Control of Ethyl Carbamate Formation in Wine from Arginine Degradation by

Objective la: Survey of commercially available arginine-degrading wine lactic acid bacteria for the ability to excrete citrulline. Several strains are being surveyed for citrulline (ethyl carbamate precursor) excretion during arginine degradation using a resting cell culture technique. Strains are grown in culture medium containing arginine to induce formation of the enzymes of the arginine-degrading pathway. Upon growth, the cells are centrifuged, washed, centrifuged again and resuspended in tartrate buffer (pH 3.6) to give a high cell density. Arginine (plus some glucose for arginine uptake) is added to the cell suspension and samples taken periodically for analysis of arginine, citrulline, and pH. This procedure enables a relatively quick (hours) assessment of many cultures, rather than using the lengthy procedure of growing cells in synthetic media/wine that may take weeks. It has been shown that the resting cell fermentation technique gives valuable results as compared with fermentation in synthetic wine. In addition to testing for citrulline excretion, citrulline utilization is being tested separately using the resting cell technique with citrulline as the substrate instead of arginine. To date, 24 commercially available arginine-degrading wine lactic acid bacteria strains have been evaluated for citrulline excretion and utilization. All the strains tested so far are capable of excreting citrulline during arginine degradation and all can utilize citrulline as a substrate except for one. Upon completion of the survey, selected strains will be used to test various factors that may affect citrulline excretion (and uptake) as outlined in the AVF El01 grant proposal. Recently, a review on arginine catabolism by wine lactic acid bacteria has been published: S.-Q. Liu and GJ. Pilone (1998): A review: Arginine metabolism in wine lactic acid bacteria and its practical significance. Journal of Applied Microbiology: 85 (3), 315-327