The development of a novel yeast-based flavour enhancer

Yeast extracts are the products derived from the hydrolysis of proteins and nucleic acids, initiated by the action of intracellular enzymes, nucleases or phosphatases following cellular autolysis. 5´-nucleotides are the primary flavour enhancers derived from this hydrolysis and are used as flavour potentiators in soups, sauces, meats and vegetarian products. Production of a commercial yeast extract requires the autolysis, extraction, hydrolysis and purification of a cell’s cytoplasmic components. Results derived from this study provide a novel process for the production of 5´-nucleotides based on the intracellular delivery of 5´-phosphodiesterase-1 (5´-PDE-1) into viable cells without autolysis and alleviating the generation of excess waste streams. Technology developed during this research includes an ion exchange protocol for barley 5´-PDE-1 (used in the manufacture of 5´-nucleotides) based on a characterisation study of the enzyme. The purified 5´-PDE-1 has a molecular weight of 236 kDa, a pI of 6.0 and a Km of 0.28 mM, consistent with literature, with an identified heat tolerance to 60oC based on the addition of Calcium and Magnesium ions during processing. Activity of the enzyme was screened spectrophotometrically at 410 nm by reaction with a reporter substrate, Thymidine 5´-monophosphate p-Nitrophenyl ester (TMP-p-NPE) with ultimate productivity assessed by Reverse Phase HPLC measuring the formation of 5´-GMP. A liposome vesicle containing PC:DOPE:DOTAP:ERGO (50:30:10:10 mol%) was used to encapsulate the extracted enzyme for endocytosis into a prepared yeast suspension with translocation of the liposome across the yeast plasma membrane identified by confocal fluorescence and transmission electron microscopy. The successful release of liposomal bound 5´-PDE-1 into the yeast cytoplasm resulted in a 26-fold increase in 5´-GMP flavouring nucleotide measured within the cell culture