Production of pyrazine flavours by mycelial fungi

Abstract

An overview of the flavour and fragrance industry has indicated that there is a great demand for flavours to have the natural, halaal and kosher status. This has opened the door for the increase in production of flavours by microbial means. Particularly with regards to pyrazine production, bacterial species such as Bacillus are commonly used. Although previous literature indicated that fungi are not as prominent pyrazine producers compared to bacteria, this study has indicated that mycelial fungi can be considered for the production of natural pyrazine flavours. Out of the 280 fungi screened, 45% showed pyrazine flavour production as indicated in chapter 2. This chapter also showed that the content of growth media can have a substantial influence on flavour production. For example, fungi grown in Cz-medium produced more of the caramel and chocolate flavours, compared to fungi that were grown in TSB, which produced more of the nutty, meaty and potato flavours. The green flavour was, however, prominent in both media, of which mostly members of the Aspergillus and Penicillium groups produced this flavour. Selected Penicillium species, including Penicillium rubrum and P. purpurogenum produced a green pepper odour that is indicative of the presence of methoxypyrazine. Chemical analytical methods as described in chapter 3 confirmed that Penicillium purpurogenum produced 2-methoxy-3-isobutylpyrazine (MIBP) and Penicillium rubrum produced both 2-methoxy-3-isobutylpyrazine (MIBP) and 2-methoxy-3,5/6-isopropylpyrazine (MIPP). Methoxypyrazines are high impact aroma chemicals that have a typical green pepper odour and a high market value. Due to the favourable characteristics of the Penicillium species in industrial fermentation processes (such as mass spore production and rapid colonization of substrates) and their ability to produce high value compounds, these fungi were selected for further studies. In order to explore the potential use of these fungi in an industrial application, the methoxypyrazines produced were quantified. Prior to quantitation, a solvent extraction method, using dichloromethane, was developed. Amongst the different pH parameters analysed acidified conditions showed the best results, where 69% MIPP and 97% MIBP were recovered from the liquid-liquid extraction and 76% MIPP and 99% MIBP were recovered from the solid-liquid extraction. Pyrazines quantified from the liquid-liquid extractions indicated that Penicillium rubrum produced 0.38 μg MIPP.L-1 and 0.88 μg MIBP.L-1, and Penicillium purpurogenum produced 0.88 μg MIPP.L-1 and 2.15 μg MIBP.L-1 (Chapter 4). Yields obtained from this study were not seen as feasible for the production of methoxypyrazines by the fungi on an industrial scale and, therefore, solid state fermentation was investigated as an option to improve the yields. Due to the availability of soy press cake as a by-product, as well as the variety of the amino acids present, it was selected as a possible substrate for pyrazine production. The results from this study, however, indicated that the nature of the substrate, such as the lipid content, fungal content and particle size of the soy press cake does not support fungal growth and makes pyrazine analyses problematic. Alternate methods of improving methoxypyrazine yields thus need to be found. A limiting factor is the lack of understanding of the metabolic pathway involved in pyrazine production by Penicillium species. By having this knowledge the fermentation process can be adapted accordingly for the optimal production of methoxypyrazines by these microorganisms. Additionally, substrates that contain the necessary precursors that are cost effective would contribute significantly in the development of an economically viable fermentation process for the production of methoxypyrazines by Penicillium species.