Catherine Cailliez-Grimal

Carnobacterium maltaromaticum: identification, isolation tools, ecology and technological aspects in dairy products.

Carnobacterium species constitute a genus of Lactic Acid Bacteria (LAB) present in different ecological niches. The aim of this article is to summarize the knowledge about Carnobacterium maltaromaticum species at different microbiological levels such as taxonomy, isolation and identification, ecology, technological aspects and safety in dairy products. Works published during the last decade concerning C. maltaromaticum have shown that this non-starter LAB (NSLAB) could present major interests in dairy product technology. Four reasons can be mentioned: i) it can grow in milk during the ripening period with no competition with starter LAB, ii) this species synthesizes different flavouring compounds e.g., 3-methylbutanal, iii) it can inhibit the growth of foodborne pathogens as Listeria monocytogenes due to its ability to produce bacteriocins, iv) it has never been reported to be involved in human diseases as no cases of human infection have been directly linked to the consumption of dairy products containing this species.

In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy.

Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3-7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

Bacterial diversity of Darfiyeh, a Lebanese artisanal raw goat's milk cheese.

In order to contribute to the preservation of the Lebanese dairy heritage, the aim of this study was to characterize the Darfiyeh cheese, a traditional variety made from raw goats milk and ripened in goats skin. Three independent batches of Darfiyeh production were analyzed after 20, 40 and 60 days of ripening. Mesophilic lactobacilli, thermophilic coccal-shaped lactic acid bacteria (LAB) and thermophilic lactobacilli were enumerated. In order to explore the Darfiyeh natural ecosystem, a combination of phenotypical and molecular approaches was applied. The latter included Polymerase Chain Reaction-temporal temperature gel electrophoresis (PCR-TTGE), classical PCR and quantitative PCR. These methods revealed the presence of Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus malodoratus, group D Streptococcus sp., Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus haemolyticus, Escherichia coli, Clostridium sp./Eubacterium tenue. Real-time PCR enabled quantification of E. faecium, with a detection of 10(7)-10(9) cfu g(-1) of product. The present molecular approaches combined with phenotypic method allowed describing the complex natural ecosystem of Darfiyeh, giving useful information for the preservation of Lebanese artisanal dairy products.

Lactic acid bacteria in dairy food: surface characterization and interactions with food matrix components.

This review gives an overview of the importance of interactions occurring in dairy matrices between Lactic Acid Bacteria and milk components. Dairy products are important sources of biological active compounds of particular relevance to human health. These compounds include immunoglobulins, whey proteins and peptides, polar lipids, and lactic acid bacteria including probiotics. A better understanding of interactions between bioactive components and their delivery matrix may successfully improve their transport to their target site of action. Pioneering research on probiotic lactic acid bacteria has mainly focused on their host effects. However, very little is known about their interaction with dairy ingredients. Such knowledge could contribute to designing new and more efficient dairy food, and to better understand relationships between milk constituents. The purpose of this review is first to provide an overview of the current knowledge about the biomolecules produced on bacterial surface and the composition of the dairy matter. In order to understand how bacteria interact with dairy molecules, adhesion mechanisms are subsequently reviewed with a special focus on the environmental conditions affecting bacterial adhesion. Methods dedicated to investigate the bacterial surface and to decipher interactions between bacteria and abiotic dairy components are also detailed. Finally, relevant industrial implications of these interactions are presented and discussed.

Interactions between two carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 on target bacteria and Caco-2 cells.

Two purified class IIa carnobacteriocins Cbn BM1 and Cbn B2, from Carnobacterium maltaromaticum CP5, were evaluated for antimicrobial activity against pathogenic, spoilage and lactic acid bacteria. Then, the presence of a synergistic mode of action of these two carnobacteriocins on Listeria sp., Enterococcus sp. and Carnobacterium sp. was investigated. A synergistic mode of action between Cbn BM1 and Cbn B2 on sensitive target bacteria was demonstrated using the FIC index method. Combinations of carnobacteriocins enhanced their antibacterial activities and MICs were significantly reduced, between 2- and 15-fold, by the addition of the second bacteriocin. To improve the safety of the bacteriocins as biopreservative agents, the cytotoxicity of the combination of theses two bacteriocins was determined on Caco-2 cell line. However, these two peptides used alone or in combination, at concentration 100-fold higher than those required for antimicrobial activity, were not cytotoxic. This suggests that the two carnobacteriocins produced by C. maltaromaticum CP5 could be potential natural agents for food preservation.

Synergistic mode of action of mesenterocins 52A and 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52.

Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain. In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both. In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin). In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FICindex < 1), but also on the two strains susceptible to only one mesenterocin. This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action. Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.

Antibacterial activity of class IIa bacteriocin Cbn BM1 depends on the physiological state of the target bacteria.

Carnobacteriocin BM1 (Cbn BM1) is a class IIa bacteriocin produced by Carnobacterium maltaromaticum CP5 isolated from a French mold ripened cheese. Numerous studies highlight variations in numerous parameters, such as bacterial membrane composition and potential, according to physiological changes. In this work, the mechanism of action of an oxidized form of Cbn BM1 was studied on C. maltaromaticum DSM20730 in log and stationary growth phases. Membrane integrity assessment and high resolution imaging by atomic force microscopy confirmed the link between physiological state and bacterial sensitivity to Cbn BM1. Indeed, these approaches enable visualizing morphological damage of C. maltaromaticum DSM20730 only in an active dividing state. To specifically address the interaction between peptide and bacterial membrane, fluorescence anisotropy measurements were conducted. Results revealed strong modifications in membrane fluidity by Cbn BM1 only for C. maltaromaticum DSM20730 in log growth phase. In a similar way, the Δψ component, but not the ΔpH component of the proton-motive force, was perturbed only for bacteria in log growth phase. These results clearly show that a class IIa bacteriocin antimicrobial mechanism of action can be modulated by the physiological state of its target bacteria.

Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28

Carnobacterium maltaromaticum strains are widely found in food including fish, meat and some dairy products. Producing a malty/chocolate like aroma due to 3-methylbutanal from the catabolism of leucine is a general characteristic of this species. In this study, we investigated metabolic routes responsible for the biosynthesis of this flavor compound from the catabolism of leucine in C. maltaromaticum LMA 28, a strain isolated from mold ripened soft cheese. Depending on the lactic acid bacterium, leucine can be converted into 3-methylbutanal following two possible metabolic pathways: either directly by α-ketoacid decarboxylase (KADC) pathway or indirectly by α-ketoacid dehydrogenase (KADH) pathway. Both KADC (41.0±3.0 nmol/mg protein/min) and KADH (1.43±0.62 nmol/mg protein/min) activities were detected and determined in vitro in C. maltaromaticum LMA 28. C. maltaromaticum LMA 28 slightly reduced the production of 3-methylbutanal from leucine in the presence of a specific inhibitor of KADH enzyme complex, i.e. sodium meta-arsenite, suggesting that both pathways were involved in vivo in leucine catabolism. Moreover the presence of genes encoding aminotransferase, glutamate dehydrogenase, α-ketoacid decarboxylase, α-ketoacid dehydrogenase and aldehyde dehydrogenase was confirmed. C. maltaromaticum is then the first lactic acid bacterium in which presence of both metabolic routes responsible for the biosynthesis of 3-methylbutanal from leucine catabolism was confirmed in vitro and in vivo as well.

Complete Chromosome Sequence of Carnobacterium maltaromaticum LMA 28.

ABSTRACT Within the lactic acid bacterium genus Carnobacterium, Carnobacterium maltaromaticum is one of the most frequently isolated species from natural environments and food. It potentially plays a major role in food product biopreservation. We report here on the 3.649-Mb chromosome sequence of C. maltaromaticum LMA 28, which was isolated from ripened soft cheese.

Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli

An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.