NMR and mushrooms : imaging post harvest senescence

The objective of the study described in this thesis was to explore the potentials of NMR for the study of water relations in harvested mushrooms ( Agaricus bisporus ). Since harvested mushrooms tend to continue their growth after harvest, their morphogenesis is heavily influenced by the external climatic conditions. Their respirative resources as well as their internal water can not be replenished after harvest and has therefore to be present in the mushroom before harvest.The main metabolic pools and changes in these pools were studied in Chapter 2. Extracts of the cap, gill and stipe of the fruiting body of the mushroom ( Agaricus biporus ) were studied by 13 C-NMR spectroscopy. This technique enables changes in the main metabolite pools to be studied simultaneously as a function of storage time, temperature and postharvest development. An earlier reported reduction in dry weight of the stipe could be explained by a decrease in mannitol content. At 274 K storage temperature no postharvest development occurred, yet mannitol content decreased. It was concluded that mannitol is probably used as a respiratory substrate in gill tissue. Proteolytic breakdown was apparent, even during storage at 274 K, but occurred preferentially in the stipe. The products were most probably used by the gill and to a lesser extent by the cap to maintain metabolic activity as demonstrated by urea-cycle activity. Changes in the content of four amino acid pools (glutamate, glutamine, alanine and aspartate) proved to be tissue-specific, as were changes in the content of mannitol, fumarate and malate.In Chapter 3, multi-echo imaging together with mono-exponential T 2 decay fitting was applied to determine reliable proton density and T 2 distributions over a mushroom. This was done at three magnetic fields strengths (9.4, 4.7 and 0.47 T) because susceptibility inhomogeneities were suspected to influence the T 2 relaxation times negatively and because the influence of susceptibility inhomogeneities increases with raising magnetic field strength. Electron microscopy was used to understand the different T 2 's for the various tissue types in mushrooms. Large influences of the tissue ultrastructure on the observed T 2 relaxation times were found and explained. Based on these results, it was concluded that imaging mushrooms at low fields (around or below 0.47 T) and short echo-times has strong advantages over its high field counterpart, especially with respect to quantitative imaging of the water balance of mushrooms. These conclusions indicate to have general validity whenever NMR-imaging contrast is influenced by susceptibility inhomogeneities.In Chapter 4, the influence of the tissue structure of mushrooms, e.g. tissue density (susceptibility inhomogeneity) and cell shape on the amplitude-, T 2 - and T 1 images was analysed. This was achieved by vacuum infiltration of the cavities in the mushroom's spongy structure with Gd-DTPA solutions and acquiring Saturation Recovery-Multi Spin Echo images (SR-MSE images).It was demonstrated that the intrinsic long T 2 values in the cap and outer stipe tissue strongly relate to the size and geometry of the highly vacuolated cells in these spongy tissues. All observed T 2 values were strongly affected by susceptibility effects. The T 2 of gill tissue was shorter than T 2 of the cap and outer stipe probably because these cells were less vacuolized and smaller in size.The calculated amplitude images were not directly influenced by susceptibility inhomogeneities as long as the observed relaxation times remained sufficient long. They reflected the water distribution in mushrooms best if short echo-times were applied in a multi spin echo imaging sequence at low magnetic field strength.In Chapter 5, the evolution and mechanism of redistribution of water in harvested mushrooms was studied using quantitative water density and T 2 MRI. It was revealed that when mushrooms did not lose water during the storage period, a redistribution of water occurred from stipe to cap and gills. When the storage condition resulted in a net loss of water, the stipe lost more water than the cap. The water density in the gill increased, probably due to development of spores.Deterioration effects (i.e. leakage of cells, decrease in osmotic water potential) were found in the outer stipe. They were not found in the cap, even at prolonged storage at 293 K and R.H. = 70%. The changes in osmotic potential were partly accounted for by changes in the mannitol concentration. Changing membrane permeability's were indicated too. Cells in the cap had a constant low membrane (water) permeability. They developed a decreasing osmotic potential (more negative), whereas the osmotic potential in the outer stipe increased, together with the permeability of cells.The NMR experiments described in this Thesis can find their future applications in the post-harvest quality control of mushrooms.