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E. SOERYAPRANATA¹, G. Ü. Yüksel², and J. R. Powers¹. (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

Bitterness is a flavor defect that can develop in Cheddar-type cheeses. Adjunct cultures are often used to advance cheese aging and reduce bitterness, but selection of adjuncts remains largely empirical. Lactobacillus helveticus WSU19 is an excellent adjunct used in the production of Cougar Gold cheese, an aged Cheddar-type cheese with little or no bitterness. Our long-range goal is to elucidate the role of peptidases from L. helveticus WSU19 in flavor development and debittering of Cougar Gold cheese. The objective of this study was to clone and characterize the endopeptidase O (pepO), endopeptidase O2 (pepO2), and endopeptidase O3 (pepO3) genes from L. helveticus WSU19. The pepO, pepO2, and pepO3 genes from L. helveticus WSU19 were identified by Southern hybridization using the corresponding genes from L. helveticus CNRZ32 as probes. Hybridization-positive genomic DNA fragments from L. helveticus WSU19 were cloned in Escherichia coli DH5α and fully sequenced. Primer walking of a 4.3-kb HindIII-SalI genomic insert from L. helveticus WSU19 revealed an open reading frame (ORF) of 1941-bp encoding for endopeptidase O (PepO) enzyme. BLAST analyses of the ORF revealed 99%, 62%, and 56% identities with PepO, PepO3, and PepO2 enzymes from L. helveticus CNRZ32, respectively. Primer walking of a 5.8-kb XbaI-SphI genomic insert and a 3.4-kb KpnI genomic insert from L. helveticus WSU19 revealed ORFs of 1944-bp and 1929-bp encoding for endopeptidase O2 (PepO2) and endopeptidase O3 (PepO3) enzymes, respectively. PepO2 and PepO3 enzymes from L. helveticus WSU19 share 98% and 99% identities with PepO2 and PepO3 enzymes from L. helveticus CNRZ32, respectively. Our present research deals with the construction of the PepO, PepO2, and PepO3-deficient derivatives of L. helveticus WSU19 and their use in Cougar Gold cheese manufacture. These derivatives will verify the importance of the endopeptidases of interest in aged cheese, allowing a systematic development of improved cultures.

Session 17, Biotechnology: General
2:30 PM - 5:30 PM, Sunday PM Room 296

2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana