Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

The long-term survival of Propionibacterium freudenreichii in a context of nutrient shortage.

AIMS: Propionibacterium freudenreichii is an actinobacterium widely used in dairy industry during the ripening process of Swiss-type cheeses and which presents probiotic properties. P. freudenreichii is reportedly a hardy bacterium, able to survive during the cheese-making process and when subjected to digestive stresses. During this study the long-term survival (LTS) of P. freudenreichii was investigated for 11 days by means of phenotypic characterization in a culture medium without the addition of any nutrients. METHODS AND RESULTS: For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. Under these conditions, all strains displayed high survival rates in the culture medium, their culturability reaching more than 9 log10 CFU ml(-1) after 2 days. After 11 days, this value ranged from 7·8 to 8·2 log10 CFU ml(-1) depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. As lysis of part of a bacterial population may be a microbial strategy to recover nutrients, in CIRM-BIA 138 (the strain with the highest population at day 11), cell lysis was assessed by quantifying intact bacterial cells using qPCR targeting the housekeeping gene tuf. No lysis was observed. CONCLUSION: Taken together, our results suggest that P. freudenreichii strains use a viable but nonculturable state to adapt to the LTS phase. SIGNIFICANCE AND IMPACT OF THE STUDY: Assessing the viability of P. freudenreichii and understanding their mechanisms for survival should be of great interest regarding their potential probiotic applications.

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.

Lactobacillus helveticus as a tool to change proteolysis and functionality in Swiss-type cheeses.

Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α(s1)-casein. The aim of this work was to investigate the proteolytic activity of 2L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α(s1)- and α(s2)-caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis.

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.

Fat properties during homogenization, spray-drying, and storage affect the physical properties of dairy powders.

Changes in fat properties were studied before, during, and after the drying process (including during storage) to determine the consequences on powder physical properties. Several methods were combined to characterize changes in fat structure and thermal properties as well as the physical properties of powders. Emulsion droplet size and droplet aggregation depended on the homogenizing pressures and were also affected by spray atomization. Aggregation was usually greater after spray atomization, resulting in greater viscosities. These processes did not have the same consequences on the stability of fat in the powders. The quantification of free fat is a pertinent indicator of fat instability in the powders. Confocal laser scanning microscopy permitted the characterization of the structure of fat in situ in the powders. Powders from unhomogenized emulsions showed greater free fat content. Surface fat was always overrepresented, regardless of the composition and process parameters. Differential scanning calorimetry melting experiments showed that fat was partially crystallized in situ in the powders stored at 20 degrees C, and that it was unstable on a molecular scale. Thermal profiles were also related to the supramolecular structure of fat in the powder particle matrix. Powder physical properties depended on both composition and process conditions. The free fat content seemed to have a greater influence than surface fat on powder physical properties, except for wettability. This study clearly showed that an understanding of fat behavior is essential for controlling and improving the physical properties of fat-filled dairy powders and their overall quality.

Variability of bacterial biofilms of the "tina" wood vats used in the ragusano cheese-making process.

Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.

Altering renneting pH changes microstructure, cell distribution, and lysis of Lactococcus lactis AM2 in cheese made from ultrafiltered milk.

The objective of this study was to investigate the lysis of a highly autolytic strain of Lactococcus lactis ssp. cremoris AM2 in a model cheese made from concentrated ultrafiltered milk. From the same initial ultrafiltered retentate inoculated with L. lactis AM2, 5 cheeses were made by the addition of rennet at different pH values (6.6, 6.2, 5.8, 5.4, and 5.2). Lysis was monitored by measurement of the release of lactate dehydrogenase, an intracellular marker enzyme, and by immunodetection of intracellular proteins with species-specific antibodies. Confocal scanning laser microscopy (CSLM) was used to investigate the cheese microstructure by staining for protein and fat. Dual staining with a bacterial viability kit with CSLM was performed to reveal the integrity and localization of the bacterial cells. Levels of soluble calcium significantly increased when the pH at which the rennet was added decreased. In cheese renneted at pH 6.6, CSLM revealed an open porous structure containing a dense protein network with fat globules of different sizes distributed in the aqueous phase. In cheese renneted at pH 5.2, the protein network was homogeneous, with a less dense protein network, and an even distribution of fat globules. On d 1, bacterial cells were organized into colonies in cheese renneted at pH 6.6, whereas in cheeses renneted at pH 5.2, bacteria were evenly dispersed as single cells throughout the protein network. Lysis was detected on d 1 in cheeses renneted at high pH values and continued to increase throughout ripening, whereas induction of lysis was delayed in cheeses renneted at lower pH values until the end of ripening. This study demonstrates that alterations in the microstructure of the cheese and the distribution of cells play a role in lysis induction of L. lactis AM2.

[Mitral commissurotomy with extracorporeal circulation. Apropos of 200 cases with a median survival of 15 years]. / Commissurotomie mitrale sous CEC. A propos de 200 cas avec un suivi moyen de 15 ans.

This is a retrospective study of 200 patients, with long-term results operated upon from October 1965 to July 1984. 83% women, 17% men. The mean age was 42.40 +/- 11.03 years. In 87% mitral stenosis was pure and 13% systolic murmur was heard. 60% were in classes III and IV and 40% in classes I and II (NYHA). Hemodynamically mean C.W.P. was 21 +/- 6.27 mm Hg and mean P.A. pressure 30 +/- 9.5 mm Hg. Right anterior thoracotomy was done in every case with canulation of femoral artery and V.C. extracorporeal circulation consisted of a bubble oxgenation (RYGG) and a Roller Pump. In 88.5% both commissures were opened and in 11.5% only the anterolateral commissure. In 58% both papillary muscles were incised, in 15% anterolateral and in 13% posteromedial papillary muscles were incised. In 17% valves or commissures were decalcified. In 5.5% a thrombus was removed from the auricle. Postoperative mortality was 0%. 13% of patients experienced a postoperative complication: 4% hemothorax, 2.5% gaz embolism without sequela, 0.5% lower extremity embolism, 3% pulmonary embolism, 2% phlebitis and 1% gastrointestinal haemorrhage. Postoperatively in 75% of cases no murmur was heard, and in 25% a systolic murmur was found over pericardium. 167 patients were assessed at a mean interval of 129.88 months. 11 patients died at a mean interval of 98.56 +/- 48.56 months with non cardiac cause in 9 cases.(ABSTRACT TRUNCATED AT 250 WORDS)