Arginine degradation by malolactic wine lactic acid bacteria and its oenological and toxicological implications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

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Malolactic fermentation (MLF) in wines is a secondary fermentation carried out by lactic acid bacteria (LAB), mostly encouraged for decreasing acidity by degradation of malic acid and for modifying flavour. During MLF, wine LAB may also degrade arginine, leading to the formation of ATP, ammonia and citrulline, among others. This is of concern to the winemaker and the consumer alike because ammonia increases the pH and thus the risk of growth by spoilage micro-organisms, and citrulline is a precursor in the formation of carcinogenic ethyl carbamate (EC). The degradation of arginine and the excretion of citrulline was studied with resting and growing cells of selected wine LAB in synthetic buffer and wine. All wine LAB tested degraded arginine and excreted citrulline, and also degraded citrulline as a sole amino acid. However, reutilization of excreted citrulline depended on the biomass condition. Arginine degradation and citrulline excretion rates had a linear, positive correlation. Arginine to citrulline conversion ratios ranged between 0.8 and 4.6% (w/w) and can be used to estimate the potential formation of citrulline from a given amount of arginine. Combining these ratios with literature data, an approximate arginine to EC conversion ratio of 0.006% (w/w) can be calculated. In Lactobacillus buchneri, arginine degradation occurred during growth and malic acid degradation, and this stimulated growth. In contrast, arginine degradation in oenococci occurred in late exponential or stationary phase and after malic acid degradation, and this did not stimulate growth. Citrulline excretion results from an unfavourable equilibrium of the citrulline degrading reaction. The pH (3.5 - 6.5) did not have a direct effect on citrulline excretion. However, the excretion of citrulline was influenced by the growth phase in which arginine degradation occurred and can be partially regarded as an overflow metabolism caused by insufficient coupling of energy formed from arginine degradation to growth. To control citrulline formation and pH increase in wine from bacterial arginine degradation, the results suggest carrying out MLF with pure oenococcal cultures and inhibiting bacterial metabolism after malic acid depletion. This is especially important in wines with high arginine concentrations and high pH values. Malic acid itself should be measured since pH increase or CO2 formation may also result from arginine degradation. Alternatively, arginine negative Oenococcus strains could be isolated and used for MLF. The excessive addition of ammonia as yeast nutrient during alcoholic fermentation and storage of wines on yeast sediments (lees) increase the potential for citrulline formation. A simple enzymatic assay for the determination of arginine was developed and this method enables a fast "risk-assessment" of must and wines.
Content removed due to copyright restriction: Mira De Orduna, R., Patchett, M. L., Liu, S. -., & Pilone, G. J. (2001). Growth and arginine metabolism of the wine lactic acid bacteria lactobacillus buchneri and oenococcus oeni at different pH values and arginine concentrations. Applied and Environmental Microbiology, 67(4), 1657-1662. De Mira Ordun~a, R., Liu, S. -., Patchett, M. L., & Pilone, G. J. (2000). Kinetics of the arginine metabolism of malolactic wine lactic acid bacteria lactobacillus buchneri CUC-3 and oenococcus oeni Lo111. Journal of Applied Microbiology, 89(3), 547-552. Mira De Orduña, R., Liu, S. -., Patchett, M. L., & Pilone, G. J. (2000). Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria. FEMS Microbiology Letters, 183(1), 31-35 Mira De Orduna, R. (2001). Quantitative determination of l-Arginine by enzymatic end-point analysis. Journal of Agricultural and Food Chemistry, 49, 549-552.
Wine fermentation, Lactic acid bacteria, Oenology