| Bitterness, a common flavor defect of cheese occurs during aging due to the accumulation of casein (CN)-derived hydrophobic peptides, such as beta-CN (f193--209) and alphaSI-CN (f1--9). Preferential hydrolysis of bitter peptides by specific starter and non-starter peptidases could result in reduction of bitterness, provided they are food-grade. In this study, two food-grade vectors, pSUW611 and pSUW711, were constructed based on the novel food-grade markers aspartate aminotransferase and alpha-galactosidase, respectively. The vector pSUW611 was determined to be stable for 100 generations of non-selective growth. Food-grade derivatives of Lactococcus lactis were constructed that expressed Lactobacillus helveticus CNRZ32 PepN 6-fold. However, the level of expression of PepN was directly related to the vector copy number. This study also evaluated the activity of three newly identified endopeptidases (PepO3, PepF and PepE2), and three previously characterized endopeptidases (PepO, PepO2 and PepE) of Lb. helveticus CNRZ32 on beta-CN (f193--209) and alphaSI-CN (f1--9) under cheese ripening conditions, (pH 5.1--5.2, 4% NaCl, and 10°C). CNRZ32 PepO2, PepO3, PepF, and PepE had unique specificities towards these peptides under these conditions, however only PepO2, PepO3 and PepE were successfully expressed under the control of pepO3 promoter, on a high copy vector in Lc. lactis. The activity of these peptidases on beta-CN (f193--209) and alphaSI-CN (f1--9) was determined in single peptide reactions, in a defined mixture of cheese-derived peptides and in Cheddar cheese serum (CCS). In CCS both PepO2 and PepO3 had high specific activity for beta-CN (f193--209), while only PepO2 had greater activity than PepE and PepO3 for alpha SI-CN (f1--9). It was concluded that: the two food-grade vectors, pSUW611 and pSUW711 have the potential to overexpress genes of interest in Lc. lactis; CCS serves as a simple cheese like model system for peptide hydrolysis studies; and two endopeptidases from Lb. helveticus CNRZ32, PepO2 and PepO3, when expressed on a high copy vector in a lactococcal strain, have the potential to reduce bitterness in cheese. |
