Efficacy of bacteriocin-based formula for reducing staphylococci, streptococci, and total bacterial counts on teat skin of dairy cows.

The use of teat dips is one of the most effective strategies to control mastitis by preventing new intramammary infections. Reducing bacterial load on teat skin helps control the spread of pathogens and spoilage and improves the quality of milk. The objective of this study was to evaluate the reduction of bacterial populations through the application of bacteriocin-based teat formulas. Teats of 12 Holstein cows received 2 different concentrations of bactofencin A, nisin, and reuterin alone or in combination, as well as iodine (positive control) and saline (negative control). Teat swabs were collected before and after application of teat formulas and analyzed for staphylococci, streptococci, and total bacteria counts. There were no differences for staphylococci, streptococci, and total bacterial counts for samples collected before application throughout the entire experiment. Reuterin-low and reuterin-high treatments reduced total bacterial count by 0.47 and 0.36 logs, respectively, whereas bactofencin A had no effect on any tested bacterial groups. Nisin-low treatment reduced staphylococci, streptococci, and total bacterial counts by 0.47, 0.30 and 0.50 logs, respectively. Nisin-high treatment resulted in 0.50, 0.50, and 0.47 log reduction for staphylococci, streptococci, and total bacterial counts. The bacteriocin consortium showed the highest reduction rates with 0.91, 0.54, and 0.90 log reductions obtained for staphylococci, streptococci, and total bacteria counts, respectively, for the low-concentration consortium. Similarly, the high-concentration consortium showed reduction rates with 0.95, 0.60, and 0.82 log reductions obtained for staphylococci, streptococci, and total bacteria counts, respectively. Thus, nisin and the bacteriocin consortium showed the most promise as a teat disinfectant by reducing staphylococci, streptococci, and total bacteria counts.

Pediococcus acidilactici UL5 and Lactococcus lactis ATCC 11454 are able to survive and express their bacteriocin genes under simulated gastrointestinal conditions.

AIMS: The aim of this work is to study the expression of stress genes and those involved in pediocin and nisin production in Pediococcus acidilactici UL5 and Lactococcus lactis ATCC11454 under simulated gastrointestinal (GI) physiological conditions. METHODS AND RESULTS: The two strains were fed to a dynamic GI model (TIM-1). Samples were taken from different compartments and analysed for strain survival as well as for the expression of pediocin PA-1 operon, nisin A production gene and stress genes using RT-qPCR. Ileal-delivered efflux showed a survival rate of 17 and 0·0007% for Ped. acidilactici and La. lactis, respectively. Pediocin operon genes from stressed cells were generally expressed at least at the same level as for unstressed cells. However, pedA is up-regulated in the effluent at 120 and 180 min. Nisin A genes were always up-regulated with particularly in the stomach after 70 min compared with control. CONCLUSIONS: Bacteriocin production of Ped. acidilactici UL5 and Lc. lactis ATCC 11454 are not affected by upper GI simulated conditions and thus could be considered as relevant probiotic candidates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the capacity of lactic acid bacteria to survive and express their bacteriocins genes under simulated GI conditions.

Growth, acid production and bacteriocin production by probiotic candidates under simulated colonic conditions.

AIMS: The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA-1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine. METHODS AND RESULTS: The three bacteriocin-producing strains were grown in Macfarlane broth and in De Man-Rogosa-Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co-culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant. CONCLUSIONS: The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.

Study of the physicochemical and biological stability of pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model.

AIMS: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified pediocin PA-1 under GIT conditions. METHODS AND RESULTS: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 × 10(7) CFU ml(-1) fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce pediocin at the same level as unstressed cells. The activity of the purified pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. CONCLUSIONS: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of pediocin was significantly poor under GIT conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified pediocin PA-1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.

In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells.

AIM: To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form. METHODS AND RESULTS: Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD(50) showed that the fibroblasts were able to tolerate up to 80 microg ml(-1) for 24 h, dropping thereafter to 62 mug ml(-1) after 72 h of contact, compared to 160 microg ml(-1) after 24 h, and 80 microg ml(-1) after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25 microg ml(-1)) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition. CONCLUSION: These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.

Comparison of different application strategies of divergicin M35 for inactivation of Listeria monocytogenes in cold-smoked wild salmon.

Cold-smoked salmon treated with divergicin M35-producing Carnobacterium divergens M35, C. divergens ATCC 35677 (a non-producer of bacteriocin), purified divergicin M35 or supernatants of C. divergens M35 culture in snow crab hepatopancreas (SCH) medium or MRS broth was challenged with Listeria monocytogenes (up to 10(3) CFU/g). Samples were stored at 4 degrees C for up to four weeks. L. monocytogenes, total bacterial and lactic acid bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amine production as well as texture, color and odor. A 2.6 log CFU/g reduction in L. monocytogenes was obtained for up to 10 days of storage in samples treated with C. divergens M35. Purified divergicin M35 (50 microg/g), SCH supernatant or MRS supernatant brought reductions of 1 log CFU/g at the beginning of storage. However, the anti-listerial activity of the supernatants lasted for 15 days compared to 3 days for purified divergicin M35. Color and texture were affected little in samples containing C. divergens M35 compared to un-inoculated samples. TVBN and biogenic amine production, particularly tyramine, remained below the maximum acceptable level in fish appreciation. These results clearly show the potential of C. divergens M35 culture as well as divergicin M35 bio-ingredient for application to the inactivation of L. monocytogenes in ready-to-eat seafood.

Quantitative study of persistence of human norovirus genome in water using TaqMan real-time RT-PCR.

AIMS: To evaluate the persistence of human norovirus (NoV) in different types of water at various temperatures using conventional and TaqMan real-time reverse transcription-PCR (RT-PCR). METHODS AND RESULTS: Water from different sources was spiked with NoV and incubated at different temperatures over a 3-month period. NoV viral RNA was amplified by one-step TaqMan real-time RT-PCR and by conventional two-step RT-PCR. NoV persisted in mineral and tap water for over 2 months at all tested temperatures but disappeared after 100 days. At 4 and -20 degrees C, viral degradation was slower than that at 25 degrees C. In river water and effluent from primary sewage treatment, a slight reduction in viral load was observed after 1 month at 4 degrees C. This is the first demonstration of medium-to-long-term survival of human NoVs in different types of water using TaqMan real-time detection. CONCLUSIONS: NoV genome may persist for long periods of time in different types of water. Quantitative TaqMan real-time RT-PCR is a sensitive system that allows accurate evaluation of the persistence of human NoVs in different water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study is one of the few to demonstrate the ability of NoV to survive for a long time in water.

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form.

AIMS: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. METHODS AND RESULTS: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 microg ml(-1)) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast's transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 microg ml(-1). This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. CONCLUSION: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.

Detection of pediocin PA-1 in food matrices using specific polyclonal antibodies.

Pediocin PA-1 was conjugated with keyhole limpet hemocyanin (KLH) and used to immunize rabbits and mice for the production of polyclonal (PAb) and monoclonal (MAb) antibodies. Titers of PAb and MAb of about 4.7 and 2.9 were obtained after three and six immunizations, respectively. An enzyme linked immunosorbent assay (ELISA) was developed for the detection and quantification of pediocin.

Growth of probiotic bacteria and bifidobacteria in a soy yogurt formulation.

Soy beverage and cows' milk yogurts were produced with Steptococcus thermophilus (ATCC 4356) and Lactobacillus delbrueckii subsp. bulgaricus (IM 025). The drop in pH during fermentation was faster in the soy beverage than in cows' milk, but the final pH values were similar. Yogurts were prepared with a yogurt starter in conjunction with either the probiotic bacteria Lactobacillus johnsonii NCC533 (La-1), Lactobacillus rhamnosus ATCC 53103 (GG) or human derived bifidobacteria. The presence of the probiotic bacteria did not affect the growth of the yogurt strains. Approximately 2 log increases in both L. rhamnosus GG and L. johnsonii La-1 were observed when each was added with the yogurt strains in both cows' milk and the soy beverage. Two of the five bifidobacteria strains grew well in the cows' milk and soy beverage during fermentation with the yogurt bacteria. High pressure liquid chromatography (HPLC) analyses showed that the probiotic bacteria and the bifidobacteria were using different sugars to support their growth, depending on whether the bacteria were growing in cows' milk or soy beverage.

Production and characterization of anti-bifidobacteria monoclonal antibodies and their application in the development of an immuno-culture detection method.

An immuno-culture method has been developed by combination of specific monoclonal antibodies and plate culture to allow detection of viable bifidobacteria. Cell wall proteins were selected as surface antigen to produce antibodies against bifidobacteria. The cell wall proteins were extracted and purified from six ATCC strains of bifidobacteria grown in MRS broth using an anaerobic system. To compare the profile of the protein extracts, all the protein solutions obtained were analyzed by SDS-PAGE. Similar bands corresponding to the major proteins of each species of bifidobacteria were observed. The proteins were tested for their immunogenicity in Balb/c mice after immunization and subsequent analysis using ELISA procedures. High immune responses were generated in mice immunized by proteins from Bifidobacterium bifidum and Bifidobacterium longum. Monoclonal antibodies were produced against B. longum and tested for their specificity, sensitivity and cross reactivity with other bifidobacteria species. All the hybridoma cells selected produced anti-B. longum antibodies cross-reacting with native and purified proteins from five other bifidobacteria species. An epitope supported by a cross-reacting protein of 58 kDa shared by bifidobacteria was revealed by western blot. This was confirmed by immune-transmission electron microscopy observations which showed the specific interaction of these antibodies with bifidobacterial cell wall proteins. Also, the antibody obtained was found to be specific for the genus Bifidobacterium and sensitive, allowing the detection of at least 10(5) target cells/ml. An immuno-culture detection approach was then developed using the selected anti-B. longum antibodies. This method was shown to be very efficient for the detection of viable cells of bifidobacteria suggesting the possibility of its use to quantify these bacteria in various food matrices.

Improvement of texture and structure of reduced-fat Cheddar cheese by exopolysaccharide-producing lactococci.

The objective of this study was to evaluate the effect of capsular and ropy exopolysaccharide (EPS)-producing strains of Lactococcus lactis ssp. cremoris on textural and microstructural attributes during ripening of 50%-reduced-fat Cheddar cheese. Cheeses were manufactured with added capsule- or ropy-forming strains individually or in combination. For comparison, reduced-fat cheese with or without lecithin added at 0.2% (wt/vol) to cheese milk and full-fat cheeses were made using EPS-nonproducing starter, and all cheeses were ripened at 7 degrees C for 6 mo. Exopolysaccharide-producing strains increased cheese moisture retention by 3.6 to 4.8% and cheese yield by 0.28 to 1.19 kg/100 kg compared with control cheese, whereas lecithin-containing cheese retained 1.4% higher moisture and had 0.37 kg/100 kg higher yield over the control cheese. Texture profile analyses for 0-d-old cheeses revealed that cheeses with EPS-producing strains had less firm, springy, and cohesive texture but were more brittle than control cheeses. However, these effects became less pronounced after 6 mo of ripening. Using transmission electron microscopy, fresh and aged cheeses with added EPS-producing strains showed a less compact protein matrix through which larger whey pockets were dispersed compared with control cheese. The numerical analysis of transmission electron microscopy images showed that the area in the cheese matrix occupied by protein was smaller in cheeses with added EPS-producing strains than in control cheese. On the other hand, lecithin had little impact on both cheese texture and microstructure; after 6 mo, cheese containing lecithin showed a texture profile very close to that of control reduced-fat cheese. The protein-occupied area in the cheese matrix did not appear to be significantly affected by lecithin addition. Exopolysaccharide-producing strains could contribute to the modification of cheese texture and microstructure and thus modify the functional properties of reduced-fat Cheddar cheese.

Production and characterization of polyclonal antibodies against cholecalciferol (vitamin D3).

Vitamin D is one of the essential vitamins in the human diet for normal growth and function. In Canada and the USA, fortified milk and milk products are the essential source of vitamin D. The adult recommended nutrient intake of vitamin D is 200 to 400 I.U. (corresponding to 5 to 10 microg) per day. Additional amounts of vitamin D do not confer benefits and may even be toxic. However, a deficiency of this vitamin leads to inadequate absorption of calcium and phosphorus and faulty mineralization of bones and teeth. Actual methods for measuring vitamin D in milk are limited in terms of sensitivity, rapidity and simplicity. The objective of this manuscript was to develop a new molecular strategy for the production, purification and characterization of polyclonal antibodies to vitamin D. Specific antibodies were raised in rabbits against vitamin D using cationized bovine serum albumin (cBSA) as a carrier protein. Anti-vitamin D antibodies were recovered from rabbit sera by sequential affinity chromatographies through Protein A/G Agarose, cBSA Sepharose and cOVA-vitamin D Sepharose columns. Although the yields of anti-vitamin D were relatively low, recovered antibodies showed high specificity and affinity to vitamin D. The purified antibody was used to develop a solid-phase enzyme immunoassay in order to determine the exact concentration of vitamin D in phosphate buffer. Using this immunoassay, approximately 35 ng of vitamin D can be detected within 3 h. The signal obtained was proportional to the amount of vitamin D in the sample analyzed. The strategy developed in this paper appears to be very promising in terms of sensitivity, rapidity and simplicity. It offers a great potential for automation and use on a routine basis for the quantification of vitamin D in fortified milk and other milk products.

A rapid and efficient method of lysis of Listeria and other gram-positive bacteria using mutanolysin.

A rapid and simple procedure is described for cell lysis for preparation of nucleic acids and intact ribosomal RNA from Gram-positive bacteria. Commercial mutanolysin (purified from Streptomyces globisporus) was used for inducing lysis. Listeria, Lactobacillus and Lactococcus strains were very sensitive to mutanolysin when compared to lysozyme. Susceptibility to mutanolysin was improved by a preliminary treatment with acetone, and sodium dodecyl sulfate reduced the efficiency of lysis when used together with mutanolysin. The procedure was also effective for recovering plasmids from these bacteria.

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains.

From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification. The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity. By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification. A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

Continuous production of mixed lactic starters containing probiotics using immobilized cell technology.

The production of a mixed lactic culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707 was studied during a 17-day continuous immobilized-cell culture at different temperatures between 32 and 37 degrees C. The two-stage fermentation system was composed of a first reactor (R1) containing cells of the two strains separately immobilized in kappa-carrageenan/locust bean gum gel beads and a second reactor (R2) operated with free cells released from the first reactor. The system allowed continuous production of a concentrated mixed culture with a strain ratio whose composition depended on temperature and fermentation time. A stable mixed culture (with a 22:1 ratio of L. diacetylactis and B. longum) was produced at 35 degrees C in the effluent of R2, whereas the mixed culture was rapidly unbalanced in favor of B. longum at a higher temperature (37 degrees C) or L. diacetylactis at a lower temperature (32 degrees C). Strain redistribution in beads originally immobilizing pure cultures of L. diacetylactis or B. longum was observed. At the end of culture, the strain ratio (7:1 L. diacetylactis/B. longum) in bulk bead samples was similar to that of individual beads. The determination of the spatial distribution of the two strains in gel beads by immunofluorescence and confocal laser-scanning microscopy showed that bead cross-contamination was limited to a 100 microm peripheral layer. Data from this study validate a previous model for population dynamics and cell release in gel beads during mixed immobilized-cell cultures.

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35.

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.

Effectiveness of commercial disinfectants for inactivating hepatitis A virus on agri-food surfaces.

Six commercial disinfectants were tested for their efficacy in inactivating hepatitis A virus in solution or attached to agri-food surfaces. Disinfectant I contains 10% quaternary ammonium plus 5% glutaraldehyde; disinfectant II contains 12% sodium hypochlorite; disinfectant III contains 2.9% dodecylbenzene sulfonic acid plus 16% phosphoric acid; disinfectant IV contains 10% quaternary ammonium; disinfectant V contains 2% iodide; and disinfectant VI contains 2% stabilized chlorine dioxide. Among these, disinfectants I and II were shown to be the most effective in inactivating hepatitis A virus in solution. The efficacy of these disinfectants was further tested against hepatitis A virus attached to common agri-food surfaces, including polyvinyl chlorine, high-density polyethylene, aluminum, stainless steel, and copper. Disinfectant II was shown to be the most effective, with a maximum inactivation level of about 3 log10. The inactivation efficacy was shown to be affected by the concentration of the active ingredient, the contact time between the disinfectant and the contaminated surfaces, and the incubation temperature. In general, hepatitis A virus was shown to be highly resistant to most disinfectants tested, and high concentrations of active ingredient were needed to achieve acceptable inactivation levels.

Inactivation of foodborne pathogens in milk using dynamic high pressure.

Improving the microbiological safety of perishable foods is currently a major preoccupation in the food industry. The aim of this study was to investigate the inactivation of three major food pathogens (Listeria monocytogenes [LSD 105-1], Escherichia coli O157:H7 [ATCC 35150], and Salmonella enterica serotype Enteritidis ATCC [13047]) by dynamic high pressure (DHP) in order to evaluate its potential as a new alternative for the cold pasteurization of milk. The effectiveness of DHP treatment against L. monocYtogenes, E. coli O157:H7, and Salmonella Enteritidis was first evaluated in 0.01 M phosphate-buffered saline (PBS) at pH 7.2 as a function of applied pressure (100, 200, and 300 MPa) and of the number of passes (1, 3, and 5) at 25 degrees C. A single pass at 100 MPa produced no significant inactivation of the three pathogens, while increasing the pressure up to 300 MPa or the number of passes to five increased inactivation. From an initial count of 8.3 log CFU/ml, complete inactivation of viable L. monocytogenes was achieved after three successive passes at 300 MPa, while 200-MPa treatments with three and five passes completely eliminated viable Salmonella Enteritidis and E. coli O157:H7, respectively. The effectiveness of DHP for the inactivation of these pathogens was compared to that of hydrostatic high pressure (HHP) using the same pressure (200 MPa, single pass at 25 degrees C). In general, two additional log reductions in viable count were obtained with DHP DHP was less effective against L. monocytogenes and E. coli O157:H7 in raw milk than in PBS. After five passes at 200 MPa, an 8.3-log reduction was obtained for E. coli O157:H7, while a reduction of about 5.8 log CFU/ml was obtained for L. monocytogenes exposed to 300 MPa for five passes. Exposing milk or buffer samples to mild heating (45 to 60 degrees C) prior to dynamic pressurization enhanced the lethal effect of DHP The inactivation of pathogens also depended on the initial bacterial concentration. The highest reduction was obtained when the bacterial load did not exceed 10(5) CFU/ml. In conclusion, DHP was shown to be very effective for the destruction of the tested pathogens. It offers a promising alternative for the cold pasteurization of milk and possibly other liquid foods.

Inhibition of Listeria monocytogenes by a combination of chitosan and divergicin M35.

The antimicrobial activities of the class IIa bacteriocin divergicin M35 and several types of chitosan against Listeria monocytogenes were quantified by agar diffusion, critical micro-dilution, and viable count and observed by electron microscopy. Antimicrobial activity of chitosan depended on its molecular mass (MM) and the pH. Three chitosans with MM values of 2, 20, and 100 kDa and 87.4% degree of deacetylation (DDA) were chosen for further study, based on high anti-listerial activity at pH 4.5. Electron microscopy suggested that the mechanism of anti-listerial activity also varied with the MM. Low-MM chitosan appeared to inhibit L. monocytogenes by affecting cell permeability and growth, whereas medium- and high-MM chitosan may form a barrier on the cell surface that prevents entry of nutrients. The minimum inhibitory concentrations (MICs) of 2, 20, and 100 kDa chitosan and divergicin M35 against a divergicin-resistant strain of L. monocytogenes (LSD 535) were 2.5, 2.5, 0.625, and 0.25 mg/mL, respectively. The combination of any of these 3 chitosans and divergicin M35 appeared to have an additive effect against L. monocytogenes, as determined by fractional inhibitory concentration (FIC) index. This study provides useful data for the development of chitosan films incorporating divergicin M35 for inhibiting L. monocytogenes in foods.