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E. SOERYAPRANATA1, J. R. Powers1, and G. Ü. Yüksel2. (1) Dept. of Food Science & Human Nutrition, Washington State Univ., 106 FSHN Bldg., PO Box 646376, Pullman, WA 99164-6376, (2) Dept. of Food Science & Toxicology, Univ. of Idaho, Agricultural Biotechnology Lab., Moscow, ID 83844-2312 Chymosin is important to initiate proteolysis of caseins during cheese ripening. Degradation of β-casein by chymosin produces the bitter peptide β-CN f193-209. Cleavage of αs1-casein by chymosin produces αs1-CN f1-23, which affects cheese flavor development. Lactobacillus helveticus WSU19 is the adjunct culture for Cougar Gold, an aged Cheddar-type cheese with strong nutty flavor. Peptidases from this organism are important in the flavor development and bitterness prevention of Cougar Gold cheese. In our previous work, five peptidase genes encoding for aminopeptidase N and endopeptidases E, O, O2, and O3 from L. helveticus WSU19 have been cloned/characterized. However, roles of these peptidases in the degradation of peptides produced by chymosin are unknown. The objective of this study was to determine specificities of selected peptidases from L. helveticus WSU19 on β-CN f193-209 and αs1-CN f1-23. Cell-free extracts (CFEs) of Escherichia coli DH5α clones expressing selected peptidase genes from L. helveticus WSU19 were prepared. Hydrolyses of β-CN f193-209 and αs1-CN f1-23 by the CFEs were conducted at 37 °C under simulated cheese conditions (pH 5.2, 4% salt). Peptide hydrolysates were desalted and analyzed by MALDI-TOF mass spectrometry. PepN cleaved tyrosine and glutamine from β-CN f193-209, producing the bitter peptides β-CN f194-209 and f195-209. No PepN activity was detected on αs1-CN f1-23. PepE cleaved αs1-CN f1-23 only. PepO, PepO2, and PepO3 exhibited postproline endopeptidase activities by cleaving glutamine and proline from β-CN f193-209, producing β-CN f197-209. PepO3 activity also produced β-CN f193-206 and f201-209. PepO and PepO2 appeared to be more active on αs1-CN f1-23 than PepO3. Our present work deals with the elucidation of the roles of selected peptidases in cheese flavor development through construction/utilization of peptidase-deficient mutants. Better understanding of the roles of peptidases from adjunct cultures is essential for the improvement of existing and the development of new cultures for cheesemaking.
Session 31, Dairy Foods: General I
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |