In most ripened cheeses, bacteria are responsible for the ripening process. Immobilized in the cheese matrix, they grow as colonies. Therefore, their distribution as well as the distance between them are of major importance for ripening steps since metabolites diffuse within the cheese matrix. No data are available to date about the spatial distribution of bacterial colonies in cheese. This is the first study to model the distribution of bacterial colonies in a food-type matrix using nondestructive techniques. We compared (i) the mean theoretical three-dimensional (3D) distances between colonies calculated on the basis of inoculation levels and considering colony distribution to be random and (ii) experimental measurements using confocal microscopy photographs of fluorescent colonies of a Lactococcus lactis strain producing green fluorescent protein (GFP) inoculated, at different levels, into a model cheese made by ultrafiltration (UF). Enumerations showed that the final numbers of cells were identical whatever the inoculation level (10 4 to 10 7 CFU/g). Bacterial colonies were shown to be randomly distributed, fitting Poisson's model. The initial inoculation level strongly influenced the mean distances between colonies (from 25 m to 250 m) and also their mean diameters. The lower the inoculation level, the larger the colonies were and the further away from each other. Multiplying the inoculation level by 50 multiplied the interfacial area of exchange with the cheese matrix by 7 for the same cell biomass. We finally suggested that final cell numbers should be discussed together with inoculation levels to take into account the distribution and, consequently, the interfacial area of colonies, which can have a significant influence on the cheese-ripening process on a microscopic scale.During cheese making, regardless of the cheese type, bacteria are immobilized in the curd during the coagulation step. It is generally accepted that 90% of the bacteria present in the milk are retained, trapped in the curd, while only 10% are lost in the whey during draining (16). In cheeses made by ultrafiltration (UF), the draining step is absent, and 100% of the cells are then retained in the curd. In any case, after immobilization by coagulation, each inoculated bacterial cell is assumed to grow, generating a colony inside the curd. Colonies are then spread within the cheese curd, and they interact with the cheese matrix during ripening. Consequently, the ripening process must take place on a microscopic scale around colonies. Only studies showing microscopic examinations of bacterial colonies in cheese either by electronic microscopy (24) or, more recently, by confocal laser scanning microscopy (7, 19) have been reported.The ripening process (proteolysis, lipolysis, amino acid catabolism, and the production of organic acids, etc.) relies on the metabolic activities of bacterial colonies, leading to the formation of flavors and textures of cheese (11,25). So far, ripening has always been described with average processes on the che...

Bacteria, either indigenous or added, are immobilized in solid foods where they grow as colonies. Since the 80's, relatively few research groups have explored the implications of bacteria growing as colonies and mostly focused on pathogens in large colonies on agar/gelatine media. It is only recently that high resolution imaging techniques and biophysical characterization techniques increased the understanding of the growth of bacterial colonies, for different sizes of colonies, at the microscopic level and even down to the molecular level. This review covers the studies on bacterial colony growth in agar or gelatine media mimicking the food environment and in model cheese. The following conclusions have been brought to light. Firstly, under unfavorable conditions, mimicking food conditions, the immobilization of bacteria always constrains their growth in comparison with planktonic growth and increases the sensibility of bacteria to environmental stresses. Secondly, the spatial distribution describes both the distance between colonies and the size of the colonies as a function of the initial level of population. By studying the literature, we concluded that there systematically exists a threshold that distinguishes micro-colonies (radius < 100–200 μm) from macro-colonies (radius >200 μm). Micro-colonies growth resembles planktonic growth and no pH microgradients could be observed. Macro-colonies growth is slower than planktonic growth and pH microgradients could be observed in and around them due to diffusion limitations which occur around, but also inside the macro-colonies. Diffusion limitations of milk proteins have been demonstrated in a model cheese around and in the bacterial colonies. In conclusion, the impact of immobilization is predominant for macro-colonies in comparison with micro-colonies. However, the interaction between the colonies and the food matrix itself remains to be further investigated at the microscopic scale.

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic-and oxidativestress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions. Lactic acid bacteria, particularly Lactococcus lactis, have a long history of use in milk fermentation, from small-scale traditional operations to well-controlled industrial applications. Recent developments of molecular tools have unraveled the genetics, physiology, and metabolism of this economically very important microorganism. However, interpretation has always been limited by the lack of knowledge regarding in situ bacterial physiology. Technological properties (e.g., acidification, proteolytic or lipolytic activity, and bacteriocin production) can easily be shown and quantified in vitro but have hardly ever been verified in a complex solid matrix, due to local intrinsic factors. There have been several successful attempts to measure the DNA and rRNA extracted directly from a solid food (or environmental) matrix in order to estimate either the predominant species and/or the overall level of metabolic activity of the species (11,33). In a few cases, the expression of genes of technological interest from extracted mRNAs has been checked (42). The global gene or protein expression of L. lactis has been characterized in milk (15, 34) but not directly in cheese, the corresponding solid dairy matrix.Over the last few years, functional-genomics approaches, including transcriptomics, have been increasingly used to obtain global gene expression profiles, thereby providing a comprehensive view of microorganism physiology. So far, such global approaches in food microbiology and in situ have been poorly documented. Recently, Bachmann et al. presented the genetic responses of L. lactis in mixed cu...

-The acidification and reduction activities of lactic starters have been followed by continuous measurement of the pH and the Eh during the course of milk fermentation. These measurements allowed the calculation of the maximum acidification and reduction rates, and the time, pH and Eh at which these values occurred. Nine strains of Lactococcus sp., 6 strains of Streptococcus thermophilus and 5 strains of Lactobacillus helveticus were studied. In general, the maximum reduction rate of the lactococci was six-fold higher than those of the streptococci and lactobacilli. On the other hand, the streptococci and the lactobacilli acidified with a higher maximum acidifying rate than those of lactococci. Consequently, it was observed that all the cultures with lactococci reached their final Eh before the end of the lactic acid fermentation, while acidifications with the streptococci or the lactobacilli finished before the end of the reduction phase. A principal components analysis clearly differentiates the three species on the basis of their aptitudes for acidification and reduction. This new approach might be used to select adequate starters for the manufacture of fermented dairy products. terminait avant la fin de la phase de réduction. L'analyse en composante principale différencie clairement les 3 espèces sur la base de leurs aptitudes acidifiantes et réductrices. Ces nouveaux paramètres pourraient être associés à ceux classiquement utilisés pour sélectionner les levains les plus adéquats pour la fabrication de produits laitiers fermentés. Acidification / réduction / Lactococcus / Streptococcus thermophilus / Lactobacillus helveticus

The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, D-lactate dehydrogenase and a-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

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Predictive models are mathematical expressions that describe the growth, survival, inactivation, or biochemical processes of foodborne bacteria. During processing of contaminated raw materials and food preparation, bacteria are entrapped into the food residues, potentially transferred to the equipment surfaces (abiotic or inert surfaces) or cross-contaminate other foods (biotic surfaces). Growth of bacterial cells can either occur planktonically in liquid or immobilized as colonies. Colonies are on the surface or confined in the interior (submerged colonies) of structured foods. For low initial levels of bacterial population leading to large colonies, the immobilized growth differs from planktonic growth due to physical constrains and to diffusion limitations within the structured foods. Indeed, cells in colonies experience substrate starvation and/or stresses from the accumulation of toxic metabolites such as lactic acid. Furthermore, the micro-architecture of foods also influences the rate and extent of growth. The micro-architecture is determined by (i) the non-aqueous phase with the distribution and size of oil particles and the pore size of the network when proteins or gelling agent are solidified, and by (ii) the available aqueous phase within which bacteria may swarm or swim. As a consequence, the micro-environment of bacterial cells when they grow in colonies might greatly differs from that when they grow planktonically. The broth-based data used for modeling (lag time and generation time, the growth rate, and population level) are poorly transferable to solid foods. It may lead to an over-estimation or under-estimation of the predicted population compared to the observed population in food. If the growth prediction concerns pathogen bacteria, it is a major importance for the safety of foods to improve the knowledge on immobilized growth. In this review, the different types of models are presented taking into account the stochastic behavior of single cells in the growth of a bacterial population. Finally, the recent advances in the rules controlling different modes of growth, as well as the methodological approaches for monitoring and modeling such growth are detailed.

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