Inactivation of food poisoning bacteria and Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment

Inactivation of the vegetative foodborne pathogens by high pressure carbon dioxide treatment (HPCT) was investigated. Pseudomonas aeruginosa IFO3445, Aeromonas hydrophila IFO13286, Salmonella enteritidis JCM3313, Salmonella typhimurium IID1000, Yersinia enterocolitica JCM1677, Escherichia coli O157:H7 ATCC35150, E. coli O157:H7 ATCC43889, Staphylococcus aureus FDA209P and Listeria monocytogenes 1/2a were used in this study. In all cases, HPCT at 35 °C, 10 MPa for 1 min reduced initial numbers by approximately 6 logs. In addition HPCT inactivation of 9 pathogenic strains of Bacillus cereus (mixture of spores and vegetative cells) was investigated. HPCT at 75 °C, 10 MPa for 120 min resulted in significant inactivation in all the strains. The effect of HPCT (30 MPa at 95 °C for up to 120 min) on the inactivation of Geobacillus stearothermophilus spores in the presence of sodium chloride (3% and 6% w/v), glucose (6% and 12% w/v) and ethanol (10% and 20% v/v) was also studied. Both sodium chloride and glucose had a protective effect and the level of inactivation was reduced. The effect was in proportion to the solute concentration. However, 10% and 20% ethanol did not significantly affect the level of inactivation.     

Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes

Flow cytometry was applied to assess the antimicrobial activity of oregano, thyme and cinnamon essential oils against Listeria monocytogenes ATCC19114, using combined staining with propidium iodide (PI) for membrane damage evaluation and carboxyfluorescein diacetate (cFDA) for esterase activity detection. The antimicrobial activity of the essential oils was also tested at different NaCl concentrations.Significant differences were observed between plate count results and flow cytometric data, which suggested the presence of a sublethally stressed subpopulation, not able to form colonies on agar plates.Following treatments, flow cytometric assessment clearly discriminated three different subpopulations: viable, dead and injured cells. Cinnamon essential oil exerted a different impact on the cellular subpopulations, with a lower overall activity and a large percentage of cells having minimally damaged membranes. On the contrary, membrane disintegration seemed to be the primary inactivation mechanism of oregano and thyme essential oils.The antimicrobial activity of the essential oils increased with NaCl concentration increase, but higher NaCl concentrations were necessary following treatments with cinnamon essential oil.Our findings suggest differences in the mode of action of cinnamon essential oil against L. monocytogenes, in comparison with thyme and oregano essential oils.     

Bacterial dynamics during the spontaneous fermentation of cassava dough in gari production

The microbial dynamics and diversity during solid substrate fermentation of cassava, a case study of gari production in West Africa, was investigated. The 16S rDNA gene sequence analysis of the PCR-Denaturing Gradient Gel Electrophoresis (PCR–DGGE) analysis of microbial community DNA and Pulsed Field Gel Electrophoresis (PFGE) of selected isolates as well as culturing techniques using different selective media were used to monitor the bacterial dynamics during cassava fermentation. The V3 variable region of the 16S gene was analyzed and the closest relatives of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus casei were identified by sequencing of the DGGE band amplimers. The DGGE amplimers also revealed the succession and dynamics of LAB; there was a progressive increase in their population proportional to the fermentation period. The analysis of the PFGE band patterns showed that five diverse species of LAB were involved in the fermentation. The representative isolate of each of the PFGE clusters was phenotypicaly identified as L. plantarum, L. fermentum and Leuconostoc mesenteroides by the API 50 CHL sugar fermentation profile. These combinations of parameters identified heterofermentative LAB as bacteria that initiated the fermentation, reduced the pH below four and increased the acidity of the fermentation mash. Information such as this is relevant for the development of starter cultures and predictability of the process for traditional fermented foods and to aid their intermediate and large scale production.     

Sigmoidal thermal inactivation kinetics of Listeria innocua in broth: Influence of strain and growth phase

Listeria innocua inactivation was studied within the temperature range 52.5–65.0 °C, comparing two different strains (10528 and 2030c) and two growth phases (exponential and stationary). Survival curves may present a sigmoidal behaviour, with an initial shoulder (L), followed by a maximum inactivation rate (k_max) period and a final tailing tendency. A Gompertz-inspired model was used to fit experimental data, and kinetic parameters (L, k_max and tail) were estimated by non-linear regression analysis. The influence of temperature, growth phase and strain on kinetic parameters was studied using a 2³ factorial experimental design. Results showed that temperature and growth phase were the most significant variables affecting the kinetic parameters. Listeria thermal inactivation varied from a log-linear tendency till a pronounced sigmoidal behaviour, depending on the studied factors.     

Sub-lethal damage in Cronobacter sakazakii subsp. sakazakii cells after different pulsed electric field treatments in infant formula milk

A study was carried out to study the possible cell damage caused by Pulsed Electric Field (PEF) to Cronobacter sakazakii subsp. sakazakii suspended in different commercial Infant Formula Milk (IFM) after different treatment conditions (15 and 35 kV/cm) associated with a minimum, medium and maximum energy input values. Post-treatment, the growth of survivors and the possible presence, recovery or death of sub-lethaly damaged cells was studied under refrigerated storage (8 °C) period of 24 h. A higher proportion of damaged cells were found at treatment conditions at which C. sakazakii shows higher resistance. PEF damaged a large proportion (80–90%) of C. sakazakii cells which then became sensitive to subsequent refrigerated storage in IFM.The greatest C. sakazakii reduction of 2.30 log cycles was achieved after 15 kV/cm–3000 μs PEF treatment followed by 24 h refrigerated storage (8 °C), primarily due to PEF but also the formation and subsequent death of damaged cells followed by 24 h refrigerated storage (8 °C) period, with consequent IFM safety applicability.     

Evaluation of growth and transfer of Staphylococcus aureus from poultry meat to surfaces of stainless steel and polyethylene and their disinfection

The growth of Staphylococcus aureus inoculated onto poultry meat was investigated under different incubation periods and temperature patterns. Transfer of this microorganism to surface materials and their disinfection was also evaluated. The evaluation of transfer was carried out by placing the contaminated meat cubes on stainless steel and polyethylene surfaces for 10 s and 10 min each, and the surfaces were disinfected with 0.5% chlorhexidine digluconate (CHXdG) for 1 and 10 min each. After 24 h, there was a significant increase of the bacteria count at 20 °C, but not at temperatures between 7 and 15 °C. Significant counts of S. aureus were transferred after a few seconds of contact of the cubes with both materials, and significant differences of transferred cell counts were not detected among the surface materials or durations of contact. The CHXdG solution was able to inactivate all the transferred cells after 10 min of exposure; however, the same result was not observed with 1-min exposure. The time of contact and the type of surface material did not influence the quantity of the transferred cells. The 0.5% CHXdG solution was effective for the disinfection of the contaminated surfaces without previous cleaning.     

The occurrence of Campylobacter spp. in Estonian broiler chicken production in 2002–2007

The aim of the present study was to monitor the occurrence of Campylobacter spp. in broiler chicken production in Estonia from 2002 to 2007. Campylobacter spp. was isolated in 163 (12.3%) of 1320 broiler chicken meat samples form 2002 to 2007 and in 115 (6.3%) of 1819 cecal samples in 2005–2007. Campylobacter jejuni was the most commonly isolated species (98.2%), followed by Campylobacter coli (1.4%) and Campylobacter lari (0.4%). The seasonal peak of Campylobacter contamination was from July to September. Our findings showed that Campylobacter contamination at all levels of broiler chicken production in Estonia was low.     

Ochratoxin A removal in synthetic media by living and heat-inactivated cells of Oenococcus oeni isolated from wines

The capacity of Oenococcus oeni to eliminate ochratoxin A (OTA) from synthetic media in different conditions was studied. Ten tested O. oeni strains removed OTA from the medium but with significant differences depending on the strain, incubation period, and initial OTA level in the medium. Mycotoxin reductions higher than 60% were recorded in 14-day cultures spiked with 2 μg OTA/l. Toxin removal was independent of bacterial viability and culture medium composition. This is the first study carried out to study OTA removal dynamics by living and heat-inactivated cells of O. oeni. The results aim that this bacterium may be a very useful tool to control OTA in food and beverages.     

Survival of Listeria innocua in Minas Traditional Serro cheese during ripening

Cheese produced with raw milk can be a risk to consumer health. It is known that lactic acid bacteria present in raw milk and in natural starters can produce antimicrobial compounds against some foodborne bacteria. This work aimed to evaluate the survival of Listeria innocua in Minas Traditional Serro cheese during cheese ripening. The cheeses were inoculated with 10¹, 10² or 10³ CFU mL⁻¹ of the bacterium and were analyzed for 60 days of ripening at 30 °C. It was observed that the time and the dose of bacteria inoculated affected (p < 0.05) the survival of L. innocua. Even when the lowest dose was inoculated, at the end of the 60 days, approximately 10² CFU mL⁻¹ of L. innocua was detected in the cheese. The lactic acid bacteria present in the milk and in the natural starter were not sufficient to guarantee the absence of L. innocua in Minas Traditional Serro cheese even after 60 days of storage, as is required by Brazilian legislation.     

Listeriosis risk assessment: Simulation modelling and “what if” scenarios applied to consumption of ready-to-eat products in a Spanish population

A one dimensional model was developed to assess the risk of listeriosis in Navarra (Spain) due to the consumption of smoked fish (salmon and trout) and sliced cooked ham (vacuum or non-vacuum-packed). Data regarding serving size, proportion of consumers, time of storage, refrigerator temperature, prevalence and counts of Listeria monocytogenes were loaded into the model in order to describe the variability, using Monte Carlo simulation. The final output of the model was the estimation of the annual number of listeriosis cases, the consumption of cooked ham being the responsible for the higher prediction of cases. Different “what if” scenarios have been studied in order to assess the likely impact of strategies in the estimated cases. Temperature storage at 4 °C through the food chain (including storage at home) has been demonstrated to be the best tool to decrease the risk of this serious illness.     

Growth of acid-adapted Listeria monocytogenes in orange juice and in minimally processed orange slices

The aim of this work was to study the growth/survival of acid-adapted cells of Listeria monocytogenes, in orange juice and in minimally processed orange slices. The L. monocytogenes OML 45 behaviour into TSB (Tryptic Soy Broth) medium was evaluated at different pH values (between 3.7 and 6.7). The acid-adapted cells were obtained maintaining L. monocytogenes in TSB at pH 5.7 for 3 h. The obtained cells were then inoculated into a diluted orange juice with a pH of 2.6. Moreover, the acid-adapted cells were inoculated into minimally processed orange slices. The growth was evaluated during storage at different temperatures. The study confirms that orange juice and minimally processed orange slices can support the acid-adapted pathogen growth.     

Survival of Listeria innocua and Listeria monocytogenes in muscle of cod (Gadus morhua L.) during salt-curing and growth during chilled storage of rehydrated product

The aim of this work was to study survival of Listeria innocua and Listeria monocytogenes in muscle of cod during salt-curing and growth during chilled storage of the rehydrated product. Fresh cod was inoculated with L. innocua and L. monocytogenes at different levels before salt-curing. After salt-curing and rehydration, the levels were within 1 log₁₀ CFU/g lower than prior to salt-curing in all experiments. During the first 5 days of storage after rehydration, growth of L. innocua was observed in 1 out of 5 experiments at 4 °C, but a 10–100-fold increase were observed in all experiments from day 5 to day 10. The growth started earlier and was more rapid when samples were stored at 7 °C. Growth of L. monocytogenes at 4 °C appeared to start earlier than for L. innocua, but a 10–100-fold increase was observed also for this bacterium. The lag phases in rehydrated products were longer than in experiments with cod muscle juice. The differences could be explained by a different level of salt stress. This work demonstrates that long term exposure to very high salt concentrations does not eliminate Listeria spp., and that Listeria being present in the fish prior to salt-curing can recover and grow in rehydrated salt-cured cod during chilled storage.     

Plantaricin MG active against Gram-negative bacteria produced by Lactobacillus plantarum KLDS1.0391 isolated from “Jiaoke”, a traditional fermented cream from China

A bacteriocin named plantaricin MG produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China is reported in this article. Plantaricin MG was purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography and reverse-phase chromatography. Mass spectrometry analysis showed the mass of plantaricin MG to be approximately 2180 Da. The bacteriocin showed a broad inhibitory activity against Gram-positive and Gram-negative bacteria including Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli. The bacteriocin was extremely heat-stable (30 min at 121 °C) and remained active after incubation at pH 2.0–10.0. It was found to be sensitive to proteolytic enzymes (pepsin, trypsin, papain, α-chymotrypsin, proteinase K, Neutrase and Alcalase). The mode of action of plantaricin MG was identified as bactericidal.     

Effect of weak acids on Listeria monocytogenes survival: Evidence for a viable but nonculturable state in response to low pH

Listeria monocytogenes is capable of surviving environmental conditions that are normally fatal to many other bacteria, enabling this pathogen to remain on foods and eventually establish infection after consumption. This study investigated the extent to which L. monocytogenes is killed by several weak acids, with particular emphasis on the commonly used food preservative, potassium sorbate. The effects of potassium sorbate were found to be highly pH dependant, with significant killing only observed at a pH of 4.0. At this pH, the order of effectiveness of the acids tested was Na benzoate > Na propionate > acetic acid > K sorbate. Temperature had a significant impact on sensitivity, with K sorbate being effective at 50 mM and elevated (37 °C) temperature, but failing to affect survival when cells were at 4 °C or 21 °C. Finally, we found that cells grown in the presence of K sorbate at pH 4.0 entered a viable but nonculturable (“VBNC”) state, but became nonviable by 24 h. In contrast, cells incubated under these conditions in the absence of K sorbate entered the VBNC state and maintained viability throughout the 24 h study period.     

Technological characterization of Lactobacillus isolates from traditional Manchego cheese for potential use as adjunct starter cultures

Thirty Lactobacillus isolates from artisanal Manchego cheeses, 10 belonging to Lactobacillus plantarum and 20 belonging to Lactobacillus paracasei subsp. paracasei, were examined for potential use as adjunct cultures. Isolates were screened for acidifying and amino-biogenic capacity, for proteolytic, lipolytic, autolytic and aminopeptidase activities, and for sensory characteristics in UHT coagulated milk. Most of the lactobacilli displayed high aminopeptidase and autolytic activities and low acidifying activity. Only one isolate was able to produce tyramine. When growing on milk 53.3% of the Lb. paracasei subsp. paracasei isolates were able to produce pleasant flavours. Strains PBL210 (Lb. plantarum) and PBL213 and PBL226 (Lb. paracasei subsp. paracasei) presented the best technological characteristics, and also were still viable and with unaltered properties after freeze-drying and storage for 12 months. For these reasons, these strains could be good candidates for inclusion as adjuncts in a starter culture to manufacture industrial Manchego cheese.     

An outbreak of Proteus mirabilis food poisoning associated with eating stewed pork balls in brown sauce, Beijing

We report an outbreak of food poisoning that occurred in August 2008 in a group of 13 persons who ate Stewed Pork Balls in Brown Sauce in Beijing. Epidemiology investigation and case-control analysis indicated that the Stewed Pork Balls in Brown Sauce was the source of food poisoning (OR 3.34, 95% CI 1.30–8.65). With the different measurements including API 20E and PCR, Dienes typing and PFGE, Proteus mirabilis was identified as a contributor to this matter, which evaluated by analyzing the collectives, which derived from contaminated food, patients, cook handlers and waiter. So this is the first report that P. mirabilis as a culprit resulted in food poisoning in Beijing.     

Survival of Listeria innocua in thermally processed orange juice as affected by vanillin addition

Presence of Listeria monocytogenes could seriously affect the safety of fruit juices. Addition of natural antimicrobials may be an alternative to enhance microbial inactivation in fruit juice thermal preservation. The response of L. innocua, surrogate for L. monocytogenes, to combined treatments involving moderate temperatures (57–61 °C) and addition of different levels of vanillin (0–1100 ppm) was assessed to find the most effective inactivation treatment in orange juice. The presence of vanillin greatly increased the bactericidal effect of the mild heat treatment. This effect considerably depended on the amount of added vanillin when working at the lowest temperature (57 °C), while at higher temperatures (60 or 61 °C) the increase in vanillin concentration did not produce a clear change in the response. Nonlinear semilogarithmic survival curves were successfully fitted using a modified version of Gompertz model and by the Weibullian model.     

Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

Solubility of β-cyclodextrin in different mixed solvents

The solubility of β-cyclodextrin (β-CD) in ionic liquid/N, N-dimethyl-acetamide (IL/DMAC) mixed solvent and in LiCl/DMAC mixed solvent was investigated at 323.15 K and 353.15 K. The results showed that the solubility of β-CD in IL/DMAC system increased with increasing temperature, but decreased in LiCl/DMAC system. β-CD had the highest solubility in IL/DMAC system with [Cl]⁻ or [Br]⁻ anions. The solubility of β-CD in LiCl/DMAC system was lower than that in IL/DMAC system with [Cl]⁻ or [Br]⁻ anions but higher than that in IL/DMAC system with weak-coordinated anions (e.g [BF₄]⁻ or [PF₆]⁻). The effects of substituent groups (benzyl and butyl) of cation and the structure of cations on β-cyclodextrin solubility were also investigated.     

A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage

By excluding the effects of organic acids and hydrogen peroxide, strain C2 producing a bacteriocin strongly inhibited Staphylococcus aureus ATCC 63589 and Escherichia coli ATCC 25922 was selected from 300 lactic acid bacteria isolated from traditional Chinese fermented cabbage. Strain C2 was identified as Lactobacillus sakei by phenotypical and physiological tests and 16S rDNA identification. This bacteriocin, which was designated sakacin C2, was purified by cold ethanol and Sephadex G50 column chromatography for further studies. The molecular weight of sakacin C2 was 5.5 kDa by Tris-Tricine SDS–PAGE, which was the largest among all known sakacins. Sakacin C2 exhibited a wide range of antimicrobial activity, strong heat stability (15 min at 121 °C) and pH stability (pH 3.0–8.0). Sakacin C2 was sensitive to protease but insensitive to lipase, α-amylase and β-amylase. These characters suggested that sakacin C2 was a novel Class II bacteriocin with a broad inhibitory spectrum and might broaden the application range of bacteriocins from LAB in the food industry.