Mathieu Millette

Modified alginate matrices for the immobilization of bioactive agents

Bioactive agents (catalase – an enzyme, and nisin – a bacteriocin) were covalently immobilized on alginate activated with sodium periodate (oxidatively converting 2,3‐dihydroxy groups into dialdehyde residues), followed or preceded by ionotropic gelation. For the same protein coupling yield, the retained enzyme activity of the immobilized enzyme (ImE) can be markedly increased by diminishing the bead diameter, a phenomenon that illustrates the role of substrate/product diffusion through the bead gel layer. When the amount of enzyme introduced for coupling was about 15 mg/100 mg of support and the bead diameter was about 100 μm, a high retained specific activity (95–98%) was obtained. Diffusion phenomena can be markedly decreased by enzyme immobilization on the surface of microbeads (obtained by gelation of activated alginate prior to immobilization). In this case, the retained activity was approx. 75% of that of the free enzyme. A slightly higher Km value of ImE suggested that the enzyme–substrate affinity was almost maintained. The profiles of ImE activities at various pH values, at various temperatures and when undergoing proteolysis showed a overall higher stability for the immbolized than that for the free enzyme. Nisin immobilized on the microbead surface, when submitted to proteolysis, conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus sake when subjected to an agar spot test, whereas free nisin totally lost its activity.

Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci

ABSTRACT This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model. Lactococcus lactis MM19 and Pediococcus acidilactici MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by P. acidilactici is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that L. lactis harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of L. lactis MM19, P. acidilactici MM33, P. acidilactici MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that L. lactis and P. acidilactici MM33A increased the concentrations of total LAB and anaerobes while P. acidilactici MM33 decreased the Enterobacteriaceae populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In L. lactis-fed mice, fecal VRE levels 1.73 and 2.50 log10 CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the P. acidilactici MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log10 CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human L. lactis and P. acidilactici nisin- and pediocin-producing strains can reduce VRE intestinal colonization.

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

Aims:  Food‐borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions.

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

Aims:  The aim of this study was to isolate bacteriocin‐producing lactic acid bacteria (LAB) from human intestine.

Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW-9595M increase IL-10 production by macrophages

Aims:  To evaluate the immunosuppressive properties of the exopolysaccharide (EPS) from high‐EPS producer Lactobacillus rhamnosus RW‐9595M on inflammatory cytokines produced by macrophages.

Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis.

To assess the potential of Lactobacillus acidophilus and Lactobacillus casei strains to increase the apoptosis of a colorectal cancer cell line in the presence of 5-fluorouracil (5-FU), LS513 colorectal cancer cells were treated for 48 h with increasing concentrations of these lactic acid bacteria (LAB) in the presence of 100 μ g/ml of 5-FU. In the presence of 108 CFU/ml of live LAB, the apoptotic efficacy of the 5-FU increased by 40%, and the phenomenon was dose dependent. Moreover, irradiation-inactivated LAB caused the same level of induction, whereas microwave-inactivated LAB reduced the apoptotic capacity of the 5-FU. When cells were treated with a combination of live LAB and 5-FU, a faster activation of caspase-3 protein was observed, and the p21 protein seems to be downregulated. These results suggest that live L. acidophilus and L. casei are able to increase the apoptosis-induction capacity of 5-FU. The mechanisms of action are still not elucidated, and more research is needed to understand them. This is the first set of experiments demonstrating that some strains of LAB can enhance the apoptosis-induction capacity of the 5-FU. Based on these results, it is possible to speculate that LAB or probiotics could be used as an adjuvant treatment during anticancer chemotherapy.

Combined Effects of Antimicrobial Coating, Modified Atmosphere Packaging, and Gamma Irradiation on Listeria innocua Present in Ready-to-Use Carrots (Daucus carota)

The objective of this study was to evaluate the effect of an edible antimicrobial coating combined with modified atmosphere (MA) packaging (60% O2, 30% CO2, and 10% N2) and gamma irradiation on peeled minicarrots inoculated with Listeria innocua. Carrots were inoculated with L. innocua (10(3) CFU/g) and then coated with an antimicrobial coating based on calcium caseinate containing trans-cinnamaldehyde. The same formulation without trans-cinnamaldehyde was used as an inactive coating. Coated and uncoated carrots were packed under the MA or under air, irradiated at 0.25 or 0.5 kGy, and stored at 4 +/- 1 degrees C for 21 days. Samples were evaluated periodically for enumeration of L. innocua. Unirradiated carrots stored under air had the highest concentrations of L. innocua after 21 days of storage: 2.23 CFU/g in the uncoated samples and 2.26 CFU/ g in samples coated with the inactive coating. These results suggest that the inactive coating did not have any antimicrobial effect against L. innocua. However, the addition of the antimicrobial coating resulted in a 1.29-log reduction in the concentration of L. innocua in carrots packed under air after 21 days of storage and a 1.08-log reduction in carrots packed under MA after 7 days of storage. After 7 days of storage, no L. innocua was detected in samples treated at 0.5 kGy under air or in samples treated at 0.25 kGy under MA. A complete inhibition of L. innocua was also observed during all storage periods in uncoated and coated samples treated at 0.5 kGy under MA. These results indicate that the combination of irradiation and MA conditions play an important role in the radiosensitization of L. innocua.

Effect of Selected Plant Essential Oils or Their Constituents and Modified Atmosphere Packaging on the Radiosensitivity of Escherichia coli O157:H7 and Salmonella Typhi in Ground Beef

Twenty-six different essential oils were tested for their efficiency to increase the relative radiosensitivity of Escherichia coli and Salmonella Typhi in medium-fat ground beef (23% fat). Ground beef was inoculated with E. coli O157:H7 or Salmonella (10(6) CFU/g), and each essential oil or one of their main constituents was added separately at a concentration of 0.5% (wt/wt). Meat samples (10 g) were packed under air or under modified atmosphere and irradiated at doses from 0 to 1 kGy for the determination of the D10-value of E. coli O157:H7, and from 0 to 1.75 kGy for the determination of the D10-value of Salmonella Typhi. Depending on the compound tested, the relative radiation sensitivity increased from 1 to 3.57 for E. coli O157:H7 and from 1 to 3.26 for Salmonella Typhi. Addition of essential oils or their constituents before irradiation also reduced the irradiation dose needed to eliminate both pathogens. In the presence of Chinese cinnamon or Spanish oregano essential oils, the minimum doses required to eliminate the bacteria were reduced from 1.2 to 0.35 and from 1.4 to 0.5 for E. coli O157:H7 and Salmonella Typhi, respectively. Cinnamon, oregano, and mustard essential oils were the most effective radiosensitizers.

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.

Influence of Antimicrobial Compounds and Modified Atmosphere Packaging on Radiation Sensitivity of Listeria monocytogenes Present in Ready-to-Use Carrots (Daucus carota)

Radiosensitization of Listeria monocytogenes was determined in the presence of trans-cinnamaldehyde, Spanish oregano, winter savory, and Chinese cinnamon on peeled minicarrots packed under air or under a modified atmosphere (60% O2, 30% CO2, and 10% N2). Samples were inoculated with L. monocytogenes HPB 2812 serovar 1/2a (106 CFU/g) and were coated separately with each active compound (0.5%, wt/wt) before being packaged under air or the modified atmosphere and irradiated at doses from 0.07 to 2.4 kGy. Results indicated that the bacterium was more resistant to irradiation under air in the absence of active compound. The dose required to reduce L. monocytogenes population by 1 log CFU (D10) was 0.36 kGy for samples packed under air and 0.17 kGy for those packed under the modified atmosphere. The active compounds evaluated in this study had an effect on the radiation sensitivity of L. monocytogenes on carrots. The most efficient compound was trans-cinnamaldehyde, where a mean 3.8-fold increase in relative radiation sensitivity was observed for both atmospheres compared with the control. The addition of winter savory and Chinese cinnamon produced a similar increase in relative radiation sensitivity but only when samples where packed under modified atmosphere conditions.