First mass spectrometry metabolic fingerprinting of bacterial metabolism in a model cheese.

Metabolic fingerprinting is an untargeted approach which has not yet been undertaken to investigate cheese. This study is a proof of concept, concerning the ability of mass spectrometry (MS) metabolic fingerprinting to investigate modifications induced by bacterial metabolism in cheese over time. An ultrafiltrated milk concentrate was used to manufacture model cheeses inoculated with Lactococcus lactis LD61. Metabolic fingerprints were acquired after 0, 8 and 48h from two different fractions of the metabolome: the water-soluble fraction using liquid chromatography-high resolution-MS and a volatile fraction using gas chromatography-MS. Metabolic fingerprints differed significantly over time. Forty-five metabolites were identified, including well-known cheese metabolites, such as 12 amino acids and 25 volatile metabolites, and less studied ones, such as four vitamins, uric acid, creatine and l-carnitine. These results showed the relevance of cheese MS fingerprinting to generate new findings and to detect even slight differences between two conditions.

Porosity of Lactococcus lactis subsp. lactis LD61 colonies immobilised in model cheese.

During cheese ripening, micro-organisms grow as immobilised colonies, metabolising substrates present in the matrix which generate products triggered by enzymatic reactions. Local limitation rates of diffusion, either in the matrix or within the bacterial colonies, can be responsible for modulation in the metabolic and enzymatic activities of micro-organisms during ripening. How bacterial colonies immobilised in cheese are porous to these diffusing solutes has never been explored. The objective of this study was to determine if fluorescent dextrans of different sizes (4.4, 70 and 155 kDa) are able to penetrate through colonies of Lactococcus lactis LD61 immobilised in solid media, either agar or model cheese. Confocal microscopic observations showed that lactococcus colonies immobilised in these two media were porous to dextrans from 4 kDa to 155 kDa. However, the rate of diffusion of the solutes was faster inside the colonies immobilised in ultrafiltered-cheese than in agar when large dextrans were considered (≥70 kDa). The colonial shape of the lactococcus strain was also shown to be lenticular in agar and spherical in the model cheese, indicating that the physical pressure exerted on the colony by the surrounding casein network was probably isotropous in the UF-cheese but not in agar. In both cases, the fact that lactococcus colonies immobilised in solid media are porous to large dextran solutes suggests that substrates or enzymes are likely also to be able to migrate inside the colonies during cheese ripening.

First assessment of diffusion coefficients in model cheese by fluorescence recovery after photobleaching (FRAP).

Mass transfer of solutes like salt, moisture and metabolites, is very important for the final quality of cheese, through the control of the brining and ripening processes. Numerous studies have reported salt and water transfer properties in cheese, but few have dealt with other solutes. Moreover, most diffusion coefficients have been obtained by macroscopic and destructive methods. We developed the fluorescence recovery after photobleaching (FRAP) technique on a confocal microscope to measure in situ and at the microscopic scale diffusion properties inside cheese. A model matrix based on ultrafiltrated milk was used. FITC-dextran molecules were chosen as models of migrant solutes. Diffusion coefficients were estimated with a modelling approach which takes into account diffusion during the bleach phase. The FITC-dextrans (4 and 20 kDa) were able to migrate in the proteinic network, but their mobility was 2.2-3 times lower than in water, depending on their size.

Spatial distribution of bacterial colonies in a model cheese.

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 µm to 250 µm) 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.

Characterization of the heat treatment undergone by milk using two inhibition ELISAs for quantification of native and heat denatured alpha-lactalbumin.

Dairy industries are interested to know the heat treatment undergone by milk for controlling the quality of drinking milks or to control their heating systems. The purpose of this work was to develop a specific and sensitive technique for classification of the heat treatment a milk has been submitted to, without disposing of the original raw milk. For this purpose, alpha-lactalbumin was chosen as a bioindicator of heat treatment, and monoclonal antibodies specific for its native or heat-denatured form were raised and used in two inhibition ELISAs. ELISA allowed differentiation among raw, pasteurized, ultrahigh-temperature-treated, and sterilized milks without even having to know the alpha-lactalbumin concentration of the original raw milk. However, this technique was more suitable for intense heat treatments such as UHT treatment and sterilization because of the heat stability of alpha-lactalbumin.

[Prevalence of latex allergy among personnel at a hospital]. / Prévalence de l'allergie au latex parmi le personnel d'un hôpital.

The aim of this study was to compile a register of all the employees of a University Hospital Centre who complained of dermatosis of the hands due to latex. Recruitment was made by their spontaneous presentation in the service of the Workers Doctor (Médecine de Travail). The prevalence of clinical signs of contact eczema or professional urticaria is 2.3% for all personnel. 2.7% for nurses, 4.4% for care assistants or ancillary staff of the hospital service. In this population, 73% of subjects have shown previous atopy. The positive predictive value of the allergy tests was 51.3%, 2.32% of ASH-ASI, 1.75% of AS, 1.47% of IDE were allergic to latex, about 1.06% of the total personnel. 80% had atopy and 36.66% had a crossed allergy (banana, kiwi, avocat, pollen). Prevalence was zero amongst the administrative officers, but not systematic enquiry was made in the professional category. The orthoergical dermatoses were more frequent amongst the ASH-ASI, though the positive predictive value of tests was less. The considerable exposure to a number of caustic substances, as well as absence of precautions such as rinsing and drying of hands may explain this. All employees with allergy to latex have been declared to have a professional illness a card that mentions this allergy has been given to them.

Ontogeny of taurocholate accumulation in the terminal ileal mucosal cells of young chicks.

The everted, ileal ring technique of Little and Lester was utilized to study taurocholate accumulation against a concentration gradient in the terminal ileal mucosal cells of chicks from hatch to 6 wk of age. The chicks had low levels of taurocholate accumulation at hatch. A three- to fourfold increase in accumulation was observed at Day 3 posthatch, but taurocholate returned to baseline levels within 2 days. This peak has not been previously reported in any other species. The age-related peak in the ileal accumulation of taurocholate was not influenced by removal of the yolk sac at hatch, oral feeding of yolk, or supplementation of the diet with taurocholate or cholestyramine. Comparison of ileal uptake of proline versus uptake of taurocholate during the 1st wk posthatch indicated that the peak in ileal taurocholate accumulation is not the result of a general increase in absorptive capacity through cell proliferation. These data suggest that the age-related changes in taurocholate absorption are genetically determined and are not responsive to external influences.