To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Despite substantial research, it is still difficult to produce high quality reducedfat Cheddar cheese. The objective of this study was to investigate how two coagulants, bovine chymosin (BC) and camel chymosin (CC) having different proteolytic activities and two starter cultures, an O-culture (O) and a thermophilic strain of Lactobacillus plus O-culture (OLb) having different abilities to release amino acids, contribute to the structure and flavour development in reduced-fat Cheddar cheese. Cheeses manufactured using the four combinations of coagulants and cultures were analysed during a 28-week ripening period for the composition of casein, peptides, free amino acids, rheological properties and for sensory properties at end of ripening. Cheeses with CC, showed less extensive primary proteolysis, lower levels of bitterness and higher stress at fracture, which correlates to a harder structure. Whereas cheeses with BC had a higher amount of peptides released by chymosin, e.g. the bitter peptide β-casein (f193-209) or by starter proteases from the chymosin-produced peptide α s1-CN (f1-23). BC cheeses were also judged to be softer by the sensory panel. Cheeses containing the OLb-culture had a higher amount of free amino acids and lower strain at fracture, which correlates to a shorter structure, and the peptide profiles of cheeses produced with BC and CC were rather similar after 28 weeks in contradiction to cheeses with O-culture. Replacing the traditional coagulant BC with CC reduced bitterness but increased hardness of the reduced-fat Cheddar cheese. Replacing O-with OLb-culture also reduced bitterness but resulted in a shorter structure. The results highlight tools

The effect of hyperosmotic shock on cell volume, vacuole volume, and intracellular pH (pH i ) of individual cells of Saccharomyces cerevisiae and Zygosaccharomyces mellis was investigated. After transfer from a high water activity (a w ) medium to low a w media, the growth latency periods of Z. mellis were shorter than those of S. cerevisiae. These results demonstrate that Z. mellis manages hyperosmotic shock better than S. cerevisiae. As a response to acute hyperosmotic shock, i.e. the first minute of perfusion with hypertonic buffers, the vacuoles shrank and the pH i decreased in both yeasts. Furthermore, in the presence of glucose, vacuole shrinkage and intracellular acidification were more pronounced in S. cerevisiae than in Z. mellis. These results may be explained by the fact that the S. cerevisiae cells shrank more than the Z. mellis cells as a response to acute hyperosmotic shock. In the presence of glucose, the vacuoles and the cells of both S. cerevisiae and Z. mellis shrank simultaneously and in proportion to a minimum level during acute hyperosmotic shock, and remained constant at this level throughout the experiment (11 min). These results indicate that vacuoles do not act as water reserves in yeasts after acute hyperosmotic shock. Finally, Z. mellis was able to maintain its pH i near normal physiological levels after acute hyperosmotic shock, whereas S. cerevisiae was not. These results suggest that pH i regulation may be important for the ability of yeasts to manage hyperosmotic shock.

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region.

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