Transport phenomena in a model cheese: the influence of the charge and shape of solutes on diffusion.

During cheese ripening, microorganisms grow as immobilized colonies, metabolizing substrates present in the matrix and generating products from enzymatic reactions. Local factors that limit the rates of diffusion, either within the general cheese matrix or near the colonies, may influence the metabolic activity of the bacteria during ripening, affecting the final quality of the cheese. The objective of this study was to determine the diffusion coefficients of solutes as a function of their different physicochemical characteristics (size, charge, and shape) in an ultrafiltrate (UF) model cheese (based on ultrafiltered milk) to enable better understanding of the ripening mechanisms. Diffusion coefficients of fluorescein isothiocyanate (FITC)-dextrans (4 kDa to 2 MDa) and FITC-labeled dairy proteins (α-lactalbumin, ß-lactoglobulin, and BSA) were measured using the technique of fluorescence recovery after photobleaching (FRAP). This study showed that macromolecules up to 2 MDa and proteins could diffuse through the UF model cheese. The larger FITC-dextrans were not more hindered by the structure of the UF model cheese compared with the smaller ones. Any decrease in the diffusion coefficients of solutes was related only to their hydrodynamic radii. The FITC-dextran diffusion data were fitted to an obstruction model, resulting in a constant obstruction factor (k ~0.42). Diffusion in the model cheese was sensitive to the physicochemical characteristics of the solute. The FITC-dairy proteins studied (rigid and negatively charged molecules) were hindered to a greater degree than the FITC-dextrans (flexible and charge-neutral molecules) in the UF model cheese. The existence of steric and electrostatic interactions between the protein matrix of the UF model cheese and the FITC-dairy proteins could explain the decrease in diffusion compared with FITC-dextrans.

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.

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.

Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus Strains improves breakdown of the pure alphas1-casein.

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and ß-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure ß-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

prtH2, not prtH, is the ubiquitous cell wall proteinase gene in Lactobacillus helveticus.

Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied, whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening.

Invited review: Proteomics of milk and bacteria used in fermented dairy products: from qualitative to quantitative advances.

Proteomics is a powerful tool that can simultaneously analyze several hundred proteins in complex mixtures, either through the use of high-resolution 2-dimensional gel electrophoresis or by mono- and multi-dimensional liquid chromatography coupled with mass spectrometry. Since the last review in 2005, proteomics has mainly been applied to describe minor proteins in the bovine milk fat globule membrane and soluble proteins in human colostrum. At least 130 new minor proteins have been identified. These proteins play roles in cell signaling, host defense, and transport as suggested by sequence homology. Proteomic approaches have also been applied to milk of other species such as donkey, horse, and marsupial. Peptides produced in food matrices that can exhibit functional or bioactive properties have been identified as have the proteases leading to their release in situ. However, the most spectacular proteomic development has been in the field of bacteria used in dairy products. Proteomics has resulted in the establishment of reference maps to detect strain-to-strain variations and to elucidate the mechanisms of in vitro and in vivo adaptation to environmental conditions. Proteomic analysis of bacteria entrapped in cheese has been achieved and revealed which predominant metabolic pathways are active depending on the strain. Proteomic approaches are often evoked as time-consuming procedures that provide a list of identified proteins without efficient quantification of each one. New quantitative proteomic methods have emerged and the most promising ones and their application to dairy products and bacteria will be presented.

RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis.

AIMS: The isolation of high-quality RNA from cheese is a prerequisite for analysis of in situ gene expression of dairy micro-organisms. METHODS AND RESULTS: A method for rapid isolation of bacterial cells from cheese using cold citrate buffer followed by mechanical cell disruption was developed. RNA was extracted from experimental ultrafiltration (UF) cheeses (at 2, 8, 24 h, 7 and 14 days) and from Cheddar cheese (from 1 day to 1 year). The quantity and quality of the extracted RNA was assessed. The transcript abundance of seven genes (tuf, gapB, purM, cysK, ldh, cit and gyrA) was estimated by reverse transcription real-time PCR. In UF cheeses, the quantity of RNA extracted increased from 0.2 to 24 microg g(-1), with an RNA Integrity Number (RIN) above 9. In the experimental Cheddar cheeses, the RNA extraction yield decreased from 67.7 microg g(-1) after 1 day to 23.7 microg g(-1) after 6 months, with RIN value above 9 during the first month. The transcript abundance of the seven genes demonstrated metabolic activity of lactococci after several weeks of ripening in both cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described produced large quantities of high-quality RNA for future whole genome expression studies in cheese.

Kluyveromyces lactis but not Pichia fermentans used as adjunct culture modifies the olfactory profiles of Cantalet cheese.

Yeasts are commonly detected in cheese. Two yeast species, Kluyveromyces lactis and Pichia fermentans, were isolated at high populations from raw-milk Cantalet cheese, a French Protected Denomination of Origin hard cheese. To investigate the interest of these 2 species as adjunct cultures to promote flavor development of Cantalet cheese, they were added at 10(5) cfu/mL to microfiltered milk. The global microbiological, biochemical, and flavor changes induced by the presence of the yeasts in cheese were determined. Adjunct yeasts were present at 10(6) cfu/g in curd, declined to 10(4) to 10(5) cfu/g in cheese, and did not influence gross composition, content of free amino acids, or content of free fatty acids. By using 8-way gas chromatography-olfactometry in parallel with gas chromatography-mass spectrometry, 30 odorous compounds of Cantalet cheese were identified. The olfactory profiles of K. lactis cheeses contained significantly greater levels of 8 odorous compounds (ethanol, ethyl hexanoate, 4 aldehydes, and 2 branched-chain acids) compared with the control and P. fermentans cheeses. Sensory analysis of cheeses flavor discriminated K. lactis cheeses on only 2 attributes (acetaldehyde and alcohol odors). This study shows that yeast contribution is species-specific and that K. lactis, at a population of 10(6) viable cells/g, can influence Cantalet cheese flavor.

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.

Efficient mechanical disruption of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity.

Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess the activity of an intracellular aminotransferase. Varied pressures (50, 100 and 200 MPa) were applied to concentrated bacterial suspensions (1.2 mg dry weight/ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650 nm, cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50 MPa was sufficient to break 80% of lactobacilli cells whereas 200 MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20 s/run), easy, continuous and highly efficient cell breaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogenetically related, and had different morphologies and cell wall compositions, we conclude that most Gram-positive bacteria may be broken efficiently by this new device.

Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus.

AIMS: Lactobacillus helveticus is an essential starter in Swiss-type cheeses such as Emmental. This study was to determine whether cell-free extracts of Lact. helveticus were able to convert free amino acids into neutral volatile aroma compounds at the pH and temperature occurring in cheese. METHODS AND RESULTS: A mix of branched-chain (Leu, Ile, Val), aromatic (Tyr, Phe) and sulphur (Met) amino acids was incubated for 7 days, at pH 5.7 and 24 degrees C, with cell-free extracts of six strains. The amino acids were all transaminated into the corresponding keto acids when an amino group acceptor (alpha-ketoglutaric acid) was provided. Phe and Tyr were transaminated the most efficiently, followed by Leu, Met, Ile and Val. Three major volatile compounds were detected by GC-MS: benzaldehyde, dimethyl disulphide and 2-methyl propanol. Whatever the strain, benzaldehyde was produced in the highest quantity (0.25-1 micromol l(-1) mg(-1) protein). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus intracellular enzymes could significantly contribute to the production of aroma compounds from amino acid catabolism.

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides.

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.

Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains.

Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis. Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb. helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro. All the cheeses were biochemically identical after pressing. The viability of Lb. helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77. Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain. Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room. Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77. As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb. helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.

Electrophoretic pattern of peptidoglycan hydrolases, a new tool for bacterial species identification: application to 10 Lactobacillus species.

Lactobacilli have been used as industrial starters for a long time, but in many cases their phenotypic identification is still neither easy nor reliable. Previously we observed that the cell wall peptidoglycan hydrolases of Lactobacillus helveticus were highly conserved enzymes; the aim of the present work was to determine whether peptidoglycan hydrolase patterns obtained by renaturing SDS-PAGE could be of interest in the identification of lactobacilli species. For that purpose, the peptidoglycan hydrolase patterns of 94 strains of lactobacilli belonging to 10 different species were determined; most of the species studied are used either in dairy, meat, bakery or vegetable fermentations: L. helveticus, L. acidophilus, L. delbrueckii, L. brevis, L. fermentum, L. jensenii, L. plantarum, L. sake, L. curvatus and L. reuteri. Within a species, the strains exhibited highly similar patterns: the apparent molecular weights of the lytic bands were identical, with only slight variations of intensity. Moreover, each species, including phylogenetically close species such as L. sake and L. curvatus, or L. acidophilus and L. helveticus, gave a different pattern. Interestingly, the closer the species were phylogenetically, the more related were their patterns. The sensitivity of the method was checked using various quantities of L. acidophilus cells: a peptidoglycan hydrolase extract of 5 x 10(6) cells was sufficient to obtain an informative pattern, as was a single colony. Finally, the method was also successfully applied to distinguish two Carnobacterium species. In conclusion, the electrophoretic pattern of peptidoglycan hydrolases is proposed as a new tool for lactobacilli identification: it is rapid, sensitive and effective even for phylogenetically close species. Furthermore, this work provides the first evidence of the potential overall taxonomic value of bacterial peptidoglycan hydrolases.

Biochemistry of S-layers.

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.

Lactobacillus helveticus: strain typing and genome size estimation by pulsed field gel electrophoresis.

Genomic DNAs of 22 strains of Lactobacillus helveticus of various geographical origins were analyzed by pulsed-field gel electrophoresis. Two endonucleases, SmaI and SgrAI, of the 19 tested produced DNA fragments useful for strain comparison. With the endonuclease SmaI, a characteristic restriction pattern was identified for 18 of the 22 strains. The percentage of similarity (Dice coefficient) between the profiles varied between 26% and 100%, and clustering was accomplished by using the unweighted pair group method with arithmetic averages (UPGMA). For the strains showing identical profiles,the high genomic similarity was confirmed when the endonuclease SgrAI was used instead of SmaI. From summation of SmaI and SgrAI fragments from three L. helveticus strains(CNRZ 241, CNRZ 303, and CIP 57.15), the genomic length was estimated at ca.1. 85-2.0 Mb.