EFFECT OF GLUCOSE-OXIDASE ON THE CONCENTR ATION OF SUCROSE, GLUCOSE AND FRUCTOSE DURING THE ENSILING OF GRASS PERUN AND ALFALFA

Wilted grass Perun (Lolium multiflorum Lam. x Festuca pratensis Huds., 2n = 28) with dry matter (DM) 33.6% and alfalfa (Medicago sativa L.) with 33.5% DM were ensiled untreated (control) or treated with both th e inoculant of Lactobacillus (Microsil) and the enzyme complex of cell ulase, hemicellulase and glucose oxidase (GO) which contained the iden tical inoculant (Bactozyme). Sucrose, glucose and fructose were extrac ted with boiling ethanol (70%, v/v) and the individual sugars were det ermined using ligand exchange chromatography on a strong cation exchan ger in Na+ form with water as an eluent at 53 degrees C. Ribose was us ed as an internal standard. Under these conditions, the high-molecular compounds are eluated with a column dead volume, disaccharides give o nly one peak, and monosaccharides are separated efficiently (Tab. I). This method is very useful for routine analyses of natural materials. The chromatograms of both grass Perun and alfalfa extracts are depicte d in Fig. 1. Among sugars tested, sucrose disappeared most rapidly in all experiments (Figs. 2 and 3). Interestingly, the hydrolysis of sucr ose was a first-order reaction characterized by a linear dependence of logarithm of the concentration on time between 3 and 30 hours of the ensiling. The calculated rate constants (Tab. II) were practically the same with respect to the experimental error. Sucrose is most probably hydrolyzed with the original microflora, and the half-time of this re action is approximately 10 hours. The changes in the glucose concentra tion (Figs. 4 and 5) reflected a complex role of this monosaccharide: its content increased by the hydrolysis of sucrose or polysaccharides and glucose was simultaneously consumed by fermentation or by the acti on of GO. The final concentration profile is determined by the relativ e rates of these individual reactions, therefore any attempt at the ap plication of a simple mathematic description need not be correct (Spic ka, 1995). The significant effect of GO was found in the initial stage of ensiling where glucose concentration was decreased by its oxidatio n to gluconic acid as it had been described previously (Rauramaa et al ., 1991). In the homofermentative stage, the rate of consumption of bo th glucose and fructose was higher in the treated silages than in the control due to the activity of an inoculant (Figs. 4-7). Surprisingly, GO seemed to have the pronounced influence on the fructose concentrat ion (Figs. 6 and 7). The content of fructose was not changed during in itial 20 h although it was formed from sucrose by hydrolysis. Furtherm ore, this observation is illustrated by the changes in the sum of gluc ose and fructose concentrations which was remained rather constant (Fi g. 8). The GO probably activated the original microflora which isomeri zed fructose to glucose within the heterofermentative stage of ensilin g. The addition of glucose oxidase has the greatest effect in the init ial hours of ensiling when glucose is rapidly oxidized to gluconic aci d. Glucose oxidase also activates the original plant microflora which isomerizes fructose to glucose. This observation had not been describe d previously.