No description available.

The proteolytic system of Lactococcus lactis starter cultures influences both flavor and the characteristic body and texture of cheese. The ability to further understand and control how different components of this proteolytic system work together to hydrolyze milk proteins would be of immense importance to the dairy industry. The goal of this research was to characterize Lactococcus lactis subsp. lactis starter bacteria with varying prt operon compositions by proteinase specificity, aminopeptidase and lipase activities, growth, and influence on cheese flavor. By using a cheese slurry system, a statistical model to predict milk protein hydrolysis patterns was developed. Lactococcus lactis subsp. lactis C20 has five plasmids of 55 (pJK550), 48 (pJK480), 43 (pJK430), 3.7 (pJK037), and 2.1 (pJK021) kilo bases. Two of these plasmids (pJK550 and pJK430) are necessary for full proteolytic capability, i.e., clotting milk in 16 h at 20°C. Plasmid pJK550 codes for a proteinase that catalyses the first step in casein degradation. Plasmid pJK430 codes for an oligopeptide transport system, which further transports peptides across the membrane for bacterial metabolism. Strains were constructed containing twelve different combinations of proteolytic phenotypes, such as Lac+PrtP+Opp+, Lac+PrtP+Opp-, Lac+PrtP-Opp+, Lac+Prt-Opp-, Lac-PrtP+Opp+, Lac-PrtP+Opp-, Lac-Prt-Opp+, and Lac-Prt-Opp-. The proteinase specificities of these strains toward milk proteins were dependent on the genotypes present. Genetically all strains showed a P1-type proteinase. Enzymatically C20 had group g proteinase specificity, whereas the rest of the strains containing the proteinase gene showed mixed group specificity. a a-Casein was only slightly hydrolyzed by all strains. B-Casein had a variable pattern, as did mixed casein and milk. K-Casein hydrolysis showed similar degradation patterns in all strains except CB06, which varied in its profile from the other strains. Sensory evaluation showed that culture had a significant effect on rancidity but not on acidity or bitterness. It also showed that the proteolytic system was associated with lipase activity in these strains. A statistical prediction model was developed that allowed strains to be classified according to their amino acid hydrolysis patterns. Mixed casein solution proved to be the best substrate for this analysis. Relationships among strains were seen more easily with canonical analysis and distance tables than by looking only at amino acid hydrolysis patterns.

La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l’absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L’expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l’est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l’expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l’expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodY / The variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon CodY