J. Colin Slaughter

The naturally occurring furanones: Formation and function from pheromone to food

Three closely related 4‐hydroxy‐3(2H)‐furanones have been found in a range of highly cooked foodstuffs where they are important flavour compounds with aroma threshold values as low as 20 μg kg‐1 water (approximately 0.14 μmol 1‐1). The compounds are formed mainly as a result of the operation of the Maillard reactions between sugars and amino acids during heating but one compound, 5‐(or 2)‐ethyl‐2‐(or 5)‐methyl‐4‐hydroxy‐3(2H)‐furanone, appears in practice to be produced by yeast, probably from a Maillard intermediate, during the fermentation stages in the production of soy sauce and beer. The compounds are also important in the flavour of strawberry, raspberry, pineapple and tomato but the route of biosynthesis is unknown. Two 3‐hydroxy‐2(5H)‐furanones, emoxyfuranone and sotolon, which are produced spontaneously from amino acids such as threonine and 4‐hydroxy‐l‐leucine are major contributors to meaty and spicy/nutty flavours in foods. The biosynthesis of 5‐(1,2‐dihydroxyethyl)‐3,4‐dihydroxy‐2(5H)‐furanone (ascorbic acid, vitamin C) and 5‐hydroxymethyl‐3,4‐dihydroxy‐2(5H)‐furanone (erythroascorbic acid) from sugars in plants and yeast, respectively, has been characterized to the enzymic level. After treatment with chlorine, humic waters contain a range of chloro‐furanones, some of which, particularly 3‐ chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX), are powerful mutagens. The furanones which occur in foods are also mutagenic to bacteria and cause DNA damage in laboratory tests. However, these compounds are, in practice, very effective anti‐carcinogenic agents in the diets of animals which are being treated with known cancer‐inducing compounds such as benzo[α]pyrene or azoxymethane. Two of the food‐derived furanones have antioxidant activity comparable to that of ascorbic acid. A biological function has been discovered for some of the furanones besides vitamin C. 5‐Methyl‐4‐hydroxy‐3(2H)‐furanone is a male pheromone in the cockroach Eurycolis florionda (Walker) and the 2,5‐dimethyl derivative deters fungal growth on strawberries and is an important component of the attractive aroma of the fruit. The red seaweed Delisea pulchra (Greville) Montagne produces a range of brominated furanones which prevent colonisation of the plant by bacteria by interfering with the acylated homoserine lactone (AHL) signalling system used by the bacteria for quorum sensing. In addition, these compounds can deter grazing by marine herbivores. It is proposed here that the evolved biological function of a number of furanones is to act as inter‐organism signal molecules in several different systems. This has resulted in two coincidental effects which are important for humans. Firstly, the easily oxidized nature of the furanones in general, which is likely to be an important property in their functioning as signal molecules, results in both mutagenic and anti‐carcinogenic activity. The balance of these two effects from compounds in the diet has yet to be fully established. Secondly, and more specifically, the 4‐hydroxy‐3(2H)‐furanones associated with fruit aromas act to attract animals to the fruit, which ensures seed dispersal. In the case of humans, the coincidental synthesis of some of these compounds in foods during preparation results in these foods appearing particularly attractive through the transferred operation of the original signalling mechanisms.

The importance of the furanones HDMF and HEMF in the flavour profile of Japanese barley miso and their production during fermentation

All 13 samples of Japanese barley miso accepted for the Kumamoto Prefecture Miso Competition of 1995 were assessed by tasting and by GC analysis using FID, SIM and olfactometric detection. Forty three compounds were separated by GC, of which 15 had a detectable aroma. Consideration of the chemical and sensory analyses of this latter group showed that only the two furanones, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), correlated significantly with the quality of miso flavour (P < 0·05). Experimental fermentations of salted barley koji and full barley miso mash with Zygosaccharomyces rouxii resulted in alcoholic fermentation in all cases investigated. In contrast, production of the three furanones, HMMF (4-hydroxy-5-monomethyl-3(2H)-furanone), HDMF and HEMF, was very sensitive to the conditions used. Negligible amounts were found in the absence of soy beans and the highest levels occurred when the full mash was incubated at 37°C for 14 days before inoculation of the yeast. When pre-incubation was carried out, the concentration of HMMF was maximal at the end of mashing and declined during fermentation whilst the amount of both HEMF and HDMF increased. The time courses were consistent with synthesis of HEMF, and then HDMF, by the yeast from HMMF.

Production of 4-hydroxyfuranones in simple media by fermentation

Both 2,5-dimethyl-3(2H)-furanone (DMHF) and 2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (EMHF) were produced at concentrations up to 4 mg l−1 and 20 mg l−1 respectively by yeast fermentation of a heated mixture of a single amino acid and a single sugar added to a yeast extract/peptone/glucose (YPG) medium. About 1 mg DMHF 1−1 was also produced from precursors in the autoclaved YPG medium but EMHF formation depended entirely on the presence of a heated ribose/amino acid mixture. Glutamate was the best precursor amino acid for both furanones. Formation of EMHF showed a positive, non-linear response to ribose/glutamate concentration from 20 to 200 mM in the heated mixture.

Biosynthesis of flavour-active furanones by Saccharomyces cerevisiae during fermentation depends on the malt type used in medium preparation

Extracts of a coloured malt contained 4-hydroxy-5-monomethyl-3(2H)-furanone (HMMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) but not 4-hydroxy-5(or 2)-ethyl-2(or 5)-methyl-3(2H)-furanone (HEMF). Extracts of a pale malt did not contain any of the furanones. HMMF and HDMF were produced by Saccharomyces cerevisiae during fermen-tation of both types of malt extract. About 0.09 mg HEMF l −1 was synthesised during fermentation of the coloured malt extract but none was produced with the pale malt extract. Final concentrations of HDMF (2.0 mgl −1) and HEMF (0.09 mgl −1) were in excess of their aroma threshold values in water (0.16 and 0.02 mgl −1 respectively) after fermen-tation of the coloured malt extract.

Effect of vacuoles on viability of Saccharomyces cerevisiae

Abstract In order to investigate the importance of the vacuole on viability, small-scale fermentation tests were conducted using diploid yeast cells constructed by mating. SKD1, a vacuole-complete strain, did not die in YPD medium after glucose was consumed. However, SKD2, a vacuole-deficient mutant, began to die within 50 h after glucose was consumed completely. In order to ascertain the effect of energy source on the viability of the vacuole-deficient mutant, fermentation was conducted with a minimal medium, YNBD medium. SKD2 died quickly as soon as glucose was consumed whereas SKD1 maintained its viability. Replacement studies with SKD2 confirmed the need for an external supply of glucose to prevent cell death. From changes of glycogen and trehalose in cells during fermentation, it was found that trehalose rather than glycogen was associated with cell death.

Sulphate availability and cysteine desulphydration activity as influences on production of hydrogen sulphide by Saccharomyces cerevisiae during growth in a defined glucose-salts medium

Saccharomycetes cerevisiae NCYC 1108 is a pantothenate-requiring yeast which produces hydrogen sulphide in a defined glucose-salts medium containing less than 0.1 mg l −1 of the vitamin. Hydrogen sulphide production is repressed by l -methionine but stimulated by several other amino acids even in complete medium, although these compounds have no effect on growth. A comparison of the cysteine pool size, hydrogen sulphide produced and cysteine desulphydration activity under several fermentation conditions does not support the current hypothesis that cysteine breakdown is a major source of external hydrogen sulphide. The results are more consistent with a metabolic blockage at the point of cysteine biosynthesis from O -acetylserine resulting in a loss of the biosynthetic sulphide component of the reaction as hydrogen sulphide.

Intracellular asparagine pool as a factor in control of ammonium uptake by Saccharomyces cerevisiae

During growth of Saccharomyces cerevisiae in defined media containing 1 m m ammonium and a single amino acid at 2 m m , asparagine, and to a lesser extent aspartic acid and glutamine, significantly delayed ammonium uptake. Addition of asparagine or glutamine to the medium during growth on ammonium caused an immediate cessation of ammonium uptake. Measurement of the pool sizes indicated that ammonium uptake responded to the internal concentration of asparagine rather than that of glutamine and, because of the speed of the response the most likely mechanism was inhibition of the ammonium permease system.

Mediation of the allosteric response of 3-phosphoglycerate dehydrogenase from peas

Earlier studies with plant extracts have shown that the first enzyme in the biosynthetic route to serine from 3-phosphoglycerate, 3-phosphoglycerate dehydrogenase, can be inhibited by low concentrations of its eventual end-product, L-serine [1,2]. In addition, the enzyme from peas can be activated by a range of amino acids including L-methionine [3,4]. However, desensitization of the pea enzyme to serine and methionine occurs on storage or on attempts at purification and this has greatly hindered the elucidation of the detailed mechanisms by which amino acids can alter the catalytic activity of 3-phosphoglycerate dehydrogenase. The results of experiments described in this paper suggest that these mechanisms are more complicated than was originally thought in that the enzyme from~etiolated pea epicotyls appears to be normally associated with a reversibly bound compound, probably a nucleotide related to AMP, which sensitises the enzyme to allosteric effectors without itself having any effect on the catalytic activity of the enzyme.

Inhibition of 3-phosphoglycerate dehydrogenase by compounds other than l-serine

Abstract 3-Phosphoglycerate dehydrogenase from etiolated pea epicotyls was not affected during in vitro assay by a range of hexose phosphates, amino group precursors and nucleotides at 1 mM but was significantly inhibited by 1 mM ATP and GTP. ADP and GDP gave slight inhibition at this concentration. NADH caused almost total inhibition at 0.45 mM.

Sulphate determination by ion chromatography in the presence of sulphite and organic acids

A method to analyse sulphate in beer in the presence of organic anions and sulphite has been developed using a Dionex Ion Chromatography system. Sulphite was stabilised in the sample by addition of acetaldehyde, 3.3% (v/v) and, by increasing the acetonitrile concentration of the eluant to 4.52 % (v/v), sulphate was separated from both malate and succinate without pre-treatment.