooling on the inactivation of Escherichia coli O157:H7 ATCC 43889. It was found that vacuum cooling causes bacterial internalization, making them harder to reach by sanitizer. However, the application of ozone during the vacuum cooling step significantly reduced (p < 0.05) population of viable internalized bacteria which otherwise remain unaffected by sodium hypochlorite and UV light surface treatments. The presence of free water inside the vessel available for evaporation during vacuum cooling was found to impact the microbial reduction during combined vacuum cooling and the gaseous ozone treatment. The efficacy of application of high ozone concentration (1.5 g/m3) short term during the vacuum cooling step in combination with low ozone concentration (0.032–0.528 g/m3) long term sanitization treatments (days) was evaluated. This combination of gaseous treatment was found more effective in microbial reduction compared to a single treatment with a strongly expressed synergistic effect. The post-treatment spinach quality evaluation, however showed an increasing degree of damage as the time of treatment increases even at low ozone concentrations.
[Show abstract][Hide abstract]ABSTRACT: A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporus via morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance liquid chromatography, and structurally analyzed using mass spectrometry (MS)
and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of
13 amino acids and an N-terminal C6 fatty acid (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA-Dhb-Leu-Orn-Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu-valinol,
where Dhb is α,β-didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1,583.0794 Da and
contains three modified amino acid residues: α,β-didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin,
was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant
bacteria, brevibacillin also exhibited low MICs (1 to 8 μg/ml) against selected foodborne pathogenic and spoilage bacteria,
such as Listeria monocytogenes, Bacillus cereus, and Alicyclobacillus acidoterrestris. Purified brevibacillin showed no sign of degradation when it was held at 80°C for 60 min, and it retained at least 50% of
its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings,
brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens
and foodborne pathogenic and spoilage bacteria.
Article · Feb 2016 · Applied and Environmental Microbiology
[Show abstract][Hide abstract]ABSTRACT: High-pressure processing (HPP) of food utilizes elevated pressures with
or without combination of heat to inactivate harmful pathogens and spoilage microorganisms
in their vegetative or spore state. Since the treatment reduces thermal
impact, pressure-treated products have better organoleptic attributes. The importance
of identifying a relevant surrogate organism for high-pressure pasteurization
and sterilization studies is highlighted. Process- and product-related factors influencing
the antimicrobial efficacy of pressure treatment are reviewed.
[Show abstract][Hide abstract]ABSTRACT: Pressure-assisted thermal processing (PATP; 500–700 MPa, 90–121 °C) offers new opportunities to sterilize low-acid foods while preserving quality attributes to an extent greater than is possible with traditional thermal processing (TP). This study was conducted to evaluate the possibility of enhancing PATP lethality against the spores of Bacillus amyloliquefaciens, by sensitizing the spores with selected antimicrobial compounds (including emphasis on the use of natural antimicrobials) prior to treatment. A spore crop of B. amyloliquefaciens TMW 2.479 Fad 82, that had previously shown high resistance to combined pressure-heat treatment, was prepared on Nutrient Agar medium supplemented with 10 mg L−1 MnSO4·H2O and incubated at 32 °C for 3 d. Spores were inoculated (at ∼10^7–10^8 CFU mL−1 inoculum level) in HEPES buffer (pH ≤ 7.0) or selected low-acid foods (pH 5.2–5.6) with or without added antimicrobial compounds. The samples were then treated at 600 MPa and 105 °C (PATP) or 0.1 MPa and 105 °C (TP) for various holding times. Among different compounds tested, low-molecular-weight chitosan, and combination of chitosan with some surfactants were most effective (P < 0.05) in enhancing the PATP and TP lethality. This study suggests that certain antimicrobials can be added to the low-acid media prior to PATP or TP treatment to enhance the efficacy of the process. The treatment allows sterilization of low-acid foods at lower process temperatures thus ensuring better preservation of quality attributes.
[Show abstract][Hide abstract]ABSTRACT: Produce safety has received much recent attention, with the emphasis being largely on discovery of how microbes invade produce. However, the sanitization operation deserves more attention than it has received. The ability of a sanitizer to reach the site of pathogens is a fundamental prerequisite for efficacy. This work addresses the transport processes of ozone (gaseous and liquid) sanitizer for decontamination of leafy greens. The liquid sanitizer was ineffective against Escherichia coli K-12 in situations where air bubbles may be trapped within cavities. A model was developed for diffusion of sanitizer into the interior of produce. The reaction rate of ozone with the surface of a lettuce leaf was determined experimentally and was used in a numerical simulation to evaluate ozone concentrations within the produce and to determine the time required to reach different locations. For aqueous ozone, the penetration depth was limited to several millimeters by ozone self-decomposition due to the significant time required for diffusion. In contrast, gaseous sanitizer was able to reach a depth of 100 mm in several minutes without depletion in the absence of reaction with surfaces. However, when the ozone gas reacted with the produce surface, gas concentration was significantly affected. Simulation data were validated experimentally by measuring ozone concentrations at the bottom of a cylinder made of lettuce leaf. The microbiological test confirmed the relationship between ozone transport, its self-decomposition, reaction with surrounding materials, and the degree of inactivation of E. coli K-12. Our study shows that decontamination of fresh produce, through direct contact with the sanitizer, is more feasible with gaseous than with aqueous sanitizers. Therefore, sanitization during a high-speed washing process is effective only for decontaminating the wash water.
[Show abstract][Hide abstract]ABSTRACT: Paenibacillin, a recently-discovered lantibiotic from Paenibacillus polymyxa OSY-DF, showed potency against Listeria monocytogenes, methicillin-resistant Staphylococcus aureus and other Gram-positive bacteria. The chemical structure of paenibacillin has been determined previously. This study was initiated to investigate the biosynthesis of paenibacillin, and to reveal unique features in its biosynthetic pathway. Paenibacillin structural gene (paeA) was identified by polymerase chain reaction (PCR) analysis. The complete biosynthetic gene cluster was revealed by whole genome sequencing of the producer strain. The paenibacillin gene cluster (11.7. kb) comprises 11 open reading frames (ORFs) encoding proteins for production, modification, regulation, immunity and transportation of the lantibiotic. Disruption of the gene encoding lantibiotic dehydratase (PaeB) completely eliminated the production of paenibacillin. The cluster includes a gene encoding a putative acetylase (PaeN), which may catalyze the N-terminal acetylation of paenibacillin during its biosynthesis. This finding supports the results of a previous chemical analysis, reporting an acetyl moiety uniquely located at paenibacillin N-terminus. Results of this study may expedite efforts to design effective lantibiotic drugs and facilitate attempts to increase the productivity of the lantibiotic-producing strain.
[Show abstract][Hide abstract]ABSTRACT: Microorganisms are widely distributed in food and contribute to food safety due to production of antagonistic substances. A new bacterial strain, OSY-7LA, was isolated from a Chinese delicacy food and exhibited strong antagonistic activity against Listeria innocua. It was identified as Bacillus atrophaeus by morphological, physiological, and biochemical properties and genetic relatedness. The culture supernatant has antimicrobial activities against the Gram-positive pathogens tested, namely, Listeria monocytogenes, Bacillus cereus and methicillin-resistant Staphylococcus aureus. The antimicrobial agents were harvested by solvent extraction and were purified by high performance liquid chromatography (HPLC). Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) were performed to identify these compounds. A protonated ion at m/z 3401.414 corresponded to the molecular mass of subtilosin, and the identity of the antimicrobial agent was confirmed by amplification of subtilosin gene (. sbo) from isolate's genomic DNA. Sodiated ions at m/z 1030.553, 1044.642 and 1058.701 were identified as C13, C14 and C15 surfactins. LC/MS analysis proved the production of plipastatin by OSY-7LA. Supplement of crude extract of OSY-7LA supernatant in Vienna sausage that was inoculated with L. innocua showed 2-log reduction after 12 and 24 h. The new strain and related antimicrobials are potentially useful in food preservation.
[Show abstract][Hide abstract]ABSTRACT: Peanut safety and quality were evaluated for different roasting technologies. Shelled raw peanuts were roasted using an oven at 163 to 204 degrees C, microwave, or oven and microwave combinations. The lethal effect of these treatments was investigated on peanuts inoculated with the Salmonella surrogate, Enterococcus faecium and stored at room temperature for 1 h, 24 h, or 7 d before roasting. Roasted peanut color, odor activity values (OAVs), descriptive sensory panel analysis, free fatty acid, and peroxide values were determined. Color and OAVs were also analyzed on 2 commercial peanut butters. OAVs were calculated using volatile levels quantified with selected ion flow tube mass spectrometry and known odor thresholds. All treatments resulted in a minimum of 3 log reduction of inoculated bacterial population. Resistance to the process was not influenced by storage of inoculated peanuts prior to treatment. Roasting by different methods produced equivalent, commercially ideal L* color. Based on the OAVs, treatments had similar volatiles important to flavor compared to the commercial samples. Descriptive sensory analysis showed no significant difference between the roasting treatments for most of the sensory attributes. Lipid oxidation was not significantly different between the roasting methods, displaying no evidence that roasting time or temperature affected lipid oxidation, when ideal color was produced. These results suggest that oven, microwave, or combination roasting should be sufficient to mitigate the threat of Salmonella contamination and produce similar color, OAVs, sensory attributes, and lipid oxidation results.
[Show abstract][Hide abstract]ABSTRACT: The purpose of this study was to investigate the effect of osmotic shock and adaptation at low water activity (aw) and the type of humectant used to lower the aw, on heat resistance of three Salmonella enterica serovars (Saintpaul 02-109, Tennessee 2053H, and Elmsbuettel 1236H). The serovars were grown (adapted) or transferred (osmotic shocked) in low-aw broths and subjected to heat treatment at 55°C for up to 45 min; samples were removed at 5-min intervals and immediately placed in an ice-water bath until plating. The aw of tryptic soy broth (TSB) was lowered by the addition of 20% (wt/wt) glycerol (aw 0.94), 4% (wt/wt) sodium chloride (NaCl; aw 0.97), or 35% sucrose (wt/wt) (aw 0.95). The type of humectant and cell adaptation significantly affected the D55°C-value. Cells merely suspended in 20% glycerol broth (i.e., nonadapted) prior to heat treatment showed a larger D55°C-value (3.0 to 3.9 min), when compared with that of cells adapted in the same medium (D55°C-values of 0.86 to 0.98 min). Interestingly, cells adapted to TSB plus glycerol were not more resistant to heat than were the controls. NaCl and sucrose showed a net protective effect for all serovars under both the adapted and nonadapted conditions, with sucrose providing the most protection. Highest D55°C-values were obtained for cultures adapted to TSB plus sucrose. Based on these results, the effect of reduced aw on thermal resistance of Salmonella serovars varies greatly, depending on medium constituents and adaptation of the pathogen in these media.
[Show abstract][Hide abstract]ABSTRACT: Paenibacterin, produced by Paenibacillus thiaminolyticus OSY-SE, is active both against Gram-negative and Gram-positive pathogens, including antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis. Paenibacterin showed relatively low cytotoxicity against a human kidney cell line (ATCC CRL-2190), with a 50% inhibitory concentration (IC50)≥109μg/mL. The cationic paenibacterin molecule binds to the negatively charged Gram-negative endotoxins in vitro, suggesting that paenibacterin can neutralise lipopolysaccharides. In a murine septic shock model, two 500μg doses of paenibacterin significantly increased the survival of mice challenged with a lethal level of P. aeruginosa. Considering that paenibacterin is effective against many strains of antibiotic-resistant pathogens, this study suggests that this antimicrobial agent is a promising candidate as a new drug.
Article · Apr 2014 · International journal of antimicrobial agents
[Show abstract][Hide abstract]ABSTRACT: Paenibacterin is a novel lipopeptide antibiotic with potent activity against Gram-negative and Gram-positive human pathogens. The antibiotic consists of a cyclic 13-residue peptide and an N-terminal C15 fatty acyl chain. To elucidate the biosynthesis of paenibacterin, we determined the whole genome sequence of the producer strain Paenibacillus thiaminolyticus OSY-SE, and the function of the peptide synthetase was confirmed experimentally. The gene cluster of paenibacterin was identified within a 52-kb DNA region, encoding thee non-ribosomal peptide synthetases, PbtA, PbtB and PbtC, and two ABC-transporters, PbtD and PbtE. Both PbtA and PbtB consist of five modules, whereas PbtC comprises thee modules. Each of these 13 modules consists of thee essential domains (condensation-adenylation-thiolation) and assembles an amino acid into the paenibacterin peptide. Selected adenylation domains in the NRPS were cloned and expressed in Escherichia coli; the substrate specificity of each recombinant A-domain was studied in vitro by protein function analysis. The presence of four epimerization domains in paenibacterin peptide synthetases suggests that Orn1, Orn4, Lys7 and Ser8 in the paenibacterin molecule have D-configuration; the absolute configuration of two ornithine residues in paenibacterin was confirmed by chiral amino acid analysis using Marfey's reagents. Taken together, the findings enabled us to propose the biosynthetic pathway of paenibacterin.
[Show abstract][Hide abstract]ABSTRACT: Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C15 fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged
lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated
the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and
thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane,
as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative
bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of
membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.
Article · Feb 2014 · Applied and Environmental Microbiology
[Show abstract][Hide abstract]ABSTRACT: To accomplish continuous flow ohmic heating of a low-acid food product, sufficient heat treatment needs to be delivered to the slowest-heating particle at the outlet of the holding section. This research was aimed at developing mathematical models for sterilization of a multicomponent food in a pilot-scale ohmic heater with electric-field-oriented parallel to the flow and validating microbial inactivation by inoculated particle methods. The model involved 2 sets of simulations, one for determination of fluid temperatures, and a second for evaluating the worst-case scenario. A residence time distribution study was conducted using radio frequency identification methodology to determine the residence time of the fastest-moving particle from a sample of at least 300 particles. Thermal verification of the mathematical model showed good agreement between calculated and experimental fluid temperatures (P > 0.05) at heater and holding tube exits, with a maximum error of 0.6 °C. To achieve a specified target lethal effect at the cold spot of the slowest-heating particle, the length of holding tube required was predicted to be 22 m for a 139.6 °C process temperature with volumetric flow rate of 1.0 × 10(-4) m(3) /s and 0.05 m in diameter. To verify the model, a microbiological validation test was conducted using at least 299 chicken-alginate particles inoculated with Clostridium sporogenes spores per run. The inoculated pack study indicated the absence of viable microorganisms at the target treatment and its presence for a subtarget treatment, thereby verifying model predictions.
[Show abstract][Hide abstract]ABSTRACT: To ensure sterility of a solid–liquid mixture processed in continuous-flow ohmic systems, the slowest-heating solid particle needs to receive sufficient heat treatment at the outlet of the holding section. We describe herein, the development of a mathematical model for solid–liquid mixtures in a commercial ohmic heater with electric field oriented perpendicular to the flow. The fastest moving particle velocity was identified using over 299 particles and a radio-frequency identification technique, and used as an input to the model for the worst-case heating scenario. Thermal verification was conducted by comparing predicted and measured fluid temperatures at heater and hold tube outlets; the model showed good agreement between calculated and experimental fluid temperatures (P > 0.05) with a maximum error of 0.4 °C. The model predicted a hold tube length of 15.85 m at 134.0 °C process temperature to achieve a target lethal effect at the cold spot of the slowest-heating particle. Using this length of hold tube, microbiological tests were conducted using at least 299 chicken/alginate particles inoculated with Clostridium sporogenes spores per run. These tests showed the absence of viable microorganisms at the target treatment and positive growth when temperatures were below target, thereby verifying model predictions.
[Show abstract][Hide abstract]ABSTRACT: Use of bacteriocins in food preservation has received great attention in recent years. The goal of this study is to characterize enterocin RM6 from Enterococcus faecalis OSY-RM6 and investigate its efficacy against Listeria monocytogenes in cottage cheese. Enterocin RM6 was purified from E. faecalis culture supernatant using ion exchange column, multiple C18-silica cartridges, followed by reverse-phase high-performance liquid chromatography. The molecular weight of enterocin RM6 is 7145.0823 as determined by mass spectrometry (MS). Tandem mass spectrometry (MS/MS) analysis revealed that enterocin RM6 is a 70-residue cyclic peptide with a head-to-tail linkage between methionine and tryptophan residues. The peptide sequence of enterocin RM6 was further confirmed by sequencing the structural gene of the peptide. Enterocin RM6 is active against Gram-positive bacteria, including L. monocytogenes, Bacillus cereus, and methicillin-resistant Staphylococcus aureus (MRSA). Enterocin RM6 (final concentration in cottage cheese, 80 AU/mL) caused a 4-log reduction in population of L. monocytogenes inoculated in cottage cheese within 30 min of treatment. Therefore, enterocin RM6 has potential applications as a potent antimicrobial peptide against foodborne pathogens in food.
[Show abstract][Hide abstract]ABSTRACT: Infection of laying hens with Salmonella enterica serovar Enteritidis leads to deposition of the pathogen into the albumen or yolk of forming eggs. Heat treatment can inactivate internalized Salmonella Enteritidis in shell eggs, but factors such as the nature and location of contamination may influence the efficacy of thermal treatments. In the current research, natural contamination was mimicked by introducing small inocula of Salmonella Enteritidis into different locations of shell eggs and incubating inoculated eggs. These pathogen-containing eggs were heated at 57°C for 40 min, and temperature within eggs was monitored at the locations of inocula. Comparison of inactivation at equivalent internal temperatures revealed similar levels of lethality regardless of inoculum location. Refrigeration between incubation and heat treatment did not increase thermal resistance of cells in albumen but decreased cell inactivation in yolk. Sequential application of heat and gaseous ozone allows for the development of a process capable of decontaminating shell eggs with minimal thermal treatment and impact on egg quality. Inoculated eggs were subjected to (i) an immersion heating process similar to that used in commercial pasteurization or (ii) immersion heating, at reduced duration, followed by vacuum (50.8 kPa) and treatment with ozone gas (maximum 160 g/m(3)) under pressure (∼187.5 kPa). All treatments tested produced greater than 5-log inactivation, which is required for "pasteurization" processes. Differences were observed in the visual quality of eggs depending on treatment parameters. Application of ozone subsequent to heating allows for a significant reduction in heating time without decreasing process lethality.