Growth and metabolism of lactic acid bacteria in a model wine system and a red wine with emphasis on carbohydrate metabolism : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Technology (Food Technology) in the Faculty of Technology at Massey University, New Zealand

Abstract

Studies were conducted to investigate the application of capillary gas­ liquid chromatography in analysis of wine carbohydrates, and the growth and metabolism of wine lactic acid bacteria in a synthetic model wine system.

1. Analysis of carbohydrates in wine using capillary gas-liquid chromatography Wine carbohydrates were analysed by capillary gas liquid chromatography of their acetate and aldononitrile acetate derivatives. A wide range of aldoses, polyols and disaccharides (30 compounds) were analysed in 55 minutes, using a single injection. All the derivatives were well­ separated except for ribose and rhamnose, which almost co-eluted. The method recovered spiked carbohydrates at 86 to 110% and had adequate reliability. This technique may be applied routinely to the analysis of other alcoholic and non-alcoholic beverages.

2. Growth and metabolism of wine lactic acid bacteria

Malic acid and pH values had determinative effects on the growth of wine lactic acid bacteria. Malic acid stimulated the growth rate and cell population of - 122 and 252 at pH 4 and allowed their growth at pH 3.2. The absence of malic acid at pH 3.2 inhibited the growth of ­ oenos 122 and 252. The stimulatory effect of malic acid on growth was more striking at pH 3.2. This effect was not caused by the pH increases

resulting from malic acid degradation. Malic acid had only a small stimulation on the growth rate of - plantarum 49 and f. parvulus 93 at pH 4 and their growth was suppressed at pH 3.5, irrespective of malic acid. These results imply that pH 3.5 is a critical value for the bacteriological stability of wine after malolactic fermentation.

This study confirmed that sugars served as the main growth substrates for wine lactic acid bacteria and polyols did not act as growth substrates, with the exception of mannitol. Glucose and trehalose were the preferred substrates for all the bacteria tested. The significance of trehalose in relation to yeast autolysis in induction of malolactic fermentation was discussed. Wine lactic acid bacteria varied in the ability to utilise substrates. Malic acid, citric acid and arginine did not serve as single energy sources.

Malolactic fermentation had a profound impact on substrate utilization by - oenos 122 and 252, yet seemed not to affect the substrate utilization of - plantarum 49 and f. parvulus 93. The presence of malic acid resulted in an increased utilization of sugars by k• oenos 122 and 252, and decreased utilization of arabinose by k• oenos 252. Trehalose utilization by - oenos 252 was not influenced by malolactic fermentation. The increased utilization of sugars may be the biological functions of malolactic fermentation.

pH exerted a marked effect on the metabolism of k• oenos 122 and 252. More sugars were utilized at pH 4 and above than at pH 3.31 and below. k• oenos 122 attacked only a very minor amount of glucose and a portion of malic and citric acids at pH below 3.31. k• oenos 252 also used only

a small quantity of sugars except for glucose, which was used completely, but degraded all malic and citric acid at pH below 3.42. These results strongly suggest that the degradation of malic acid, citric acid and arginine required the presence of fermentable sugars. This implies that the absence of fermentable sugars in wine may prevent malolactic fermentation. These results also justify the benefits of malolactic fermentation at low pH values (below 3.3).

The role of wine lactic acid bacteria in formation of biogenic amines was clarified. - olantarum 49 was the only organism which reduced the levels of tyrosine and phenylalanine dramatically, indicating that this bacterium may be a potential producer of tyramine and phenylethylamine. - parvulus 93 did not markedly decrease the levels of any amino acids. Arginine was catabolised only by - 122 and 252 with the formation of ornithine and ammonia. Arginine was not degraded at low pH values (below 3.5), suggesting that arginine may not play any role in energy supply at low pH values. - oenos 122 and 252 did not significantly reduce the concentrations of other amino acids.

The role of malolactic fermentation may lie in energy generation. Two potential energy-yielding mechanisms of malolactic reaction were proposed: ATP production through pyruvic acid cleavage (substrate level phosphorylation, pseudo-malolactic fermentation) and chemiosmotic ATP synthesis via formation of extra lactic acid (non-substrate level phosphorylation, real malolactic fermentation). It is speculated that ­ oenos 122 may employ the pyruvic acid cleavage pathway and generation of superfluous lactic acid may be adopted by - oenos 252, - plantarum 49 and - parvulus 93. The biological function of the extra lactic acid

could be accounted for by the chemiosmotic theory that postulates energy (ATP) production through efflux of metabolic end-products (e.g., lactic acid) The origin of the superfluous lactic acid remains to be investigated. These findings suggest that the criteria for selection of starter cultures be redefined.