Production of Japanese soy-sauce flavours

The salt-tolerant yeasts Zygosaccharomyces rouxii and Candida versatilis are important for the formation of flavour in Japanese soy-sauce processes. In these processes Z. rouxii produces the flavour components ethanol, higher alcohols and 4-hydroxyfuranones, while C. versatilis is responsible for the production of phenolic flavour components, such as 4-ethylguaiacol and 4-ethylphenol. These yeast-derived flavour components give, amongst other components, the characteristic flavour to Japanese soy sauce.Little is known, however, about the flavour formation by the salt-tolerant yeasts, making the process difficult to control. Therefore, in this study, the metabolism of the salt-tolerant yeasts was investigated. Especially, much attention has been paid to the formation of higher alcohols by Z. rouxii . The higher-alcohols formation in Z. rouxii is strongly related to its amino-acid metabolism, whereinα-keto acids are key intermediates.The separate effects of the amino acids threonine, cystathionine, and the branched-chain amino acids (isoleucine, valine and leucine) on the metabolism of Z. rouxii were determined. The exogenous addition of these amino acids appeared to have large effects on the higher-alcohols production by Z. rouxii . For the production of the higher alcohols isobutyl alcohol, active amyl alcohol and isoamyl alcohol the Ehrlich pathway appeared to be very important. In this pathway, uptake and transamination of amino acids results in the higher-alcohols formation. The added amino acids also clearly influenced the formation and conversion rate of theα-keto acidα-ketobutyrate, as appeared from measured enzyme activities. This influence did not result in the accumulation ofα-ketobutyrate, because theα-ketobutyrate pool size in Z. rouxii appeared to be tightly regulated. Furthermore, independent of the amino acids added, Z. rouxii produced ethanol under aerobic conditions (Crabtree effect), which is in contrast to what is described in the literature. In addition, the supply of threonine severely inhibited the growth of Z. rouxii .In order to clarify the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Z. rouxii was investigated. It was shown that the poor growth of Z. rouxii in the presence of threonine was due to a lack of methionine, which was caused by a blocked methionine synthesis. Threonine seemed to block this synthesis in Z. rouxii by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii was not inhibited by the herbicide sulfometuron methyl (SMM) that is a well-known growth inhibitor of various plants, bacteria and yeasts like Saccharomyces cerevisiae . The insensitivity of Z. rouxii growth to SMM appeared to be caused by the fact that the activity of the enzyme acetohydroxy acid synthase in Z. rouxii , unlike that in S. cerevisiae , was not affected by SMM. On the other hand, the activity of the enzyme threonine deaminase in Z. rouxii was similarly regulated as that in S. cerevisiae . Based on these observations it was concluded that the aspartate-derived amino-acid metabolism in Z. rouxii is only partly like that in S. cerevisiae .The steady-state culture characteristics of Z. rouxii were investigated in this study as well. It was confirmed that Z. rouxii , like S. cerevisiae , showed the Crabtree effect. It appeared that Z. rouxii started to produce ethanol at a lower dilution rate than S. cerevisiae (0.1 versus 0.3 h -1), while also the maximum specific growth rate of Z. rouxii was lower than that of S. cerevisiae (0.17 versus 0.45 h -1). For this investigation, the acceleration-stat (A-stat) cultivation method in which the dilution rate is continuously changed with a constant acceleration rate was used. The A-stat cultivation can be much less time-consuming than the widely used chemostat cultivation, especially at high acceleration rates. However, at very high acceleration rates the A-stat does not provide steady-state culture characteristics. The highest acceleration rate for estimating the steady-state culture characteristics of Z. rouxii was determined to be 0.001 h -2. For higher acceleration rates, an increased difference between A-stat and chemostat culture at a given dilution rate was observed. This observation for Z. rouxii was confirmed with simulations for S. cerevisiae . Moreover these simulations showed that, for estimating the steady-state culture characteristics with the A-stat, both the metabolic adaptation rate of the yeast and the rate at which the environmental substrate concentrations change should be taken into consideration. At an acceleration rate of 0.001 h -2, the A-stat proved to be advantageous to the chemostat, because the A-stat provided much more data in the same time than the chemostat.The A-stat cultivation was also used to study the concomitant extracellular accumulation ofα-keto acids and higher alcohols by Z. rouxii . Allα-keto acids from the aspartate-derived amino-acid metabolism, exceptα-ketobutyrate, could be extracellularly accumulated by exogenous supplying the amino acids valine, leucine, threonine and methionine. From this study it was concluded that in Z. rouxii valine, leucine and methionine were converted via similar Ehrlich pathways as in S. cerevisiae , while for the conversion of threonine both the Ehrlich and amino-acid biosynthetic pathways in Z. rouxii were used. Additionally the Ehrlich pathway appeared to be the only pathway for the formation of the higher alcohol methionol in Z. rouxii .Another problem associated with the yeast flavour formation in Japanese soy-sauce processes is that the flavour formation is normally very slow. For this reason the conventional batch process takes about 6 months. In literature the development of a new continuous process using immobilized salt-tolerant yeasts resulted in a 90% reduction of the process time. However, the new immobilized-cell process seemed not very suitable for long-term operation, because alginate was used as immobilization material. The reason for this is that alginate is sensitive to abrasion and chemically unstable towards the high salt concentration in the soy-sauce medium (about 17% (w/v)). To replace alginate, a chemically crosslinked polyethylene-oxide gel was investigated in this study.The problem of the chemically crosslinked polyethylene-oxide gel was that Z. rouxii cells did not survive the immobilization procedure. The absence of survival appeared to be caused by the toxic effect of the crosslinker used for making this gel. Therefore, a new immobilization procedure, in which direct contact between the crosslinker and yeast was circumvented, was developed. For both Z. rouxii and C. versatilis the survival percentages in the newly developed polyethylene-oxide gel were high and comparable to those in alginate. Unlike alginate gel, the new polyethylene-oxide gel showed, during rheological studies, to be insensitive to abrasion, even in the presence of high salt concentrations.The insensitivity to abrasion of the new polyethylene-oxide gel was confirmed during cultivations in a stirred-tank reactor with varying high salt concentrations (12.5-17% (w/v)). In this reactor no abrasion of polyethylene-oxide gel particles was observed for several days, while alginate gel beads were completely destroyed within one day. However, the polyethylene-oxide particles appeared to stick together, making long-term processing difficult. It also appeared during these cultivations that Z. rouxii and C. versatilis immobilized in the polyethylene-oxide particles were capable of producing characteristic soy-sauce flavours. Therefore, it was concluded that the application of polyethylene-oxide gel in long-term soy-sauce processes is attractive provided that the stickiness of the particles can be controlled.However, from a comparison of literature data it was also concluded that the application of immobilized salt-tolerant yeasts instead of free yeasts cells in the continuous process hardly accelerates the flavour formation. For accelerating the flavour formation the continuous microfiltration membrane reactor seems to be more promising. Finally, it was concluded that this study and other recent research has enhanced the understanding of the yeast flavour formation during Japanese soy-sauce processes, which facilitates process control.</p