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...