1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark3 University of Copenhagen
Induction of carbohydrate acting enzymes from Aspergillus nidulans
Starches are used to enhance aroma perception in low-fat foods. Aroma compounds can bind physically to the starch in grooves on the surface or they can form complexes inside amylose helices. This study has been divided into two parts: one part regarding binding of aromas to starches and their aroma-release, and another part regarding stimulation of a fungal secretome using different carbohydrates. In the first part, nine aromas and one aroma-mixture were mixed with nine different starches, including genetically modified starches. The objective of this sub-project was to bind aromas to the starches to 15 weight-percent. Aroma binding was tested on both amorphous starches and on native starch granules. A series of aldehydes and alcohols were also tested for binding to the starches. The aromas with the highest volatility were positively retained by starch, whereas for aromas with a lower volatility the starch had a negative effect on retention. No trends were observed that could relate aroma binding or retention to physical qualities of the starch such as amylose or phosphate content. Still, the physical state of the starch was shown to influence retention of some aromas, possibly by diffusion through the outer layers of the starch granule or binding to grooves on the surface. Chemical reactivity of some of the aromas also influenced the retention. Analysing a series of aldehydes and alcohols revealed an influence of the size of the molecules on retention. Hexanal showed a remarkable drop in retention for all starches, and pentanol showed a favoured retention by native starch granules. The aroma compounds bound to the starch proved very difficult to release as only a minute fraction of the aroma added could be released from the starch, even under conditions favouring aroma release, as monitored by GC-MS and solvent extractions. Addition of water to the extraction lowered the amount of aroma released, and addition of a starch-degrading enzyme, α-amylase, did not significantly change the amount extracted. Studies by differential scanning calorimetry and wide-angle X-ray scattering did not show complete complexation of aromas in the amylose helices, but instead changes were observed that could be evidence of partial complexation. This complexation is not exclusive to the amylose helices, but also appears to include interactions in the amylopectin double helices. In particular, one of the analysed aromas showed a very noticeable reduction in melting temperature, but showed only a minor reduction in melting enthalpy and no evidence of amylose complexation. Using an enzyme-discovery approach in the second sub-project, the industrially relevant fungus Aspergillus nidulans was stimulated with different carbohydrates. Stimulation with starch induced expression of starch-degrading enzymes, while stimulation with the hemicellulose xylan induced expression of xylanases. One particular hypothetical protein was ubiquitously expressed. This protein had no apparent homology with known proteins, but may iv be involved in attacking other organisms as a weak homology with other proteins involved in membrane attack was detected. Degradation of secreted proteins was observed in some cultures. Identification of a glycoside hydrolase family 61 using xylan as carbon-source was not successful despite previous evidence for the induction of this enzyme.